Sequential invasion assays identify carbonic anhydrase IX as a driver of invasiveness in epithelial triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with poor prognosis and diverse response to treatment that is mostly attributed to heterogeneity of the disease that includes a diverse set of tumor cells at various stages of the epithelial-to-mesenchymal transition. Despite advances in our understanding of TNBC biology, isolating the phenotypically distinct cell subpopulations and their molecular drivers of invasiveness remains a major challenge. In this study, we used sequential invasion assays in Boyden chambers coated with growth factor-reduced Matrigel to isolate invasive subpopulations from model migratory mesenchymal-like and proliferative epithelial TNBC cell lines. We ascertained phenotypic heterogeneity of the invasive subpopulations by assessing markers of invasiveness, drug response, growth in three-dimensional cultures, and proteomic analysis. We demonstrated that isolated invasive subpopulations of epithelial cells are E-cadherin-low, whereas those from mesenchymal-like TNBC cells are vimentin-high. The isolated invasive subpopulations are chemotherapy-resistant, stem cell-like cells that express distinct druggable drivers of invasiveness, including carbonic anhydrase 9 (CA9, encoded by the CAIX gene) in the invasive subpopulations of epithelial TNBC cells. Downregulation of CAIX in the invasive subpopulations of epithelial cells resulted in decreased cell proliferation, invasiveness, and sensitivity to chemotherapy. Together, this study demonstrates that targeting specific drivers of invasiveness, such as CA9, in the invasive subpopulations of epithelial cells may provide viable options for novel therapeutic strategies for metastatic TNBC.NEW & NOTEWORTHY This study addressed a major challenge in studying phenotypically distinct cell populations within bulk tumors or cell lines by using sequential invasion assays to reliably isolate vimentin-high mesenchymal-like and E-cadherin-low epithelial invasive subpopulations of TNBC cells. The isolated invasive subpopulations from these phenotypically distinct TNBC cell types are chemotherapy-resistant, stem cell-like, and mammosphere-forming tumor cells that express distinct druggable drivers of invasiveness, including CAIX in the heterogeneous invasive subpopulations of epithelial TNBC cells.