Triple negative breast cancer initiating cell subsets differ in functional and molecular characteristics and in γ‐secretase inhibitor drug responses

Increasing evidence suggests that stem‐like cells mediate cancer therapy resistance and metastasis. Breast tumour‐initiating stem cells (T‐ISC) are known to be enriched in CD44 + CD24 neg/low cells. Here, we identify two T‐ISC subsets within this population in triple negative breast cancer (TNBC) lines and dissociated primary breast cancer cultures: CD44 + CD24 low+ subpopulation generates CD44 + CD24 neg progeny with reduced sphere formation and tumourigenicity. CD44 + CD24 low+ populations contain subsets of ALDH1 + and ESA + cells, yield more frequent spheres and/or T‐ISC in limiting dilution assays, preferentially express metastatic gene signatures and show greater motility, invasion and, in the MDA‐MB‐231 model, metastatic potential. CD44 + CD24 low+ but not CD44 + CD24 neg express activated Notch1 intracellular domain (N1‐ICD) and Notch target genes. We show N1‐ICD transactivates SOX2 to increase sphere formation, ALDH1+ and CD44 + CD24 low+ cells. Gamma secretase inhibitors (GSI) reduced sphere formation and xenograft growth from CD44 + CD24 low+ cells, but CD44 + CD24 neg were resistant. While GSI hold promise for targeting T‐ISC, stem cell heterogeneity as observed herein, could limit GSI efficacy. These data suggest a breast T‐ISC hierarchy in which distinct pathways drive developmentally related subpopulations with different anti‐cancer drug responsiveness.