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

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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.

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References

  • Arendt LM, Keller PJ, Skibinski A, Goncalves K, Naber SP, Buchsbaum RJ, Gilmore H, Come SE, Kuperwasser C (2014) Anatomical localization of progenitor cells in human breast tissue reveals enrichment of uncommitted cells within immature lobules. Breast Cancer Res 16(5):453. https://doi.org/10.1186/s13058-014-0453-3

    Article  Google Scholar 

  • Atkinson RL, Zhang M, Diagaradjane P, Peddibhotla S, Contreras A, Hilsenbeck SG, Woodward WA, Krishnan S, Chang JC, Rosen JM (2010) Thermal enhancement with optically activated gold nanoshells sensitizes breast cancer stem cells to radiation therapy. Sci Transl Med 2(55):55–79. https://doi.org/10.1126/scitranslmed.3001447

    Article  Google Scholar 

  • Bartstra RW, Bentvelzen PA, Zoetelief J, Mulder AH, Broerse JJ, van Bekkum DW (2000) The effects of fractionated gamma irradiation on induction of mammary carcinoma in normal and estrogen-treated rats. Radiat Res 153(5 Pt 1):557–569

    Article  ADS  Google Scholar 

  • Brenner AV, Preston DL, Sakata R, Sugiyama H, de Gonzalez AB, French B, Utada M, Cahoon EK, Sadakane A, Ozasa K, Grant EJ, Mabuchi K (2018) Incidence of breast cancer in the life span study of atomic bomb survivors: 1958–2009. Radiat Res. https://doi.org/10.1667/RR15015.1

    Article  Google Scholar 

  • Brill B, Boecher N, Groner B, Shemanko CS (2008) A sparing procedure to clear the mouse mammary fat pad of epithelial components for transplantation analysis. Lab Anim 42(1):104–110. https://doi.org/10.1258/la.2007.06003e

    Article  Google Scholar 

  • Broerse JJ, Hennen LA, van Zwieten MJ (1985) Radiation carcinogenesis in experimental animals and its implications for radiation protection. Int J Radiat Biol Relat Stud Phys Chem Med 48(2):167–187

    Article  Google Scholar 

  • Burger PE, Gupta R, Xiong X, Ontiveros CS, Salm SN, Moscatelli D, Wilson EL (2009) High aldehyde dehydrogenase activity: a novel functional marker of murine prostate stem/progenitor cells. Stem Cells 27(9):2220–2228. https://doi.org/10.1002/stem.135

    Article  Google Scholar 

  • Clifton KH (1986) Thyroid and mammary radiobiology: radiogenic damage to glandular tissue. Br J Cancer Suppl 7:237–250

    Google Scholar 

  • Clifton KH, Tanner MA, Gould MN (1986) Assessment of radiogenic cancer initiation frequency per clonogenic rat mammary cell in vivo. Cancer Res 46(5):2390–2395

    Google Scholar 

  • Dicello JF, Christian A, Cucinotta FA, Gridley DS, Kathirithamby R, Mann J, Markham AR, Moyers MF, Novak GR, Piantadosi S, Ricart-Arbona R, Simonson DM, Strandberg JD, Vazquez M, Williams JR, Zhang Y, Zhou H, Huso D (2004) In vivo mammary tumourigenesis in the Sprague–Dawley rat and microdosimetric correlates. Phys Med Biol 49(16):3817–3830

    Article  Google Scholar 

  • Dong Q, Wang D, Bandyopadhyay A, Gao H, Gorena KM, Hildreth K, Rebel VI, Walter CA, Huang C, Sun LZ (2013) Mammospheres from murine mammary stem cell-enriched basal cells: clonal characteristics and repopulating potential. Stem Cell Res 10(3):396–404. https://doi.org/10.1016/j.scr.2013.01.007

    Article  Google Scholar 

  • Dontu G, Wicha MS (2005) Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia 10(1):75–86. https://doi.org/10.1007/s10911-005-2542-5

    Article  Google Scholar 

  • Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17(10):1253–1270. https://doi.org/10.1101/gad.1061803

    Article  Google Scholar 

  • Douglas BG, Fowler JF (1976) The effect of multiple small doses of X rays on skin reactions in the mouse and a basic interpretation. Radiat Res 66(2):401–426

    Article  ADS  Google Scholar 

  • Facchino S, Abdouh M, Chatoo W, Bernier G (2010) BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery. J Neurosci 30(30):10096–10111. https://doi.org/10.1523/JNEUROSCI.1634-10.2010

    Article  Google Scholar 

  • Gould MN, Clifton KH (1977) The survival of mammary cells following irradiation in vivo: a directly generated single-dose-survival curve. Radiat Res 72(2):343–352

    Article  ADS  Google Scholar 

  • Hindupur SK, Balaji SA, Saxena M, Pandey S, Sravan GS, Heda N, Kumar MV, Mukherjee G, Dey D, Rangarajan A (2014) Identification of a novel AMPK-PEA15 axis in the anoikis-resistant growth of mammary cells. Breast Cancer Res 16(4):420. https://doi.org/10.1186/s13058-014-0420-z

    Article  Google Scholar 

  • Hu Y, Smyth GK (2009) ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347(1–2):70–78. https://doi.org/10.1016/j.jim.2009.06.008

    Article  Google Scholar 

  • Imaoka T, Nishimura M, Iizuka D, Daino K, Takabatake T, Okamoto M, Kakinuma S, Shimada Y (2009) Radiation-induced mammary carcinogenesis in rodent models: what’s different from chemical carcinogenesis? J Radiat Res 50(4):281–293. https://doi.org/10.1269/jrr.09027

    Article  Google Scholar 

  • Imaoka T, Nishimura M, Daino K, Hosoki A, Takabatake M, Nishimura Y, Kokubo T, Morioka T, Doi K, Shimada Y, Kakinuma S (2019) Prominent dose-rate effect and its age dependence of rat mammary carcinogenesis induced by continuous gamma-ray exposure. Radiat Res 191(3):245–254. https://doi.org/10.1667/RR15094.1

    Article  Google Scholar 

  • Inomata K, Aoto T, Binh NT, Okamoto N, Tanimura S, Wakayama T, Iseki S, Hara E, Masunaga T, Shimizu H, Nishimura EK (2009) Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation. Cell 137(6):1088–1099. https://doi.org/10.1016/j.cell.2009.03.037

    Article  Google Scholar 

  • Inoue H, Ohsawa I, Murakami T, Kimura A, Hakamata Y, Sato Y, Kaneko T, Takahashi M, Okada T, Ozawa K, Francis J, Leone P, Kobayashi E (2005) Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun 329(1):288–295. https://doi.org/10.1016/j.bbrc.2005.01.132

    Article  Google Scholar 

  • Insinga A, Cicalese A, Faretta M, Gallo B, Albano L, Ronzoni S, Furia L, Viale A, Pelicci PG (2013) DNA damage in stem cells activates p21, inhibits p53, and induces symmetric self-renewing divisions. Proc Natl Acad Sci USA 110(10):3931–3936. https://doi.org/10.1073/pnas.1213394110

    Article  ADS  Google Scholar 

  • Keller PJ, Arendt LM, Kuperwasser C (2011) Stem cell maintenance of the mammary gland: it takes two. Cell Stem Cell 9(6):496–497. https://doi.org/10.1016/j.stem.2011.11.008

    Article  Google Scholar 

  • Kudo KI, Takabatake M, Nagata K, Nishimura Y, Daino K, Iizuka D, Nishimura M, Suzuki K, Kakinuma S, Imaoka T (2020) Flow cytometry definition of rat mammary epithelial cell populations and their distinct radiation responses. Radiat Res 194(1):22–37. https://doi.org/10.1667/RR15566.1

    Article  ADS  Google Scholar 

  • Mandal PK, Blanpain C, Rossi DJ (2011) DNA damage response in adult stem cells: pathways and consequences. Nat Rev Mol Cell Biol 12(3):198–202. https://doi.org/10.1038/nrm3060

    Article  Google Scholar 

  • Miyoshi-Imamura T, Kakinuma S, Kaminishi M, Okamoto M, Takabatake T, Nishimura Y, Imaoka T, Nishimura M, Murakami-Murofushi K, Shimada Y (2010) Unique characteristics of radiation-induced apoptosis in the postnatally developing small intestine and colon of mice. Radiat Res 173(3):310–318. https://doi.org/10.1667/RR1905.1

    Article  ADS  Google Scholar 

  • Niwa O, Barcellos-Hoff MH, Globus RK, Harrison JD, Hendry JH, Jacob P, Martin MT, Seed TM, Shay JW, Story MD, Suzuki K, Yamashita S (2015) ICRP Publication 131: Stem cell biology with respect to carcinogenesis aspects of radiological protection. Ann ICRP 44(3–4):7–357. https://doi.org/10.1177/0146645315595585

    Article  Google Scholar 

  • R Development Core Team (2012) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna

    Google Scholar 

  • Rauner G, Barash I (2012) Cell hierarchy and lineage commitment in the bovine mammary gland. PLoS One 7(1):e30113. https://doi.org/10.1371/journal.pone.0030113

    Article  ADS  Google Scholar 

  • Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414(6859):105–111. https://doi.org/10.1038/35102167

    Article  ADS  Google Scholar 

  • Reynolds BA, Weiss S (1996) Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175(1):1–13. https://doi.org/10.1006/dbio.1996.0090

    Article  Google Scholar 

  • Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  Google Scholar 

  • Serikawa T, Mashimo T, Takizawa A, Okajima R, Maedomari N, Kumafuji K, Tagami F, Neoda Y, Otsuki M, Nakanishi S, Yamasaki K, Voigt B, Kuramoto T (2009) National BioResource Project-Rat and related activities. Exp Anim 58(4):333–341. https://doi.org/10.1538/expanim.58.333

    Article  Google Scholar 

  • Shellabarger CJ, Chmelevsky D, Kellerer AM (1980) Induction of mammary neoplasms in the Sprague–Dawley rat by 430 keV neutrons and X-rays. J Natl Cancer Inst 64(4):821–833

    Google Scholar 

  • Sleeman KE, Kendrick H, Robertson D, Isacke CM, Ashworth A, Smalley MJ (2007) Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland. J Cell Biol 176(1):19–26

    Article  Google Scholar 

  • Sotiropoulou PA, Candi A, Mascre G, De Clercq S, Youssef KK, Lapouge G, Dahl E, Semeraro C, Denecker G, Marine JC, Blanpain C (2010) Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death. Nat Cell Biol 12(6):572–582. https://doi.org/10.1038/ncb2059

    Article  Google Scholar 

  • Spaas JH, Chiers K, Bussche L, Burvenich C, Van de Walle GR (2012) Stem/progenitor cells in non-lactating versus lactating equine mammary gland. Stem Cells Dev 21(16):3055–3067. https://doi.org/10.1089/scd.2012.0042

    Article  Google Scholar 

  • Tang J, Fernandez-Garcia I, Vijayakumar S, Martinez-Ruis H, Illa-Bochaca I, Nguyen DH, Mao JH, Costes SV, Barcellos-Hoff MH (2014) Irradiation of juvenile, but not adult, mammary gland increases stem cell self-renewal and estrogen receptor negative tumors. Stem Cells 32(3):649–661. https://doi.org/10.1002/stem.1533

    Article  Google Scholar 

  • Tao L, Roberts AL, Dunphy KA, Bigelow C, Yan H, Jerry DJ (2011) Repression of mammary stem/progenitor cells by p53 is mediated by Notch and separable from apoptotic activity. Stem Cells 29(1):119–127. https://doi.org/10.1002/stem.552

    Article  Google Scholar 

  • Tiberio R, Marconi A, Fila C, Fumelli C, Pignatti M, Krajewski S, Giannetti A, Reed JC, Pincelli C (2002) Keratinocytes enriched for stem cells are protected from anoikis via an integrin signaling pathway in a Bcl-2 dependent manner. FEBS Lett 524(1–3):139–144

    Article  Google Scholar 

  • Villadsen R, Fridriksdottir AJ, Ronnov-Jessen L, Gudjonsson T, Rank F, LaBarge MA, Bissell MJ, Petersen OW (2007) Evidence for a stem cell hierarchy in the adult human breast. J Cell Biol 177(1):87–101. https://doi.org/10.1083/jcb.200611114

    Article  Google Scholar 

  • Visvader JE, Clevers H (2016) Tissue-specific designs of stem cell hierarchies. Nat Cell Biol 18(4):349–355. https://doi.org/10.1038/ncb3332

    Article  Google Scholar 

  • Visvader JE, Stingl J (2014) Mammary stem cells and the differentiation hierarchy: current status and perspectives. Genes Dev 28(11):1143–1158. https://doi.org/10.1101/gad.242511.114

    Article  Google Scholar 

  • Weeden CE, Asselin-Labat ML (2018) Mechanisms of DNA damage repair in adult stem cells and implications for cancer formation. Biochim Biophys Acta 1864(1):89–101. https://doi.org/10.1016/j.bbadis.2017.10.015

    Article  Google Scholar 

  • Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM (2007) WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA 104(2):618–623

    Article  ADS  Google Scholar 

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Acknowledgements

The authors thank Masami Ootawara, Harumi Osada and the staff of the Laboratory Animal and Genome Sciences Section for technical assistance, and Dr. Benjamin J. Blyth for critical reviewing of data. The transgenic rat strain LEW-Tg(CAG-EGFP)1Ys was kindly provided by the National BioResource Project for the Rat (https://www.anim.med.kyoto-u.ac.jp/NBR/).

Funding

This work was supported in part by the Ministry of the Environment, Japan, via the Study of the Health Effects of Radiation, and in part by Japanese Society for Promotion of Science via JSPS KAKENHI (Grant number JP15H02824).

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Author notes

  1. Ayaka Hosoki

    Present address: Healios KK Kobe Research Institute, Kobe, Japan

  2. Yukiko Nishimura & Yoshiya Shimada

    Present address: Institute for Environmental Sciences, Aomori, Japan

  3. Ayaka Hosoki belonged to NIRS before it was organized as QST.

Authors and Affiliations

  1. Fukushima Project Headquarters, National Institute of Radiological Sciences (NIRS), Chiba, Japan

    Ayaka Hosoki

  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

    Mari Ogawa, Yukiko Nishimura, Mayumi Nishimura, Kazuhiro Daino, Shizuko Kakinuma & Tatsuhiko Imaoka

  3. Executive Board, QST, Chiba, Japan

    Yoshiya Shimada

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  1. Ayaka Hosoki
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Hosoki, A., Ogawa, M., Nishimura, Y. et al. The effect of radiation on the ability of rat mammary cells to form mammospheres. Radiat Environ Biophys 59, 711–721 (2020). https://doi.org/10.1007/s00411-020-00869-4

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