The effect of radiation on the ability of rat mammary cells to form mammospheres

As classical transplantation repopulation assays for studying the radiobiology of rat mammary stem/progenitor cells are extremely time-consuming, this study aimed to characterize the radiobiological properties of mammospheres, spherical clumps of mammary cells formed under non-adherent culture conditions, which are a simple and widely used technique for assessing progenitor cell activity. Rat mammary cells were dissociated and used in transplantation repopulation assays and for the formation of mammospheres. Immunofluorescence for cytokeratin 14 and 18 was used to identify basal and luminal mammary epithelial cells, respectively. Incorporation of 5-bromo-2’-deoxyuridine (BrdU) was used to evaluate cell proliferation. The repopulating activity of the transplanted primary rat mammary cells demonstrated their radiosensitivity, reproducing previous data, with a significant reduction in repopulating activity at ≥2 Gy. Cells constituting rat mammospheres were positive for either cytokeratin 14 or 18, with occasional double-positive cells. Both proliferation and aggregation contributed to sphere formation. Cells obtained from the spheres showed lower repopulating activity after transplantation than primary cells. When primary cells were irradiated and then used for sphere formation, the efficiency of sphere formation was significantly decreased at 8 Gy but not at ≤6 Gy, indicating radioresistance of the formation process. Irradiation at 8 Gy reduced the proliferation of cells during sphere formation, whereas the cellular composition of the resulting spheres was unaffected. Thus, mammosphere formation assays may measure a property of putative mammary progenitors that is different from what is measured in the classic transplantation repopulation assay in radiobiology