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