We previously discussed the impact of prolonging cryopreservation of breast cancer stem cells on the biological functioning of the cells (Karimi-Busheri et al. 2010a
). In that report we showed that cryopreservation of breast cancer stem cells does not influence viability, proliferation, basic DNA repair ability, and self-renewal. The only difference we observed was the presence of a significantly higher number of mammospheres undergoing senescence. In this study we focused on the impact of prolonged storage on the molecular integrity and global gene expression patterns of breast and lung cancer stem cells.
Contrary to our previous findings (Karimi-Busheri et al. 2010a
) that cryopreservation will not generate any observable changes in biological activity of the cells, including proliferation and self-renewal, here we report significant alterations at the molecular level of the post-thawed cells. To prevent the onset of senescence associated with long term culture (Kuilman et al. 2010
) and adaptation to culture conditions, post-thawed cells were in culture for two passages before being analyzed. Furthermore, to increase assay reproducibility all the assays and equipments used in our laboratories, in particular our Attune Acoustic Focusing Cytometer from Applied Biosystems (Foster City, CA), are routinely calibrated to the FDA standard of quality to produce consistent results as a requirement for an ongoing lung cancer Phase III clinical trial of belagenpumatucel-L (Lucanix(r)) in our company. Down-regulation of the expression of seven out of nine genes following 12 months freezing could be considered as a dominant pattern in lungospheres following cryopreservation.
As the cell surface proteins play a major role in stem cell fate the majority of genes selected for this study, with the exception of ALDHA1 and HLA, were cell surface markers. As all the cell surface genes studied were down-regulated that might indicate the expected susceptibility of the stem cell membrane to cryopreservation. Previous reports have mainly focused on cell viability but a few reports, including the influence of cryopreservation on amniotic fluid-derived stem cells and human limbal epithelial stem cells (Seo et al. 2011
; Vasania et al. 2011
) also confirm alterations in molecular profiling of stem cells. Of considerable interest is the result that the cryopreserved human limbal epithelial stem cells express negative immunoregulatory molecules and are non-immunogenic in nature (Vasania et al. 2011
). The finding clearly questions the survival of these cells in an allogeneic environment. This could be a significant factor influencing the outcome of any research on stem cells that is intended to be developed for storage, good manufacturing practice, and the manufacturing of clinical grade stem cells.
Global gene expression analysis of fresh versus post-thawed breast and lung cancer stem cells also confirmed a significant amount of down regulation in freeze-thaw cells. Interestingly, gene expression changes in both cell lines are independent from each other and no one gene is shared between the two population sets. There are, however, similarities between both breast and lung cancer stem cell populations in the biological functions distribution and canonical pathways. Genes involved in genetic disorders and cancer rank the top two in the disease category affected by long term storage of the cells. Within the molecular and cellular function category, cell death and cellular growth and proliferation pathways are the most predominantly affected among the two populations. For the physiological system development and function category breast and lung stem cell populations share the highest changes in the tissue development pathway.
Analysis of canonical pathways between breast and lung cancer stem cell reveals interesting results. In post-thawed lung cancer stem cells, the majority of pathways showing significant alteration in gene expression are the pathways involved in cell cycle, mitosis, and ATM that also regulates a number of pathways involved in DNA repair, cell cycle, and apoptosis (Smith et al. 2010
). In contrast, in breast cancer stem cells canonical pathways influenced by cryopreservation are mainly associated with metabolism. Our results clearly display the profound impact of long term storage of cancer stem cells under cryopreserved conditions. The influence has a wide spectrum and is cell type dependent. Down-regulation of gene expression is a dominant pattern among post-thawed cells. In adherent and non-stem cell populations greater passaging after thawing could return the cells to normal but in the case of stem cells, with the possibility of senescence (Karimi-Busheri et al. 2010a
; Kuilman et al. 2010
), this could be problematic. Changing freezing conditions and reducing the amount of DMSO (Seo et al. 2011
) could be an alternative that remains to be tested in different panels of tissues and cells-derived stem cells.
In conclusion, we observed direct impact of cryopreservation on the molecular integrity of cancer stem cells, in particular on the cell surface membrane. Similar results were also confirmed by global gene expression analysis on two different sets of stem cell populations all indicating a significant down regulation following long term storage of the cells. Our findings could have significant implications in the emerging field of cancer stem cells since cell surface markers, biological functional and canonical pathways play a major role towards understanding cancer initiation and progression.
Finally, we believe current protocols for cryopreservation of cancer stem cells could substantially influence the activity and function of genes and accordingly we encourage employing rigorous research on methodology for freezing and utilizing cancer stem cells following long-term storage. These results are an alarming signals suggesting that cancer stem cells could be more sensitive to long-term storage and recovering process and part of the discrepancies in the results obtained by different investigators on biomarkers and genetic profiling of cancer stem cells might be in fact attributed to the freezing process.