Ink4a/Arf(−/−) and HRAS(G12V) transform mouse mammary cells into triple-negative breast cancer containing tumorigenic CD49f(−) quiescent cells

Intratumoral heterogeneity within individual breast tumors is a well-known phenomenon that may contribute to drug resistance. This heterogeneity is dependent on several factors, such as types of oncogenic drivers and tumor precursor cells. The purpose of our study was to engineer a mouse mammary tumor model with intratumoral heterogeneity by using defined genetic perturbations. To achieve this, we used mice with knockout (−/−) of Ink4a/Arf, a tumor suppressor locus; these mice are known to be susceptible to non-mammary tumors such as fibrosarcoma. To induce mammary tumors, we retrovirally introduced an oncogene, HRAS(G12V), into Ink4a/Arf(−/−) mammary cells in vitro, and those cells were inoculated into syngeneic mice mammary fat pads. We observed 100% tumorigenesis. The tumors formed were negative for estrogen receptor, progesterone receptor, and HER2. Further, they had pathological features similar to those of human triple-negative breast cancer (e.g. pushing borders, central necrosis). The tumors were found to be heterogeneous and included two subpopulations: CD49f(−) quiescent cells and CD49f(+) cells. Contrary to our expectation, CD49f(−) quiescent cells had high tumor-initiating potential and CD49f(+) cells had relatively low tumor-initiating potential. Gene expression analysis revealed that CD49f(−) quiescent cells overexpressed epithelial-to-mesenchymal transition-driving genes, reminiscent of tumor-initiating cells and claudin-low breast cancer. Our animal model with intratumoral heterogeneity, derived from defined genetic perturbations, allows us to test novel molecular targeted drugs in a setting that mimics the intratumoral heterogeneity of human triple-negative breast cancer.