How Storage Alters Cancer's Most Dangerous Cells
The very process meant to preserve cancer stem cells for future study may be changing them in ways we never anticipated.
Imagine a powerful tool used in laboratories worldwide to fight cancer—a technique so standard that we rarely question its effects. Cryopreservation, the process of freezing biological material at ultra-low temperatures, has been a cornerstone of cancer research for decades, allowing scientists to store and study cancer stem cells, the very drivers of tumor growth. Yet, what if this fundamental procedure was subtly altering these cells, potentially skewing research results and our understanding of cancer itself?
Recent research reveals a startling truth: the long-term storage of breast and lung cancer stem cells doesn't just pause their biological clock—it actively rewires their genetic programming, potentially impacting how we develop cancer treatments.
To understand why this discovery matters, we must first grasp what makes cancer stem cells so significant. Cancer stem cells (CSCs) represent a small but powerful subpopulation within tumors that possess remarkable abilities reminiscent of normal stem cells: self-renewal, differentiation, and the capacity to drive tumor growth 2 . Think of them as the "command centers" of cancer—while they may be few in number, they wield disproportionate power.
Unlike regular cancer cells that might be eliminated by chemotherapy, CSCs possess built-in survival mechanisms that make them resistant to conventional treatments 7 .
They can lie dormant for extended periods before awakening to regenerate tumors, making them primarily responsible for cancer recurrence and metastasis 2 .
When we study these cells, we're studying the very roots of cancer—which is why any alteration to their natural state during storage could have far-reaching implications for research outcomes.
A crucial experiment conducted in 2013 set out to answer a previously overlooked question: what happens to cancer stem cells during long-term frozen storage? Researchers focused on breast cancer stem cells (from the MCF7 cell line) and lung cancer stem cells (from A549 and H460 cell lines), carefully comparing their molecular profiles before and after cryopreservation 1 4 .
They first cultured cancer stem cells as three-dimensional "lungospheres" and "mammospheres," structures that enrich for stem-like properties and better mimic tumor conditions than traditional flat cultures 4 .
The cells were prepared using a standard freezing method, suspended in a solution containing 15% dimethyl sulfoxide (DMSO)—a common cryoprotectant—and slowly cooled to -80°C before long-term storage in liquid nitrogen 4 .
After twelve months of storage, the cells were carefully thawed, allowed to recover through two growth cycles, and then subjected to comprehensive analysis comparing them to never-frozen control cells 4 .
The researchers didn't just check if the cells survived—they dug deeper, examining specific biomarkers and the entire genetic landscape of the thawed cells.
The findings revealed consistent changes that raised important questions about standard cryopreservation practices.
Cancer stem cells are identified by specific protein markers on their surfaces—molecular "badges" that help scientists isolate and study them. The experiment examined several key biomarkers and found a troubling pattern of down-regulation, meaning these identification markers became less apparent after freezing and thawing 1 4 .
| Biomarker | Normal Role in Cancer Stem Cells | Effect of Cryopreservation |
|---|---|---|
| CD24 & CD38 | Potent biomarkers for lung cancer stem cells 4 | Significant down-regulation 1 |
| EpCAM | Used as a biomarker for a wide range of cancer stem cells 4 | Significant down-regulation 1 |
| ALDH | Marks stem-like cells with high tumorigenicity 2 | Significant down-regulation 1 |
Visualization of down-regulation in key cancer stem cell biomarkers
Beyond specific biomarkers, the researchers employed advanced microarray technology to analyze the entire genetic profile of the cells. The results were striking: global gene expression in post-thaw breast and lung cancer stem cells showed significant down-regulation compared to their never-frozen counterparts 1 4 . This wasn't just a few genes here and there—the very activity of the cells' genetic machinery had shifted substantially.
Perhaps most importantly, the study identified specific biological pathways that were altered by the freezing process. Analyzing canonical pathways revealed significant disturbances in genes involved in 1 :
Controls cellular division and proliferation
Directs the process of cell division
Coordinates DNA damage repair
| Cellular Pathway | Normal Function | Impact of Cryopreservation |
|---|---|---|
| Cell Cycle | Regulates cellular division and proliferation | Significant alteration in gene expression 1 |
| Mitosis | Directs the process of cell division | Significant alteration in gene expression 1 |
| ATM Pathway | Coordinates DNA damage repair | Significant alteration in gene expression 1 |
The implications of these genetic changes extend far beyond theoretical concerns. If cancer stem cells' molecular profiles are altered by storage, then experiments conducted with these cells may not accurately reflect their true biology.
Alterations in critical pathways for cell division and DNA repair might artificially change how these cells respond to experimental treatments, potentially skewing drug testing results 1 .
These findings highlight an uncomfortable reality: some of what we know about cancer stem cells might be influenced by how we've stored them before study.
This research relied on several critical laboratory tools and reagents that enabled the precise study of cancer stem cells before and after cryopreservation.
| Research Tool | Specific Example | Function in Research |
|---|---|---|
| Cell Lines | MCF7 (breast), A549 & H460 (lung) | Provided standardized cancer stem cell sources for experimentation 4 |
| Cryoprotectant | Dimethyl sulfoxide (DMSO) | Protected cells from ice crystal formation during freezing 4 |
| Biomarker Antibodies | CD24, CD38, EpCAM, ALDH | Enabled identification and sorting of cancer stem cell populations 4 |
| Culture Media | Serum-free media with growth factors | Supported growth and maintenance of stem cell properties 4 |
| Analysis Platform | Ilumina Human HT-12 Expression BeadChips | Allowed comprehensive analysis of global gene expression 4 |
The revelation that standard cryopreservation protocols substantially influence cancer stem cells has sparked important conversations in the research community. While cryopreservation remains an essential tool, scientists are now exploring ways to improve these methods.
The development of DMSO-free freezing media represents another promising approach to avoid the gene expression changes associated with this cryoprotectant 6 .
Scientists are paying closer attention to post-thaw recovery conditions, recognizing that how cells are handled after thawing may be as important as the freezing process itself 3 . Some researchers have begun using Rho-associated kinase (ROCK) inhibitors to improve long-term post-thaw recovery of stem cells 3 .
The discovery that long-term storage alters the molecular integrity of breast and lung cancer stem cells represents both a challenge and an opportunity for cancer research. It reminds us that even our most standard laboratory techniques deserve periodic scrutiny, and that advancing our understanding of cancer requires not just studying the disease, but continuously improving how we study it.
As researchers refine cryopreservation methods to better preserve the true nature of cancer stem cells, we move closer to more accurate disease models and more effective treatments. The frozen cells in laboratory tanks hold incredible potential—unlocking their secrets requires ensuring that what we recover from the deep freeze truly represents what we put in.