Tumor analysis: Freeze-thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells

Objective: The use of novel methods to characterize living tumor cells relies on well-conceived biobanks. Herein, we raised the question of whether the composition of fresh and freeze/thawed dissociated tumor samples is comparable in terms of quantitative and qualitative profiling. Results: Breast cancer is a heterogeneous disease, encompassing luminal A and B, basal/triple-negative breast cancer (TNBC), and ERBB2-like tumors. We examined living cells dissociated from TNBC and found that a classical freeze/thaw protocol leads to a marked reduction in the number of CD45^-CD44^LowCD24^Low tumor cells. This, in turn, changed the percentage of tumor cells with certain CD44/CD24 expression patterns and changed the percentage of tumor-infiltrating immune cells. These cryopreservation-driven alterations in cellular phenotype make it impossible to compare fresh and frozen samples from the same patient directly. Moreover, the freeze/thaw process changed the transcriptomic signatures of triple-negative cancer stem cells in such a manner that hierarchical clustering no longer ranked them according to expected inter-individual differences. Overall, this study suggests that all analyses of living tumor cells should be conducted only using freshly dissociated tumors if we are to generate a robust scoring system for prognostic/predictive markers.