SOX9 in Immunity: Conserved Targets and Divergent Functions Across Species

Abstract

The transcription factor SOX9 is a pivotal, yet dualistic, regulator of the immune system, playing context-dependent roles in cancer immune escape, inflammatory diseases, and tissue repair. This article synthesizes foundational and contemporary research to explore the conservation of SOX9's immunological targets and functions across different species. We delve into the methodologies uncovering its genomic binding landscape, analyze challenges in translating findings due to functional divergence, and present a comparative validation of its role as a biomarker and therapeutic target in pan-cancers. Aimed at researchers and drug development professionals, this review underscores the necessity of a nuanced, cell-type-specific understanding of SOX9 for developing effective immunotherapeutic strategies.

SOX9: A Janus-Faced Transcription Factor in the Immune Landscape

Protein Structure and Functional Domains

SOX9 is a member of the SOX (SRY-related HMG-box) family of transcription factors, which share a highly conserved high mobility group (HMG) domain. This 509-amino acid protein contains several critical functional domains that determine its DNA-binding capacity, dimerization potential, and transcriptional activity [1] [2].

Table 1: SOX9 Protein Domains and Their Functions

Domain	Position	Key Functions	Molecular Interactions
Dimerization Domain (DIM)	N-terminal	Facilitates self-dimerization and heterodimerization	Enables DNA binding and transactivation of tissue-specific genes [2]
HMG Box	Central	Sequence-specific DNA binding, nuclear localization, DNA bending	Recognizes (A/T)(A/T)CAA(A/T)G motif; induces L-shaped DNA bending [1] [2]
Transactivation Domain Middle (TAM)	Middle	Synergistic transcriptional activation	Works with TAC to augment transcriptional potential [3]
Transactivation Domain C-terminal (TAC)	C-terminal	Primary transcriptional activation, β-catenin inhibition	Interacts with MED12, CBP/p300, TIP60, WWP2 coactivators [2]
PQA-rich Domain	C-terminal	Enhances transactivation	No autonomous transactivation capability [2]

The HMG domain serves dual roles: it facilitates DNA binding to the consensus sequence AGAACAATGG (with AACAAT as the core binding element) and directs nuclear localization through embedded nuclear localization and export signals [3] [2]. SOX9 can function as either a monomer or dimer, with dimerization occurring through the DIM domain and being essential for its function in chondrogenesis, while it operates as a monomer in testicular Sertoli cells [2].

Figure 1: SOX9 Domain Structure - Schematic representation of functional domains in the SOX9 protein

Fundamental Roles in Development

SOX9 serves as a master regulator of development across all three germ layers, with particularly critical functions in chondrogenesis, sex determination, and organogenesis. Its versatile functions are modulated through post-translational modifications, binding partner availability, and tissue-specific chromatin accessibility [1] [2].

Chondrogenesis and Skeletal Development

During skeletal development, SOX9 is essential for mesenchymal condensation prior to chondrogenesis and subsequent chondrocyte differentiation [1]. It activates numerous cartilage-specific genes including COL2A1, COL9A1, COL11A2, and Acan (aggrecan), while simultaneously repressing hypertrophic markers like COL10A1 just prior to chondrocyte hypertrophy [1] [2]. The essential nature of SOX9 in cartilage development is evidenced by its association with campomelic dysplasia, a human skeletal malformation syndrome, when mutated [1] [4].

Sex Determination

In male sexual development, SOX9 plays a pivotal role downstream of SRY (sex-determining region Y)[ccitation:9]. SOX9 activates FGF9 and forms feedforward loops with both FGF9 and PGD2 to promote Sertoli cell differentiation and testis cord formation [4]. With steroidogenic factor 1 (SF1), SOX9 regulates transcription of the anti-Müllerian hormone (AMH) gene, which is crucial for regression of Müllerian ducts in male embryonic development [4].

Organogenesis

SOX9 functions as a critical determinant in the development of multiple organs including pancreas, intestine, heart, lung, liver, and nervous system [2]. In the pancreas, SOX9 maintains pancreatic progenitor cells and regulates endocrine differentiation [2] [5]. Recent research has revealed that in mature pancreatic beta cells, SOX9 regulates alternative splicing, with its depletion disrupting splicing and impairing insulin secretion [5]. In the nervous system, SOX9 promotes gliogenesis and neural stem cell survival while negatively regulating neurogenesis [4].

Table 2: SOX9 Roles in Organ Development and Key Target Genes

Organ/Tissue	Key Developmental Functions	Validated Target Genes
Cartilage	Chondrogenic mesenchymal condensation, chondrocyte differentiation	SOX5, SOX6, COL2A1, ACAN, COMP [2]
Testis	Sertoli cell differentiation, repression of ovarian pathway	SOX8, SOX10, AMH, FGF9, GDNF [2]
Pancreas	Pancreatic progenitor maintenance, endocrine differentiation	NEUROG3, FGFR2B, PTF1A [2]
Intestine	Progenitor maintenance, epithelial cell differentiation	CDX2, LGR5 [1] [2]
Nervous System	Neural stem cell maintenance, glial specification, astrocyte differentiation	NFIA, PDGFRA [2]
Heart	Heart valve development, progenitor cell proliferation, ECM regulation	HAPLN1, ACAN, ELN, POSTN [2]

SOX9 in Immunity and Inflammation

SOX9 exhibits a dual, context-dependent role in immunology, functioning as a "double-edged sword" in immune regulation [3]. It participates in both innate and adaptive immunity, with significant implications for cancer immunotherapy and inflammatory diseases.

Immunomodulatory Functions

In cancer immunity, SOX9 frequently promotes immunosuppression by shaping the tumor microenvironment. Bioinformatics analyses reveal that SOX9 expression negatively correlates with infiltration of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, while positively correlating with neutrophils, macrophages, activated mast cells, and naive/activated T cells in colorectal cancer [3]. SOX9 overexpression negatively correlates with genes associated with CD8+ T cell function, NK cells, and M1 macrophages, while showing positive correlation with memory CD4+ T cells [3].

SOX9 contributes to immune evasion in latent cancer cells by maintaining cellular stemness, thereby preserving long-term survival and tumor-initiating capabilities while avoiding immune surveillance [6]. In glioblastoma, however, high SOX9 expression associates with better prognosis in specific subgroups and correlates with immune cell infiltration and checkpoint expression [7].

Role in Inflammatory Diseases and Tissue Repair

Beyond its immunosuppressive roles, SOX9 also contributes beneficially to tissue repair and regeneration. Increased SOX9 levels help maintain macrophage function and contribute to cartilage formation and tissue regeneration [3]. In osteoarthritis, SOX9 exhibits protective functions through its roles in cartilage maintenance and repair [3]. Prostaglandin E2 (PGE2) plays a role in immunomodulation and tissue regeneration by activating SOX9 expression in endogenous renal progenitor cells [6].

Figure 2: SOX9's Dual Immunological Roles - The transcription factor exhibits context-dependent pro- and anti-immunity functions

Conservation of SOX9 Targets Across Species

Comparative analyses of SOX9 binding patterns reveal significant conservation differences between developmental processes and across species, with important implications for translational research.

Cell Type-Specific Conservation Patterns

Chromatin immunoprecipitation sequencing (ChIP-seq) studies comparing SOX9 targets in mouse and chicken demonstrate that SOX9 binding regions show higher conservation in chondrogenesis than in gonad development [8] [9]. In developing limb buds, SOX9 predominantly binds to intronic and distal regions, with frequent SOX palindromic repeats identified in these binding regions [9]. Conversely, in male gonads, SOX9 binds more frequently to proximal upstream regions of genes with fewer palindromic SOX motifs [9].

The conservation of SOX9 binding regions is significantly higher in limb bud genes compared to male gonad genes [8] [9]. SOX9 target genes show high similarity in chondrocytes but not in Sertoli cells between mouse and chicken, indicating that the regulatory targets of SOX9 in testis development differ more substantially between species [9].

Table 3: Comparative Analysis of SOX9 Binding in Mouse and Chicken

Parameter	Limb Bud (Chondrogenesis)	Male Gonad (Sex Determination)
Binding Region Preference	Intronic and distal regions [9]	Proximal upstream regions [9]
SOX Palindromic Motif Frequency	19.65% of binding regions [9]	8.72% of binding regions [9]
Cross-Species Conservation	High conservation between mouse and chicken [8]	Lower conservation between mouse and chicken [8]
SOX9 Target Similarity	High similarity in chondrocytes [9]	Low similarity in Sertoli cells [9]

Experimental Approaches and Research Toolkit

Key Methodologies for SOX9 Research

Chromatin immunoprecipitation sequencing (ChIP-seq) has been instrumental in mapping SOX9 binding regions genome-wide. Typical protocols involve cross-linking cells with formaldehyde, chromatin shearing, immunoprecipitation with SOX9-specific antibodies, library preparation, and high-throughput sequencing [8] [9]. For comparative analyses across species, researchers typically perform ChIP-seq on homologous tissues (e.g., limb buds and gonads) from model organisms at equivalent developmental stages, followed by integrative analysis with RNA sequencing data to identify functional target genes [9].

Conditional gene knockout models utilizing Cre-loxP systems have been essential for delineating SOX9 functions in specific tissues and developmental stages. The Ins-Cre;Sox9fl/fl model enables deletion of Sox9 in insulin-positive cells, revealing its role in pancreatic beta cell function [5]. Similarly, the MIP-CreERT;Sox9-/- model allows tamoxifen-inducible deletion of Sox9 in adult beta cells, demonstrating its ongoing requirement in mature cells [5].

Essential Research Reagents

Table 4: Key Research Reagents for SOX9 Investigation

Reagent/Tool	Type	Research Applications	Key Functions
SOX9 Antibodies	Biological reagent	ChIP-seq, immunohistochemistry, Western blot	Target protein detection and localization [9]
Sox9-floxed mice (Sox9fl/fl)	Animal model	Conditional gene knockout studies	Tissue-specific Sox9 deletion [5]
Cre recombinase lines	Animal model	Cell-type specific gene manipulation	Spatial and temporal control of gene recombination [5]
Adenovirus-Cre vectors	Viral vector	In vitro gene deletion	Efficient Sox9 deletion in primary cells [5]
RNAscope probes	Molecular probe	Single-mRNA detection	Precise transcript localization [5]
N-Acetyltyramine Glucuronide-d3	N-Acetyltyramine Glucuronide-d3, CAS:1429623-59-5, MF:C₁₆H₁₈D₃NO₈, MW:358.36	Chemical Reagent	Bench Chemicals
1-Benzyl-1-methylbiguanide Hydrochloride	1-Benzyl-1-methylbiguanide Hydrochloride, CAS:2123-07-1, MF:C₁₀H₁₆ClN₅, MW:241.72	Chemical Reagent	Bench Chemicals

Figure 3: SOX9 Comparative Analysis Workflow - Experimental pipeline for studying SOX9 binding conservation across species and tissues

Concluding Perspectives

SOX9 represents a paradigm of transcriptional versatility, coordinating diverse developmental programs and immunological processes through context-dependent mechanisms. The conservation of its functions in chondrogenesis across vertebrate species contrasts with its divergent roles in sex determination, highlighting both conserved and evolutionarily plastic regulatory networks. In immunology, SOX9's dual nature as both promoter and suppressor of immune responses presents challenges and opportunities for therapeutic targeting.

Future research directions include elucidating the precise mechanisms governing SOX9's context-dependent functions, particularly its role in immune cell differentiation and function, and developing strategies to modulate its activity for cancer therapy and tissue regeneration. The integration of comparative genomics with functional studies across multiple species and tissue types will continue to reveal fundamental insights into this multifunctional transcription factor and its applications in biomedical research.

The transcription factor SOX9 exemplifies a paradigm of functional duality in immunology, acting as a critical regulator in both pathological and physiological processes. This review synthesizes recent evidence demonstrating how SOX9 facilitates tumor immune evasion while simultaneously promoting tissue repair and macrophage function. Through comparative analysis of experimental data across cancer, fibrotic, and infection models, we reveal how SOX9's context-dependent functions are governed by distinct molecular mechanisms and cellular partners. The conservation of these dual roles across species underscores SOX9's fundamental importance in immune regulation and highlights its potential as a therapeutic target for both oncology and regenerative medicine. Our integrated analysis provides a framework for understanding how SOX9 maintains immunological balance and how its dysregulation contributes to disease pathogenesis.

SOX9 (SRY-Box Transcription Factor 9), a member of the SOX family of transcription factors, has emerged as a critical player with opposing functions in immune regulation. Initially recognized for its roles in embryonic development, chondrogenesis, and sex determination, SOX9 is now increasingly appreciated for its complex immunological functions [10]. This transcription factor operates as a "double-edged sword" in immunity—on one hand promoting immune escape in cancer by impairing immune cell function, while on the other hand maintaining macrophage function and facilitating tissue regeneration and repair [10]. This review systematically examines SOX9's dual nature by comparing its mechanisms across disease contexts, analyzing experimental approaches for studying its functions, and evaluating its conservation across species. Understanding these opposing roles is crucial for developing targeted therapeutic strategies that can either inhibit or enhance SOX9 activity based on clinical context.

SOX9 Structure and Functional Domains: Implications for Immune Regulation

The functional complexity of SOX9 arises from its multi-domain structure, which enables diverse protein interactions and regulatory capacities. SOX9 contains several functionally specialized domains: a dimerization domain (DIM), a high mobility group (HMG) box DNA-binding domain, two transcriptional activation domains (TAM and TAC), and a proline/glutamine/alanine (PQA)-rich domain [10] [11]. The HMG domain facilitates sequence-specific DNA binding and nuclear localization through embedded nuclear localization signals [10]. The transcriptional activation domains TAM and TAC interact with various cofactors to enhance SOX9's transcriptional activity, while the PQA-rich domain stabilizes the protein and enhances transactivation [11]. Post-translational modifications, particularly phosphorylation at serine residues S64, S181, and S211, further regulate SOX9's activity and nuclear localization [11]. This structural complexity allows SOX9 to participate in diverse transcriptional programs depending on cellular context, contributing to its dual functions in immunity.

Table 1: SOX9 Protein Domains and Their Functional Roles in Immune Regulation

Domain	Location	Key Functions	Role in Immune Processes
Dimerization Domain (DIM)	N-terminal	Facilitates homo- and heterodimerization with SOXE proteins	Enables cooperative transcription with immune-related partners
HMG Box	Central	DNA binding, sequence recognition, nuclear localization	Binds to promoters of immune genes; contains NLS/NES for nucleocytoplasmic shuttling
Transactivation Domain Middle (TAM)	Central	Transcriptional activation through cofactor recruitment	Recruits transcriptional co-activators in immune cells
Transactivation Domain C-terminal (TAC)	C-terminal	Primary transcriptional activation domain	Critical for β-catenin inhibition during differentiation processes
PQA-rich Domain	C-terminal	Protein stabilization, enhancement of transactivation	Modulates protein stability in inflammatory environments

SOX9 as a Promoter of Immune Escape in Cancer

Mechanisms of SOX9-Mediated Immune Evasion

In the tumor microenvironment, SOX9 facilitates immune escape through multiple interconnected mechanisms. SOX9 enables tumor cells to evade immune surveillance by maintaining cancer stemness, regulating immune cell infiltration, and modulating immune checkpoint molecules. Research has demonstrated that SOX9 helps tumor cells maintain a stem-like state and evade innate immunity by remaining dormant for extended periods [12]. This dormancy allows latent cancer cells with high SOX2 and SOX9 expression to avoid immune monitoring under immunotolerant conditions, preserving their long-term survival and tumor-initiating capabilities [6]. Single-cell RNA sequencing analyses in head and neck squamous cell carcinoma (HNSCC) have revealed that SOX9+ tumor cells are significantly enriched in tumors resistant to combined anti-PD-1 and anti-LAG-3 immunotherapy [13]. Furthermore, SOX9 directly regulates annexin A1 (Anxa1) expression, which mediates apoptosis of formyl peptide receptor 1 (Fpr1)+ neutrophils through the Anxa1-Fpr1 axis [13]. This pathway promotes mitochondrial fission, inhibits mitophagy by downregulating Bnip3 expression, and ultimately prevents neutrophil accumulation in tumor tissues, creating an immunosuppressive microenvironment.

SOX9 Regulation of Tumor Immune Microenvironment

SOX9 significantly influences the composition and function of the tumor immune microenvironment. Bioinformatics analyses of data from The Cancer Genome Atlas reveal that SOX9 expression correlates with specific patterns of immune cell infiltration [10]. In colorectal cancer, SOX9 expression negatively correlates with infiltration levels of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, while showing positive correlation with neutrophils, macrophages, activated mast cells, and naive/activated T cells [10]. Similarly, in prostate cancer, SOX9 contributes to an "immune desert" microenvironment characterized by decreased effector immune cells (CD8+CXCR6+ T cells and activated neutrophils) and increased immunosuppressive cells (Tregs, M2 macrophages, and anergic neutrophils) [10]. This imbalance ultimately promotes tumor immune escape. The interaction between SOX9 and long-term androgen deprivation therapy in prostate cancer may further weaken anti-tumor immune responses by enriching a subpopulation of club cells characterized by high SOX9 and low AR expression [10].

Table 2: SOX9-Mediated Immune Evasion Mechanisms Across Cancer Types

Cancer Type	Immune Evasion Mechanism	Key Findings	Experimental Evidence
Head and Neck SCC	Anxa1-Fpr1 axis-mediated neutrophil apoptosis	SOX9+ tumor cells enriched in anti-PD-1/anti-LAG-3 resistant tumors; reduces Fpr1+ neutrophil accumulation	scRNA-seq, transgenic mouse models, flow cytometry [13]
Colorectal Cancer	Altered immune cell infiltration	Negative correlation with B cells, resting T cells; positive correlation with neutrophils, macrophages	TCGA data analysis, bioinformatics [10]
Prostate Cancer	Creation of "immune desert" microenvironment	Decreased CD8+CXCR6+ T cells; increased Tregs, M2 macrophages, anergic neutrophils	scRNA-seq, spatial transcriptomics [10]
Breast Cancer	Maintenance of cancer stemness and dormancy	SOX9 promotes immune evasion by sustaining stemness in latent cancer cells	In vitro models, animal studies [6]
Multiple Solid Tumors	Regulation of immune checkpoint molecules	Correlated with expression of various immune checkpoints	Pan-cancer analysis, correlation studies [14]

Experimental Models for Studying SOX9 in Cancer Immunity

Research on SOX9's role in cancer immune evasion employs diverse experimental approaches. The 4-nitroquinoline 1-oxide (4NQO)-induced HNSCC mouse model has been particularly valuable for studying resistance mechanisms to immunotherapy [13]. In this model, mice with established tumors are treated with combination immunotherapy (anti-PD-1 plus anti-LAG-3), and resistant versus sensitive tumors are compared using single-cell RNA sequencing, magnetic resonance imaging, and histopathological analysis [13]. Tumor size changes are monitored according to Response Evaluation Criteria in Solid Tumors (RECIST), with tumors growing more than 20% larger than original size classified as resistant [13]. For mechanistic studies, transgenic mouse models with conditional SOX9 knockout or overexpression in specific cell types, combined with flow cytometry analysis of immune cell populations, help elucidate SOX9's cell-autonomous versus non-autonomous functions in shaping the tumor immune landscape [13].

SOX9 as a Facilitator of Tissue Repair and Macrophage Function

SOX9 in Tissue Repair and Regeneration

Beyond its pathological role in cancer, SOX9 serves essential functions in tissue homeostasis and repair across multiple organ systems. In schistosomiasis-induced liver damage, SOX9 is progressively expressed in myofibroblasts within granulomas and surrounding hepatocytes following infection [15]. SOX9-deficient mice demonstrate significantly diminished granuloma size and fail to produce a robust extracellular matrix barrier around parasite eggs, resulting in more diffuse liver injury and scattered immune cell distribution [15]. This compromised barrier function leads to uncontrolled tissue damage, highlighting SOX9's critical role in containing injury and facilitating structured repair. Similarly, in hepatic stellate cells, SOX9 coordinates extracellular matrix production during fibrotic responses to various insults, including carbon tetrachloride (CCl4) exposure and bile duct ligation [15]. These findings position SOX9 as a central regulator of tissue integrity during damage response, with its absence resulting in disorganized repair processes and exacerbated tissue dysfunction.

SOX9 Regulation of Macrophage Function and Immune Homeostasis

SOX9 significantly influences macrophage populations and function during tissue repair. In schistosomiasis infection models, SOX9 loss alters hepatic immune cell composition, increasing neutrophil and monocyte proportions while expanding Ly6clo monocyte populations [15]. Infected SOX9-deficient mice also display exaggerated Type 2 inflammation with pronounced eosinophilia and reduced CD4+ T cell proportions [15]. These immunological disturbances correlate with defective granuloma formation and impaired tissue repair, suggesting that SOX9 helps maintain appropriate immune activation thresholds necessary for effective tissue restoration. The increased levels of SOX9 help maintain macrophage function, contributing to cartilage formation, tissue regeneration, and repair [10]. This immunomodulatory function extends beyond infection models, as prostaglandin E2 (PGE2) contributes to immunomodulation and tissue regeneration by activating SOX9 expression in endogenous renal progenitor cells [6], indicating a conserved mechanism across tissue types.

Experimental Approaches for Studying SOX9 in Tissue Repair

Investigating SOX9's protective functions requires specialized disease models and analytical techniques. Schistosoma mansoni infection in mice provides a well-established model for studying granulomatous inflammation and tissue repair [15]. In this system, researchers utilize global inducible SOX9-deficient mouse models combined with detailed histomorphometric analysis of granuloma size, extracellular matrix deposition (using picrosirius red staining), and immune cell characterization through immunohistochemistry and flow cytometry [15]. Lineage tracing approaches help identify SOX9-expressing cell populations, revealing that during infection, SOX9 is upregulated not only in activated hepatic stellate cells and cholangiocytes but also in injured hepatocytes, particularly at the periphery of granulomas [15]. These techniques demonstrate SOX9's multifunctional role across different cell types during tissue repair processes, with its expression progressively increasing as fibrosis establishes during the infection time course.

Table 3: SOX9 in Tissue Repair and Regeneration Across Disease Models

Disease/Injury Model	SOX9-Positive Cell Types	Repair Function	Consequence of SOX9 Loss
Schistosomiasis-induced liver damage	Myofibroblasts, hepatocytes, cholangiocytes	Granuloma organization, ECM barrier formation	Diminished granuloma size, diffuse liver injury, scattered immune cells [15]
Hepatic stellate cell activation	Activated hepatic stellate cells	Production of fibrotic ECM components	Reduced collagen deposition, decreased HSC activation [15]
Cartilage formation and OA	Chondrocytes, progenitor cells	Cartilage formation, maintenance	Impaired chondrogenesis, defective tissue repair [10]
Renal tissue regeneration	Renal progenitor cells	Progenitor cell proliferation, differentiation	Not specified (PGE2 activates SOX9) [6]

SOX9 Conservation Across Species: Implications for Immune Cell Research

The conservation of SOX9 across species underscores its fundamental role in biological processes, including immune regulation. The SOX9 gene has been mapped to human chromosome 17q and mouse chromosome 11q, with similar genomic organization in both species [11]. The protein's functional domains, particularly the HMG box DNA-binding domain, exhibit high evolutionary conservation, enabling cross-species functional studies [11]. This conservation permits researchers to utilize mouse models to investigate SOX9 functions with reasonable translational relevance to human biology. In both humans and mice, SOX9 operates within the SOXE subgroup alongside SOX8 and SOX10, sharing homologous regions in the HMG, DIM, TAM, and TAC domains that facilitate similar protein interactions and transcriptional regulatory functions across species [11]. The conservation extends to SOX9's role in immune processes, as demonstrated by similar immune-related phenotypes in SOX9-deficient mice and corresponding human pathological conditions, particularly in tissue repair and inflammation resolution processes.

Research Reagent Solutions for Studying SOX9 in Immunology

Investigating SOX9's dual immunological roles requires specialized research tools and experimental approaches. The table below summarizes key reagents and their applications in SOX9 immune function research.

Table 4: Essential Research Reagents for Investigating SOX9 Immune Functions

Reagent/Category	Specific Examples	Research Applications	Function in SOX9 Studies
Animal Models	Global inducible SOX9-deficient mice; Cell-specific SOX9 knockout mice	In vivo functional studies	Determine SOX9 requirement in immune processes across tissues [13] [15]
scRNA-seq Platforms	10X Genomics; Single-cell RNA sequencing	Tumor microenvironment analysis	Identify SOX9+ cell populations in resistant vs. sensitive tumors [13]
Immunotherapy Agents	Anti-PD-1; Anti-LAG-3; Combination therapy	Therapy resistance models	Study SOX9 role in immunotherapy resistance [13]
Infection Models	Schistosoma mansoni mouse infection	Tissue repair and fibrosis studies	Analyze SOX9 in granuloma formation and immune cell recruitment [15]
Flow Cytometry Antibodies	Immune cell markers: CD45, CD3, CD4, CD8, F4/80, Ly6G, etc.	Immune phenotyping	Characterize immune cell changes in SOX9-deficient settings [13] [15]
Histology Reagents	α-SMA antibodies; Picrosirius red; SOX9 IHC reagents	Tissue analysis	Localize SOX9 expression and assess fibrosis/repair [15]
Bioinformatics Tools	TCGA/GTEx data analysis; LinkedOmics; Metascape	Human data mining	Correlate SOX9 with immune signatures across cancers [10] [14]

Comparative Analysis: Contextual Factors Determining SOX9's Immunological Role

The opposing functions of SOX9 in immune escape versus tissue repair are determined by specific contextual factors including cell type, disease state, and microenvironmental cues. In cancer contexts, SOX9 predominantly localizes to malignant epithelial cells and promotes stemness, dormancy, and immunosuppression [13]. In contrast, during tissue repair and infection, SOX9 expression occurs predominantly in stromal cells (myofibroblasts, hepatic stellate cells) and parenchymal cells (hepatocytes) where it facilitates structured repair and immune coordination [15]. The duration of SOX9 expression also appears critical—transient SOX9 activation supports controlled tissue repair, while persistent SOX9 expression in chronic conditions (such as cancer or progressive fibrosis) drives pathology [10] [15]. The cellular partners of SOX9 differ between contexts; in cancer immune evasion, SOX9 interacts with ANXA1 to suppress neutrophils [13], while in tissue repair, it cooperates with extracellular matrix components and immune modulators to coordinate regeneration [15]. Understanding these contextual determinants is essential for developing targeted therapeutic strategies that can selectively inhibit SOX9's pathological functions while preserving or enhancing its beneficial roles.

The dual nature of SOX9 in immunology presents both challenges and opportunities for therapeutic development. In oncology, SOX9 inhibition represents a promising strategy to counteract immune evasion and enhance immunotherapy efficacy [13]. Conversely, in degenerative, fibrotic, or infectious diseases, carefully controlled SOX9 activation might promote proper tissue repair and immune regulation [10] [15]. Future research should focus on developing context-specific SOX9 modulators that can distinguish between its pathological and protective functions. The conservation of SOX9's dual roles across species supports the translational relevance of preclinical findings while highlighting the need for careful model selection in drug development. As our understanding of SOX9's immunological functions continues to evolve, this transcription factor represents a compelling target for manipulating immune responses in both cancer and regenerative medicine.

Visual Appendix: SOX9 Signaling Pathways

Diagram 1: SOX9 Signaling in Immune Evasion versus Tissue Repair. This diagram contrasts SOX9's pathological role in promoting cancer immunotherapy resistance through neutrophil suppression (top) with its protective role in facilitating organized tissue repair during infection (bottom).

Diagram 2: Experimental Approaches for Investigating SOX9 Immune Functions. This workflow outlines key disease models, analytical methods, and research applications for studying SOX9's dual immunological roles.

The SRY-box transcription factor 9 (SOX9) is a pivotal regulator of embryonic development, cell differentiation, and stem cell maintenance, with evolving roles in immunological processes. As a transcription factor containing a high mobility group (HMG) box DNA-binding domain, SOX9 recognizes specific DNA sequences and orchestrates complex gene expression programs [9] [16]. Beyond its well-characterized functions in chondrogenesis and sex determination, emerging evidence positions SOX9 as a significant modulator of immune cell biology within the tumor microenvironment and inflammatory contexts [3]. This transcription factor exhibits a dual nature in immune regulation—acting as both an activator and repressor across different immune cell lineages—thereby contributing to the intricate balance of immune responses [3]. Its expression and function in T cells, B cells, and macrophages reveal a complex picture of cell type-specific regulation that influences both developmental processes and pathological conditions, including cancer immunology and inflammation.

The conservation of SOX9 targets across species presents a fascinating aspect of its biology. While SOX9's functions in chondrogenesis are well conserved, its regulatory targets exhibit both conservation and divergence in different cellular contexts [9]. Understanding SOX9's immunoregulatory functions provides not only fundamental biological insights but also potential therapeutic avenues for manipulating immune responses in disease states, particularly in oncology where SOX9 contributes to the immunosuppressive tumor microenvironment [17] [14].

Comparative Analysis of SOX9 Across Immune Cell Lineages

Table 1: SOX9 Expression and Functional Roles Across Immune Cell Lineages

Immune Cell Lineage	SOX9 Expression Level/Pattern	Primary Functional Role	Key Target Genes/Pathways	Experimental Evidence
T Cells	Cooperates with c-Maf in specific subsets [3]	Modulates lineage commitment of early thymic progenitors; influences αβ vs. γδ T cell balance [3]	Activates Rorc, Il17a, and Blk (key Tγδ17 effector genes) [3]	Genetic manipulation in murine models [3]
B Cells	High in Germinal Center B Cells (GCB): 6.75 RPKM vs. 0.29 in naïve B cells [18]	Critical for germinal center reaction and terminal differentiation; loss may promote lymphomagenesis [18]	Binds enhancers of PRDM1, CCND2, CDC25B, BCOR, DNMT3A [18]	ChIP-seq in human tonsilar GCB; RNAi in mouse BCL1 lymphoma cells [18]
Macrophages	Not directly expressed but induces M2 polarization via TGF-β secretion [19]	Drives M2 polarization and creates immunosuppressive microenvironment [19]	TGF-β secretion; promotes SOX9-dependent EMT in cancer cells via C-jun/SMAD3 [19]	Co-culture of lung cancer cells with macrophages; clinical NSCLC sample analysis [19]

Table 2: SOX9-Associated Immune Correlations in Human Cancers

Cancer Type	Correlation with Immune Cell Infiltration	Association with Immune Checkpoints	Clinical/Prognostic Significance
Colorectal Cancer	Negative correlation: B cells, resting mast cells, resting T cells, monocytes, plasma cells, eosinophils [3]	Not specified	SOX9 as characteristic gene for early and late diagnosis [3]
Pan-Cancer Analysis	Positive correlation: neutrophils, macrophages, activated mast cells, naive/activated T cells [3]	Negative correlation with CD8+ T cell, NK cell, M1 macrophage function genes; positive with memory CD4+ T cells [3]	High SOX9 associated with worst OS in LGG, CESC, THYM [17]
Glioblastoma	Correlated with immune infiltration and checkpoint expression [14]	Involved in immunosuppressive tumor microenvironment [14]	Diagnostic and prognostic biomarker, particularly in IDH-mutant cases [14]
Prostate Cancer	"Immune desert" microenvironment: decreased CD8+CXCR6+ T cells, increased Tregs and M2 macrophages [3]	Androgen deprivation therapy enriches SOX9-high club cells with low AR [3]	Promotes tumor immune escape [3]

Molecular Mechanisms and Signaling Pathways

The TGF-β/SOX9 Axis in Macrophage-Mediated Tumor Progression

The interaction between tumor-associated macrophages (TAMs) and SOX9 expression in cancer cells represents a critical pathway in tumor progression. In non-small cell lung cancer (NSCLC), TAMs secrete transforming growth factor-beta (TGF-β), which activates the C-jun/SMAD3 pathway in cancer cells, leading to increased SOX9 expression [19]. This upregulation of SOX9 promotes epithelial-to-mesenchymal transition (EMT), a key process in tumor metastasis characterized by loss of E-cadherin and gain of vimentin expression [19]. The SOX9-dependent EMT enhances tumor cell proliferation, migration, and invasion capabilities. Crucially, knockdown experiments demonstrate that SOX9 is essential for this TGF-β-mediated EMT phenotype, as its inhibition prevents the morphological and molecular changes associated with EMT, reducing tumor cell migration and invasion [19].

This pathway forms a vicious cycle in the tumor microenvironment. Lung cancer cells promote M2 polarization in macrophages, indicated by increased secretion of TGF-β and IL-10, while TAMs in turn enhance SOX9 expression in cancer cells [19]. Clinical validation of this pathway comes from analysis of NSCLC patient samples, revealing a positive correlation between TAM density (CD163+ macrophages) and SOX9 expression in tumor cells [19]. Patients with high co-expression of both CD163 and SOX9 experience significantly shorter overall and disease-free survival compared to those with low expression of either marker alone, underscoring the clinical relevance of this pathway [19].

Diagram Title: TGF-β/SOX9 Axis in Macrophage-Mediated Cancer Progression

SOX9 in Germinal Center B Cell Differentiation

In B cell biology, SOX9 plays a crucial role in germinal center reactions and terminal differentiation. Comprehensive enhancer profiling revealed SOX9 as a novel transcription factor in germinal center B cells (GCB), with its expression dramatically increased more than 20-fold in GCB (6.75 RPKM) compared to naïve B cells (0.29 RPKM) [18]. SOX9 binds to 1,668 upstream distal enhancer regions associated with 963 genes, regulating critical pathways including cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2), and MAPK signaling (MAP2K3, MAP3K7) [18].

A particularly significant SOX9 target is PRDM1, a transcription factor that controls the transition from GCB to plasma cells, positioning SOX9 as a regulator of B cell terminal differentiation [18]. Interestingly, SOX9 expression is lost in most diffuse large B cell lymphoma (DLBCL) cell lines and primary malignant non-Hodgkin's lymphoma cases [18]. Functional experiments demonstrate that SOX9 knockdown increases colony-forming ability in mouse BCL1 lymphoma cells, suggesting that loss of SOX9 expression may contribute to lymphomagenesis by potentially blocking terminal differentiation of mature GCB [18].

Experimental Approaches and Methodologies

Key Experimental Protocols for Studying SOX9 in Immune Regulation

Chromatin Immunoprecipitation Sequencing (ChIP-seq) has been instrumental in mapping SOX9 binding sites across immune cell genomes. The standard protocol involves cross-linking proteins to DNA, chromatin shearing, immunoprecipitation with SOX9-specific antibodies, and high-throughput sequencing [9] [18]. In B cell studies, this approach identified SOX9 binding to distal enhancer regions -5 to -100 kb from transcription start sites [18]. For data analysis, MEME-ChIP and DREME software packages are used for de novo motif discovery within SOX9-bound regions, revealing enriched DNA motifs including consensus SOX binding sequences ((A/T)(A/T)CAA(A/T)G) and CAGA repeats [9].

Macrophage-Cancer Cell Co-culture Systems have elucidated the TGF-β/SOX9 axis. The typical methodology involves using human monocytic THP-1 cells differentiated into macrophages, which are then co-cultured with lung adenocarcinoma cell lines (A549 and H1299) either directly or using macrophage-conditioned media [19]. After 24-48 hours of co-culture, cancer cells typically transition to an EMT-like phenotype. The experimental readouts include Western blot analysis for SOX9, E-cadherin, and vimentin protein expression; RT-qPCR for mRNA quantification; and functional assays for cell migration and invasion [19]. To specifically test TGF-β involvement, researchers use recombinant TGF-β and TGF-β receptor inhibitors to confirm pathway specificity [19].

RNA Interference for SOX9 Knockdown has been employed to establish causal relationships in multiple immune-related contexts. In macrophage studies, SOX9 knockdown in lung cancer cells using RNAi prevented TGF-β-mediated EMT, maintaining epithelial characteristics despite macrophage co-culture [19]. In lymphoma research, shRNA-mediated SOX9 knockdown in mouse BCL1 lymphoma cells enhanced colony-forming ability in methylcellulose assays, supporting its role as a potential tumor suppressor in lymphomagenesis [18]. These experiments typically include controls for off-target effects and measurement of knockdown efficiency via Western blot and qPCR.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for Studying SOX9 in Immune Cells

Reagent Category	Specific Examples	Application/Function
Cell Lines	THP-1 (human monocytic), A549/H1299 (lung adenocarcinoma), BCL1 (mouse lymphoma), 22RV1/PC3 (prostate cancer) [17] [19] [18]	In vitro modeling of SOX9-immune interactions; transformation assays
Antibodies	Anti-SOX9 (ChIP-seq, WB, IHC), Anti-CD163 (TAM marker), Anti-E-cadherin (EMT marker), Anti-vimentin (EMT marker) [17] [19]	Protein detection, localization, and quantification; cell phenotyping
Cytokines/Chemicals	Recombinant TGF-β, TGF-β receptor inhibitors, Cordycepin (CD) [17] [19]	Pathway activation/inhibition; SOX9 expression modulation
Animal Models	Ins-Cre;Sox9fl/fl mice, MIP-CreERT;Sox9-/- mice, Xenograft models [17] [5]	In vivo functional validation; glucose tolerance testing
3,6-Bis-O-benzyl-D,L-myo-inositol	3,6-Bis-O-benzyl-D,L-myo-inositol, CAS:111408-68-5, MF:C₂₀H₂₄O₆, MW:360.4	Chemical Reagent
2,2'-(1,2-Diaminoethane-1,2-diyl)diphenol	2,2'-(1,2-Diaminoethane-1,2-diyl)diphenol Research Chemical	High-purity 2,2'-(1,2-Diaminoethane-1,2-diyl)diphenol for research applications. This product is For Research Use Only (RUO). Not for human or veterinary use.

Cross-Species Conservation of SOX9 Functions

The conservation of SOX9 targets exhibits remarkable cell type specificity. Comparative ChIP-seq analyses between mouse and chicken embryos demonstrated that SOX9 binding regions in chondrocytes show high conservation, while those in Sertoli cells show significantly lower conservation [9]. This pattern suggests that SOX9's regulatory functions in immune cells may also exhibit species-specific differences, an important consideration for translational research.

The structural and functional domains of SOX9 itself are highly conserved across species [16]. The protein contains several critical domains: a dimerization domain (DIM), the HMG box DNA-binding domain, two transcriptional activation domains (TAM and TAC), and a proline/glutamine/alanine (PQA)-rich domain [3] [16]. The HMG domain facilitates both DNA binding and nuclear localization, while the transcriptional activation domains interact with various cofactors to regulate target gene expression [3]. This structural conservation underscores SOX9's fundamental role in developmental and regulatory processes across vertebrate species.

Discussion and Therapeutic Implications

The dual nature of SOX9 in immune regulation presents both challenges and opportunities for therapeutic intervention. In cancer contexts, SOX9 primarily exhibits oncogenic properties, promoting immune escape through multiple mechanisms [17] [3]. Its expression correlates with immunosuppressive microenvironments across various cancers, characterized by reduced cytotoxic T cells and increased regulatory T cells and M2 macrophages [19] [3]. This makes SOX9 an attractive target for overcoming immunosuppression in oncology.

The natural compound cordycepin (CD), an adenosine analog, demonstrates promising SOX9-targeting effects. Experimental studies show that CD inhibits both protein and mRNA expression of SOX9 in a dose-dependent manner in prostate cancer (22RV1, PC3) and lung cancer (H1975) cell lines [17]. This SOX9 inhibition likely contributes to CD's documented anticancer effects, suggesting a potential therapeutic strategy for targeting SOX9 in immune-evasive cancers.

For inflammatory diseases and tissue repair, the picture is more complex. SOX9 contributes to maintaining macrophage function and promotes cartilage formation and tissue regeneration [3]. This beneficial role in tissue homeostasis suggests that therapeutic SOX9 modulation would require careful context-specific approaches—inhibition in cancer versus potential activation in degenerative conditions. Further research is needed to develop cell type-specific delivery systems that can leverage SOX9's diverse functions for therapeutic benefit while minimizing off-target effects.

The conservation of SOX9's structural domains across species, coupled with the cell type-specific conservation of its targets, highlights both challenges and opportunities for drug development. While the core molecular machinery is conserved, species-specific differences in regulatory networks necessitate careful preclinical model selection. As research continues to unravel the complexities of SOX9 in immune regulation, its potential as a therapeutic target across cancer, inflammatory diseases, and tissue regeneration continues to grow.

The transcription factor SOX9 serves as a master regulator in multiple developmental pathways, with two of its most critical roles being chondrogenesis and gonad development. Despite its fundamental importance across vertebrates, the evolutionary conservation of its functions differs remarkably between these two biological processes. This guide provides a detailed comparison of SOX9's conserved role in skeletal development versus its divergent functions in sex determination, drawing primarily from experimental evidence in mouse and chicken models. The objective analysis presented herein focuses on comparative genomic and molecular studies to elucidate the mechanisms underlying this functional dichotomy, providing essential context for researchers investigating SOX9 in immune cells and other systems where its conservation patterns may inform experimental design and therapeutic targeting.

Comparative Analysis of SOX9 Binding Landscapes

Cell Type-Specific SOX9 Binding Patterns

Comprehensive chromatin immunoprecipitation sequencing (ChIP-seq) analyses of developing mouse and chicken embryos have revealed fundamental differences in how SOX9 interacts with the genome across tissue types. These studies demonstrate that SOX9 exhibits tissue-specific binding patterns that are remarkably consistent across species, suggesting evolutionary constraints that operate differently in chondrogenesis versus gonad development.

Table 1: Genomic Distribution of SOX9 Binding Regions in Mouse and Chicken [9]

Genomic Location	Mouse Limb Bud (%)	Mouse Male Gonad (%)	Chicken Limb Bud (%)	Chicken Male Gonad (%)
0-10 kb Upstream	32.4	51.9	Similar to mouse	Similar to mouse
Intronic Regions	Higher than gonads	Lower than limb buds	Higher than gonads	Lower than limb buds
Distal/Other Regions	Higher than gonads	Lower than limb buds	Higher than gonads	Lower than limb buds

The consistent pattern across both species reveals that SOX9 favors intronic and distal regulatory regions in chondrogenic contexts, while preferentially binding to proximal promoter regions in gonadal contexts. This fundamental difference in genomic engagement likely contributes to the differential conservation of SOX9 functions across tissues.

DNA Binding Motif Conservation

Further illuminating the conservation differences, analysis of enriched DNA sequences within SOX9 binding regions reveals distinct motif preferences between tissue types:

Table 2: SOX9 Binding Motif Characteristics Across Tissues and Species [9]

Binding Motif Feature	Limb Bud (Both Species)	Male Gonad (Both Species)
SOX Palindromic Motif	19.65% frequency	8.72% frequency
Consensus SOX Motif	Present (E-value: 1.8e-25 in mouse)	Present (E-value: 7.5e-10 in mouse)
CAGA Repeats	Identified in both species	Identified in both species
CCAAT Motif	Present (E-value: 4.8e-17 in mouse)	Present (E-value: 1.6e-53 in mouse)

The significantly higher frequency of SOX palindromic repeats in limb bud genes across both species indicates conserved homodimer formation in chondrogenesis, while gonad development appears to utilize different protein complex formations.

SOX9 Regulatory Divergence Between Tissues

Functional Conservation in Chondrogenesis

Conserved Transcriptional Programs

The high conservation of SOX9 function in chondrogenesis is evidenced by its maintained regulatory relationships with core cartilage-specific genes across evolutionary distance. In both mouse and chicken models, SOX9 demonstrates conserved binding patterns at key chondrogenic loci including COL2A1 (collagen type II alpha 1 chain), COL11A2 (collagen type XI alpha 2 chain), and HAPLN1 (hyaluronan and proteoglycan link protein 1) [9]. These genes encode critical components of the cartilage extracellular matrix and their regulation by SOX9 appears indispensable for proper skeletal development across vertebrate species.

The mechanistic basis for this conservation involves SOX9's role as a pioneer transcription factor capable of binding closed chromatin and initiating chromatin remodeling events that activate the chondrogenic program [20]. This pioneering activity appears to be a deeply conserved feature of SOX9 function in skeletal development, as evidenced by studies in zebrafish demonstrating similar regulatory relationships [9].

Experimental Evidence from Cross-Species Studies

Direct comparative analysis of SOX9 target genes in developing chondrocytes of mouse and chicken reveals significant overlap, with the majority of core cartilage matrix genes maintaining SOX9 regulation across this evolutionary distance [9]. The conservation of SOX9 binding regions was quantitatively demonstrated to be significantly higher in limb bud genes compared to gonad genes, with phylogenetic analyses showing stronger sequence conservation in chondrocyte-specific enhancers compared to testis-specific regulatory elements [9] [8].

Divergent Functions in Gonad Development

Evolutionary Plasticity in Sex Determination

While SOX9 maintains a critical role in testis development across vertebrates, its specific functions, regulatory targets, and position within sex determination cascades show remarkable evolutionary divergence. The contrast between conserved chondrogenic function and divergent gonad development roles illustrates the evolutionary plasticity of transcription factor networks in reproductive biology compared to structural development.

In mammals, SOX9 lies directly downstream of SRY (sex-determining region Y) and activates the expression of AMH (anti-Müllerian hormone) and other genes essential for testicular differentiation [16] [21]. However, in birds, which lack SRY, AMH expression actually precedes SOX9 upregulation during gonad development, suggesting a repositioning within the regulatory hierarchy [9]. This represents a fundamental rewiring of the sex determination network despite SOX9's maintained importance in testis differentiation.

Species-Specific Target Gene Regulation

Comprehensive analysis combining ChIP-seq with RNA expression data from Sertoli cells at equivalent developmental stages in mouse and chicken reveals low similarity in SOX9 target genes [9] [8]. While both species utilize SOX9 to promote testis development, the specific genomic targets and regulated genes show considerable divergence, indicating different mechanistic implementations of a conserved overall outcome.

This target gene divergence is further evidenced by studies in non-avian/mammalian systems. In medaka fish (Oryzias latipes), SOX9 ortholog knockout actually causes female-to-male sex reversal, directly opposing the mammalian phenotype [9]. Similarly, in Xenopus tropicalis, SOX9 is not expressed during testis development at all [9], demonstrating that even the necessity of SOX9 for testis formation varies across vertebrates.

Experimental Approaches and Methodologies

Key Experimental Protocols

The comparative analyses referenced in this guide primarily employed cutting-edge genomic techniques to map SOX9 interactions and functions across species and tissues:

Chromatin Immunoprecipitation Sequencing (ChIP-seq):

• Purpose: Genome-wide identification of SOX9 binding regions in specific tissues and developmental stages [9]
• Typical Protocol: Crosslink proteins to DNA → shear chromatin → immunoprecipitate with SOX9-specific antibody → reverse crosslinks → purify and sequence DNA → map reads to reference genome to identify enriched regions [9]
• Key Considerations: Use of validated, specific antibodies; appropriate controls (input DNA, IgG controls); biological replicates; species-specific genome alignment

RNA Sequencing (RNA-seq) for Target Gene Identification:

• Purpose: Transcriptome profiling to identify SOX9-dependent genes when combined with ChIP-seq data [9]
• Typical Protocol: Isolate RNA from specific cell populations (e.g., FACS-purified chondrocytes or Sertoli cells) → prepare sequencing libraries → high-throughput sequencing → differential expression analysis [9]
• Integration with ChIP-seq: Combined analysis identifies direct SOX9 targets versus indirectly regulated genes

Cross-Species Comparative Genomics:

• Purpose: Assessment of evolutionary conservation of SOX9 binding regions and target genes [9]
• Methodologies: Sequence alignment of regulatory regions; phylogenetic footprinting; motif conservation analysis; synteny analysis [9] [22]

SOX9 Comparative Analysis Workflow

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Research Reagents for SOX9 Functional Studies

Reagent/Category	Specific Examples	Function/Application
SOX9 Antibodies	Validated ChIP-grade anti-SOX9	Immunoprecipitation for ChIP-seq; immunohistochemistry; Western blot
Animal Models	Mouse (Mus musculus), Chicken (Gallus gallus), Japanese flounder (Paralichthys olivaceus)	Cross-species comparative studies; evolutionary analysis
Cell Isolation Systems	Fluorescence-activated cell sorting (FACS)	Purification of specific cell types (chondrocytes, Sertoli cells)
Genomic Tools	Species-specific genome assemblies; chromatin state annotations	Reference for sequencing alignment; epigenetic context interpretation
Small Molecule Inhibitors	Cordycepin (adenosine analog)	SOX9 expression modulation; functional perturbation studies [17]
Di-2-thienylglycolic Acid Potassium Salt	Di-2-thienylglycolic Acid Potassium Salt, MF:C₁₀H₇KO₃S₂, MW:278.39	Chemical Reagent
21-Dehydro Betamethasone 17-Propionate	21-Dehydro Betamethasone 17-Propionate|C25H31FO6	21-Dehydro Betamethasone 17-Propionate (C25H31FO6), a betamethasone impurity. Explore its research applications. For Research Use Only. Not for human or veterinary use.

Implications for Immune Cell Research

The dual nature of SOX9 as both a conserved developmental regulator and a context-dependent transcriptional modulator has significant implications for its roles in immunity. Recent evidence positions SOX9 as a "double-edged sword" in immunology, mirroring its tissue-dependent functions in development [3].

In cancer immunology, SOX9 frequently exhibits oncogenic properties, promoting tumor immune escape by impairing immune cell function [3]. Bioinformatic analyses reveal SOX9 expression negatively correlates with infiltration of B cells, resting T cells, monocytes, and plasma cells in colorectal cancer, while positively correlating with neutrophils, macrophages, and activated mast cells [3]. This immune modulatory capacity may reflect SOX9's evolutionary plasticity in gonad development, where its specific functions are adaptable to cellular context.

Conversely, in inflammatory diseases like osteoarthritis, SOX9 demonstrates protective functions, maintaining macrophage function and promoting tissue repair [3]. This dichotomy suggests that SOX9's conserved structural role in chondrogenesis may extend to maintenance of tissue integrity in inflammatory contexts. The differential conservation patterns observed in developmental systems may thus inform mechanistic studies of SOX9 in immunity, particularly regarding its potential as a therapeutic target in both cancer and inflammatory diseases.

The comparative analysis of SOX9 function in mouse and chicken models reveals a fundamental principle in evolutionary biology: transcription factors can maintain highly conserved roles in some developmental processes while exhibiting significant functional divergence in others. SOX9's deep conservation in chondrogenesis contrasts sharply with its evolutionary plasticity in gonad development, providing a powerful paradigm for understanding how gene regulatory networks evolve. For researchers investigating SOX9 in immune contexts, these developmental patterns offer valuable insights into its potential as both a therapeutic target and biomarker, with the recognition that its functions will likely be highly context-dependent and may exhibit both conserved and species-specific elements in different immunological processes.

Unveiling the SOX9 Regulome: From ChIP-seq to Single-Cell RNA Sequencing

The transcription factor SOX9 plays critical roles in development, cancer, and immunity, yet its genome-wide binding landscape varies significantly across cellular and species contexts. Chromatin Immunoprecipitation Sequencing (ChIP-seq) has emerged as a powerful tool for mapping SOX9 targets, revealing both conserved and cell-type-specific regulatory networks. This guide compares experimental approaches for SOX9 target identification, analyzes the conservation of SOX9 targets between species and cell types, and examines the implications for immune regulation and cancer biology. We integrate quantitative data from multiple studies to provide a structured comparison of SOX9 binding patterns in immune versus non-immune contexts, along with detailed methodological protocols for researchers investigating SOX9-mediated transcriptional programs.

SOX9 (SRY-related HMG-box 9) is a pivotal transcription factor belonging to the SOX family of proteins characterized by a highly conserved high-mobility group (HMG) box domain that facilitates DNA binding [7] [3]. This domain encodes a 79-amino-acid region that recognizes specific DNA sequences, enabling SOX9 to function as a key transcriptional regulator in diverse biological processes [7]. Structurally, SOX9 contains several functional domains organized from N- to C-terminus: a dimerization domain (DIM), the HMG box domain, two transcriptional activation domains (TAM and TAC), and a proline/glutamine/alanine (PQA)-rich domain [3]. The HMG domain directs nuclear localization and facilitates DNA binding, while the transcriptional activation domains interact with various cofactors to enhance SOX9's transcriptional activity [3].

Beyond its established roles in embryonic development, chondrogenesis, and sex determination, SOX9 has gained significant attention for its functions in cancer biology and immune regulation [3] [12]. SOX9 exhibits context-dependent dual functions—acting as both an activator and repressor—across diverse cell types, contributing to the regulation of numerous biological processes [3]. In cancer, SOX9 is frequently overexpressed and plays crucial roles in tumor progression, stemness maintenance, therapy resistance, and immune evasion [7] [3] [23]. The complex biology of SOX9, particularly its emerging role in immunological processes, necessitates precise genome-wide mapping of its binding sites to understand its mechanistic actions across different biological contexts.

ChIP-seq methodology for SOX9 target identification

Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq) provides a powerful method for identifying genome-wide binding sites of transcription factors like SOX9. Below, we detail the core experimental protocol and key methodological considerations for SOX9 ChIP-seq studies.

Core experimental protocol

The standard ChIP-seq protocol for SOX9 involves crosslinking cells to preserve protein-DNA interactions, followed by chromatin fragmentation, immunoprecipitation with SOX9-specific antibodies, and library preparation for high-throughput sequencing. A dual-crosslinking approach using both disuccinimidyl glutarate (DSG) and formaldehyde has been shown to improve the efficiency of chromatin immunoprecipitation for chromatin-binding proteins, potentially offering advantages for SOX9 studies [24]. This method enhances the preservation of protein-DNA interactions by first using a reversible amine crosslinker (DSG) followed by formaldehyde crosslinking.

After crosslinking, chromatin is typically fragmented to 200-500 bp fragments using sonication or enzymatic digestion. Immunoprecipitation is then performed using validated SOX9 antibodies, followed by decrosslinking, DNA purification, and library construction. Critical quality control steps include measuring DNA concentration and fragment size distribution, while experimental validation often involves quantitative PCR at known binding sites before proceeding to sequencing.

Methodological variations and considerations

Research indicates significant methodological variations in SOX9 ChIP-seq protocols across different biological contexts. A comparative analysis of SOX9 binding in developing limb buds and male gonads from mouse and chicken embryos revealed that SOX9 binding patterns differ substantially between tissue types [25]. In both species, SOX9 bound to intronic and distal regions more frequently in limb buds, while proximal upstream regions were preferentially targeted in male gonads [25].

The resolution of binding site mapping can be enhanced through complementary assays. For instance, CUT&RUN (Cleavage Under Targets and Release Using Nuclease) sequencing has been employed to temporally assay SOX9 binding to chromatin during cellular reprogramming events [20]. This approach demonstrated that SOX9 can bind to closed chromatin before accessibility changes, highlighting its potential pioneer factor activity [20].

Table 1: Key Methodological Variations in SOX9 Genomic Mapping Studies

Method	Biological Context	Key Findings	Advantages
ChIP-seq	Limb buds vs. male gonads (mouse, chicken)	Cell-type-specific binding patterns; SOX palindromic repeats more frequent in limb buds [25]	Comprehensive genome-wide coverage
CUT&RUN	Epidermal stem cell reprogramming	SOX9 binds closed chromatin before accessibility changes [20]	Higher resolution; requires fewer cells
ATAC-seq combined with TF perturbation	Facial progenitor cells (CNCCs)	Identified dosage-sensitive SOX9 targets [26] [27]	Reveals functional, dosage-sensitive targets
Multi-omics integration	Ovarian cancer	SOX9 binds promoters of DNA damage repair genes [28]	Connects binding to functional outcomes

Comparative analysis of SOX9 targets across species and cell types

Conservation patterns in non-immune contexts

Comparative ChIP-seq analyses between mouse and chicken embryos have revealed intriguing patterns of SOX9 target conservation across species. In developing limb buds, which represent a classical non-immune context, SOX9 binding regions show significantly higher conservation between mouse and chicken compared to male gonads [25]. This suggests that the regulatory functions of SOX9 in chondrogenesis are more evolutionarily conserved than its roles in sex determination.

The genomic distribution of SOX9 binding sites also varies by cell type. In both species, SOX9 preferentially binds intronic and distal regions in limb buds more frequently than in male gonads, where proximal upstream regions are more commonly targeted [25]. Additionally, SOX palindromic repeats were identified more frequently in SOX9 binding regions associated with limb bud genes compared to male gonad genes, suggesting distinct mechanistic modes of DNA recognition and binding in different tissue contexts [25].

Integration of ChIP-seq data with transcriptomic profiling (RNA sequencing) in developing chondrocytes and Sertoli cells confirmed that SOX9 target genes exhibit high similarity in chondrocytes but not in Sertoli cells between the two species [25]. This fundamental difference in target conservation has important implications for extrapolating findings from model organisms to human biology, particularly for musculoskeletal development versus reproductive biology.

SOX9 targets in immune and cancer contexts

While comprehensive ChIP-seq data specifically mapping SOX9 targets in immune cells remains limited in the available literature, numerous studies have indirectly elucidated SOX9's immunological roles through functional and bioinformatic analyses. In cancer biology, SOX9 has been implicated in regulating genes that shape the tumor immune microenvironment. ChIP-seq analysis in ovarian cancer cells revealed that SOX9 binds to promoters of key DNA damage repair genes (SMARCA4, UIMC1, and SLX4), thereby regulating DNA damage processes [28]. This function has significant implications for therapy resistance and potentially for antigen presentation in immune responses.

SOX9 also plays a role in immune evasion mechanisms, though the direct targets mediating these effects remain to be fully mapped. Evidence suggests that SOX9 expression correlates with immune checkpoint expression and immune cell infiltration in various cancers [7] [12]. In glioblastoma, SOX9 expression correlates with immune cell infiltration and expression of immune checkpoints, indicating its involvement in the immunosuppressive tumor microenvironment [7]. Furthermore, SOX9 helps tumor cells maintain a stem-like state and evade innate immunity by remaining dormant for extended periods [12].

Table 2: Conservation of SOX9 Targets Across Biological Contexts

Biological Context	Level of Conservation	Key Genomic Features	Functional Implications
Chondrogenesis (limb buds)	High between mouse and chicken	Preferential binding to intronic/distal regions; SOX palindromic repeats [25]	conserved regulatory programs in skeletal development
Sex Determination (male gonads)	Low between mouse and chicken	Preferential binding to proximal upstream regions [25]	Species-specific regulatory mechanisms
Cancer Contexts	Varies by cancer type	Promoter binding of DDR genes; enhancer binding in stem-like cells [23] [28]	Therapy resistance; immune modulation
Immune Regulation	Limited direct data	Correlation with immune gene expression [7] [12]	Tumor immune evasion; microenvironment shaping

Dosage sensitivity of SOX9 targets

Recent studies have revealed that SOX9 exhibits dosage-sensitive effects, with important implications for both developmental disorders and cancer. Innovative approaches combining degradation tag (dTAG) systems with ATAC-seq have enabled precise modulation of SOX9 levels in human embryonic stem cell-derived cranial neural crest cells (CNCCs), allowing identification of dosage-sensitive targets [26] [27].

Features of dosage-sensitive SOX9 targets

Research indicates that most SOX9-dependent regulatory elements are buffered against small decreases in SOX9 dosage, but a subset shows heightened sensitivity [26]. These sensitive regulatory elements are often directly and primarily regulated by SOX9 and are enriched for specific sequence features. Low-affinity binding motifs distributed throughout regulatory elements drive sensitive responses, while high-affinity motifs that allow for heterotypic transcription factor co-binding tend to buffer against quantitative changes in SOX9 dosage [27].

Dosage-sensitive SOX9 targets are functionally significant, as they preferentially affect chondrogenesis and craniofacial development [26]. In fact, sensitive regulatory elements and genes are associated with Pierre Robin sequence (PRS)-like craniofacial shape variation, explaining why specific phenotypes are particularly sensitive to SOX9 dosage reduction [26]. This dosage sensitivity also extends to cancer contexts, where subtle changes in SOX9 levels can drive significant transcriptional reprogramming toward stem-like states associated with therapy resistance [23].

Pioneer factor activity and chromatin remodeling

SOX9 exhibits pioneer factor activity, enabling it to bind cognate motifs in closed chromatin and initiate fate switching in stem cells [20]. During cellular reprogramming, SOX9 binding to closed chromatin occurs before accessibility changes, with nearly 30% of SOX9 binding sites located in closed chromatin prior to activation [20]. This pioneer activity involves SOX9 recruiting histone and chromatin modifiers to remodel and open chromatin for transcription, while simultaneously redistributing co-factors away from previous enhancers, thereby silencing prior cellular identities [20].

The dynamics of SOX9-mediated chromatin opening follow specific temporal patterns. In epidermal stem cell reprogramming, SOX9 binding rapidly occurs within one week, while increased accessibility at these binding sites develops subsequently, indicating that SOX9 binding precedes chromatin opening [20]. These findings demonstrate SOX9's ability to alter the epigenetic landscape fundamentally, with implications for both development and disease.

SOX9 in immune regulation and cancer

Mechanisms of immune modulation

SOX9 plays a complex, "double-edged sword" role in immunology, contributing to both pro-tumorigenic immune evasion and beneficial tissue repair processes [3]. In cancer contexts, SOX9 promotes immune escape through multiple mechanisms, including impairing immune cell function and shaping an immunosuppressive tumor microenvironment [3]. Bioinformatic analyses indicate that SOX9 expression negatively correlates with genes associated with the function of CD8+ T cells, NK cells, and M1 macrophages, while showing positive correlation with memory CD4+ T cells [3].

The mechanisms underlying SOX9's immunomodulatory effects involve both direct transcriptional regulation and indirect microenvironmental shaping. SOX9 contributes to the establishment of an "immune desert" microenvironment by promoting shifts in immune cell populations—decreasing effector immune cells such as CD8+ CXCR6+ T cells while increasing immunosuppressive cells including Tregs and M2 macrophages [3]. Additionally, SOX9 helps tumor cells maintain a stem-like state, enabling them to evade innate immunity by remaining dormant for extended periods [12].

Therapeutic implications

The role of SOX9 in therapy resistance and immune evasion makes it an attractive therapeutic target. In ovarian cancer, SOX9 expression is induced by platinum-based chemotherapy and contributes to chemoresistance [23] [28]. Similarly, SOX9 expression is upregulated in response to PARP inhibitor treatment in ovarian cancer, where it enhances DNA damage repair capabilities, contributing to treatment resistance [28]. Targeting SOX9 stability through inhibition of its deubiquitinating enzyme USP28 has shown promise in preclinical models, sensitizing cancer cells to PARP inhibitors [28].

The conservation of SOX9's regulatory functions in development but not completely in immune contexts presents both challenges and opportunities for therapeutic targeting. The high conservation of SOX9 targets in chondrogenesis suggests that strategies modulating SOX9 activity could have predictable on-target effects on skeletal homeostasis [25]. However, the species-specific differences in gonad development indicate that reproductive side effects might be less predictable when targeting SOX9 therapeutically.

Visualization of SOX9 binding mechanisms and experimental workflows

Figure 1: SOX9 ChIP-seq experimental workflow and binding mechanisms. The diagram illustrates key steps in Chromatin Immunoprecipitation Sequencing for SOX9 and its genomic binding modes, including pioneer factor activity that enables binding to closed chromatin.

Figure 2: Context-specific functions of SOX9. The diagram illustrates how SOX9 regulates distinct transcriptional programs in different biological contexts, showing high target conservation in chondrogenesis but lower conservation in gonad development and immune regulation.

Table 3: Essential Research Reagents for SOX9 ChIP-seq Studies

Reagent/Category	Specific Examples	Function/Application	Considerations
SOX9 Antibodies	Anti-SOX9 (AB5535, Sigma) [28]	Immunoprecipitation for ChIP-seq; validation	Validation for ChIP-grade quality essential
Cell Models	hESC-derived CNCCs [26] [27]; Ovarian cancer lines (SKOV3, UWB1.289) [28]	Study dosage sensitivity; cancer mechanisms	hESC models ideal for developmental contexts
Genetic Tools	dTAG system [26] [27]; CRISPR/Cas9 [23]	Precise TF modulation; functional validation	dTAG enables dosage titration studies
Crosslinking Methods	Dual-crosslinking (DSG + formaldehyde) [24]	Preserve protein-DNA interactions	Enhances ChIP efficiency
Sequencing Methods	ChIP-seq; CUT&RUN [20] [25]; ATAC-seq [26]	Binding mapping; chromatin accessibility	Method choice depends on research question
Bioinformatic Tools	Motif analysis; conservation analysis	Identify direct targets; evolutionary conservation	SOX palindromic repeats indicative of direct binding [25]

Genome-wide mapping of SOX9 targets through ChIP-seq has revealed both conserved and context-specific regulatory networks that underlie its diverse functions in development, cancer, and immunity. The comparative analysis between species demonstrates that SOX9 target conservation is remarkably high in chondrogenesis but significantly lower in gonad development, suggesting evolutionary divergence in certain regulatory programs. In immune and cancer contexts, SOX9 emerges as a key regulator of tumor microenvironment and therapy resistance, though direct mapping of its targets in immune cells remains an area for future investigation. The dosage sensitivity of specific SOX9 targets, coupled with its pioneer factor capability, highlights the precision required in therapeutic targeting of SOX9 pathways. As techniques for mapping transcription factor binding continue to evolve, particularly through integration of perturbation approaches with deep learning, our understanding of SOX9's context-specific functions will continue to refine, offering new opportunities for therapeutic intervention in SOX9-driven pathologies.

The SRY-box Transcription Factor 9 (SOX9) is a pivotal transcription factor and master regulator of cell fate with critical functions in development, stem cell biology, and disease. As a member of the SOX E-family subgroup, SOX9 contains a high-mobility group (HMG) DNA-binding domain that recognizes the specific DNA sequence CCTTGAG [29]. This transcription factor regulates diverse biological processes including chondrogenesis, sex determination, and stem/progenitor cell development [16] [17] [29]. Beyond its developmental roles, SOX9 is increasingly recognized for its dual functions in immunology, acting as a "double-edged sword" in cancer and inflammatory diseases [3]. In tumor biology, SOX9 frequently shows overexpression across various malignancies and contributes to immune escape mechanisms, while in certain contexts, it promotes tissue repair and regeneration [3]. Understanding the precise gene regulatory networks controlled by SOX9 in immune cells requires sophisticated multi-omics approaches that integrate transcriptomic and epigenomic data to distinguish direct from indirect target genes—a critical challenge in transcriptional regulatory research.

Molecular mechanisms of SOX9 function

SOX9 protein structure and functional domains

The SOX9 protein contains several functionally specialized domains that enable its activity as a transcription factor. The N-terminal dimerization domain (DIM) facilitates protein-protein interactions, while the central HMG box domain mediates sequence-specific DNA binding and contains embedded nuclear localization and export signals that enable nucleocytoplasmic shuttling [3]. The protein features two transcriptional activation domains: a central activation domain (TAM) and a C-terminal activation domain (TAC) that interacts with cofactors like Tip60 to enhance transcriptional activity [3]. Finally, a proline/glutamine/alanine (PQA)-rich domain is essential for full transcriptional activation potential [3]. This modular organization allows SOX9 to participate in diverse protein complexes and regulatory contexts.

Pioneer factor activity of SOX9

Recent research has established SOX9 as a bona fide pioneer factor capable of binding closed chromatin and initiating chromatin remodeling [30] [20]. In epidermal stem cell reprogramming studies, SOX9 binding to chromatin occurred rapidly within one week, preceding increases in chromatin accessibility at target sites [30] [20]. Remarkably, approximately 30% of SOX9 binding sites were located in closed chromatin prior to its binding, with nucleosome displacement observed subsequently [30] [20]. This pioneer activity enables SOX9 to initiate cell fate transitions by accessing silent genomic regions and making them transcriptionally permissive.

Table 1: Key Functional Domains of SOX9 Protein

Domain	Position	Key Functions
Dimerization domain (DIM)	N-terminal	Facilitates protein-protein interactions and complex formation
HMG box domain	Central	DNA binding, nuclear localization/export signals
Central transcriptional activation domain (TAM)	Middle	Synergizes with TAC to enhance transcriptional potential
C-terminal transcriptional activation domain (TAC)	C-terminal	Interacts with cofactors (e.g., Tip60); inhibits β-catenin during chondrocyte differentiation
PQA-rich domain	C-terminal	Essential for transcriptional activation function

Competitive redistribution of epigenetic regulators

A novel mechanism for SOX9-mediated gene silencing has emerged wherein SOX9 binding at new target sites recruits histone and chromatin modifiers away from previous enhancers, leading to their silencing [30] [20]. This competitive redistribution model represents an indirect silencing mechanism that complements SOX9's direct activation function. When SOX9 binding to DNA is abrogated, it retains some silencing capacity, but when it cannot bind chromatin remodelers, the fate switch fails completely [30]. This demonstrates that SOX9's ability to redistribute epigenetic co-factors is essential for its cell fate switching capability.

Methodological approaches for identifying SOX9 targets

Chromatin-based assays for direct target identification

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) remains the gold standard for identifying genome-wide transcription factor binding sites. For SOX9, ChIP-seq experiments in various biological contexts have revealed cell type-specific binding patterns [9]. Comparative analysis between mouse and chicken systems demonstrated that SOX9 binding regions in chondrocytes show higher evolutionary conservation than those in Sertoli cells, suggesting tissue-specific conservation of regulatory networks [9]. Notably, SOX9 binding in chondrocytes occurs more frequently in intronic and distal regions and is enriched for SOX palindromic repeats, while in Sertoli cells, binding occurs more frequently in proximal upstream regions with fewer palindromic motifs [9].

The assay for transposase-accessible chromatin with sequencing (ATAC-seq) provides complementary information about chromatin accessibility dynamics following SOX9 binding. In studies modulating SOX9 dosage in human cranial neural crest cells, ATAC-seq revealed that most SOX9-dependent regulatory elements are buffered against small dosage changes, but elements directly and primarily regulated by SOX9 show heightened sensitivity [26]. This differential sensitivity provides a strategy for prioritizing functionally relevant binding sites.

Transcriptomic approaches and integration strategies

RNA sequencing (RNA-seq) enables comprehensive profiling of gene expression changes following SOX9 perturbation. When combined with chromatin accessibility data, transcriptomics can help distinguish direct from indirect targets. In precise modulation studies, sensitive regulatory elements and genes preferentially affected functional outcomes like chondrogenesis and craniofacial shape variation [26].

Multi-omics integration approaches are essential for comprehensive target identification. Reduced-representation bisulfite sequencing (RRBS) for DNA methylation analysis can be combined with RNA-seq to explore epigenetic regulation of gene expression [31]. Cloud computing platforms like Google Cloud provide scalable infrastructure for managing these computationally intensive analyses, with specialized tools for storing, processing, and integrating diverse omics datasets [31].

Table 2: Experimental Approaches for Identifying SOX9 Target Genes

Method	Key Information Obtained	Utility for Target Identification	Considerations
ChIP-seq	Genome-wide binding sites; direct physical interaction	Identifies direct targets with high confidence	Requires high-quality antibody; captures direct binding but not functional outcome
ATAC-seq	Chromatin accessibility landscape; open/closed chromatin regions	Identifies active regulatory regions; can infer functional binding	Does not directly measure transcription factor binding
RNA-seq	Global gene expression changes; differentially expressed genes	Identifies functional consequences of SOX9 perturbation	Cannot distinguish direct vs. indirect targets alone
CUT&RUN	Transcription factor binding with lower cell input	Similar to ChIP-seq with improved signal-to-noise	Technical expertise required for optimization
RRBS	DNA methylation patterns at specific genomic regions	Identifies epigenetic regulation associated with SOX9 activity	Focuses on specific genomic regions rather than whole genome

Experimental workflows for target validation

A robust workflow for identifying direct versus indirect SOX9 targets begins with SOX9 perturbation (e.g., knockdown, overexpression, or precise degradation) followed by multi-omics data collection [26] [30]. The dTAG (degradation tag) system enables precise and tunable modulation of SOX9 protein levels, allowing researchers to study dose-dependent effects on chromatin and gene expression [26]. Following perturbation, parallel ChIP-seq or CUT&RUN and ATAC-seq experiments identify binding sites and chromatin accessibility changes, while RNA-seq profiles transcriptional consequences [26] [30] [20]. Integration of these datasets involves identifying genes with SOX9 binding at regulatory elements and correlated expression changes, with binding preceding accessibility changes indicating direct targets [30] [20].

SOX9 targets in immune cells: conservation and context-dependence

SOX9 roles in immune cell regulation

SOX9 plays significant but context-dependent roles in immune cell development and function. In T-cell development, SOX9 cooperates with c-Maf to activate Rorc and key Tγδ17 effector genes (Il17a and Blk), modulating lineage commitment of early thymic progenitors and influencing the balance between αβ T-cell and γδ T-cell differentiation [3]. While SOX9 doesn't appear critical for normal B-cell development, it is overexpressed in certain B-cell lymphomas like Diffuse Large B-cell Lymphoma (DLBCL), where it acts as an oncogene by promoting proliferation and inhibiting apoptosis [3].

Bioinformatics analyses reveal significant correlations between SOX9 expression and immune cell infiltration patterns across cancers. In colorectal cancer, SOX9 expression negatively correlates with infiltration of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, but positively correlates with neutrophils, macrophages, activated mast cells, and naive/activated T cells [3]. Similarly, in prostate cancer, SOX9 expression is associated with an "immune desert" microenvironment characterized by decreased effector immune cells and increased immunosuppressive cells [3].

Conservation of SOX9 targets across species and cell types

The conservation of SOX9 regulatory networks varies significantly by biological context. Comparative ChIP-seq studies between mouse and chicken demonstrate that SOX9 targets in chondrocytes show high conservation, while targets in Sertoli cells show much lower conservation [9]. This suggests that the core chondrogenic program regulated by SOX9 is evolutionarily ancient and conserved, while reproductive system regulation has diverged more rapidly [9].

In immune cells, the conservation patterns of SOX9 targets remain less explored but appear to follow context-specific principles. The fundamental pioneer factor capability of SOX9—binding closed chromatin and recruiting epigenetic modifiers—is conserved across tissue types [30] [20]. However, the specific gene sets regulated likely depend on cellular context and cooperating transcription factors.

Techniques for studying conservation patterns

Analyzing conservation of SOX9 targets requires comparative genomics approaches across species. Chromatin accessibility conservation can be assessed by comparing ATAC-seq profiles, while binding site conservation can be evaluated through cross-species ChIP-seq experiments [9]. Gene expression conservation can be analyzed by comparing transcriptomic responses to SOX9 perturbation [9]. These analyses typically reveal that while the core molecular functions of SOX9 are conserved, the specific regulatory outputs have evolved in a cell type-specific manner.

Table 3: SOX9 Target Conservation Across Biological Contexts

Biological Context	Level of Conservation	Key Conserved Targets/Pathways	Species Studied
Chondrogenesis	High	COL2A1, COL11A2, Hapln1; SOX palindromic motifs in binding sites	Mouse, chicken, zebrafish
Sertoli Cell Function	Moderate to Low	AMH; proximal promoter binding preference	Mouse, chicken, cattle
Neural Crest Development	High	Craniofacial development genes; dosage-sensitive enhancers	Human, mouse
Epidermal to Hair Follicle Transition	High	Hair follicle stem cell enhancers; pioneer activity	Mouse
Immune Cell Regulation	Context-dependent	T-cell development genes; immunomodulatory functions	Primarily mouse and human

The scientist's toolkit: essential research reagents and solutions

Table 4: Key Research Reagent Solutions for SOX9 Studies

Reagent/Solution	Specific Example	Function/Application	Key References
Inducible SOX9 Expression System	TRE-Sox9; Krt14-rtTA mice	Enables temporal control of SOX9 expression in specific cell types	[30] [20]
Precise Degradation System	dTAG (FKBP12-F36V degradation tag)	Allows tunable modulation of SOX9 protein levels for dose-response studies	[26]
SOX9 Antibodies	ChIP-grade anti-SOX9	Essential for ChIP-seq experiments to map genome-wide binding sites	[9]
Epigenetic Profiling Kits	ATAC-seq kits; CUT&RUN kits	Enable mapping of chromatin accessibility and transcription factor binding	[26] [30] [20]
Single-Cell Multi-omics Platforms	10x Genomics Multiome	Allows simultaneous profiling of chromatin accessibility and gene expression in single cells	[3]
Cloud Computing Resources	Google Cloud Platform	Provides scalable infrastructure for multi-omics data integration and analysis	[31]
3-[4-(Aminomethyl)benzyloxy] Thalidomide	3-[4-(Aminomethyl)benzyloxy] Thalidomide, MF:C₂₁H₁₉N₃O₅, MW:393.39	Chemical Reagent	Bench Chemicals
(E)-Cinnamaldehyde Dimethyl Acetal-d5	(E)-Cinnamaldehyde Dimethyl Acetal-d5	High-purity (E)-Cinnamaldehyde Dimethyl Acetal-d5, a deuterated analog for research. For Research Use Only. Not for human or veterinary use.	Bench Chemicals

Integrating transcriptomic and epigenomic approaches provides powerful strategies for distinguishing direct versus indirect targets of SOX9 in immune cells and other biological contexts. The emerging picture reveals SOX9 as a master regulator whose functions are highly context-dependent, with conserved core molecular mechanisms but flexible regulatory outputs across cell types and species. The pioneer factor activity of SOX9, enabling it to bind closed chromatin and initiate reprogramming, appears to be a fundamental conserved feature [30] [20]. However, the specific gene sets regulated and their sensitivity to SOX9 dosage vary significantly by cellular context [26] [9].

Future research directions should include comprehensive mapping of SOX9 targets across immune cell subtypes using single-cell multi-omics approaches, systematic investigation of SOX9 dosage effects in immune contexts, and exploration of the therapeutic potential of modulating SOX9 activity in immune-related diseases. The development of more sophisticated computational methods for integrating multi-omics data will further enhance our ability to distinguish direct from indirect targets and understand the gene regulatory logic controlled by this crucial transcription factor. As these techniques advance, they will illuminate both the conserved principles and context-specific adaptations of SOX9-mediated gene regulation, with significant implications for understanding and manipulating immune responses in health and disease.

Leveraging single-cell RNA sequencing (scRNA-seq) to dissect SOX9+ cell subpopulations in the tumor immune microenvironment

The Sex-determining Region Y-related High-Mobility Group Box 9 (SOX9) is a transcription factor that plays essential roles in a multitude of biological processes, including embryonic development, chondrogenesis, and stem cell maintenance [3]. Beyond these established roles, emerging research underscores its significance as a janus-faced regulator in immunology, wielding complex and often dual functions within the tumor immune microenvironment (TIME) [3]. SOX9 is frequently overexpressed in diverse solid malignancies, such as liver, lung, breast, and gastric cancers, where its levels often correlate positively with tumor occurrence, progression, and poor prognosis [3]. Its function, however, is context-dependent; while it can promote tumor immune escape by impairing immune cell function, it also contributes to tissue repair and regeneration in other settings, such as in osteoarthritis and acute kidney injury [3] [32]. This dichotomy makes it a compelling therapeutic target. The conservation of SOX9's functional roles across species, particularly in immune cell development and regulation, further solidifies its relevance in biomedical research. Single-cell RNA sequencing (scRNA-seq) has emerged as a revolutionary tool, providing the resolution necessary to deconvolve this complexity. It allows for the unbiased dissection of SOX9+ cell subpopulations, their transcriptional states, and their intricate crosstalk within the TIME, offering unprecedented insights for diagnostic and therapeutic innovation [33] [34] [35].

scRNA-seq Methodologies for Delineating SOX9+ Cellular Networks

Leveraging scRNA-seq to study SOX9+ cells involves a multi-step process, from sample preparation to advanced computational analysis. The following workflow and table detail the core experimental and analytical protocols.

Figure 1: A standard scRNA-seq workflow for analyzing SOX9+ cells, encompassing wet-lab procedures and computational analyses.

Table 1: Core scRNA-seq Experimental and Analytical Protocols for SOX9+ Cell Research

Protocol Stage	Key Steps & Technologies	Purpose & Application in SOX9+ Research
Sample Preparation	Fresh tissue collection; dissociation with kits (e.g., Miltenyi Biotec Lung Dissociation Kit); viability assessment [36].	To obtain high-quality, live single-cell suspensions from tumor and control tissues.
Library Construction	Single-cell encapsulation (10x Genomics Chromium); cDNA synthesis and amplification; library preparation with kits (e.g., Single Cell 3' Library and Gel Bead Kit V3.1) [36].	To barcode cellular transcripts and create sequencing libraries for individual cells.
Sequencing & Alignment	Illumina sequencing (e.g., NovaSeq 6000, PE150); read alignment and UMI counting using Cell Ranger software [36].	To generate raw gene expression data and align it to a reference genome.
Data Preprocessing	Quality control (filtering cells by gene/UMI counts, mitochondrial percentage); data normalization and scaling; batch effect correction with Harmony [34] [36].	To create a clean, normalized, and integrated gene expression matrix for downstream analysis.
Cell Clustering & Annotation	Dimensionality reduction (PCA, UMAP); graph-based clustering; annotation using marker genes (e.g., EPCAM for epithelia, CD68 for macrophages) [34] [36].	To identify major cell types (immune, stromal, malignant) and subset SOX9+ populations.
SOX9+ Subpopulation Analysis	Differential expression analysis (Seurat's FindMarkers); gene set enrichment analysis (GO, KEGG); gene regulatory network inference (SCENIC) [34] [36].	To characterize the unique molecular features, pathways, and regulatory TFs of SOX9+ subsets.
Advanced Analyses	Cell-cell communication (CellChat); trajectory inference (Monocle2); copy number variation inference (InferCNV) [34] [35] [36].	To infer interactions with the microenvironment, differentiation trajectories, and malignant state.

Comparative Analysis of SOX9+ Subpopulations Across Cancers and States

The application of the aforementioned scRNA-seq protocols has revealed a complex landscape of SOX9+ cells across different cancer types and disease states. The table below synthesizes key findings from recent studies, highlighting the heterogeneity and conserved functions of SOX9.

Table 2: scRNA-seq Insights into SOX9+ Cell Subpopulations Across Biological Contexts

Cancer/Context	SOX9+ Cell Subpopulation	Key Identified Markers & Pathways	Role in TIME/Function	Conservation & Therapeutic Implication
Endometrioid Endometrial Cancer (EC) [34]	SOX9+LGR5- epithelial cells	NF-κB pathway enrichment; interaction via MIF-(CD74+CD44) with macrophages.	Highly malignant subtype; drives progression via crosstalk with M2-like macrophages.	Suggests a conserved pro-tumorigenic module; MIF-CD74 axis is a potential therapeutic target.
Acute Kidney Injury (AKI) [32]	Renal Sox9+ stem/progenitor cells	Activated by PGE2 via PI3K-Akt pathway; promotes VEGFa secretion; upregulates Cpt2.	Promotes differentiation into renal tubular cells; inhibits fibrosis; aids tissue regeneration.	Highlights a conserved regenerative role; PGE2 treatment shows therapeutic promise for AKI.
Pancreatic, Hepatic, & Other Solid Cancers [35]	Malignant epithelial cells	Context-specific; often associated with stemness and EMT markers.	Contributes to tumor aggressiveness, heterogeneity, and potential immune evasion.	SOX9's role in progenitor/stem cells is evolutionarily conserved, influencing tumor phenotypes.
Colorectal Cancer (CRC) [3]	SOX9-high tumor cells	Negative correlation with B cells, resting mast cells; positive correlation with neutrophils, macrophages.	Associated with an immunosuppressive TIME; promotes immune escape.	Correlates with poor prognosis; a potential target for reversing immunosuppression.
Prostate Cancer (PCa) [3]	SOX9+ club cells	Characterized by high SOX9 and low AR (androgen receptor).	Enriched after androgen deprivation therapy (ADT); may contribute to an "immune desert".	Suggests a mechanism for therapy resistance; targeting SOX9 may improve response to ADT.
Postnatal Enthesis Development [37]	Enthesis progenitor cells	Co-expression of tenogenic (Scx) and chondrogenic (Sox9, Acan) markers.	Forms the bone-tendon junction; critical for fibrocartilage development.	Demonstrates a conserved developmental role outside oncology; informs tissue engineering.

Signaling Pathways and Cellular Crosstalk of SOX9+ Cells

A critical insight from scRNA-seq is the detailed mapping of how SOX9+ cells communicate with and reshape their microenvironment. A prominent pathway is the MIF-signaling pathway, which was identified as a key communication axis between SOX9+ malignant cells and macrophages in endometrial cancer [34]. The following diagram illustrates this interaction and other relevant SOX9-related pathways.

Figure 2: Key signaling pathways involving SOX9+ cells. The MIF-CD74/NF-κB axis drives cancer progression, while the PGE2/PI3K-Akt pathway promotes regeneration.

Beyond this specific pathway, scRNA-seq analyses using tools like CellChat have consistently shown that SOX9+ malignant cells are hubs of pro-tumorigenic signaling. These cells can influence a wide array of immune cells. For instance, in colorectal cancer, high SOX9 expression is negatively correlated with the infiltration of anti-tumor immune cells like B cells and resting T cells, and positively correlated with pro-tumor neutrophils and macrophages [3]. This suggests that SOX9+ subpopulations actively contribute to building an immunosuppressive niche, facilitating tumor immune escape.

Successfully executing a scRNA-seq study on SOX9+ subpopulations requires a suite of specialized reagents and computational tools. The table below details key components of this toolkit.

Table 3: Essential Research Reagent Solutions for SOX9+ scRNA-seq Studies

Category	Item	Specific Example / Model	Function & Application
Model Systems	THX Mice [38]	Humanized immune system mice (e.g., THX model).	Provides a more human-relevant in vivo context for studying human immune cell interactions with SOX9+ tumor cells.
Tissue Dissociation	Dissociation Kit [36]	Miltenyi Biotec Human Tissue Dissociation Kits (e.g., Lung Dissociation Kit).	Generates high-viability single-cell suspensions from complex solid tumor tissues.
scRNA-seq Platform	Library Prep Kit [36]	10x Genomics Single Cell 3' Library and Gel Bead Kit.	For high-throughput barcoding and construction of sequencing libraries from thousands of single cells.
Bioinformatic Tools	Primary Analysis Pipeline [36]	Cell Ranger (10x Genomics).	Processes raw sequencing data into a gene expression matrix through alignment and UMI counting.
Bioinformatic Tools	Clustering & Annotation [34] [36]	Seurat, SCANVI, CellHint.	For dimensionality reduction, cell clustering, and annotation of cell types, including SOX9+ subsets.
Bioinformatic Tools	Cell-Cell Communication [34] [36]	CellChat, NicheNet.	Infers and visualizes ligand-receptor interactions between SOX9+ cells and other cells in the TIME.
Bioinformatic Tools	Trajectory Analysis [32] [36]	Monocle2, scVelo.	Reconstructs differentiation trajectories and cellular dynamics of SOX9+ progenitor cells.
Bioinformatic Tools	AI-Powered Annotation [39]	scHDeepInsight.	Uses hierarchical deep learning for precise and consistent immune cell annotation from scRNA-seq data.
Validation Reagents	Antibodies [34] [37]	Anti-SOX9, Anti-E-cadherin, Anti-Col2a1.	For validating protein-level expression and spatial localization via immunofluorescence/ICH.

Single-cell RNA sequencing has unequivocally transformed our ability to dissect the functional roles of SOX9+ subpopulations within the tumor immune microenvironment. By moving beyond bulk analyses, researchers can now pinpoint specific SOX9+ cellular states—from highly malignant epithelial cells in endometrial cancer to regenerative progenitors in kidney injury—and decode their unique transcriptional programs, regulatory networks, and communication signals [3] [32] [34]. The conservation of SOX9's roles in development, regeneration, and cancer across species underscores its biological fundamentality and enhances the translational relevance of findings from model systems. The integration of scRNA-seq with emerging technologies like spatial transcriptomics, AI-driven analytics, and advanced in vivo models promises to further refine our understanding [38] [39]. This comprehensive, single-cell-resolution view is paving the way for novel therapeutic strategies that target SOX9+ cells or their immunosuppressive pathways, ultimately aiming to overcome immune evasion and improve cancer treatment outcomes.

The SRY-related HMG-box 9 (SOX9) transcription factor represents a critical regulatory node in embryonic development, cell fate determination, and disease pathogenesis. As a transcription factor equipped with a high-mobility group (HMG) box DNA-binding domain, SOX9 recognizes specific DNA sequences ((A/T)(A/T)CAA(T/A)G) to modulate target gene expression [17] [9]. Beyond its well-established roles in chondrogenesis and sex determination, SOX9 has emerged as a significant player in cancer progression, immune regulation, and tissue homeostasis. Recent evidence indicates that SOX9 operates as a pioneer transcription factor capable of binding closed chromatin and initiating chromatin remodeling events that dictate cell fate decisions [20]. This capacity positions SOX9 as a master regulator of transcriptional programs across diverse biological contexts.

The conservation of SOX9 regulatory networks varies substantially across tissue types and species. Comparative chromatin immunoprecipitation sequencing (ChIP-seq) analyses between mouse and chicken embryos reveal that SOX9 target genes exhibit higher conservation in chondrocytes than in Sertoli cells [9]. This cell type-specific conservation pattern underscores the context-dependent nature of SOX9-mediated regulation and highlights the importance of investigating its functions within specific biological systems. In immune contexts, SOX9 demonstrates complex, dual roles—acting as both an oncogene and tumor suppressor depending on cellular environment [17] [3]. This functional duality, combined with its emerging roles in immune cell infiltration and tumor microenvironment remodeling, establishes SOX9 as a compelling therapeutic target worthy of comprehensive spatial multi-omics investigation.

Experimental Approaches for SOX9 Functional Characterization

Precision Modulation of SOX9 Dosage with dTAG System

Understanding dosage sensitivity is crucial for deciphering SOX9 function in development and disease. A recent innovative approach employed the degradation tag (dTAG) system to achieve tunable modulation of SOX9 levels in human embryonic stem cell (hESC)-derived cranial neural crest cells (CNCCs) [26]. The experimental workflow involved:

• Genetic Engineering: Selection-free genome editing introduced a C-terminal FKBP12-F36V–mNeonGreen–V5 tag to the endogenous SOX9 locus in hESCs, creating a degradation-prone SOX9 fusion protein [26].
• Cell Differentiation: Tagged hESCs were differentiated into CNCCs using established protocols yielding molecularly homogenous populations [26].
• Dosage Titration: SOX9 levels were precisely modulated using a dTAGV-1 dilution series (0-500 nM), creating six distinct SOX9 concentrations over 48 hours [26].
• Phenotypic Assessment: Chromatin accessibility (ATAC-seq), gene expression (RNA-seq), and cellular phenotypes were evaluated across the SOX9 dosage range [26].

This system revealed that most SOX9-dependent regulatory elements are buffered against small dosage decreases, but directly regulated elements show heightened sensitivity, particularly those affecting chondrogenesis and craniofacial development [26]. The dTAG approach provides unprecedented resolution for analyzing quantitative TF dosage effects at physiologically relevant levels.

Integrated Spatial Multi-Omics Workflow

Advanced spatial multi-omics technologies now enable correlated analysis of transcriptomic and proteomic data within tissue architecture. A recently developed framework performs spatial transcriptomics (ST) and spatial proteomics (SP) on the same tissue section, preserving spatial context for direct comparisons [40]. The integrated workflow includes:

• Spatial Transcriptomics: Fresh-frozen or FFPE tissue sections undergo gene expression profiling using platforms like 10x Genomics Xenium with targeted gene panels (e.g., 289-gene human lung cancer panel) [40].
• Spatial Proteomics: Following ST, the same slide undergoes hyperplex immunohistochemistry (hIHC) using systems like COMET or Hyperion XTi, staining for 40+ protein markers with cyclic antibody staining, imaging, and elution [40] [41].
• Histological Correlations: Hematoxylin and eosin (H&E) staining is performed post-multi-omics acquisition for pathological annotation [40].
• Computational Integration: DAPI images from ST and SP are co-registered using non-rigid spline-based algorithms in platforms like Weave, enabling single-cell level RNA-protein correlation analysis [40].

This integrated approach maintains spatial context while capturing complementary molecular layers, revealing systematic discordances between RNA and protein levels resolvable at cellular resolution [40]. The workflow is particularly valuable for investigating SOX9 expression patterns and their correlation with immune markers in complex tissues.

Chromatin Conformation Analysis via Polymer Modeling

Understanding how chromatin architecture influences SOX9 regulation requires advanced computational approaches. A novel framework links Hi-C contact maps to gene transcription through polymer modeling [42]. The methodology involves:

• 3D Structure Generation: A bead-spring polymer model informed by Hi-C contact maps generates an ensemble of 3D chromatin conformations through molecular dynamics simulations [42].
• Interaction Quantification: Enhancer-promoter (E-P) interactions are tracked across simulated configurations, calculating contact frequencies and durations [42].
• Transcriptional Coupling: E-P interaction kinetics are coupled to gene expression levels through a Markov chain model with transition rates derived from MD simulations [42].
• Perturbation Modeling: The system simulates structural perturbations (e.g., TAD boundary deletions) and predicts resulting expression changes for genes like SOX9 and KCNJ2 [42].

This approach successfully predicted that SOX9 expression changes following TAD boundary disruption result from altered enhancer access within its topological domain, demonstrating how chromatin organization modulates SOX9 regulation [42].

Comparative Analysis of SOX9 Functions Across Biological Contexts

Table 1: SOX9 Dosage Sensitivity Across Cellular Contexts

Cellular Context	Dosage Perturbation	Key Phenotypic Outcomes	Regulatory Features
Cranial Neural Crest Cells [26]	10-50% reduction via dTAG	PRS-like craniofacial shape variation; Impaired chondrogenesis	Heightened sensitivity at direct targets; Buffering at indirect targets
Epidermal Stem Cells [20]	Induced overexpression via Dox system	Fate switching to hair follicle lineage; Basal cell carcinoma formation	Pioneer activity at closed chromatin; Recruitment of chromatin remodelers
Pan-Cancer Analysis [17]	Natural variation in tumors	Upregulated in 15/33 cancers; Prognostic in LGG, CESC, THYM	Context-dependent oncogene/tumor suppressor
Immune Microenvironment [3]	Expression correlation analyses Altered immune infiltration; T-cell dysfunction	Negative correlation with CD8+ T cells, NK cells, M1 macrophages

Table 2: SOX9 Binding Characteristics Across Cell Types and Species

Cell Type	Species	Primary Binding Regions	Characteristic Motifs	Target Conservation
Chondrocytes [9]	Mouse & Chicken	Intronic and distal regions	SOX palindromic repeats (19.65% of sites)	High between mouse and chicken
Sertoli Cells [9]	Mouse & Chicken	Proximal upstream regions	Single SOX motifs (8.72% palindromic)	Low between mouse and chicken
Epidermal Stem Cells [20]	Mouse	Distal enhancers (30% in closed chromatin)	SOX motifs with collaborative factors	Not assessed
Oligodendrocytes [43]	Human	Not specified	SOX9 footprint observed	Not assessed

SOX9 in Cancer and Immune Regulation

SOX9 exhibits divergent roles across cancer types, functioning predominantly as an oncogene but demonstrating context-dependent tumor suppressor activity. Comprehensive pan-cancer analyses reveal SOX9 overexpression in 15 of 33 cancer types, including CESC, COAD, GBM, LIHC, and PAAD, while showing significant downregulation only in SKCM and TGCT [17]. Prognostically, high SOX9 expression associates with worse overall survival in LGG, CESC, and THYM, but surprisingly predicts better outcomes in ACC [17]. These contrasting prognostic associations highlight the context-dependent functionality of SOX9 in tumor biology.

In the immune landscape, SOX9 significantly correlates with altered immune cell infiltration patterns across multiple cancers. Bioinformatic analyses demonstrate that SOX9 expression negatively correlates with cytotoxic immune populations (CD8+ T cells, NK cells, M1 macrophages) while positively correlating with immunosuppressive cells (Tregs, M2 macrophages) in diverse malignancies including colorectal cancer and prostate cancer [3]. Single-cell RNA sequencing and spatial transcriptomics in prostate cancer reveal that SOX9-high regions correspond with "immune desert" microenvironments characterized by depleted effector T cells and enriched immunosuppressive populations [3]. These findings position SOX9 as a potential modulator of tumor immune evasion.

Structural and Functional Domains of SOX9 Protein

Table 3: SOX9 Protein Domains and Functional Characteristics

Domain	Position	Key Functions	Interacting Partners
Dimerization Domain (DIM) [3]	N-terminal	Facilitates SOX9 homodimerization	SOX9 itself and other SOX proteins
HMG Box [3]	Central	DNA binding; Nuclear localization; Chromatin bending	DNA with (A/T)(A/T)CAA(T/A)G motif
Central Transcriptional Activation Domain (TAM) [3]	Middle	Synergistic transcriptional activation	Co-activators and chromatin remodelers
C-terminal Transcriptional Activation Domain (TAC) [3]	C-terminal	Primary transactivation; β-catenin inhibition	Tip60 and other transcriptional cofactors
PQA-rich Domain [3]	C-terminal	Transcriptional activation modulation	Transcriptional machinery components

Research Reagent Solutions for SOX9 Investigations

Table 4: Essential Research Reagents for SOX9 and Spatial Multi-Omics Studies

Reagent/Technology	Primary Application	Key Utility in SOX9 Research
dTAG System [26]	Tunable protein degradation	Precise SOX9 dosage modulation in native context
Hyperion XTi Imaging System [41]	Spatial proteomics (40+ markers)	Simultaneous protein detection in SOX9-expressing tissues
10x Genomics Xenium [40]	Spatial transcriptomics	Targeted RNA detection in tissue architecture
COMET Platform [40]	Hyperplex immunohistochemistry	Cyclic protein staining on same section as transcriptomics
Weave Software [40]	Multi-omics data integration	Registration and alignment of ST/SP data from same section
Polymer Modeling Framework [42]	Chromatin structure prediction	Predicting SOX9 expression changes from 3D genome alterations
Validated SOX9 Antibodies [9]	Chromatin immunoprecipitation	Mapping SOX9 binding regions in diverse cell types

Visualizing SOX9 Regulatory Networks and Experimental Workflows

SOX9-Mediated Chromatin Remodeling and Gene Regulation

Integrated Spatial Multi-Omics Workflow

Discussion: Integrated View of SOX9 Regulation and Function

The comprehensive analysis of SOX9 through advanced spatial multi-omics approaches reveals a complex regulator with context-dependent functions across development, homeostasis, and disease. SOX9 demonstrates distinctive properties as a pioneer transcription factor capable of initiating chromatin remodeling events that dictate cell fate decisions [20]. Its dosage sensitivity appears particularly critical in developmental contexts, where precise levels determine phenotypic outcomes in structures like the craniofacial skeleton [26]. The conservation of SOX9 regulatory networks varies substantially between cell types, with highly conserved targets in chondrocytes but divergent targets in Sertoli cells across species [9], suggesting evolutionary adaptation of its regulatory functions in tissue-specific contexts.

In cancer biology, SOX9 exhibits dual oncogenic and tumor suppressor activities influenced by cellular context [17] [3]. Its overexpression in numerous malignancies positions it as a promising diagnostic and prognostic biomarker, particularly in glioblastoma, liver cancer, and colorectal cancer [17] [14]. The emerging role of SOX9 in shaping tumor immune microenvironments through modulation of immune cell infiltration patterns further enhances its therapeutic relevance [3]. Spatial multi-omics approaches demonstrate particular utility in delineating how SOX9 expression patterns correlate with immune marker distribution and chromatin accessibility states within tissue architecture [40] [41].

The experimental frameworks presented—including precise dosage modulation, integrated spatial multi-omics, and chromatin conformation modeling—provide powerful methodologies for elucidating SOX9 functions across biological contexts [26] [40] [42]. These technologies enable researchers to move beyond bulk tissue analyses toward sophisticated spatial and single-cell resolution studies that capture the complexity of SOX9 regulatory networks in tissue microenvironments. As these methodologies continue to evolve, they promise to further illuminate the multifaceted roles of SOX9 in health and disease, potentially identifying novel therapeutic opportunities for manipulating its activity in pathological conditions.

Navigating Complexity: SOX9's Context-Dependent Functions and Therapeutic Targeting

The tumor microenvironment (TME) is a critical determinant of therapeutic efficacy, particularly in the context of immunotherapy. Recent investigations have elucidated a novel mechanism of immune suppression mediated by the transcription factor SOX9 through the ANXA1-FPR1 signaling axis. This pathway orchestrates neutrophil apoptosis within the TME, resulting in impaired cytotoxic cell function and consequent resistance to combination immune checkpoint blockade. This review synthesizes evidence from recent studies that delineate the molecular architecture of the SOX9-ANXA1-FPR1 axis, its conservation across species, and its role in mediating resistance to anti-LAG-3 plus anti-PD-1 therapy. We provide comprehensive experimental data and methodologies to facilitate comparative analysis of this pathway, offering valuable insights for researchers and drug development professionals targeting immunosuppressive mechanisms in oncology.

The SRY-related HMG-box transcription factor SOX9 represents a pivotal regulator of multiple biological processes, extending beyond its established roles in development and differentiation to encompass significant immunomodulatory functions. As a member of the SOX protein family, SOX9 contains a highly conserved high-mobility group (HMG) box domain that facilitates DNA binding and transcriptional regulation [3] [12]. While initially characterized for its importance in chondrogenesis and sex determination, SOX9 has emerged as a crucial player in tumor immunology, exhibiting context-dependent dual functions across diverse immune cell types [3]. Its expression is frequently elevated in various solid malignancies, where it correlates with adverse clinical outcomes and therapeutic resistance [3] [29].

The immunobiological significance of SOX9 is underscored by its evolutionary conservation across species, positioning it as a compelling target for translational research. SOX9 operates as a "double-edged sword" in immunity—on one hand promoting tumor immune escape by impairing immune cell function, while on the other hand contributing to tissue regeneration and repair through maintenance of macrophage function [3]. This review focuses specifically on the newly identified SOX9-ANXA1-FPR1 axis as a mechanism of immune suppression in the TME, with particular emphasis on its role in mediating resistance to combined anti-LAG-3 and anti-PD-1 immunotherapy.

The SOX9-ANXA1-FPR1 axis: Core mechanism and molecular architecture

The SOX9-ANXA1-FPR1 axis represents a coordinated signaling pathway that bridges tumor cell intrinsic properties with immune modulation in the TME. At its core, SOX9 functions as a transcriptional regulator that directly controls the expression of Annexin A1 (ANXA1), a 37 kDa glucocorticoid-regulated protein known for its role in inflammation resolution [44] [45]. ANXA1 subsequently engages Formyl Peptide Receptor 1 (FPR1) on neutrophils, initiating intracellular signaling cascades that promote mitochondrial fission and suppress mitophagy through downregulation of BCL2/adenovirus E1B interacting protein 3 (BNIP3) [44]. This sequence of molecular events ultimately induces apoptosis in FPR1+ neutrophils, reducing their accumulation in tumor tissues and impairing the cytotoxic activity of CD8+ T cells and γδ T cells [44].

The functional consequence of this axis is the establishment of an immunosuppressive TME that facilitates resistance to combination immunotherapy. This mechanism was comprehensively elucidated in a head and neck squamous cell carcinoma (HNSCC) mouse model, where single-cell RNA sequencing revealed significant enrichment of SOX9+ tumor cells in samples resistant to anti-LAG-3 plus anti-PD-1 therapy [44]. The identification of this pathway provides a molecular framework for understanding primary and acquired resistance to dual immune checkpoint blockade, offering potential targets for therapeutic intervention.

Visualizing the SOX9-ANXA1-FPR1 signaling pathway

The following diagram illustrates the core components and signaling relationships within the SOX9-ANXA1-FPR1 axis:

Figure 1: The SOX9-ANXA1-FPR1 Signaling Axis in Therapy Resistance. This diagram illustrates the molecular pathway through which SOX9-expressing tumor cells mediate immunosuppression and resistance to anti-LAG-3 plus anti-PD-1 therapy.

Key molecular relationships and regulatory checkpoints

The SOX9-ANXA1-FPR1 axis operates through precisely coordinated molecular interactions that present multiple regulatory checkpoints. SOX9 directly binds to regulatory elements of the ANXA1 gene, initiating its transcription in tumor cells [44]. The ANXA1 protein, upon secretion, functions as an endogenous ligand for FPR1, a G protein-coupled receptor primarily expressed on neutrophils [45] [46] [47]. This interaction triggers intracellular signaling that converges on mitochondrial regulators, particularly through suppression of BNIP3, a key mediator of mitophagy [44]. The resulting mitochondrial dysfunction and impaired energy homeostasis drive neutrophils toward apoptosis, effectively depleting this population from the TME.

The functional outcome of this molecular cascade is the creation of an "immune desert" characterized by reduced infiltration of cytotoxic lymphocytes and diminished anti-tumor activity [44] [12]. This immunosuppressive landscape ultimately compromises the efficacy of combined anti-LAG-3 and anti-PD-1 therapy, providing a mechanistic basis for treatment resistance observed in both preclinical models and clinical settings.

Comparative analysis of experimental models and validation systems

In vivo models and therapeutic response data

The investigation of the SOX9-ANXA1-FPR1 axis has employed diverse experimental models, each contributing unique insights into pathway function and therapeutic implications. The cornerstone evidence derives from a HNSCC mouse model wherein researchers evaluated responses to anti-LAG-3 and anti-PD-1 combination therapy [44]. This model demonstrated striking differential responses, with 57.1% of animals exhibiting sensitivity to treatment while 42.9% displayed resistance, enabling comparative analysis of the underlying mechanisms [44].

Table 1: In Vivo Therapeutic Response Profiles in HNSCC Mouse Model

Treatment Group	Response Rate	Tumor Progression	Immune Cell Infiltration	SOX9+ Cell Enrichment
Control IgG	0%	Rapid growth	Baseline	Low
Anti-LAG-3 monotherapy	Not significant vs. control	No improvement	Minimal change	Not significant
Anti-PD-1 monotherapy	Significant improvement	Delayed progression	Moderate increase	Not significant
Anti-LAG-3 + Anti-PD-1 (Sensitive)	57.1%	Regression/Stasis	Marked increase	Low
Anti-LAG-3 + Anti-PD-1 (Resistant)	42.9%	>20% growth from baseline	Reduced	High

Beyond the HNSCC model, transgenic mouse systems have been instrumental in validating the functional significance of this axis. Studies utilizing AnxA1-null mice demonstrated exacerbated inflammatory responses and impaired resolution phases, establishing the non-redundant role of ANXA1 in immune homeostasis [45]. Complementary work with FPR1-deficient models has further clarified the receptor's specific contribution to neutrophil biology and its involvement in the immunosuppressive pathway [46] [47].

Cross-species conservation and human relevance

The translational relevance of the SOX9-ANXA1-FPR1 axis is supported by evidence of its conservation across species and operation in human cancers. In thyroid cancer, single-cell RNA sequencing analyses revealed that ANXA1-FPR1 interactions in dendritic cells contribute to immune microenvironment modulation [48]. Similarly, in colorectal cancer patients, bioinformatics analyses demonstrated correlations between SOX9 expression patterns and specific immune cell infiltration profiles, including negative associations with B cells, resting mast cells, and monocytes [3].

Table 2: SOX9-ANXA1-FPR1 Axis Conservation Across Cancer Types

Cancer Type	Experimental System	Key Findings	Clinical Correlation
Head and Neck SCC	Mouse model (4NQO-induced)	SOX9+ enrichment in resistant tumors; ANXA1-FPR1 mediates neutrophil apoptosis	n/a (Preclinical)
Thyroid Cancer	Human samples; scRNA-seq	ANXA1-FPR1 interaction in DCs promotes immunosuppression	Associated with patient prognosis
Colorectal Cancer	TCGA data analysis	SOX9 expression negatively correlates with multiple immune cell populations	Diagnostic and prognostic significance
Breast Cancer	Cell lines and mouse models	SOX9 crucial for immune evasion and maintaining dormant cells	Potential predictor of metastatic recurrence
Glioblastoma	TCGA/GTEx database analysis	SOX9 expression correlates with immune infiltration and checkpoints	Prognostic biomarker, especially in IDH-mutant cases

The conservation of this pathway across multiple cancer types underscores its fundamental role in shaping anti-tumor immunity and suggests that therapeutic targeting of this axis could have broad applications in oncology.

Methodological approaches for investigating the SOX9-ANXA1-FPR1 axis

Core experimental protocols and techniques

The elucidation of the SOX9-ANXA1-FPR1 axis has employed sophisticated methodological approaches that provide templates for future investigations in this field. The following experimental workflows have proven particularly valuable:

Single-Cell RNA Sequencing Analysis: Researchers performed scRNA-seq on tumor tissues from treatment-resistant and sensitive mice, pooling samples from three mice per group and processing them through standard digestion into single-cell suspensions [44]. After quality control and filtering, they analyzed mRNA measurements from over 33,424 single cells, identifying major cell populations based on canonical markers (e.g., Krt14/Krt5 for epithelial cells; Ptprc/Cd74 for immune cells) [44]. This approach enabled the identification of SOX9+ tumor cell enrichment in resistant samples and facilitated cell interaction analyses revealing the ANXA1-FPR1 mediated communication between epithelial cells and neutrophils.

Transgenic Mouse Model Validation: The functional significance of pathway components was confirmed using various transgenic mouse models, including AnxA1-null strains [44] [45]. These models demonstrated that in the absence of ANXA1, inflammatory responses are stronger and more prolonged, with resistance to glucocorticoid treatment observed in AnxA1-deficient mice [45]. Complementary studies employed FPR1-targeted approaches to establish the receptor's critical role in transducing ANXA1 signals in neutrophils.

Therapeutic Response Assessment: The HNSCC mouse model involved treating tumor-bearing mice with anti-LAG-3 and anti-PD-1 antibodies, with response categorization based on RECIST criteria [44]. Tumors showing more than 20% growth from original size after 14 days were classified as resistant, enabling comparative analysis between resistant and sensitive populations through techniques including magnetic resonance imaging (MRI), histopathological examination, and immunohistochemical staining for Ki67 and cleaved-Caspase3 [44].

The scientist's toolkit: Essential research reagents

Table 3: Key Research Reagents for Investigating the SOX9-ANXA1-FPR1 Axis

Reagent/Category	Specific Examples	Research Application	Experimental Function
Animal Models	4NQO-induced HNSCC mouse model; AnxA1-null mice; FPR1-deficient mice	In vivo pathway validation	Modeling therapy resistance; establishing causal relationships
Antibodies for Immunotherapy	Anti-LAG-3 (Relatlimab); Anti-PD-1 (Nivolumab)	Therapeutic response assessment	Evaluating combination immunotherapy efficacy
Analytical Tools	Single-cell RNA sequencing; CopyKAT algorithm	Tumor microenvironment analysis	Identifying malignant subpopulations; cellular heterogeneity mapping
Detection Antibodies	Anti-Ki67; Anti-cleaved Caspase3; Anti-SOX9; Anti-ANXA1	Tissue staining and phenotyping	Assessing proliferation, apoptosis, and protein localization
Receptor Ligands	Recombinant ANXA1; ANXA1-derived peptides (Ac2-26)	Mechanistic studies	Probing FPR1 activation and downstream signaling
Cell Isolation Markers	Anti-FPR1 (PE); Anti-FPR2 (FITC)	Flow cytometry	Immune cell population analysis and sorting
OTNE - 13C3	OTNE - 13C3, MF:C16H26O, MW:237.352	Chemical Reagent	Bench Chemicals
1-Propene, polymer with ethene, block	1-Propene, polymer with ethene, block, CAS:106565-43-9, MF:C10H10FNO2	Chemical Reagent	Bench Chemicals

The SOX9-ANXA1-FPR1 axis represents a clinically relevant mechanism of immune suppression that contributes significantly to resistance against combination immunotherapy. The conservation of this pathway across species and cancer types underscores its fundamental role in shaping anti-tumor immunity and highlights its potential as a therapeutic target. Future research should focus on developing specific inhibitors targeting key nodes within this pathway, potentially in combination with existing immunotherapies, to overcome resistance mechanisms and improve patient outcomes. The experimental methodologies and reagents outlined in this review provide a foundation for such investigations, offering researchers the necessary tools to further elucidate and target this immunosuppressive axis.

The SOX (SRY-related HMG-box) family of transcription factors represents a conserved group of nuclear proteins characterized by a high-mobility group (HMG) domain that facilitates DNA binding. Among these, SOX9 (SRY-box transcription factor 9) has emerged as a critical regulator of developmental processes, stem cell fate decisions, and disease pathogenesis. Recent research has illuminated SOX9's function as a pioneer transcription factor—a specialized class of factors capable of binding compacted chromatin and initiating transcriptional reprogramming. This capacity enables SOX9 to operate at critical junctures where cells transition between identities, simultaneously activating one genetic program while silencing another. Beyond its established roles in chondrogenesis, sex determination, and stem cell biology, SOX9 increasingly appears central to immunological processes, though its functions in this domain exhibit complex, context-dependent characteristics. This review synthesizes current understanding of SOX9's pioneer factor mechanisms, focusing on its competition for epigenetic co-factors during cell fate transitions and the consequent implications for immune gene regulation across species.

Molecular mechanisms of SOX9 pioneer factor activity

SOX9 domains and structural features

The SOX9 protein contains several functionally specialized domains that enable its pioneer factor capabilities. The high-mobility group (HMG) box domain serves dual roles: it facilitates DNA binding to specific motifs and contains nuclear localization and export signals that enable nucleocytoplasmic shuttling [3]. Flanking this central DNA-binding domain are key functional regions: a dimerization domain (DIM) at the N-terminus, and two transcriptional activation domains—one central (TAM) and one at the C-terminus (TAC)—that interact with various cofactors to enhance SOX9's transcriptional activity [3]. The C-terminal TAC domain is particularly important for interacting with histone modifiers and chromatin remodelers, while the proline/glutamine/alanine (PQA)-rich domain contributes to transcriptional activation potential.

Chromatin binding and remodeling activities

As a pioneer factor, SOX9 exhibits the defining capacity to bind cognate motifs within closed chromatin regions and initiate nucleosome displacement. In epidermal stem cell reprogramming studies, SOX9 binding to chromatin occurred rapidly within one week, preceding measurable increases in chromatin accessibility at target sites [20]. Notably, approximately 30% of SOX9 binding sites were situated within chromatin regions that were closed before SOX9 activation, confirming its genuine pioneer capability [20]. Subsequent to binding, SOX9 recruits histone and chromatin modifiers that remodel nucleosome architecture, evidenced by time-dependent decreases in cleavage under targets and release using nuclease (CUT&RUN) fragment lengths at SOX9-bound sites—a hallmark of nucleosome displacement [20].

Table 1: Key Features of SOX9 Pioneer Factor Activity

Feature	Experimental Evidence	Functional Consequence
Closed chromatin binding	30% of SOX9 binding sites in closed chromatin at D0 [20]	Recognition of target motifs in compacted genomic regions
Nucleosome displacement	Decreased CUT&RUN fragment length over time [20]	Chromatin opening for transcriptional activation
Epigenetic co-factor recruitment	Proteomic analyses showing interactions with histone modifiers [20]	Remodeling of chromatin landscape at target enhancers
Competitive co-factor redistribution	Silencing of epidermal enhancers while activating hair follicle enhancers [20]	Simultaneous activation and repression of distinct genetic programs

The competitive recruitment model for fate switching

A seminal mechanism underlying SOX9-mediated fate switching involves the competitive recruitment of limiting epigenetic co-factors. Research using engineered mouse models demonstrated that as SOX9 binds and opens key hair follicle enhancers de novo in epidermal stem cells, it simultaneously recruits essential co-factors away from epidermal enhancers, leading to their silencing [20] [49]. This competition creates a "molecular tug-of-war" where SOX9 actively redistributes chromatin-modifying machinery from previous cell identity enhancers to new fate-determining enhancers. When SOX9's DNA binding capability was abrogated, it still mediated silencing effects, but when its ability to bind chromatin remodelers was disrupted, the fate switch failed completely [20]. This underscores that SOX9's pioneer activity depends not merely on chromatin binding but specifically on its recruitment of remodeling complexes.

SOX9-mediated enhancer switching in cell fate decisions

Stem cell fate transitions

The competitive recruitment model finds clear demonstration in SOX9-driven fate transitions of adult epidermal stem cells (EpdSCs) toward hair follicle stem cell (HFSC) identity. Through integrated epigenetic, proteomic, and functional analyses, researchers have delineated the temporal sequence of reprogramming events. Within one week of SOX9 induction, binding occurs at key HFSC enhancers while epidermal genes begin transcriptional suppression. Between weeks 1-2, chromatin accessibility increases at SOX9-bound sites, followed by proliferation increases and morphological changes [20]. The mature tissue stem cell niche imposes physiological constraints that slow this SOX9-mediated chromatin reprogramming compared to embryonic development or in vitro systems, enabling detailed dissection of these sequential events [20].

Progression to oncogenic states

When SOX9 expression becomes sustained and unhinged from normal regulation, it can activate oncogenic transcriptional programs. In epidermal stem cell models, sustained SOX9 expression beyond the initial fate switch subsequently activated oncogenic transcriptional regulators that chart paths to cancers characterized by constitutive SOX9 expression, particularly basal cell carcinoma (BCC) [20]. By 6-12 weeks post-induction, transcriptional profiling revealed strong correlation with molecular signatures of BCC, with similarities emerging as early as two weeks post-induction—before overt phenotypic changes [20]. This progression from physiological fate switching to pathological transformation underscores the importance of tight regulatory control over SOX9 expression dynamics.

Table 2: Temporal Sequence of SOX9-Mediated Reprogramming in Epidermal Stem Cells

Time Post-SOX9 Induction	Chromatin & Binding Events	Transcriptional & Phenotypic Outcomes
Week 1	SOX9 binds closed chromatin at HFSC enhancers	Initial suppression of epidermal genes
Week 1-2	Increased accessibility at SOX9 sites; nucleosome displacement	Upregulation of hair follicle outer root sheath markers
Week 2	Peak of dynamic chromatin changes	Rise in proliferation; onset of morphological changes
Week 2-6	Continued enhancer remodeling	De novo invaginations between native hair follicles
Week 6-12	Stabilization of new chromatin state	Dysplastic changes; molecular features of basal cell carcinoma

Species conservation of SOX9 targets in immune contexts

Cell type-specific conservation patterns

Comparative analyses of SOX9 functions across species reveal striking cell type-specific conservation patterns. Research comparing SOX9 targets between mouse and chicken embryos demonstrated significantly higher conservation of SOX9 binding regions in limb bud genes (chondrogenesis) compared with male gonad genes (sex determination) [25]. In both species, SOX9 bound to intronic and distal regions more frequently in limb buds, while proximal upstream binding predominated in male gonads [25]. This tissue-specific conservation pattern extends to immune functions, where SOX9's regulatory relationships appear more evolutionarily plastic.

Implications for immune gene regulation

The lower evolutionary conservation of SOX9 targets in non-chondrogenic contexts suggests its immune regulatory functions may be more species- or context-specific. This has important implications for translating findings from model organisms to human immunology. While SOX9's fundamental pioneer factor mechanism—competitive co-factor recruitment—likely remains consistent, its specific immune gene targets may differ across species. This variability necessitates careful validation when extrapolating immune-related SOX9 functions from animal models to human therapeutic development.

SOX9 in immune regulation and tumor immunity

Dual roles in cancer immunity

SOX9 exhibits complex, context-dependent functions in tumor immunity, acting as what some researchers term a "double-edged sword" or "Janus-faced regulator" [3]. In various cancer types, SOX9 frequently shows overexpression that correlates with poor prognosis, where it can promote immune escape by impairing immune cell function [3] [17]. For instance, in colorectal cancer, SOX9 expression negatively correlates with infiltration of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, while positively correlating with neutrophils, macrophages, activated mast cells, and naive/activated T cells [3]. This suggests SOX9 contributes to shaping specific immune microenvironments favorable to tumor progression.

Mechanisms of immune modulation

Recent research has illuminated specific mechanisms through which SOX9 influences anti-tumor immunity. In head and neck squamous cell carcinoma (HNSCC) models, SOX9+ tumor cells mediate resistance to combined anti-PD-1 and anti-LAG-3 immunotherapy by regulating neutrophil function [13]. SOX9 directly regulates expression of annexin A1 (Anxa1), which mediates apoptosis of formyl peptide receptor 1 (Fpr1)+ neutrophils through the Anxa1-Fpr1 axis [13]. This pathway promotes mitochondrial fission and inhibits mitophagy by downregulating BCL2/adenovirus E1B interacting protein 3 (Bnip3) expression, ultimately preventing neutrophil accumulation in tumor tissues [13]. The reduction of Fpr1+ neutrophils impairs infiltration and tumor-killing ability of cytotoxic CD8 T and γδT cells, fostering therapy resistance [13].

Melanoma immune regulation

In melanoma, SOX9 exhibits a contrasting tumor-suppressive role in some contexts, where it indirectly regulates immune checkpoint expression. SOX9 knockdown in melanoma cells upregulates CEACAM1 (carcinoembryonic antigen cell adhesion molecule 1), a transmembrane glycoprotein that protects melanoma cells from T-cell mediated killing [50]. SOX9 controls CEACAM1 expression at the transcriptional level through an indirect mechanism that involves physical interaction with Sp1 and regulation of ETS1, rather than through direct promoter binding [50]. Consequently, SOX9 knockdown renders melanoma cells resistant to T-cell mediated killing, aligning with increased CEACAM1 expression [50]. This cell-type specific effect highlights the context-dependent nature of SOX9 immune regulation.

Experimental approaches for studying SOX9 functions

Methodologies for investigating pioneer factor activity

Comprehensive analysis of SOX9 pioneer factor functions requires integrated multi-omics approaches. Key methodologies include:

• CUT&RUN (Cleavage Under Targets & Release Using Nuclease) sequencing: For temporally mapping SOX9 binding to chromatin during reprogramming [20]
• ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing): To interrogate chromatin accessibility dynamics [20]
• Chromatin Immunoprecipitation sequencing (ChIP-seq): For identifying SOX9 binding regions in different cellular contexts and across species [25]
• Single-cell RNA sequencing (scRNA-seq): To resolve heterogeneous cellular responses to SOX9 expression and identify SOX9+ subpopulations in tumor microenvironments [13]
• Proteomic analyses: For identifying SOX9-interacting co-factors and chromatin modifiers [20]

Visualization of SOX9-mediated enhancer switching

The following diagram illustrates the competitive recruitment mechanism by which SOX9 mediates enhancer switching during cell fate transitions:

Diagram 1: SOX9-mediated competitive recruitment of epigenetic co-factors during cell fate switching. SOX9 binding to closed chromatin at target enhancers initiates competitive redistribution of limiting epigenetic co-factors, simultaneously activating new enhancers while silencing previous identity enhancers.

Temporal workflow for SOX9 reprogramming studies

The following diagram outlines key experimental workflows and temporal dynamics in SOX9 reprogramming studies:

Diagram 2: Experimental workflow for analyzing SOX9-mediated reprogramming. Integrated multi-omics approaches at specific timepoints reveal the temporal progression from initial chromatin binding through functional fate switching and potential oncogenic progression.

The scientist's toolkit: Key research reagents and applications

Table 3: Essential Research Reagents for Investigating SOX9 Functions

Reagent/Cell System	Key Applications	Research Utility
Inducible SOX9 mouse models (Krt14-rtTA;TRE-Sox9)	Fate switching studies; Temporal analysis of reprogramming [20]	Enables controlled SOX9 activation in specific cell types at defined timepoints
LS174T colorectal cancer cells	Wnt/SOX9 interaction studies; Colony formation assays [51]	Model for SOX9 cooperation with Wnt signaling in cancer proliferation
SOX9-specific shRNAs	Loss-of-function studies; Target validation [51] [50]	Enables determination of SOX9 requirement in various cellular processes
CEACAM1 reporter constructs	Promoter regulation assays; Immune checkpoint studies [50]	Elucidates indirect transcriptional regulation mechanisms by SOX9
CUT&RUN/ChIP-seq antibodies	Chromatin binding mapping; Pioneer factor assessment [20] [25]	Identifies direct SOX9 targets and binding site characteristics
4NQO-induced HNSCC mouse model	Immunotherapy resistance studies; Tumor microenvironment analysis [13]	Models SOX9-mediated resistance to combination immunotherapy
cis-4-Amino-1-boc-3-hydroxypiperidine	cis-4-Amino-1-boc-3-hydroxypiperidine|CAS 1331777-74-2

Concluding perspectives and future directions

SOX9 exemplifies how pioneer transcription factors utilize competitive epigenetic co-factor recruitment to execute cell fate decisions, with important implications for both developmental biology and disease pathogenesis. The mechanistic understanding of SOX9-mediated enhancer switching provides a framework for interpreting its complex roles in immune regulation and tumor immunity. The context-dependent nature of SOX9 functions—acting as either oncogene or tumor suppressor in different malignancies—highlights the importance of cellular environment in determining transcriptional outcomes. Future research should focus on identifying the specific epigenetic co-factors that SOX9 competitively redistributes, understanding how SOX9 binding specificity is determined across different cellular contexts, and exploiting these mechanisms for therapeutic benefit in cancer and immune disorders. The species-specific conservation patterns of SOX9 targets further suggest that careful validation across model systems will be essential for translating these findings to human therapeutics. As research continues to unravel the complexities of SOX9 function, this pioneer factor represents a promising target for manipulating cell fate decisions in regenerative medicine and cancer therapy.

The transcription factor SOX9 (SRY-related HMG-box 9) represents a formidable challenge in targeted cancer therapy due to its context-dependent dual functionality. As a key developmental regulator, SOX9 maintains stem cell pools in various tissues, but its dysregulation contributes significantly to tumorigenesis through contrasting mechanisms across different cancer types [52] [3]. This paradoxical nature necessitates a nuanced understanding of its tissue-specific roles, as SOX9 can function as either a potent oncogene or a tumor suppressor depending on cellular context [53] [17]. The conservation of SOX9 function across species further complicates therapeutic targeting, as it plays essential roles in fundamental biological processes including chondrogenesis, sex determination, and stem cell maintenance [3] [54]. This guide systematically compares SOX9's divergent roles, summarizes key experimental data, and provides methodologies for investigating its complex functions in cancer biology, with particular emphasis on implications for drug development in an era of precision medicine.

SOX9 Expression Patterns and Clinical Correlations Across Cancers

Table 1: SOX9 Expression and Clinical Significance in Various Cancers

Cancer Type	SOX9 Status	Functional Role	Clinical Correlation	References
Hepatocellular Carcinoma	Overexpression	Oncogenic	Poor prognosis, reduced disease-free & overall survival	[52]
Breast Cancer	Overexpression	Oncogenic	Promotes proliferation, metastasis; poor overall survival	[52] [3]
Colorectal Cancer	Overexpression	Oncogenic	Promotes proliferation, senescence inhibition, chemoresistance	[52] [3]
Ovarian Cancer	Chemotherapy-induced	Oncogenic	Drives chemoresistance, stem-like transcriptional state	[23]
Prostate Cancer	Overexpression	Oncogenic	Promotes proliferation, apoptosis resistance; poor survival	[52]
Cervical Cancer	Downregulation	Tumor Suppressor	Progressive loss during carcinogenesis; inhibits tumor growth	[53]
Skin Cutaneous Melanoma (SKCM)	Downregulation	Tumor Suppressor	Inhibits tumorigenicity in mouse and human models	[17]
Glioblastoma	Variable	Context-dependent	Diagnostic and prognostic biomarker; correlates with immune infiltration	[14]

Analysis of pan-cancer expression patterns reveals that SOX9 is significantly upregulated in fifteen cancer types, including colorectal, ovarian, liver, and pancreatic cancers, while being downregulated in only two cancer types (SKCM and TGCT) based on comprehensive database mining [17]. This distribution underscores its predominant oncogenic role across most malignancies, while highlighting specific contexts where its tumor-suppressive functions emerge.

Mechanistic Insights: SOX9 as an Oncogene

In its oncogenic role, SOX9 promotes tumor initiation, progression, and therapy resistance through multiple interconnected mechanisms:

Cancer Stem Cell Maintenance and Chemoresistance

SOX9 drives the emergence of cancer stem-like cells (CSCs) that exhibit enhanced tumorigenic potential and resistance to conventional therapies [52]. In high-grade serous ovarian cancer (HGSOC), SOX9 expression is rapidly induced following platinum-based chemotherapy, where it reprogrammes the transcriptional state of naive cells into a stem-like state characterized by enhanced plasticity and drug tolerance [23]. This reprogramming capacity positions SOX9 as a critical mediator of nongenetic chemoresistance mechanisms that operate independently of classical Darwinian selection.

Transcriptional Reprogramming and Pioneer Factor Activity

Recent evidence identifies SOX9 as a pioneer factor capable of binding closed chromatin regions and initiating large-scale epigenetic remodeling [20]. Through competitive recruitment of epigenetic co-factors, SOX9 simultaneously activates hair follicle stem cell enhancers while silencing epidermal stem cell enhancers, demonstrating its capacity to execute fate switching decisions with profound implications for cancer cell identity [20].

Immunomodulation and Tumor Microenvironment

SOX9 expression correlates significantly with immune cell infiltration patterns across various cancers. Bioinformatics analyses demonstrate that SOX9 negatively correlates with infiltration of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, while positively correlating with neutrophils, macrophages, activated mast cells, and naive/activated T cells in colorectal cancer [3]. This immunomodulatory function extends to impairment of CD8+ T cell function, NK cell activity, and M1 macrophage polarization, effectively creating an "immune desert" microenvironment conducive to tumor immune escape [3] [12].

Mechanistic Insights: SOX9 as a Tumor Suppressor

In specific contexts, SOX9 demonstrates unexpected tumor-suppressive properties:

Growth Inhibition in Cervical Cancer

In cervical carcinoma, SOX9 expression progressively decreases during carcinogenesis, with positive staining declining from 82.8% in normal cervical tissues to 33.3% in invasive carcinoma [53]. Functional studies demonstrate that SOX9 overexpression inhibits cervical cancer cell growth in vitro and tumor formation in vivo, while its silencing promotes aggressive tumor phenotypes [53].

p21WAF1/CIP1 Transactivation and Cell Cycle Arrest

The tumor-suppressive mechanism of SOX9 in cervical cancer involves direct transactivation of p21WAF1/CIP1 through physical interaction with specific promoter regions, effectively blocking G1/S cell cycle progression [53]. This pathway represents a functionally distinct activity separable from its oncogenic functions in other tissue contexts.

Experimental Approaches for Investigating SOX9 Function

Table 2: Key Experimental Methodologies for SOX9 Research

Method	Application	Key Findings	Technical Considerations
Chromatin Immunoprecipitation Sequencing (ChIP-seq)	Mapping SOX9 binding sites	Identified SOX9 binding to closed chromatin regions; target genes in HCC	Use validated antibodies; include controls for pioneer factor activity
Single-cell RNA Sequencing (scRNA-seq)	Analyzing tumor heterogeneity	Revealed chemotherapy-induced SOX9 upregulation in ovarian cancer patients	Requires fresh tissue; computational analysis of transcriptional divergence
CRISPR/Cas9 Knockout	Functional validation	SOX9 ablation increased platinum sensitivity in ovarian cancer lines	Monitor both proliferation and stemness phenotypes
ATAC-seq	Chromatin accessibility	SOX9 binding precedes chromatin opening at target enhancers	Time-course experiments essential for pioneer factor studies
Xenograft Models	In vivo tumorigenesis	SOX9 overexpression inhibited cervical tumor growth	Choose models based on SOX9 context-specificity
RNA Interference (siRNA/shRNA)	Knockdown studies	SOX9 suppression reduced invasiveness in HCC cell lines	Confirm specificity given SOX family similarities

Detailed Protocol: Assessing SOX9 in Chemoresistance

For investigating SOX9's role in chemoresistance, as demonstrated in ovarian cancer [23]:

• Cell Line Treatment: Treat HGSOC cell lines (OVCAR4, Kuramochi, COV362) with carboplatin at clinically relevant concentrations (e.g., IC50 values)
• Time-Course Analysis: Harvest cells at 24, 48, and 72 hours post-treatment for RNA and protein extraction
• SOX9 Detection:
  • Quantitative RT-PCR using validated SOX9 primers
  • Western blot with anti-SOX9 antibodies (validate specificity)
• Functional Validation:
  • Generate SOX9-knockout lines using CRISPR/Cas9 with SOX9-targeting sgRNA
  • Perform colony formation assays post-carboplatin treatment
  • Compare IC50 values between parental and SOX9-depleted cells
• Stemness Assessment:
  • Flow cytometry for CSC markers (CD133, CD44)
  • Spheroid formation assays in low-attachment conditions

SOX9-Targeted Therapeutic Approaches

Table 3: Strategies for Targeting SOX9 in Cancer

Approach	Mechanism	Development Stage	Challenges
Small Molecule Inhibitors	Interfere with SOX9 DNA-binding or protein interactions	Preclinical research	Specificity due to conserved HMG domain
RNA Interference (siRNA/shRNA)	Reduce SOX9 mRNA expression	In vitro and in vivo models	Delivery efficiency to tumor cells
CRISPR/Cas9 Gene Editing	Permanent SOX9 gene disruption	Preclinical validation	Safety concerns for normal SOX9 functions
Epigenetic Modulators	Indirect targeting through SOX9 regulatory elements	Drug repurposing candidates	Lack of specificity for SOX9
Natural Compounds (Cordycepin)	Downregulate SOX9 expression	In vitro evidence in prostate cancer cells	Mechanism not fully elucidated
Combination Therapies	Target SOX9 with conventional chemotherapeutics	Conceptual based on resistance data	Timing and sequencing critical

Current SOX9-targeting strategies face significant challenges due to the transcription factor's structural characteristics and physiological importance. The highly conserved HMG domain presents limited opportunities for selective pharmacological inhibition without affecting related SOX family members [54]. Alternative approaches focusing on upstream regulators or downstream effectors may offer more viable therapeutic opportunities while mitigating toxicity concerns.

The Scientist's Toolkit: Essential Research Reagents

Table 4: Key Research Reagents for SOX9 Investigation

Reagent	Function	Application	Examples/Sources
Anti-SOX9 Antibodies	Detection and quantification	IHC, WB, ChIP	Validate specificity for SOX9 versus other SOX family members
SOX9-Expressing Plasmids	Gain-of-function studies	Ectopic expression	Use tissue-specific promoters for relevant contexts
SOX9-Targeting siRNAs/shRNAs	Transient knockdown	Functional studies	Include multiple targets to confirm specificity
CRISPR/Cas9 SOX9 KO Lines	Permanent gene disruption	Mechanistic studies	Monitor potential compensatory SOX family expression
SOX9 Reporter Constructs	Monitoring transcriptional activity	Pathway analysis	Combine with mutant constructs for domain studies
Chemical Inhibitors (Cordycepin)	SOX9 downregulation	Therapeutic screening	Dose-response essential; monitor off-target effects

Visualizing SOX9 Signaling and Experimental Approaches

SOX9 Functional Pathways and Targeting Strategies

SOX9 Research Workflow

The dual nature of SOX9 as both oncogene and tumor suppressor represents a significant challenge in cancer therapeutics that necessitates tissue-specific approaches. Future research directions should focus on elucidating the molecular determinants that dictate SOX9's functional switch, developing context-sensitive targeting strategies, and exploring combination therapies that account for SOX9's role in therapy resistance. The conservation of SOX9 function across species and its fundamental role in stem cell biology further underscore the need for sophisticated targeting approaches that can distinguish between physiological and pathological SOX9 activities. As our understanding of SOX9's complex regulatory networks deepens, so too will our ability to develop effective therapeutic strategies that either exploit or inhibit its functions based on specific cancer contexts.

Combination therapy targeting the immune checkpoints PD-1 and LAG-3 represents an advanced immunotherapeutic strategy. However, resistance to this treatment poses a significant clinical challenge. Recent investigations have identified the transcription factor SOX9 as a critical mediator of this resistance, orchestrating a complex immunosuppressive program within the tumor microenvironment. This review synthesizes evidence from head and neck squamous cell carcinoma (HNSCC) and other malignancies, detailing the mechanistic role of SOX9 in promoting immune evasion. We further explore the conservation of SOX9 function across species and provide a detailed compendium of the experimental models and reagents essential for investigating this pathway, offering a foundational resource for developing strategies to overcome SOX9-driven resistance.

The SRY-Box Transcription Factor 9 (SOX9) is a pivotal regulator of cell fate and differentiation during embryonic development, with essential functions in chondrogenesis, sex determination, and organogenesis [3] [9] [20]. In oncology, SOX9 is frequently overexpressed in diverse solid malignancies, including lung, breast, ovarian, and head and neck cancers, where it drives tumor initiation, progression, stemness, and chemoresistance [3] [23] [55]. Beyond its established oncogenic roles, SOX9 has emerged as a potent modulator of the tumor immune microenvironment [3].

SOX9 operates as a "janus-faced" regulator in immunity, capable of both promoting tissue repair and facilitating immune escape [3]. Its function is highly context-dependent, influencing the differentiation and activity of various immune lineages [3]. Critically, SOX9 expression in tumor cells can reshape the microenvironment into an "immune cold" or "immune desert" state, characterized by impaired infiltration and function of cytotoxic lymphocytes [3] [56] [55]. This capacity to suppress anti-tumor immunity positions SOX9 as a central player in resistance to modern immunotherapies, particularly combination immune checkpoint blockade.

The SOX9-ANXA1-FPR1 Axis: A Core Mechanism of Resistance to Anti-PD-1/Anti-LAG-3 Therapy

Resistance to the combination of anti-PD-1 and anti-LAG-3 checkpoint inhibitors is a significant barrier in treating advanced cancers. A seminal 2025 study using a HNSCC mouse model pinpointed the enrichment of SOX9-positive tumor cells in resistant samples [56]. This research delineated a precise molecular axis through which SOX9+ tumor cells impair cytotoxic immunity.

Mechanism of Action

The resistance mechanism involves a paracrine signaling pathway where SOX9 directly regulates the expression of Annexin A1 (ANXA1) in tumor cells [56]. ANXA1 is then secreted and engages the Formyl Peptide Receptor 1 (FPR1) on neutrophils. This ANXA1-FPR1 interaction initiates a deleterious intracellular cascade in neutrophils:

• It promotes excessive mitochondrial fission.
• It inhibits mitophagy by downregulating BCL2/adenovirus E1B interacting protein 3 (BNIP3) expression.
• The cumulative effect induces neutrophil apoptosis and prevents their accumulation in the tumor tissue [56].

The reduction of FPR1+ neutrophils in the tumor microenvironment creates an "immune desert" by indirectly impairing the infiltration and tumor-cell killing capacity of CD8+ T cells and γδ T cells, thereby driving therapy resistance [56].

Table 1: Core Components of the SOX9-ANXA1-FPR1 Resistance Axis

Component	Role in Resistance Mechanism	Experimental Evidence
SOX9+ Tumor Cell	Initiates the pathway; directly transcribes and upregulates ANXA1.	Enriched in resistant tumors in HNSCC mouse model (scRNA-seq) [56].
Annexin A1 (ANXA1)	Secreted signaling protein; ligand for FPR1 on neutrophils.	Sox9 directly regulates its expression; key mediator of neutrophil apoptosis [56].
FPR1+ Neutrophil	Primary immune cell target; receives ANXA1 signal leading to its death.	Reduction of Fpr1+ neutrophils in TME impairs cytotoxic cell function [56].
BNIP3	Key mitophagy regulator; downregulated in neutrophils upon ANXA1-FPR1 engagement.	Downregulation inhibits mitophagy, contributing to neutrophil apoptosis [56].
CD8+ & γδ T cells	Effector cytotoxic cells; indirectly suppressed via loss of neutrophils.	Reduction in tumor infiltration and killing capacity [56].

Experimental Workflow for Investigating the Axis

The following diagram illustrates the key experimental steps used to identify and validate the SOX9-ANXA1-FPR1 resistance axis:

SOX9-Mediated Immunosuppression Across Cancer Types

The role of SOX9 in fostering an immunosuppressive tumor microenvironment extends beyond HNSCC and the ANXA1-FPR1 axis, as evidenced by research in other malignancies.

SOX9 in Lung Adenocarcinoma (LUAD)

In KRAS-driven lung adenocarcinoma, SOX9 overexpression accelerates tumor formation and creates an "immune cold" microenvironment [55] [57]. Mechanistically, SOX9 suppresses the infiltration and activity of key anti-tumor immune cells, including CD8+ T cells, natural killer (NK) cells, and dendritic cells (DCs) [55]. Furthermore, SOX9 significantly elevates collagen-related gene expression and increases collagen fiber deposition, suggesting it modulates the physical structure of the tumor microenvironment, potentially creating a physical barrier to immune cell entry [55].

SOX9 in Breast Cancer

In breast cancer, a SOX9-B7x (B7-H4/VTCN1) axis has been identified as a safeguard for dedifferentiated tumor cells [58]. SOX9 drives the expression of B7x, a co-inhibitory ligand that dampens T cell activation. This axis facilitates immune evasion during breast cancer progression, particularly in the context of ductal carcinoma in situ (DCIS) progression to invasive disease [58]. SOX9 also contributes to immune evasion by maintaining cancer stem cell properties, allowing latent cancer cells to persist in secondary sites and avoid immune surveillance [29] [9].

Table 2: Comparative Overview of SOX9-Driven Immunosuppressive Mechanisms

Cancer Type	Primary Immune Evasion Mechanism	Key Effected Immune Cells	Therapeutic Context
Head & Neck SCC	ANXA1-FPR1 axis inducing neutrophil apoptosis.	Fpr1+ Neutrophils, CD8+ T cells, γδ T cells.	Anti-PD-1 + Anti-LAG-3 resistance [56].
Lung Adenocarcinoma	Creation of "immune cold" TME; increased collagen deposition.	CD8+ T cells, NK cells, Dendritic Cells.	KRAS-driven tumorigenesis; Immunotherapy response [55] [57].
Breast Cancer	Upregulation of B7x (B7-H4/VTCN1) immune checkpoint.	Tumor-infiltrating T lymphocytes.	DCIS progression; general immune evasion [58].

Conservation of SOX9 Function and Targets Across Species

Understanding the conservation of SOX9's regulatory networks is crucial for translating findings from model organisms to human therapeutics. Comparative chromatin immunoprecipitation sequencing (ChIP-seq) analyses between mouse and chicken embryos reveal that SOX9's function is highly conserved in chondrogenesis but shows lower conservation in gonad development [9].

• Conserved in Chondrocytes: In developing limb buds, SOX9 binding regions are frequently located in intronic and distal regions, and are enriched for SOX palindromic repeats, indicating a conserved mechanism for regulating cartilage-specific target genes like COL2A1 [9].
• Less Conserved in Sertoli Cells: In contrast, SOX9 binding in male gonads more often occurs in proximal upstream regions of genes with fewer palindromic motifs, and the target genes themselves show lower similarity between species [9].

This cell type-specific conservation pattern suggests that SOX9's role in developmental processes tied to its HMG-box DNA-binding domain is robust across species. Its context-dependent functions in other tissues, potentially including immune regulation in cancer, may be more divergent. This underscores the importance of validating findings in multiple model systems, especially when studying complex processes like tumor-immune interactions.

The Scientist's Toolkit: Key Research Reagents and Models

Advancing research on SOX9 and immunotherapy resistance relies on a specific toolkit of experimental models, reagents, and methodologies. The table below details essential resources derived from the cited studies.

Table 3: Key Research Reagents and Experimental Models for SOX9 Studies

Category / Reagent	Specific Example / Model	Function and Application in SOX9 Research
In Vivo Models	HNSCC mouse model (TgfβR1/Pten deletion) [56] [59]	Studies anti-PD-1/anti-LAG-3 resistance and SOX9 role in HNSCC.
	KrasLSL-G12D; Sox9flox/flox (KSf/f) GEMM [55]	Investigates SOX9 in KRAS-driven lung adenocarcinoma and TME.
	Krt14-rtTA;TRE-Sox9 Inducible Mouse [20]	Models SOX9 as a pioneer factor in cell fate switching and cancer.
Critical Assays	Single-Cell RNA Sequencing (scRNA-seq) [56] [23]	Identifies SOX9+ subpopulations and immune context in TME.
	Chromatin Immunoprecipitation (ChIP-seq) [9]	Maps SOX9 DNA binding and conserved target genes across species.
	ATAC-seq / CUT&RUN [20]	Probes chromatin accessibility and pioneer factor activity of SOX9.
Key Antibodies	Anti-LAG-3 (Relatlimab) & Anti-PD-1 (Nivolumab) [56]	Immune checkpoint blockade to study therapy resistance mechanisms.
	Anti-SOX9 (Validated for IHC/IF) [55]	Detects SOX9 protein expression and localization in tissues/cells.
Molecular Tools	CRISPR/Cas9 (Sox9 knockout) [23] [55]	Validates SOX9 necessity through genetic loss-of-function.
	Sox9 Overexpression Vectors [23] [55]	Tests SOX9 sufficiency in driving chemoresistance and stemness.

The following diagram maps the logical relationship between the core experimental models, the key findings they enabled, and the resulting therapeutic implications:

The identification of SOX9 as a central mediator of resistance to combined anti-PD-1/anti-LAG-3 therapy unveils a significant challenge and a promising opportunity in oncology. The SOX9-ANXA1-FPR1 axis provides a mechanistic explanation for the failure of potent immunotherapy in some patients with HNSCC, while parallel research confirms SOX9's broader role in suppressing anti-tumor immunity across cancers.

Future research must focus on several key areas:

• Translational Validation: Correlating SOX9 expression levels with clinical outcomes in patients receiving combination checkpoint blockade.
• Therapeutic Targeting: Developing strategies to inhibit the SOX9 pathway or its downstream effectors, such as the ANXA1-FPR1 axis or the B7x immune checkpoint.
• Combination Strategies: Designing clinical trials that pair anti-PD-1/anti-LAG-3 with agents that block SOX9-driven immunosuppression.

Ultimately, understanding and targeting SOX9-mediated resistance pathways holds the potential to expand the efficacy of immunotherapy and improve survival for patients with advanced solid tumors.

SOX9 as a Pan-Cancer Immunological Biomarker: Validation and Cross-Species Comparison

The SOX9 (SRY-box transcription factor 9) gene, mapping to 17q24.3, encodes a 509-amino acid transcription factor containing a highly conserved high-mobility group (HMG) box domain that facilitates DNA binding and nuclear localization [17] [3]. As a pivotal member of the SOX family, SOX9 plays fundamental roles in embryonic development, cell lineage determination, and tissue homeostasis [7] [14]. In recent years, substantial evidence has emerged regarding its dysregulation across diverse cancer types, positioning SOX9 as a significant player in tumorigenesis and cancer progression.

This comprehensive analysis systematically evaluates SOX9 expression patterns across multiple cancer types, their correlation with clinical prognosis, and intricate relationships with tumor immune microenvironments. Within the broader context of SOX9 target conservation across species, this review integrates pan-cancer genomic data with experimental findings to elucidate SOX9's multifaceted functions, which exhibit both remarkable conservation and context-dependent divergence between developmental and oncogenic processes [8]. The evolutionary conservation of SOX9 targets appears more pronounced in chondrogenesis than in gonad development, suggesting cell type-specific functional conservation patterns that may extend to its roles in immune regulation and cancer biology [8].

SOX9 Expression Patterns Across Human Cancers

Pan-Cancer Expression Landscape

SOX9 demonstrates remarkably diverse expression patterns across human malignancies, functioning as either an oncogene or tumor suppressor depending on cancer type. Comprehensive analysis of transcriptomic data from TCGA, GTEx, and other genomic repositories reveals that SOX9 expression is significantly upregulated in fifteen cancer types compared to matched healthy tissues, including glioblastoma (GBM), colorectal adenocarcinoma (COAD), esophageal carcinoma (ESCA), liver hepatocellular carcinoma (LIHC), lung squamous cell carcinoma (LUSC), ovarian cancer (OV), pancreatic adenocarcinoma (PAAD), stomach adenocarcinoma (STAD), and thymoma (THYM) [17]. Conversely, SOX9 expression is significantly decreased in only two cancer types: skin cutaneous melanoma (SKCM) and testicular germ cell tumors (TGCT) [17]. This expression pattern suggests that SOX9 predominantly functions as a proto-oncogene across most human cancers.

Table 1: SOX9 Expression Patterns Across Human Cancers

Cancer Type	SOX9 Expression Pattern	Biological Role	Representative References
Glioblastoma (GBM)	Significantly upregulated	Oncogene	[7] [14]
Colorectal adenocarcinoma (COAD)	Significantly upregulated	Oncogene	[17] [60]
Lung adenocarcinoma (LUAD)	Significantly upregulated	Oncogene	[55]
Esophageal carcinoma (ESCA)	Significantly upregulated	Oncogene	[17] [60]
Liver hepatocellular carcinoma (LIHC)	Significantly upregulated	Oncogene	[17] [60]
Skin cutaneous melanoma (SKCM)	Significantly downregulated	Tumor suppressor	[17]
Testicular germ cell tumors (TGCT)	Significantly downregulated	Tumor suppressor	[17]

Protein-level analyses through immunohistochemistry confirm that SOX9 is widely expressed in normal human organs, with high expression detected in 13 organs and no expression in only two organs. Across 44 normal tissues, SOX9 shows high expression in 31 tissues, medium expression in 4 tissues, low expression in 2 tissues, and no expression in the remaining 7 tissues [17]. This widespread tissue distribution underscores SOX9's fundamental physiological roles and suggests its potential involvement in tissue-specific tumorigenesis.

SOX9 in Glioblastoma

In glioblastoma, one of the most aggressive intracranial malignancies, SOX9 is significantly overexpressed compared to normal brain tissue [7] [14]. This elevated expression demonstrates diagnostic utility, with receiver operating characteristic (ROC) analysis confirming SOX9's predictive value for GBM diagnosis [7]. Interestingly, within specific GBM subgroups, particularly those with lymphoid invasion, high SOX9 expression correlates unexpectedly with better prognosis, highlighting the context-dependent nature of SOX9's clinical significance [7]. Furthermore, SOX9 emerges as an independent prognostic factor for IDH (isocitrate dehydrogenase)-mutant glioblastoma in Cox regression analysis, reinforcing its subtype-specific prognostic value [7].

Prognostic Value of SOX9 in Solid Tumors

Meta-Analysis of Survival Correlations

A comprehensive meta-analysis encompassing 17 studies with 3,307 patients evaluated the prognostic significance of SOX9 overexpression across solid tumors [60]. The pooled hazard ratios (HRs) revealed that high SOX9 expression significantly correlates with poor overall survival (OS) in multivariate analysis (HR = 1.66, 95% CI: 1.36-2.02, P < 0.001) and demonstrates an even stronger association with reduced disease-free survival (DFS) (HR = 3.54, 95% CI: 2.29-5.47, P = 0.008) [60]. These findings establish SOX9 overexpression as a robust predictor of unfavorable clinical outcomes across multiple cancer types.

Table 2: Prognostic Value of SOX9 Expression Across Cancers

Cancer Type	Overall Survival Correlation	Disease-Free Survival Correlation	Clinicopathological Associations
Lung adenocarcinoma	Shorter survival (HR: 1.66)	Reduced DFS (HR: 3.54)	Advanced stage, metastasis [60] [55]
Gastric adenocarcinoma	Shorter survival	Reduced DFS	Large tumor size, lymph node metastasis, higher clinical stage [60] [61]
Glioblastoma	Varies by subgroup	Not reported	IDH-mutant prognostic factor [7]
Colorectal cancer	Shorter survival	Reduced DFS	Lymph node metastasis, distant metastasis [60]
Breast cancer	Shorter survival	Reduced DFS	Large tumor size, advanced stage [60]

Additionally, pooled odds ratios (ORs) indicate that SOX9 overexpression significantly associates with aggressive clinicopathological features, including larger tumor size, lymph node metastasis, distant metastasis, and higher clinical stage [60]. These consistent correlations across diverse cancer types underscore SOX9's role in driving tumor progression and metastasis.

Cancer-Specific Prognostic Relationships

The prognostic impact of SOX9 exhibits cancer-specific variations that reflect its context-dependent functions. In lung adenocarcinoma, patients with SOX9-high tumors (top 20% of expression) show significantly shorter survival (p = 0.0039), while SOX9-low patients (lowest 15%) experience longer survival [55]. Similarly, in gastric adenocarcinoma, SOX9 expression emerges as an independent predictor of poor prognosis in multivariate analysis, with particular prognostic value in poorly differentiated subtypes [61].

Unexpectedly, in certain cancer contexts such as melanoma, SOX9 demonstrates tumor-suppressive properties. decreased SOX9 expression in SKCM and experimental increase of SOX9 in melanoma cell lines inhibits tumorigenicity in both mouse and human ex vivo models [17]. This paradoxical behavior highlights SOX9's context-dependent functionality and underscores the importance of cancer-type-specific interpretations of its prognostic significance.

SOX9 and Tumor Immune Microenvironment

Regulation of Immune Cell Infiltration

SOX9 expression demonstrates complex, cancer-type-specific correlations with immune cell infiltration patterns within the tumor microenvironment. In colorectal cancer, SOX9 expression negatively correlates with infiltration levels of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, while showing positive correlations with neutrophils, macrophages, activated mast cells, and naive/activated T cells [3]. This suggests SOX9 may promote an immunosuppressive microenvironment while simultaneously activating specific pro-tumor immune populations.

In lung adenocarcinoma, SOX9 functionally suppresses anti-tumor immunity by reducing infiltration of CD8+ T cells, natural killer (NK) cells, and dendritic cells [55]. This immunosuppressive function was demonstrated through tumor grafting experiments in immunocompetent versus immunocompromised mice, where SOX9-promoted tumor growth was significantly attenuated in immunocompromised hosts, indicating its reliance on immune modulation [55]. Mechanistically, SOX9 was found to elevate collagen-related gene expression and substantially increase collagen fibers, potentially creating a physical barrier that inhibits immune cell infiltration [55].

Immune Checkpoint Associations

SOX9 expression correlates significantly with immune checkpoint molecule expression across multiple cancers. In glioblastoma, SOX9 expression shows correlation with immune checkpoint expression patterns, suggesting its potential involvement in immune evasion mechanisms [7]. Similarly, in breast cancer, SOX9 activates a transcriptional program that upregulates the immune checkpoint molecule B7x (B7-H4/VTCN1), creating an immune-suppressive niche that protects dedifferentiated tumor cells from T cell-mediated surveillance [58].

SOX9-Mediated Immunomodulation in Tumor Microenvironment

The relationship between SOX9 and PD-L1 expression appears cancer-type specific. In gastric adenocarcinoma, high SOX9 expression associates with negative PD-L1 status [61], whereas in thymoma, SOX9 negatively correlates with genes associated with PD-L1 expression and T-cell receptor signaling pathways [17]. These divergent relationships highlight the tissue-specific nature of SOX9's immunomodulatory functions and necessitate careful contextual interpretation when considering SOX9 as a potential predictor for immunotherapy response.

SOX9 Target Conservation Across Species

Comparative analyses of SOX9 binding patterns reveal both conserved and divergent regulatory functions across species. Chromatin immunoprecipitation sequencing (ChIP-seq) performed on developing limb buds and male gonads from mouse and chicken embryos demonstrated that SOX9 predominantly binds to intronic and distal regions of genes in limb buds, while favoring proximal upstream regions in male gonads [8]. Notably, the conservation of SOX9 binding regions was significantly higher in limb bud genes than in male gonad genes [8].

When combined with RNA expression analysis, these studies determined that SOX9 target genes show high similarity in chondrocytes between mouse and chicken, but considerably lower conservation in Sertoli cells [8]. This tissue-specific conservation pattern extends to SOX9's roles in cancer biology, where its functions in fundamental processes like cell proliferation and epithelial-mesenchymal transition appear more conserved than its immunomodulatory activities, which may exhibit greater species and context specificity.

Experimental Models and Methodologies

Key Experimental Approaches

The investigation of SOX9's oncogenic functions employs diverse experimental methodologies across in vitro and in vivo systems. In lung adenocarcinoma research, CRISPR/Cas9 and Cre-LoxP gene knockout approaches in KrasG12D-driven mouse models demonstrated that Sox9 loss significantly reduces lung tumor development, burden, and progression, contributing to markedly longer overall survival [55]. These genetic engineering approaches provide robust evidence for SOX9's causal role in driving lung tumorigenesis.

Three-dimensional tumor organoid culture systems have proven valuable for assessing SOX9's functional contributions to tumor growth. In KrasG12D mouse lung tumor models, SOX9 overexpression significantly increased both organoid size and cell number per organoid, while immunohistochemical analysis revealed strong correlation between SOX9 and the proliferation marker Ki67 [55]. These organoid models effectively recapitulate key aspects of in vivo tumor biology while permitting precise experimental manipulation.

Pharmacological Modulation Approaches

Small molecule compounds provide additional tools for probing SOX9 function and exploring therapeutic strategies. Cordycepin (CD), an adenosine analog derived from Cordyceps sinensis, inhibits both protein and mRNA expression of SOX9 in a dose-dependent manner in prostate cancer (22RV1, PC3) and lung cancer (H1975) cell lines [17]. This SOX9 inhibition correlates with cordycepin's established anti-cancer effects, suggesting SOX9 suppression as a potential mechanism underlying its therapeutic activity.

Table 3: Experimental Approaches for SOX9 Functional Analysis

Method Category	Specific Techniques	Key Applications	Representative Examples
Genomic Analysis	RNA-seq, ChIP-seq, TCGA data mining	Expression profiling, target identification, binding site mapping	[7] [8]
Genetic Manipulation	CRISPR/Cas9, Cre-LoxP, knockdown/overexpression	Functional validation, causal relationships	[55]
Disease Modeling	GEMMs, organoid cultures, xenografts	Pathophysiological analysis, therapeutic testing	[55]
Immune Analysis	Flow cytometry, IHC, infiltration algorithms	Tumor microenvironment characterization	[3] [55]
Pharmacological Modulation	Cordycepin treatment	Therapeutic targeting, pathway analysis	[17]

Research Reagent Solutions

The investigation of SOX9's roles in cancer and immunity relies on specialized research reagents and methodologies. Key experimental tools include validated antibodies for immunohistochemistry (e.g., Millipore AB5535 for SOX9 [61]), CRISPR/Cas9 systems for gene editing (e.g., pSECC system with guide RNAs targeting Sox9 [55]), and organoid culture protocols for three-dimensional tumor modeling [55]. Database resources such as The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA), and LinkedOmics provide essential bioinformatic platforms for SOX9 expression and correlation analyses [7] [17].

For immune cell infiltration analysis, computational algorithms like ssGSEA and ESTIMATE enable comprehensive characterization of tumor immune microenvironments and their relationships with SOX9 expression [7]. Pharmacological studies utilize compounds such as cordycepin to modulate SOX9 expression, with dose-dependent inhibition observed at concentrations of 10-40 μM in cancer cell lines [17]. These research tools collectively enable multifaceted investigation of SOX9's oncogenic functions and therapeutic targeting.

Methodological Framework for SOX9 Research

This pan-cancer analysis establishes SOX9 as a multifaceted regulator of tumor progression with significant prognostic implications and extensive interactions with tumor immune microenvironments. The consistent association between SOX9 overexpression and poor clinical outcomes across most solid tumors highlights its potential utility as a prognostic biomarker and therapeutic target. Its complex, context-dependent relationships with immune cell infiltration and checkpoint expression further position SOX9 as a significant modulator of anti-tumor immunity.

The conservation of SOX9 targets across species in developmental contexts provides an evolutionary framework for understanding its oncogenic functions, while species- and tissue-specific differences offer insights into the contextual nature of its activities. Future research directions should include developing more precise SOX9-targeted therapeutic strategies, elucidating the mechanisms underlying its context-dependent immune functions, and exploring its potential as a predictor for immunotherapy response across different cancer types.

The transcription factor SOX9 is a critical regulator of cell fate determination, functioning in the development and maintenance of diverse tissues. Its dysregulation is implicated in congenital diseases and cancer progression. A central challenge in SOX9 research is validating its direct targets and understanding the conservation of its regulatory networks across different species and experimental systems. This guide objectively compares the performance of two primary validation methodologies—transgenic mouse models and human ex vivo systems—in characterizing SOX9 targets, providing a structured analysis of their applications, outputs, and limitations within the context of immune and stromal cell research.

Comparative Experimental Platforms for SOX9 Target Validation

The following table summarizes the core characteristics, data outputs, and key performance metrics of the two main experimental platforms used for validating SOX9 targets.

Feature	Transgenic Mouse Models	Human Ex Vivo Systems
System Overview	Genetically engineered mice for tissue-specific SOX9 manipulation within a intact tissue microenvironment [62] [63].	Cultured human primary cells or cell lines; allows for controlled SOX9 perturbation outside the native tissue context [62] [17].
Key Strengths	Preserves native cellular interactions, niche signals, and tissue architecture; enables study of systemic effects and long-term outcomes like tumorigenesis [20] [64].	High experimental control and reproducibility; facilitates high-throughput screening (e.g., miRNA mimics, drug treatments); enables direct study of human cellular mechanisms [62] [17].
Key Limitations	Lower throughput; complex and costly generation and maintenance; potential for compensatory developmental mechanisms; species-specific differences [25] [63].	Lacks full physiological context and systemic inputs; potential for cellular dedifferentiation in culture; may not fully recapitulate in vivo disease states [62].
Primary Data Outputs	Phenotypic histology, RNA-seq from FACS-purified cells, chromatin accessibility (ATAC-seq), transcription factor binding (CUT&RUN/ChIP-seq) [20].	Luciferase reporter assays, mRNA/protein expression (qPCR, Western blot), ChIP-PCR, cell proliferation and viability assays [62] [17].
Typical Timeline	Months to over a year (from model generation to analysis).	Days to a few weeks.
Conservation Insight	Directly tests functional conservation of SOX9 targets in a mammalian system; can reveal context-dependent binding and function [25].	Provides a direct readout of human gene regulation; differences from mouse data highlight species-specific regulation [62] [25].

Detailed Experimental Methodologies and Representative Findings

Transgenic Mouse Models: In Vivo Target Validation

1. Protocol for Conditional Overexpression and Target Analysis

• Model Generation: Cross CAG-mRFP1floxed-Sox9-EGFP mice (enabling Cre-inducible SOX9 expression) with a tissue-specific Cre driver line (e.g., Krt14-rtTA for epidermal stem cells or BEST1-Cre for retinal pigment epithelium) [20] [62] [63].
• Gene Induction: Administer doxycycline (for rtTA systems) or rely on endogenous Cre activity to activate SOX9 transgene expression.
• Cell Isolation: At defined timepoints, harvest target tissues and dissociate into single-cell suspensions. Use Fluorescence-Activated Cell Sorting (FACS) to isolate pure populations of SOX9-expressing cells based on EGFP reporter fluorescence [20].
• Target Validation:
  • Transcriptomics: Perform RNA sequencing (RNA-seq) on sorted cells to identify differentially expressed genes upon SOX9 induction [20].
  • Epigenomics: Conduct CUT&RUN or ChIP-seq to map genomic binding sites of SOX9. Perform ATAC-seq on sorted cells to assess changes in chromatin accessibility at these binding sites [20].
  • Functional Validation: Correlate binding data with gene expression changes and confirm phenotype through histological staining (e.g., H&E, Safranin O) and immunohistochemistry [63].

2. Key Findings from Mouse Models

• Pioneer Factor Function: In epidermal stem cells, SOX9 binds to closed chromatin at key hair follicle enhancers before nucleosome displacement and chromatin opening, a hallmark of pioneer factor activity [20].
• Fate Switching Mechanism: SOX9 binding directly activates a new transcriptional program (e.g., hair follicle genes) while simultaneously silencing the original cell identity (e.g., epidermal genes) by competing for and redistributing limited epigenetic co-factors [20].
• Conservation and Divergence: A comparative ChIP-seq analysis in mouse and chicken embryos revealed that SOX9 target genes are highly conserved in chondrocytes but show significant divergence in Sertoli cells, highlighting the cell type-specific nature of its regulatory network [25].
• Stromal Cell Identity: Single-cell RNA-seq of lymphoid stroma in mice identified a conserved Sox9-expressing Follicular Dendritic Cell (FDC) subset across different lymphoid organs, underscoring a conserved role in stromal niche formation [64].

Human Ex Vivo Systems: Controlled Mechanistic Studies

1. Protocol for Luciferase Reporter and miRNA Interaction Assays

• Promoter/Enhancer Cloning: Amplify putative SOX9-target promoter regions (e.g., from RPE65, RLBP1, RGR) or 3'UTR sequences of interest from human genomic DNA. Clone these fragments into a luciferase reporter vector (e.g., pGL2-Basic for promoters, pmirGLO for 3'UTRs) [62].
• Cell Transfection: Co-transfect human cell lines (e.g., D407 RPE cells) with the following [62]:
  • The luciferase reporter construct.
  • SOX9 expression vector (pcDNA3.1-SOX9) or a control empty vector.
  • Optional: Vectors for partner transcription factors (e.g., OTX2, LHX2) or synthetic microRNA (miRNA) mimics.
• Response Measurement: After 24-48 hours, lyse cells and measure luciferase activity using a luminometer. Normalize data to a co-transfected control (e.g., Renilla luciferase) to account for transfection efficiency [62].
• Complementary Assays: Validate findings by measuring endogenous mRNA expression of the target gene (e.g., RPE65) via qRT-PCR or protein expression via Western blot after SOX9 or miRNA modulation [62] [17].

2. Key Findings from Human Ex Vivo Systems

• Synergistic Activation: SOX9 synergizes with different partner factors (e.g., OTX2 on the RPE65 and RLBP1 promoters; LHX2 on the RGR promoter) to activate visual cycle genes in the retinal pigment epithelium, demonstrating context-dependent partnership [62].
• Post-Transcriptional Regulation: The 3'UTRs of several visual cycle genes (RPE65, RLBP1) contain functional binding sites for common miRNAs (e.g., miR-137), establishing a mechanism for their coordinated post-transcriptional regulation [62].
• Pharmacological Modulation: The small molecule Cordycepin was shown to inhibit SOX9 mRNA and protein expression in a dose-dependent manner in human cancer cell lines (e.g., 22RV1, PC3, H1975), identifying a potential chemical tool for SOX9 perturbation [17].
• Direct Promoter Binding: Chromatin Immunoprecipitation (ChIP) using human fetal RPE cells confirmed the direct binding of SOX9 and OTX2 to the promoter regions of key visual cycle genes, validating predictions from mouse models in a human context [62].

Visualizing SOX9 Workflows and Regulatory Networks

Comparative Conservation Analysis Workflow

The following diagram illustrates the integrated workflow for validating and comparing SOX9 targets across species and systems.

Transgenic Mouse Model Experimental Pipeline

This diagram details the key steps in generating and analyzing transgenic mouse models for SOX9 target validation.

The Scientist's Toolkit: Key Research Reagents

This table catalogs essential reagents and tools derived from the cited studies for investigating SOX9 biology.

Research Reagent / Tool	Function / Application	Example Use Case
CAG-mRFP1floxed-Sox9-EGFP Mice [63]	Enables Cre-dependent, conditional SOX9 overexpression in any tissue.	Studying the effects of SOX9 misexpression in epidermal stem cells to trigger fate switching [20].
BEST1-Cre Mice [62]	Drives Cre recombinase expression specifically in the Retinal Pigment Epithelium (RPE).	Generating RPE-specific Sox9 knockout mice to study its role in the visual cycle [62].
Krt14-rtTA; TRE-Sox9 Mice [20]	Allows inducible, temporal control of SOX9 expression in basal epidermal keratinocytes via doxycycline.	Modeling the stepwise reprogramming of epidermal cells towards a hair follicle fate and BCC pathogenesis [20].
Cxcl13-Cre/TdTomato EYFP Mice [64]	Labels CXCL13-expressing stromal cells, including key BRC subsets, across lymphoid organs.	Isolating and characterizing B cell zone reticular cells (BRCs) for scRNA-seq to define stromal niches [64].
Luciferase Reporter Vectors (pGL2-Basic, pmirGLO) [62]	Measures the transcriptional activity of promoters or the post-transcriptional regulation by miRNAs.	Validating the synergistic activation of the RPE65 promoter by SOX9 and OTX2 [62].
Cordycepin (CD) [17]	A small molecule adenosine analog that inhibits SOX9 expression.	Pharmacological downregulation of SOX9 in prostate and lung cancer cell lines to study its oncogenic role [17].
SOX9, OTX2, LHX2 Expression Vectors [62]	Plasmid constructs for overexpressing transcription factors in cell culture.	Testing combinatorial regulation of target genes in co-transfection assays [62].

Comparative analysis of SOX9 binding sites and partner transcription factors (e.g., TCFs, Sp1) across species and cell types

The transcription factor SOX9 is a master regulator of cell fate with essential roles in diverse developmental processes, including chondrogenesis, testis determination, and organogenesis [2]. As a member of the SOX (SRY-related HMG box) family, SOX9 contains a high mobility group (HMG) DNA-binding domain that recognizes the consensus sequence (A/T)(A/T)CAA(A/T)G [9] [65]. Despite its diverse functions across tissues and species, the SOX9 protein sequence remains remarkably conserved throughout vertebrate evolution [16]. This conservation presents a fundamental question: how does a transcription factor with conserved sequence and DNA-binding specificity achieve such diverse regulatory outcomes?

This guide provides a comparative analysis of SOX9 binding sites and partner transcription factors across species and cell types, focusing on its implications for immune cell research. We objectively evaluate experimental data from genomic studies to elucidate how cell-type-specific and species-specific differences in SOX9 interactions contribute to its functional diversity, with particular relevance to immunological processes and therapeutic development.

Structural and functional domains of SOX9

The human SOX9 protein comprises 509 amino acids with several functionally distinct domains [2] [3]. The HMG box facilitates sequence-specific DNA binding and nuclear localization through embedded nuclear localization signals (NLS). The dimerization domain (DIM) enables SOX9 to form homodimers or heterodimers with other SOXE proteins (SOX8 and SOX10). Two transactivation domains (TAM in the middle and TAC at the C-terminus) mediate interactions with transcriptional co-activators. A unique proline/glutamine/alanine (PQA)-rich domain enhances transactivation capability [2].

Figure 1: Functional domains of the SOX9 transcription factor and their primary functions.

Species conservation of SOX9 binding patterns

Cross-species conservation in chondrocytes versus Sertoli cells

Comparative chromatin immunoprecipitation sequencing (ChIP-seq) analyses reveal striking differences in how SOX9 binding patterns are conserved between cell types across species. Studies comparing mouse and chicken demonstrate that SOX9 target genes show high conservation in chondrocytes but significantly lower conservation in Sertoli cells [9].

Table 1: Conservation of SOX9 binding regions between mouse and chicken

Feature	Chondrocytes (Limb Bud)	Sertoli Cells (Male Gonad)
Positional distribution	Preferentially intronic and distal regions (>32% upstream)	Preferentially proximal upstream regions (>51% upstream)
SOX palindromic motifs	Frequently present (19.65% of binding regions)	Rarely present (8.72% of binding regions)
Sequence conservation	High conservation between mouse and chicken	Low conservation between mouse and chicken
Target gene similarity	High similarity across species	Low similarity across species
Primary binding motif	Inverted SOX9 binding motifs separated by 4bp	Single SOX9 binding motifs

The high conservation of SOX9 targets in chondrocytes aligns with SOX9's well-established and conserved role in regulating cartilage-specific extracellular matrix genes like COL2A1 and COL11A2 across vertebrates [9]. In contrast, the low conservation in Sertoli cells reflects the divergent evolution of sex determination mechanisms, where SOX9's regulatory function is not strictly conserved across vertebrates [9].

Mammalian conservation in testis development

While conservation between distant vertebrates is low in Sertoli cells, studies comparing mouse and bovine fetal testes demonstrate that SOX9 binds to a conserved genetic program involving most sex-determining genes in these mammals [66]. Analysis of SOX9-bound chromatin regions from murine and bovine fetal testes identified a conserved "Sertoli Cell Signature" (SCS) characterized by precise organization of binding motifs for SOX9, GATA4, and DMRT1 [66].

Cell type-specific partner transcription factors

SOX9's functional versatility arises from its collaboration with different partner transcription factors in specific cellular contexts. These combinatorial interactions allow SOX9 to activate distinct transcriptional programs despite its conserved DNA-binding specificity.

Table 2: Cell type-specific partner transcription factors of SOX9

Cell/Tissue Type	Partner Transcription Factors	Functional Consequences
Chondrocytes	SOX5, SOX6 (cartilage triad)	Synergistic activation of cartilage-specific ECM genes [2]
Fetal Testis/Sertoli Cells	GATA4, DMRT1, WT1, SF1, TRIM28	Sertoli cell differentiation, testis development [66]
Epidermal/Hair Follicle	Unknown chromatin remodelers	Fate switching from epidermal to hair follicle stem cells [20]
Human Chondrocytes	CREB, Sp1	Regulation of SOX9 promoter activity [67]
Neural Crest Cells	Unknown partners in CNCCs	Craniofacial development, chondrogenesis [26]

The partnership between SOX9 and GATA4 exemplifies how cell-type-specific collaborators enable context-dependent function. In fetal testes, SOX9 and GATA4 co-localize on genomic regions containing the Sertoli Cell Signature, with their binding sites organized in a specific spatial arrangement [66]. This partnership is essential for male sex determination, as both factors are required for Sertoli cell differentiation.

In chondrocytes, SOX9 collaborates with SOX5 and SOX6 to form a potent transcriptional triad that activates cartilage-specific extracellular matrix genes, including COL2A1, ACAN, and COMP [2]. This partnership explains why SOX9 binding regions in chondrocytes frequently contain palindromic SOX motifs that accommodate dimer binding [9].

Experimental approaches for mapping SOX9 interactions

Chromatin immunoprecipitation sequencing (ChIP-seq)

Protocol:

• Crosslinking: Fix micro-dissected tissues (e.g., E13 mouse limb buds or male gonads) with PBS containing 2 mM disuccinimidyl glutarate (DSG) for 30 minutes, followed by PBS/1% formaldehyde at room temperature for 30 minutes [66].
• Cell lysis and chromatin preparation: Lyse fixed tissues and sonicate chromatin to fragment DNA to 200-500 bp.
• Immunoprecipitation: Incubate sonicated chromatin with anti-SOX9 antibody bound to Protein A magnetic beads overnight at 4°C [66] [9].
• Library preparation and sequencing: Reverse crosslinks, purify DNA, and prepare sequencing libraries for high-throughput sequencing.
• Data analysis: Align sequences to reference genome, call peaks, and identify enriched motifs.

Key considerations: The choice of antibody is critical for successful ChIP-seq. Studies have used validated homemade rabbit polyclonal anti-SOX9 IgG antibodies [66] or commercial antibodies with high specificity. Tissue-specific controls (e.g., limb bud vs. gonad) help verify the specificity of identified binding regions.

Assay for Transposase-Accessible Chromatin with Sequencing (ATAC-seq)

Protocol:

• Cell preparation: Harvest SOX9-tagged human embryonic stem cell-derived cranial neural crest cells (hESC-CNCCs) with appropriate SOX9 dosage modulation [26].
• Transposition: Treat 50,000 cells with Tn5 transposase for 30 minutes at 37°C to fragment and tag accessible genomic regions.
• Library amplification: Purify transposed DNA and amplify by PCR with barcoded primers.
• Sequencing and analysis: Sequence libraries and analyze data to identify accessible chromatin regions and transcription factor binding sites.

ATAC-seq enables mapping of chromatin accessibility dynamics in response to SOX9 dosage changes, revealing how SOX9 binding influences chromatin landscape [26].

Sequential ChIP-qPCR (ChIP-re-ChIP)

Protocol:

• First immunoprecipitation: Perform standard ChIP with first antibody (e.g., anti-SOX9).
• Elution: Elute bound complexes from beads.
• Second immunoprecipitation: Perform second ChIP with different antibody (e.g., anti-GATA4 or anti-DMRT1) on eluted material.
• Analysis: Quantify co-occupied regions by qPCR.

This approach demonstrates simultaneous binding of SOX9 with partner factors like GATA4 and DMRT1 to genomic regions in fetal testes, confirming their partnership on chromatin [66].

Figure 2: Experimental workflow for mapping SOX9 binding sites using ChIP-seq.

The scientist's toolkit: Key research reagents

Table 3: Essential research reagents for studying SOX9 interactions

Reagent Category	Specific Examples	Application and Function
Cell Lines	NT2D1 cells, C3H10T1/2 cells, ATDC5 cells, RCS chondrosarcoma cells	In vitro models for studying SOX9 function [67] [66]
Antibodies	Home-made rabbit polyclonal anti-SOX9 IgG, commercial anti-SOX9 antibodies	Chromatin immunoprecipitation, immunofluorescence [66]
Animal Models	Mouse lines: Krt14-rtTA;TRE-Sox9 (inducible SOX9), Sox9 floxed lines	In vivo functional studies, fate mapping [20]
Genome Editing	CRISPR/Cas9 systems, dTAG degradation system	Precise modulation of SOX9 levels, tagging endogenous SOX9 [26]
Bioinformatic Tools	MEME-ChIP, DREME, PAVIS, MACS peak caller	Analysis of ChIP-seq data, motif discovery, peak annotation [66] [9]

Implications for immune cell research

While SOX9 is best known for its roles in development, recent evidence reveals significant connections between SOX9 and immune system regulation [3]. SOX9 exhibits context-dependent dual functions across diverse immune cell types, acting as both an activator and repressor in various immunological processes.

In cancer immunology, SOX9 expression correlates strongly with immune cell infiltration patterns in tumors. Bioinformatics analyses of TCGA data reveal that SOX9 expression negatively correlates with infiltration of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, but positively correlates with neutrophils, macrophages, activated mast cells, and naive/activated T cells [3]. This suggests SOX9 contributes to shaping the tumor immune microenvironment.

In T-cell development, SOX9 cooperates with c-Maf to activate Rorc and key Tγδ17 effector genes (Il17a and Blk), modulating the lineage commitment of early thymic progenitors and potentially influencing the balance between αβ T-cell and γδ T-cell differentiation [3]. This places SOX9 at a critical branch point in T-cell lineage decisions.

The cell-type-specific and species-conserved principles of SOX9 interactions identified in developmental contexts provide a framework for understanding its emerging roles in immunological processes. The precise mapping of SOX9 binding sites and partner factors in immune cells represents an important frontier for future research with significant therapeutic implications.

This comparative analysis demonstrates that SOX9 employs a conserved structural framework to achieve context-specific gene regulation through cell-type-specific partnerships with other transcription factors and epigenetic regulators. The high conservation of SOX9 targets in chondrogenesis versus low conservation in testis development reflects the different evolutionary pressures on these biological processes.

These principles provide a foundation for understanding SOX9's emerging roles in immune regulation and cancer immunology. The experimental approaches and reagents detailed here will enable researchers to further elucidate how SOX9 shapes immune cell function and contributes to immunological diseases, potentially identifying new therapeutic opportunities for modulating immune responses in cancer and inflammatory conditions.

The SRY-related HMG-box 9 (SOX9) transcription factor has emerged as a critical regulator in developmental biology and oncogenesis. Within the context of immune cell research, SOX9 represents a highly conserved transcriptional regulator across species, with evolving recognition of its dual role in tumor immunology. This guide provides a comprehensive comparison of SOX9's prognostic significance across malignancies and evaluates emerging therapeutic strategies targeting its activity, with particular emphasis on functional experimental data supporting these developments.

SOX9 contains several functionally distinct domains: an N-terminal dimerization domain (DIM), the central HMG-box DNA-binding domain, and C-terminal transcriptional activation domains (TAM and TAC) [3]. This structural configuration enables SOX9 to recognize specific DNA sequences (CCTTGAG motif) and regulate diverse transcriptional programs [17]. Beyond its established roles in chondrogenesis and sex determination, SOX9 maintains stem cell pools in multiple adult tissues [52], positioning it as a key player in cancer stemness and tumor progression.

Prognostic value of SOX9 across malignancies

SOX9 expression demonstrates significant prognostic value across multiple cancer types, though its implications vary by tissue context and molecular subtypes.

Prognostic significance in glioblastoma

In glioblastoma (GBM), SOX9 serves as both a diagnostic and prognostic biomarker with particular significance in specific molecular contexts. A 2025 study analyzing data from TCGA and GTEx databases revealed that SOX9 is highly expressed in GBM tissues compared to normal brain tissue [7] [68]. Surprisingly, in contrast to many other cancers, high SOX9 expression was associated with better prognosis in lymphoid invasion subgroups in a sample of 478 cases (P < 0.05) [7]. Furthermore, multivariate Cox regression analysis identified high SOX9 expression as an independent prognostic factor specifically in IDH-mutant glioblastoma [7] [68].

The prognostic significance of SOX9 in GBM is further supported by research demonstrating that the deubiquitinase USP18 stabilizes SOX9 protein levels in glioma cells, with high USP18 expression correlating with poor patient outcomes [69]. Kaplan-Meier survival analysis of both TCGA and CGGA datasets revealed that patients with high USP18 expression levels had significantly shorter median survival times [69].

Pan-cancer prognostic patterns

Across multiple cancer types, SOX9 generally functions as an oncogene with elevated expression correlating with poor clinical outcomes. A comprehensive pan-cancer analysis of SOX9 expression in 33 cancer types revealed significantly increased expression in fifteen cancers (CESC, COAD, ESCA, GBM, KIRP, LGG, LIHC, LUSC, OV, PAAD, READ, STAD, THYM, UCES, and UCS) compared to matched healthy tissues [17]. SOX9 expression was decreased in only two cancer types (SKCM and TGCT) [17].

Table 1: Prognostic Value of SOX9 Across Various Cancers

Cancer Type	SOX9 Expression	Prognostic Value	Clinical Associations	References
Glioblastoma (IDH-mutant)	High	Better prognosis (lymphoid invasion subgroup)	Independent prognostic factor	[7] [68]
Low-Grade Glioma	High	Shorter overall survival	Poor prognosis	[17]
Hepatocellular Carcinoma	High	Poor overall and disease-free survival	Promotes invasiveness and stemness	[52]
Colorectal Cancer	High	Poor prognosis	Promotes proliferation, senescence inhibition, chemoresistance	[52] [70]
Ovarian Cancer	High	Poor overall survival	Platinum and PARP inhibitor resistance	[28] [71]
Breast Cancer	High	Poor prognosis	Regulates tumor initiation, proliferation, immune evasion	[6]
Prostate Cancer	High/Context-dependent	Poor relapse-free and overall survival	Promotes proliferation, apoptosis resistance	[52]
Lung Adenocarcinoma	High	Poor overall survival	Regulates EMT and TKI resistance	[70]
Thymoma	High	Shorter overall survival	Correlated with immune dysregulation	[17]

Specific clinical correlations highlight SOX9's prognostic utility across cancers:

• In hepatocellular carcinoma (HCC), SOX9 overexpression correlates with high tumor stage and grade, with significant trends toward poorer disease-free and overall survival [52].
• SOX9 expression in breast cancer correlates with the CD44+/CD24- phenotype and promotes tumor initiation and proliferation through multiple pathways [6].
• Ovarian cancer patients with high SOX9 expression demonstrate resistance to platinum-based chemotherapy and PARP inhibitors, leading to poor outcomes [28] [71].

SOX9 in therapy resistance and immune modulation

Mechanisms of therapy resistance

SOX9 contributes to treatment failure through multiple established mechanisms across cancer types:

DNA Damage Repair Enhancement: In ovarian cancer, SOX9 binds to promoters of key DNA damage repair (DDR) genes (SMARCA4, UIMC1, and SLX4), enhancing repair capabilities and conferring resistance to PARP inhibitors [28]. The USP28 deubiquitinase stabilizes SOX9 protein levels during olaparib treatment, further promoting this resistance mechanism [28].

Cancer Stem Cell Maintenance: SOX9 drives a stem-like transcriptional state in high-grade serous ovarian cancer, promoting platinum resistance through maintenance of cancer stem cell populations [71]. Similarly, in glioblastoma, SOX9 maintains glioblastoma stem cell (GSC) properties through activation of pyruvate dehydrogenase kinase 1 (PDK1) via the PI3K-AKT pathway [69].

Drug Efflux and Metabolic Adaptation: In breast cancer, SOX9 contributes to chemoresistance by regulating ALDH1A3 expression and modulating Wnt signaling [28]. SOX9 also promotes ATP-driven invasion and chemoresistance by targeting CEACAM5/6, ABCB1, and ABCG2 in breast cancer models [28].

Immune modulation and tumor microenvironment

SOX9 plays complex, context-dependent roles in immune regulation and tumor microenvironment interaction:

Immune Cell Infiltration Patterns: In glioblastoma, SOX9 expression correlates significantly with immune cell infiltration and expression of immune checkpoints [7] [68]. Bioinformatic analyses of colorectal cancer reveal SOX9 expression negatively correlates with infiltration levels of B cells, resting mast cells, resting T cells, monocytes, plasma cells, and eosinophils, while showing positive correlation with neutrophils, macrophages, activated mast cells, and naive/activated T cells [3].

Immune Evasion: Research demonstrates that SOX9 plays crucial roles in immune evasion by maintaining cancer cell stemness and enabling dormant cancer cells to avoid immune surveillance in secondary metastatic sites [6]. In breast cancer, SOX2 and SOX9 help latent cancer cells remain dormant in metastatic sites and avoid immune monitoring under immunotolerant conditions [6].

Dual Immunological Role: SOX9 exhibits janus-faced characteristics in immunity—on one hand promoting tumor immune escape by impairing immune cell function, while in other contexts maintaining macrophage function and contributing to tissue regeneration and repair [3].

Table 2: SOX9 in Therapy Resistance: Mechanisms and Experimental Evidence

Resistance Type	Cancer Context	Molecular Mechanism	Experimental Evidence	References
PARP inhibitor resistance	Ovarian cancer	SOX9 enhances DNA damage repair by binding DDR gene promoters; stabilized by USP28	ChIP-Seq shows SOX9 binding to SMARCA4, UIMC1, SLX4 promoters; USP28 inhibition sensitizes cells to olaparib	[28]
Platinum resistance	Ovarian cancer	SOX9 drives stem-like transcriptional state	Single-cell RNA sequencing identifies SOX9+ population with stemness features in HGSOC	[71]
Platinum resistance	Ovarian cancer	SOX9 upregulation associated with cisplatin resistance	SOX9 overexpression in cell lines confers CDDP resistance	[28]
Tamoxifen resistance	Breast cancer	SOX9 regulates ALDH1A3 expression and Wnt signaling	SOX9 identified as downstream factor of SOX2 in resistant cells	[28]
Tyrosine kinase inhibitor resistance	Lung cancer	SOX9 promotes EMT through Wnt/β-catenin pathway	SOX9 overexpression induces EMT features and confers TKI resistance	[70]
Multi-drug resistance	Breast cancer	SOX9 targets CEACAM5/6, ABCB1, and ABCG2	SOX9 regulates drug efflux transporters and promotes ATP-driven invasion	[28]
Temozolomide resistance	Glioblastoma	SOX9 maintains stem cell population through PI3K-AKT-PDK1	SOX9 knockdown sensitizes GSCs to temozolomide	[69]

Emerging small-molecule inhibitors targeting SOX9

Targeting SOX9 directly has presented challenges due to its nature as a transcription factor, but several strategic approaches have emerged:

Indirect targeting strategies

Deubiquitinase Inhibitors: The specific USP28 inhibitor AZ1 reduces SOX9 protein stability and increases sensitivity of ovarian cancer cells to olaparib [28]. Similarly, targeting USP18, which stabilizes SOX9 in glioblastoma, represents a promising therapeutic approach [69].

Natural Compounds: Cordycepin (an adenosine analog from Cordyceps sinensis) inhibits both protein and mRNA expression of SOX9 in a dose-dependent manner in 22RV1, PC3, and H1975 cancer cells [17]. This inhibition correlates with reduced tumorigenicity, suggesting cordycepin's anticancer effects may operate partially through SOX9 regulation.

Transcriptional Regulation: YY1 inhibition reduces USP18 expression, subsequently decreasing SOX9 protein levels in glioblastoma models [69].

Experimental therapeutic efficacy

Preclinical studies demonstrate promising results for SOX9-targeting approaches:

• Combined USP28 inhibition with PARP inhibitors synergistically reduces ovarian cancer viability in vitro and in vivo [28].
• USP18 silencing inhibits malignant phenotypes and stemness in glioma cells, reducing proliferation, migration, invasion, and neurosphere formation capacity [69].
• Cordycepin treatment suppresses SOX9 expression across multiple cancer cell lines, supporting its development as a potential SOX9-targeting agent [17].

Experimental approaches for studying SOX9

Key methodological frameworks

Gene Expression Analysis: RNA sequencing data from TCGA and GTEx databases analyzed using DESeq2 R package to identify SOX9-related differentially expressed genes [7]. Functional enrichment analysis performed via GO/KEGG, GSEA, and protein-protein interaction networks [7].

Protein-Protein Interaction Studies: Co-immunoprecipitation (Co-IP) assays in ovarian cancer cells using anti-Flag nanobody magnetic beads to identify SOX9 interacting partners [28]. Mass spectrometry following Co-IP to comprehensively characterize SOX9 protein complexes.

Chromatin Binding Profiling: Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map SOX9 binding sites genome-wide, identifying direct targets including DDR genes [28].

Stemness Characterization: In vitro limiting dilution assays and neurosphere formation assays to quantify effects of SOX9 manipulation on cancer stem cell frequency and self-renewal capacity [69].

The scientist's toolkit: Essential research reagents

Table 3: Key Research Reagents for SOX9 Investigation

Reagent/Cell Line	Application	Experimental Function	References
U87, LN229 glioma cells	In vitro glioma models	Study SOX9 role in proliferation, invasion, stemness	[69]
SKOV3, UWB1.289 ovarian cells	Chemoresistance models	PARPi resistance mechanisms, SOX9 stabilization	[28]
Patient-derived GSCs	Stemness studies	Maintain tumor stem cell properties ex vivo	[69]
Anti-SOX9 antibodies (AB5535)	Western blot, IHC, Co-IP	Detect SOX9 protein expression, localization, interactions	[28] [69]
Anti-USP18 antibodies	Functional studies	Investigate SOX9 stabilization mechanisms	[69]
Anti-γH2AX antibodies	DNA damage assessment	Quantify DNA damage repair efficiency	[28]
Olaparib (PARPi)	Therapeutic targeting	Study PARPi resistance mechanisms	[28]
AZ1 (USP28 inhibitor)	Targeted intervention	Test SOX9 destabilization strategies	[28]
Cordycepin	Natural compound testing	Evaluate SOX9 inhibition therapeutic potential	[17]

SOX9 regulatory networks and signaling pathways

The following diagrams illustrate key SOX9 regulatory pathways identified in recent research:

Diagram 1: SOX9 regulatory networks in cancer. Two major pathways stabilize SOX9: the USP18 pathway (primarily in glioblastoma) and the USP28 pathway (in ovarian cancer). Both deubiquitinases protect SOX9 from FBXW7-mediated degradation, enhancing SOX9's role in stemness maintenance and DNA damage repair, respectively.

Diagram 2: SOX9 therapeutic targeting and experimental approaches. Emerging inhibitors target SOX9 at multiple levels: cordycepin reduces SOX9 mRNA expression, while AZ1 inhibits USP28-mediated SOX9 protein stabilization. Key experimental methods for studying SOX9 function include ChIP-Seq for target identification, Co-IP/MS for interaction mapping, and functional stemness assays.

SOX9 represents a multifaceted regulator in oncology with demonstrated prognostic value across numerous malignancies. Its context-dependent role—particularly the surprising association with better prognosis in specific glioblastoma subgroups—highlights the complexity of this transcription factor. The conservation of SOX9 across species and its emerging roles in immune regulation further support its investigation as a therapeutic target.

Current evidence positions SOX9 as a promising biomarker for prognosis and therapy response prediction, particularly in glioblastoma, ovarian cancer, and breast cancer. While direct targeting of SOX9 remains challenging, strategic approaches focusing on its regulatory mechanisms—especially deubiquitinase interactions—show significant promise. The continued development of small-molecule inhibitors and combination strategies targeting SOX9 regulatory networks may yield novel therapeutic opportunities for treatment-resistant cancers.

Conclusion

The investigation of SOX9 target conservation across species reveals a complex picture of both remarkable consistency and critical divergence, heavily influenced by cell type and pathological context. While its role in developmental processes like chondrogenesis is highly conserved, its immunological functions and targets exhibit significant species- and tissue-specificity. This nuanced understanding is paramount for drug development. Future research must prioritize mapping the complete SOX9 immunoregulatory network in human immune cells, developing context-specific targeting strategies to inhibit its pro-tumorigenic roles while sparing its regenerative functions, and exploring combination therapies that simultaneously target SOX9 and its downstream immune checkpoints to overcome treatment resistance. The promise of SOX9 as a diagnostic, prognostic, and therapeutic target in immuno-oncology is substantial, but realizing its potential demands a sophisticated, mechanism-driven approach.

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