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Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice

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Abstract

Background

Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive (ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data.

Methods

We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24midCD49fhi)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity.

Results

Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24+CD49fhi) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity.

Conclusions

Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.

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Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

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Acknowledgements

This research was supported by an Australian Postgraduate Award scholarship for GD, Pfizer Australia and Victorian Cancer Agency (VCA) Clinical Research Fellowships for MS, an NBCF Early Career fellowship, NHMRC New Investigator grant and VCA Early Career fellowship for KB, and the Australian Cancer Research Foundation (for the Peter Mac Flow Cytometry and Centre for Advanced Histology and Microscopy core facilities). We thank the following Peter MacCallum Cancer Centre core facilities for provision of instrumentation, training and general support: Flow Cytometry, Centre for Advanced Histology and Microscopy and Animal House core facilities.

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Author notes

  1. Mark Shackleton and Kara L Britt are Co-senior authors.

Authors and Affiliations

  1. Department of Anatomy and Developmental Biology, Monash University, Melbourne, VIC, Australia

    Genevieve V. Dall, Yashar Seyed-Razavi & Gail P. Risbridger

  2. Breast Cancer Risk and Prevention Laboratory, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC, Australia

    Genevieve V. Dall, Jessica Vieusseux & Kara L. Britt

  3. Pathology and Pathogenesis Group, CSIRO Australian Animal Health Laboratory, Geelong, VIC, Australia

    Nathan Godde

  4. Immune Signaling Laboratory, Peter MacCallum Cancer Centre, 305 Grattan St, Melbourne, VIC, Australia

    Mandy Ludford-Menting & Sarah M. Russell

  5. Centre for Micro-Photonics, Swinburne University, Hawthorn, VIC, Australia

    Mandy Ludford-Menting & Sarah M. Russell

  6. Helen Diller Comprehensive Cancer Center, University of California, San Francisco, CA, USA

    Alan Ashworth

  7. The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia

    Nathan Godde, Sarah M. Russell, Robin L. Anderson, Gail P. Risbridger, Mark Shackleton & Kara L. Britt

  8. Metastasis Research Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, VIC, 3084, Australia

    Robin L. Anderson

  9. School of Cancer Medicine, La Trobe University, Bundoora, VIC, Australia

    Robin L. Anderson

  10. Cancer Development and Treatment, Alfred Hospital, Monash University, Melbourne, VIC, Australia

    Mark Shackleton

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Contributions

GD: Concept and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript. JV: Collection and/or assembly of data, Data analysis and interpretation, final approval of manuscript. YS-R: Collection and/or assembly of data, Data analysis and interpretation, final approval of manuscript. NG: Collection and/or assembly of data, final approval of manuscript. ML-M: Collection and/or assembly of data, Data analysis and interpretation, final approval of manuscript. SR: Financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. AA: Data analysis and interpretation, final approval of manuscript. RA: Financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. GR: Financial support, data analysis and interpretation, manuscript writing, final approval of manuscript. MS: Concept and design, data analysis and interpretation, manuscript writing, final approval of manuscript. KB: Concept and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.

Corresponding author

Correspondence to Kara L. Britt.

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Conflicts of interest

Alan Ashworth has the following disclosures: A.A. is a shareholder in Tango Therapeutics and consultant for AtlasMDX, Third Rock Ventures, Pfizer, ProLynx and Bluestar, a SAB Member of Genentech and Gladiator, and receives grant support from Sun Pharma and AstraZeneca. A.A. holds patents on the use of PARP inhibitors held jointly with AstraZeneca from which he has benefitted financially (and may do so in the future) through the ICR Rewards to Inventors Scheme.

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10549_2020_5804_MOESM1_ESM.tif

Supplementary file1 Figure S1. Mouse model of early parity. Related to Figure 1. (A) Schematic of the mouse model used to mimic early parity in women, which is associated with decreased breast cancer risk. Mice were mated when young (6 weeks old) and the mammary epithelial cells analysed and/or isolated 10 weeks after involution. (B) Mammary glands from age-matched nulliparous control mice (left) and from mice at day 4 involution (involution), day 21 involution (repair) and 10 weeks after involution (10wks post Inv) were stained for morphology evaluation (H&E), mast cell infiltration using toluidine blue, and Ki67 to assess proliferation. (TIF 13302 kb)

10549_2020_5804_MOESM2_ESM.tif

Supplementary file2 Figure S2. Gating strategy used to assess mammary cell populations via flow cytometry. Related to Figure 1 and 3 (A) Gating strategy used to identify morphologically normal (i), single (ii), viable (iii) lineage negative (iv) mammary cells. CD24 and CD49f are then used together to distinguish luminal, basal and CD49fhi MaSC-enriched cells as well as the stroma (v). B We stained cells with both CD29 (i) and CD49f (ii) (as well as the other lineage and epithelial markers) and used flowlogic to analyse the populations. When coloured as populations in the analysis using CD29 and CD24, we could backgate and assess where the CD24 and CD29 populations sit on the CD24 and CD49f plot. The luminal and basal populations isolated with CD29 and CD24 were similarly isolated with CD49f and CD24. Moreover the CD29hi population labels the same cells as the CD49f hi population. (TIF 25521 kb)

10549_2020_5804_MOESM3_ESM.tif

Supplementary file3 Figure S3 Outgrowth size does not affect results of transplant assay from uniparous mice. Related to Table 1. (A) Results of mammary fat pad transplant assay with unfractionated cells isolated from uniparous and nulliparous mice. (B) Results of mammary fat pad transplant assay with MaSCs isolated from uniparous and nulliparous mice. For both tables circles show proportions of filling of each engrafted fat pad with repopulated epithelium. ELDA (Hu and Smyth, 2009) was used to determine frequencies of repopulating cells (MRCs) in cell suspensions from which aliquots were drawn for injection. Results show stem cell enrichment when all outgrowths are considered, or when only extensive outgrowths filling more than 25% of the fat pad are considered. (TIF 26856 kb)

10549_2020_5804_MOESM4_ESM.tif

Supplementary file4 Figure S4 Outgrowth size does not affect results of transplant assay from multiparous mice. Related to Table 3. Results of mammary fat pad transplant assay with MaSCs isolated from multiparous and nulliparous mice. Circles show proportions of filling of each engrafted fat pad with repopulated epithelium. ELDA (Hu and Smyth, 2009) was used to determine frequencies of repopulating cells (MRCs) in cell suspensions from which aliquots were drawn for injection. Results show stem cell enrichment when all outgrowths are considered, or when only extensive outgrowths filling more than 25% of the fat pad are considered. (TIF 25523 kb)

10549_2020_5804_MOESM5_ESM.docx

Supplementary file5 Supplementary Table 1. Results from primary transplantation using CD49fhi vs CD49flo cells (DOCX 13 kb)

10549_2020_5804_MOESM6_ESM.docx

Supplementary file6 Supplementary Table 2. Results from primary transplantation of CD49flo cells of parous and nulliparous mice (DOCX 13 kb)

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Dall, G.V., Vieusseux, J., Seyed-Razavi, Y. et al. Parity reduces mammary repopulating activity but does not affect mammary stem cells defined as CD24 + CD29/CD49fhi in mice. Breast Cancer Res Treat 183, 565–575 (2020). https://doi.org/10.1007/s10549-020-05804-1

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