The very process meant to preserve cancer cells for future study may be fundamentally altering them, creating a hidden challenge for researchers seeking cures.
In laboratories worldwide, millions of tiny vials containing precious cancer cells rest in frozen slumber within liquid nitrogen tanks, preserved at temperatures colder than the dark side of the moon. This process, known as cryopreservation, has been a cornerstone of modern biological science since the mid-20th century, allowing researchers to pause cellular life for years or even decades 2 . For cancer researchers, these frozen time capsules represent hope—potential keys to understanding how cancer grows, spreads, and resists treatment.
Recent research has uncovered a troubling paradox: the long-term storage of breast and lung cancer stem cells—the very cells believed to drive tumor growth and recurrence—may significantly alter their fundamental biological nature 1 .
This discovery raises critical questions for cancer research and forces us to reconsider what happens when we put life on ice.
The process of preserving cells or tissues at extremely low temperatures, typically using liquid nitrogen at -196°C.
The preservation of the original molecular structure and function of biological specimens during storage.
To appreciate why these findings matter, we must first understand what makes cancer stem cells so special—and so dangerous. Unlike regular cancer cells, cancer stem cells possess remarkable abilities: they can self-renew, differentiate into various cell types, and survive treatments that kill ordinary cancer cells 2 .
Think of them as the "architects" of cancer—while regular cancer cells form the bulk of a tumor, it's the cancer stem cells that design the blueprint for growth, metastasis, and recurrence.
These elusive cells are identified by specific protein markers on their surfaces—molecular fingerprints like CD24, CD38, EpCAM, and ALDH that help scientists distinguish them from other cells 1 5 .
Breast Cancer
Multiple Cancers
Brain & Colon Cancer
Epithelial Cancers
In 2013, a team of researchers set out to answer a fundamental but overlooked question: what happens to cancer stem cells during long-term frozen storage? Their findings, published in the journal Cell and Tissue Banking, revealed more changes than anyone had anticipated 1 .
Cancer stem cells were separated from regular cancer cells using specific growth conditions and identification of surface markers 2 .
The cells were frozen using standard laboratory protocols with a protective solution containing 15% dimethyl sulfoxide (Me₂SO) and stored in liquid nitrogen for 12 months 2 .
After the year-long storage, cells were carefully thawed and allowed to recover in culture for approximately eight days 2 .
The researchers conducted multiple tests comparing the frozen-thawed cells to never-frozen controls, examining gene expression, protein markers, and global genetic profiles 1 .
| Marker | Normal Role | Change After Freezing |
|---|---|---|
| CD24 | Cell adhesion and signaling | Down-regulated |
| CD38 | Cell communication | Down-regulated |
| EpCAM | Epithelial cell adhesion | Down-regulated |
| ALDH | Enzyme involved in cell differentiation | Down-regulated |
| HLA | Immune recognition | Down-regulated |
| Pathway | Normal Function | Impact of Alteration |
|---|---|---|
| Cell Cycle | Controls cell division | Disruption may change growth patterns |
| Mitosis | Process of cell division | Alterations may affect replication |
| ATM Pathway | DNA damage repair | Changes may impact genetic stability |
Understanding how scientists study these cells helps appreciate the significance of these findings. Modern cancer stem cell research relies on sophisticated tools and techniques:
Identifies and separates cells based on surface markers
Prevents stem cells from differentiating during culture
Growth factor that maintains stem cells in undifferentiated state
Cryoprotectant that prevents ice crystal formation during freezing
Technology for analyzing global gene expression patterns
Test measuring ALDH enzyme activity, a stem cell marker
A 2023 study published in npj Precision Oncology identified a new subpopulation of breast cancer stem cells (CD79A+CD24-PANCK+) located near exhausted immune cells, suggesting complex relationships between cancer stem cells and their microenvironment 8 .
The discovery that cryopreservation alters cancer stem cells has ripple effects far beyond basic biology. If we're studying cells that have been fundamentally changed by freezing, could this be affecting the accuracy of our research and the effectiveness of therapies developed from that research?
The alterations in DNA damage repair pathways are particularly concerning, as these pathways influence how cells respond to chemotherapy and radiation 1 . If freezing changes these response mechanisms, drugs tested on previously frozen cells might not work the same way on fresh tumors in patients.
The discovery that long-term storage alters cancer stem cells represents both a challenge and an opportunity for cancer research. It reveals a critical variable that may have influenced decades of findings—a hidden iceberg in the sea of cancer science. Yet, by recognizing this factor, researchers can develop better methods to preserve these cells more faithfully, ensuring that the samples they study tomorrow truly reflect the cancer that exists in patients today.
"Current protocols for long-term storage of lung and breast cancer stem cells may substantially influence the activity and function of genes" 1 .
As we continue to unravel the mysteries of cancer stem cells, this research reminds us of a fundamental truth in science: sometimes the tools we depend on to advance knowledge require as much scrutiny as the questions we seek to answer. In the quest to conquer cancer, every variable matters—even the cold preservation that we once assumed pressed pause on life without consequences.