Stromal Contributions to Radiation Resistance of Breast Cancer Stem Cells

Abstract

Breast cancer that is refractory to radiation therapy causes significant morbidity and mortality. Tumor-initiating cells (TICs), also called cancer stem cells (CSCs), display a radioresistant phenotype. Normal stem cells depend on stromal cells for the formation of a stem cell “niche”, but the role of the stromal cells in breast TIC function and treatment resistance is not well understood. We sought to define the effects of stromal fibroblasts on breast TIC self-renewal and resistance to IR (ionizing radiation). For investigations into breast cancer TIC and stromal fibroblast physiology we utilized MMTV-Wnt-1 transgenic mice, which spontaneously generate mammary tumors. These tumors can be dissociated into single cell suspensions and subjected to fluorescence-activated cell sorting (FACS) for in vitro colony assays or molecular analyses. The TIC population in this model has been previously identified based on cell surface markers and has been validated using in vivo limiting dilution analyses. Using this model system, we isolated normal and tumor-associated fibroblasts from primary normal and tumor murine breast tissues by FACS, and their effects on TICs were assayed in a 3D co-culture system. We show that co-culture with primary stromal fibroblasts promotes the expansion of mouse mammary TICs in vitro and maintains their ability to form new tumors in vivo. At clinically relevant doses of IR, DNA double strand breaks are mitigated and clonogenic growth of TICs is enhanced by co-culture with the stromal fibroblasts. Consistent with these observations, the TICs that are grown in co-culture maintain a more basal differentiation phenotype and harbor lower levels of reactive oxygen species (ROS) than TICs alone. The reduction in ROS found in the co-cultured TICs is a consequence of fewer mitochondria and less active oxidative phosphorylation in these cells. Working with primary tissues, we have demonstrated that stromal fibroblasts from mammary tumors play an important role in promoting TIC proliferation and resistance to IR. Our work has led to a better characterization of the unique microenvironment within the TIC-stromal niche. Future endeavors into pharmacological disruption of the supportive cues within the niche could lead to novel radiosensitizers and CSC-directed therapies.