Proteolysis-a characteristic of tumor-initiating cells in murine metastatic breast cancer

Abstract

Tumor initiating cells (TICs) have been identified and functionally characterized in hematological malignancies as well as in solid tumors such as breast cancer. In addition to their high tumor-initiating potential, TICs are founder cells for metastasis formation and are involved in chemotherapy resistance. In this study we explored molecular pathways which enable this tumor initiating potential for a cancer cell subset of the transgenic MMTV-PyMT mouse model for metastasizing breast cancer. The cell population, characterized by the marker profile CD24⁺CD90⁺CD45⁻, showed a high tumorigenicity compared to non-CD24⁺CD90⁺CD45⁻ cancer cells in colony formation assays, as well as upon orthotopic transplantation into the mammary fat pad of mice. In addition, these orthotopically grown CD24⁺CD90⁺CD45⁻ TICs metastasized to the lungs. The transcriptome of TICs freshly isolated from primary tumors by cell sorting was compared with that of sorted non-CD24⁺CD90⁺CD45⁻ cancer cells by RNA-seq. In addition to more established TIC signatures, such as epithelial-to-mesenchymal transition or mitogen signaling, an upregulated gene set comprising several classes of proteolytic enzymes was uncovered in the TICs. Accordingly, TICs showed high intra- and extracellular proteolytic activity. Application of a broad range of protease inhibitors to TICs in a colony formation assay reduced anchorage independent growth and had an impact on colony morphology in 3D cell culture assays. We conclude that CD24⁺CD90⁺CD45^- cells of the MMTV- PyMT mouse model possess an upregulated proteolytic signature which could very well represent a functional hallmark of metastatic TICs from mammary carcinomas.