The effect of radiation on the ability of rat mammary cells to form mammospheres
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ORIGINAL ARTICLE
The effect of radiation on the ability of rat mammary cells to form mammospheres Ayaka Hosoki1,4 · Mari Ogawa2 · Yukiko Nishimura2,5 · Mayumi Nishimura2 · Kazuhiro Daino2 · Shizuko Kakinuma2 · Yoshiya Shimada3,5 · Tatsuhiko Imaoka2 Received: 20 March 2020 / Accepted: 29 August 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract 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 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 unaffectes. 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. Keywords Mammary gland · Progenitor cells · Non-adherent culture · Mammosphere · Rat
Introduction
Ayaka Hosoki belonged to NIRS before it was organized as QST. * Tatsuhiko Imaoka [email protected] 1
Fukushima Project Headquarters, National Institute of Radiological Sciences (NIRS), Chiba, Japan
2
Department of Radiation Effects Research, NIRS, National Institutes for Quantum and Radiological Science and Technology (QST), 4‑9‑1, Anagawa, Inage‑ku, Chiba 263‑8555, Japan
3
Executive Board, QST, Chiba, Japan
4
Present Address: Healios KK Kobe Research Institute, Kobe, Japan
5
Present Address: Institute for Environmental Sciences, Aomori, Japan
Tissue stem cells have the capacity for self-renewal, are long-lived, continuously generate descendant cells, and are recruited in response to specific physiological demands for tissue regeneration. By contrast, progenitor cells
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