Highlights Breast cancer is a complex disease arising from the association and cumulation of multiple genetic alterations and environmental factors likely to alter cellular functions. The implication of stem cells in breast cancer’s origin and development has been debated for many years. Analogies between stem cells and cancer cells can be drawn on many levels. Importantly not all cancer cells are stem cells or exhibit properties similar to stem cells. There is no universal cell surface marker of the breast cancer stem cells, imputable to the diversity of the cell of origin, the heterogeneity and fluidity of the microenvironments, and a plethora of possible mutational events. Breast cancer stem cell targeting should hold a great promise for preventing metastasis, reducing the risk of drug resistance, and decreasing recurrence. However, only a few clinical trials assess the effectiveness of treatments on this specific cancer cell population. Additional considerations for the targeting of breast cancer stem cells need to be given to the tumor microenvironment. It is known to promote self-renewal, plasticity, and resistance to treatments. The fate of breast cancer stem cells is indissociable from its environment.

Cryoablation is an emerging type of treatment for cancer. The sensitization of tumors using cryosensitizing agents prior to treatment enhances ablation efficiency and may improve clinical outcomes. Water efflux, which is regulated by aquaporin channels, contributes to cancer cell damage achieved through cryoablation. An increase in aquaporin (AQP) 3 is cryoprotective, whereas its inhibition augments cryodamage. The present study aimed to investigate aquaporin (AQP1, AQP3 and AQP5) gene expression and cellular localization in response to cryoinjury. Cultured breast cancer cells (MDA-MB-231 and MCF-7) were exposed to freezing to induce cryoinjury. RNA and protein extracts were then analyzed using reverse transcription-quantitative PCR and western blotting, respectively. Localization of aquaporins was studied using immunocytochemistry. Additionally, cells were transfected with small interfering RNA to silence aquaporin gene expression and cell viability was assessed using the Sulforhodamine B assay. Cryoinjury did not influence gene expression of AQPs, except for a 4-fold increase of AQP1 expression in MDA-MD-231 cells. There were no clear differences in AQP protein expression for either cell lines upon exposure to frozen and non-frozen temperatures, with the exception of fainter AQP5 bands for non-frozen MCF-7 cells. The exposure of cancer cells to freezing temperatures altered the localization of AQP1 and AQP3 proteins in both MCF-7 and MDA-MD-231 cells. The silencing of AQP1, AQP3 and AQP5 exacerbated MDA-MD-231 cell damage associated with freezing compared with control siRNA. This was also observed with AQP3 and AQP5 silencing in MCF-7 cells. Inhibition of aquaporins may potentially enhance cryoinjury. This cryosensitizing process may be used as an adjunct to breast cancer cryotherapy, especially in the border area targeted by cryoablation where freezing temperatures are not cold enough to induce cellular damage.

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