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The Cellular Organization of the Mammary Gland: Insights From Microscopy

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Abstract

Despite rapid advances in our knowledge of the cellular heterogeneity and molecular regulation of the mammary gland, how these relate to 3D cellular organization remains unclear. In addition to hormonal regulation, mammary gland development and function is directed by para- and juxtacrine signaling among diverse cell-types, particularly the immune and mesenchymal populations. Precise mapping of the cellular landscape of the breast will help to decipher this complex coordination. Imaging of thin tissue sections has provided foundational information about cell positioning in the mammary gland and now technological advances in tissue clearing and subcellular-resolution 3D imaging are painting a more complete picture. In particular, confocal, light-sheet and multiphoton microscopy applied to intact tissue can fully capture cell morphology, position and interactions, and have the power to identify spatially rare events. This review will summarize our current understanding of mammary gland cellular organization as revealed by microscopy. We focus on the mouse mammary gland and cover a broad range of immune and stromal cell types at major developmental stages and give insights into important tissue niches and cellular interactions.

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References

  1. Cowin P, Wysolmerski J. Molecular mechanisms guiding embryonic mammary gland development. Csh Perspect Biol. 2010;2:a003251. https://doi.org/10.1101/cshperspect.a003251.

    Article  CAS  Google Scholar 

  2. Sakakura T, Sakagami Y, Nishizuka Y. Dual origin of mesenchymal tissues participating in mouse mammary gland embryogenesis. Dev Biol. 1982;91:202–7. https://doi.org/10.1016/0012-1606(82)90024-0.

    Article  CAS  PubMed  Google Scholar 

  3. Lee MY, Racine V, Jagadpramana P, et al. Ectodermal influx and cell hypertrophy provide early growth for all murine mammary rudiments, and are differentially regulated among them by Gli3. Plos One. 2011;6:e26242. https://doi.org/10.1371/journal.pone.0026242.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Heuberger B, Fitzka I, Wasner G, Kratochwil K. Induction of androgen receptor formation by epithelium-mesenchyme interaction in embryonic mouse mammary gland. Proc National Acad Sci. 1982;79:2957–61. https://doi.org/10.1073/pnas.79.9.2957.

    Article  CAS  Google Scholar 

  5. Cunha GR, Young P, Christov K, et al. Mammary phenotypic expression induced in epidermal cells by embryonic mammary mesenchyme. Cells Tissues Organs. 1995;152:195–204. https://doi.org/10.1159/000147698.

    Article  CAS  Google Scholar 

  6. Kratochwil K. Organ specificity in mesenchymal induction demonstrated in the embryonic development of the mammary gland of the mouse. Dev Biol. 1969;20:46–71. https://doi.org/10.1016/0012-1606(69)90004-9.

    Article  CAS  PubMed  Google Scholar 

  7. Foley J, Dann P, Hong J, et al. Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development. Dev Camb Engl. 2001;128:513–25.

    CAS  Google Scholar 

  8. Macias H, Hinck L. Mammary gland development. Wiley Inderdisciplinary Reviews: Developmental Biology. 2012;1:533–57. https://doi.org/10.1002/wdev.35.

    Article  CAS  Google Scholar 

  9. Hovey RC, Trott JF, Vonderhaar BK. Establishing a Framework for the Functional Mammary Gland: From Endocrinology to Morphology. J Mammary Gland Biol. 2002;7:17–38. https://doi.org/10.1023/a:1015766322258.

    Article  Google Scholar 

  10. Dulbecco R, Henahan M, Armstrong B. Cell types and morphogenesis in the mammary gland. Proc National Acad Sci. 1982;79:7346–50. https://doi.org/10.1073/pnas.79.23.7346.

    Article  CAS  Google Scholar 

  11. Howard BA, Gusterson BA. Human Breast Development. J Mammary Gland Biol. 2000;5:119–37. https://doi.org/10.1023/a:1026487120779.

    Article  CAS  Google Scholar 

  12. Ewald AJ, Brenot A, Duong M, et al. Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis. Dev Cell. 2008;14:570–81. https://doi.org/10.1016/j.devcel.2008.03.003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gouon-Evans V, Rothenberg ME, Pollard JW. Postnatal mammary gland development requires macrophages and eosinophils. Development. 2000;127:2269–82.

    Article  CAS  Google Scholar 

  14. Nguyen AV, Pollard JW. Colony Stimulating Factor-1 Is Required to Recruit Macrophages into the Mammary Gland to Facilitate Mammary Ductal Outgrowth. Dev Biol. 2002;247:11–25. https://doi.org/10.1006/dbio.2002.0669.

    Article  CAS  PubMed  Google Scholar 

  15. Plaks V, Boldajipour B, Linnemann JR, et al. Adaptive immune regulation of mammary postnatal organogenesis. Dev Cell. 2015;34:493–504. https://doi.org/10.1016/j.devcel.2015.07.015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lilla J, Werb Z. Mast cells contribute to the stromal microenvironment in mammary gland branching morphogenesis. Dev Biol. 2010;337:124–33.

    Article  CAS  Google Scholar 

  17. Wilson GJ, Fukuoka A, Love SR, et al. Chemokine receptors coordinately regulate macrophage dynamics and mammary gland development. Development. 2020;147:dev187815. https://doi.org/10.1242/dev.187815.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chakrabarti R, Celià-Terrassa T, Kumar S, et al. Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche. Science. 2018;260:eaan4153. https://doi.org/10.1126/science.aan4153.

    Article  CAS  Google Scholar 

  19. Reed JR, Schwertfeger KL. Immune cell location and function during post-natal mammary gland development. J Mammary Gland Biol. 2010;15:329–39. https://doi.org/10.1007/s10911-010-9188-7.

    Article  Google Scholar 

  20. Hammer AM, Sizemore GM, Shukla VC, et al. Stromal PDGFR-α activation enhances matrix stiffness, impedes mammary ductal development, and accelerates tumor growth. Neoplasia. 2017;19:496–508. https://doi.org/10.1016/j.neo.2017.04.004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Koledova Z, Zhang X, Streuli C, et al. SPRY1 regulates mammary epithelial morphogenesis by modulating EGFR-dependent stromal paracrine signaling and ECM remodeling. Proc Natl Acad Sci. 2016;113:E5731–40. https://doi.org/10.1073/pnas.1611532113.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Peuhu E, Kaukonen R, Lerche M, et al. SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland. The EMBO Journal. 2017;36:165–82. https://doi.org/10.15252/embj.201694387.

    Article  CAS  PubMed  Google Scholar 

  23. Chua A, Hodson L, Moldenhauer L, et al. Dual roles for macrophages in ovarian cycle-associated development and remodelling of the mammary gland epithelium. Development. 2010;137:4229–38. https://doi.org/10.1242/dev.059261.

    Article  CAS  PubMed  Google Scholar 

  24. Fata JE, Chaudhary V, Khokha R. Cellular turnover in the mammary gland is correlated with systemic levels of progesterone and not 17-estradiol during the estrous cycle. Biol Reprod. 2001;65:680–8. https://doi.org/10.1095/biolreprod65.3.680.

    Article  CAS  PubMed  Google Scholar 

  25. Andres A-C, Strange R. Apoptosis in the estrous and menstrual cycles. J Mammary Gland Biol. 1999;4:221–8. https://doi.org/10.1023/a:1018737510695.

    Article  CAS  Google Scholar 

  26. Need EF, Atashgaran V, Ingman WV, Dasari P. Hormonal regulation of the immune microenvironment in the mammary Gland. J Mammary Gland Biol. 2014;19:229–39. https://doi.org/10.1007/s10911-014-9324-x.

    Article  Google Scholar 

  27. Naylor MJ, Ormandy CJ. Mouse strain-specific patterns of mammary epithelial ductal side branching are elicited by stromal factors. Dev Dynam. 2002;225:100–5. https://doi.org/10.1002/dvdy.10133.

    Article  CAS  Google Scholar 

  28. Nandi S, Bern HA. Relation between mammary-gland responses to lactogenic hormone combinations and tumor susceptibility in various strains of mice. J Natl Cancer Inst. 1959;24:907–31. https://doi.org/10.1093/jnci/24.4.907.

    Article  Google Scholar 

  29. Brisken C, Park S, Vass T, et al. A paracrine role for the epithelial progesterone receptor in mammary gland development. Proc Natl Acad Sci. 1998;95:5076–81. https://doi.org/10.1073/pnas.95.9.5076.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Brisken C, Kaur S, Chavarria TE, et al. Prolactin controls mammary gland development via direct and indirect mechanisms. Dev Biol. 1999;210:96–106. https://doi.org/10.1006/dbio.1999.9271.

    Article  CAS  PubMed  Google Scholar 

  31. Pollard JW, Hennighausen L. Colony stimulating factor 1 is required for mammary gland development during pregnancy. Proc Natl Acad Sci. 1994;91:9312–6.

    Article  CAS  Google Scholar 

  32. McNeilly AS. Physiology of lactation. J Biosoc Sci. 1977;9:5–21. https://doi.org/10.1017/s0021932000023804.

    Article  Google Scholar 

  33. Stevenson AJ, Vanwalleghem G, Stewart TA, et al. Multiscale imaging of basal cell dynamics in the functionally mature mammary gland. Proc Natl Acad Sci. 2020;117:26822–32. https://doi.org/10.1073/pnas.2016905117.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Masedunskas A, Chen Y, Stussman R, et al. Kinetics of milk lipid droplet transport, growth, and secretion revealed by intravital imaging: lipid droplet release is intermittently stimulated by oxytocin. Mol Biol Cell. 2017;28:935–46. https://doi.org/10.1091/mbc.e16-11-0776.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Watson CJ, Kreuzaler PA. Remodeling mechanisms of the mammary gland during involution. Int J Dev Biol. 2011;55:757–62. https://doi.org/10.1387/ijdb.113414cw.

    Article  PubMed  Google Scholar 

  36. Li M, Liu X, Robinson G, et al. Mammary-derived signals activate programmed cell death during the first stage of mammary gland involution. Proc Natl Acad Sci. 1997;94:3425–30. https://doi.org/10.1073/pnas.94.7.3425.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Marti A, Feng Z, Altermatt HJ, Jaggi R. Milk accumulation triggers apoptosis of mammary epithelial cells. Eur J Cell Biol. 1997;73:158–65.

    CAS  PubMed  Google Scholar 

  38. Kreuzaler PA, Staniszewska AD, Li W, et al. Stat3 controls lysosomal-mediated cell death in vivo. Nat Cell Biol. 2011;13:303–9. https://doi.org/10.1038/ncb2171.

    Article  CAS  PubMed  Google Scholar 

  39. Sargeant T, Lloyd-Lewis B, Resemann H, et al. Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization. Nat Cell Biol. 2014;16:1057–68.

    Article  CAS  Google Scholar 

  40. Dawson CA, Pal B, Vaillant F, et al. Tissue-resident ductal macrophages survey the mammary epithelium and facilitate tissue remodelling. Nat Cell Biol. 2020;22:546–58. https://doi.org/10.1038/s41556-020-0505-0.

    Article  CAS  PubMed  Google Scholar 

  41. Lund LR, Rømer J, Thomasset N, et al. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development. 1996;122:181–93.

    Article  CAS  Google Scholar 

  42. Martinson HA, Jindal S, Durand-Rougely C, et al. Wound healing‐like immune program facilitates postpartum mammary gland involution and tumor progression. Int J Cancer. 2015;136:1803–13. https://doi.org/10.1002/ijc.29181.

    Article  CAS  PubMed  Google Scholar 

  43. Stein T, Morris JS, Davies CR, et al. Involution of the mouse mammary gland is associated with an immune cascade and an acute-phase response, involving LBP, CD14 and STAT3. Breast Cancer Res. 2003;6:R75. https://doi.org/10.1186/bcr753.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Betts CB, Pennock ND, Caruso BP, et al. Mucosal immunity in the female murine mammary gland. J Immunol. 2018;201:ji1800023. https://doi.org/10.4049/jimmunol.1800023.

    Article  CAS  Google Scholar 

  45. O’Brien J, Martinson H, Durand-Rougely C, Schedin P. Macrophages are crucial for epithelial cell death and adipocyte repopulation during mammary gland involution. Development. 2012;139:269–75. https://doi.org/10.1242/dev.071696.

    Article  CAS  PubMed  Google Scholar 

  46. Green KA, Lund LR. ECM degrading proteases and tissue remodelling in the mammary gland. Bioessays. 2005;27:894–903. https://doi.org/10.1002/bies.20281.

    Article  CAS  PubMed  Google Scholar 

  47. Lyons TR, O’Brien J, Borges VF, et al. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nature medicine. 2011;17:1109–15. https://doi.org/10.1038/nm.2416.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Stewart TA, Hughes K, Hume DA, Davis FM. Developmental stage-specific distribution of macrophages in mouse mammary gland. Front Cell Dev Biol. 2019;7:250. https://doi.org/10.3389/fcell.2019.00250.

    Article  PubMed  PubMed Central  Google Scholar 

  49. Wuidart A, Sifrim A, Fioramonti M, et al. Early lineage segregation of multipotent embryonic mammary gland progenitors. Nat Cell Biol. 2018;20:666–76. https://doi.org/10.1038/s41556-018-0095-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Lilja AM, Rodilla V, Huyghe M, et al. Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland. Nat Cell Biol. 2018;20:677–87. https://doi.org/10.1038/s41556-018-0108-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Fu N, Rios AC, Pal B, et al. Identification of quiescent and spatially restricted mammary stem cells that are hormone responsive. Nat Cell Biol. 2017;19:164–76. https://doi.org/10.1038/ncb3471.

    Article  CAS  PubMed  Google Scholar 

  52. Ying Z, Beronja S. Embryonic barcoding of equipotent mammary progenitors functionally identifies breast cancer drivers. Cell Stem Cell. 2020;26:403–19. https://doi.org/10.1016/j.stem.2020.01.009.

    Article  CAS  Google Scholar 

  53. Spike BT, Engle DD, Lin JC, et al. A mammary stem cell population identified and characterized in late embryogenesis reveals similarities to human breast cancer. Cell Stem Cell. 2012;10:183–97. https://doi.org/10.1016/j.stem.2011.12.018.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Rios AC, Fu NY, Lindeman GJ, Visvader JE. In situ identification of bipotent stem cells in the mammary gland. Nature. 2014;506:322–7. https://doi.org/10.1038/nature12948.

    Article  CAS  PubMed  Google Scholar 

  55. Davis FM, Lloyd-Lewis B, Harris OB, et al. Single-cell lineage tracing in the mammary gland reveals stochastic clonal dispersion of stem/progenitor cell progeny. Nat Commun. 2016;7:13053. https://doi.org/10.1038/ncomms13053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Lloyd-Lewis B, Davis FM, Harris OB, et al. Neutral lineage tracing of proliferative embryonic and adult mammary stem/progenitor cells. Development. 2018;145:dev164079. https://doi.org/10.1242/dev.164079.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Lloyd-Lewis B, Davis FM, Harris OB, et al. Imaging the mammary gland and mammary tumours in 3D: optical tissue clearing and immunofluorescence methods. Breast Cancer Res. 2016;18:127. https://doi.org/10.1186/s13058-016-0754-9.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Amerongen R van, Bowman A, Nusse R. Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell. 2012;11:387–400.

    Article  Google Scholar 

  59. Rodilla V, Dasti A, Huyghe M, et al. Luminal progenitors restrict their lineage potential during mammary gland development. Plos Biol. 2015;13:e1002069. https://doi.org/10.1371/journal.pbio.1002069.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kimata K, Sakakura T, Inaguma Y, et al. Participation of two different mesenchymes in the developing mouse mammary gland: synthesis of basement membrane components by fat pad precursor cells. J Embryol Exp Morph. 1985;89:243–57.

    CAS  PubMed  Google Scholar 

  61. Dunbar ME, Dann PR, Robinson GW, et al. Parathyroid hormone-related protein signaling is necessary for sexual dimorphism during embryonic mammary development. Dev Camb Engl. 1999;126:3485–93.

    CAS  Google Scholar 

  62. Watson CJ, Khaled WT. Mammary development in the embryo and adult: a journey of morphogenesis and commitment. Development. 2008;135:995–1003. https://doi.org/10.1242/dev.005439.

    Article  CAS  PubMed  Google Scholar 

  63. Robinson GW, Karpf ABC, Kratochwil K. Regulation of mammary gland development by tissue interaction. J Mammary Gland Biol. 1999;4:9–19. https://doi.org/10.1023/a:1018748418447.

    Article  CAS  Google Scholar 

  64. Inaguma Y, Kusakabe M, Mackie EJ, et al. Epithelial induction of stromal tenascin in the mouse mammary gland: From embryogenesis to carcinogenesis. Dev Biol. 1988;128:245–55. https://doi.org/10.1016/0012-1606(88)90288-6.

    Article  CAS  PubMed  Google Scholar 

  65. Chiquet-Ehrismann R, Mackie EJ, Pearson CA, Sakakura T. Tenascin: an extracellular matrix protein involved in tissue interactions during fetal development and oncogenesis. Cell. 1986;47:131–9. https://doi.org/10.1016/0092-8674(86)90374-0.

    Article  CAS  PubMed  Google Scholar 

  66. Aufderheide E, Chiquet-Ehrismann R, Ekblom P. Epithelial-mesenchymal interactions in the developing kidney lead to expression of tenascin in the mesenchyme. J Cell Biol. 1987;105:599–608. https://doi.org/10.1083/jcb.105.1.599.

    Article  CAS  PubMed  Google Scholar 

  67. Parmar H, Cunha GR. Epithelial–stromal interactions in the mouse and human mammary gland in vivo. Endocrine-related Cancer. 2004;11:437–58.

    Article  CAS  Google Scholar 

  68. Jäppinen N, Félix I, Lokka E, et al. Fetal-derived macrophages dominate in adult mammary glands. Nat commun. 2019;10:281. https://doi.org/10.1038/s41467-018-08065-1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Williams JM, Daniel CW. Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev biol. 1983;97:274–90. https://doi.org/10.1038/nature04496.

    Article  CAS  PubMed  Google Scholar 

  70. Visvader JE. Keeping abreast of the mammary epithelial hierarchy and breast tumorigenesis. Genes Dev. 2009;23:2563–77. https://doi.org/10.1101/gad.1849509.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Sreekumar A, Toneff MJ, Toh E, et al. WNT-mediated regulation of FOXO1 constitutes a critical axis maintaining pubertal mammary stem cell homeostasis. Dev Cell. 2017;43:436–48.e6. https://doi.org/10.1016/j.devcel.2017.10.007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Keymeulen AV, Rocha AS, Ousset M, et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature. 2011;479:189. https://doi.org/10.1038/nature10573.

    Article  CAS  PubMed  Google Scholar 

  73. Scheele CLGJ, Hannezo E, Muraro MJ, et al. Identity and dynamics of mammary stem cells during branching morphogenesis. Nature. 2017;542:313. https://doi.org/10.1038/nature21046.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Paine I, Chauviere A, Landua J, et al. A geometrically-constrained mathematical model of mammary gland ductal elongation reveals novel cellular dynamics within the terminal end bud. PLoS comput biol. 2016;12:e1004839. https://doi.org/10.1371/journal.pcbi.1004839.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Keymeulen AV, Fioramonti M, Centonze A, et al. Lineage-restricted mammary stem cells sustain the development, homeostasis, and regeneration of the estrogen receptor positive lineage. Cell Reports. 2017;20:1525–32. https://doi.org/10.1016/j.celrep.2017.07.066.

    Article  CAS  PubMed  Google Scholar 

  76. Wang C, Christin JR, Oktay MH, Guo W. Lineage-biased stem cells maintain estrogen-receptor-positive and -negative mouse mammary luminal lineages. Cell Reports. 2017;18:2825–35. https://doi.org/10.1016/j.celrep.2017.02.071.

    Article  CAS  PubMed  Google Scholar 

  77. Bai L, Rohrschneider LR. s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Gene Dev. 2010;24:1882–92. https://doi.org/10.1101/gad.1932810.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Mailleux AA, Overholtzer M, Schmelzle T, et al. BIM regulates apoptosis during mammary ductal morphogenesis, and its absence reveals alternative cell death mechanisms. Dev Cell. 2007;12:221–34. https://doi.org/10.1016/j.devcel.2006.12.003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Srinivasan K, Strickland P, Valdes A, et al. Netrin-1/Neogenin interaction stabilizes multipotent progenitor cap cells during mammary gland morphogenesis. Dev Cell. 2003;4:371–82. https://doi.org/10.1016/s1534-5807(03)00054-6.

    Article  CAS  PubMed  Google Scholar 

  80. Silberstein GB, Daniel CW. Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct. Dev Biol. 1982;90:215–22. https://doi.org/10.1016/0012-1606(82)90228-7.

    Article  CAS  PubMed  Google Scholar 

  81. Ewald AJ, Huebner RJ, Palsdottir H, et al. Mammary collective cell migration involves transient loss of epithelial features and individual cell migration within the epithelium. J Cell Sci. 2012;125:2638–54. https://doi.org/10.1242/jcs.096875.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Neumann NM, Perrone MC, Veldhuis JH, et al. Coordination of receptor tyrosine kinase signaling and interfacial tension dynamics drives radial intercalation and tube elongation. Dev Cell. 2018;45:67–82.e6. https://doi.org/10.1016/j.devcel.2018.03.011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Huebner RJ, Neumann NM, Ewald AJ. Mammary epithelial tubes elongate through MAPK-dependent coordination of cell migration. Development. 2016;143:983–93. https://doi.org/10.1242/dev.127944.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Keller R. Shaping the vertebrate body plan by polarized embryonic cell movements. Science. 2002;298:1950–4. https://doi.org/10.1126/science.1079478.

    Article  CAS  PubMed  Google Scholar 

  85. Sumbal J, Belisova D, Koledova Z. Fibroblasts: The grey eminence of mammary gland development. Semin Cell Dev Biol. 2020. https://doi.org/10.1016/j.semcdb.2020.10.012.

    Article  PubMed  Google Scholar 

  86. Schwertfeger KL, Xian W, Kaplan AM, et al. A critical role for the inflammatory response in a mouse model of preneoplastic progression. Cancer research. 2006;66:5676–85. https://doi.org/10.1158/0008-5472.CAN-05-3781.

    Article  CAS  PubMed  Google Scholar 

  87. Sferruzzi-Perri AN, Robertson SA, Dent LA. Interleukin-5 transgene expression and eosinophilia are associated with retarded mammary gland development in mice. Biol Reprod. 2003;69:224–33. https://doi.org/10.1095/biolreprod.102.010611.

    Article  CAS  PubMed  Google Scholar 

  88. Ingman WV, Wyckoff J, Gouon-Evans V, et al. Macrophages promote collagen fibrillogenesis around terminal end buds of the developing mammary gland. Dev Dynam. 2006;235:3222–9. https://doi.org/10.1002/dvdy.20972.

    Article  CAS  Google Scholar 

  89. Howlin J, McBryan J, Martin F. Pubertal mammary gland development: Insights from mouse models. J Mammary Gland Biol. 2006;11:283–97. https://doi.org/10.1007/s10911-006-9024-2.

    Article  Google Scholar 

  90. Fata JE, Werb Z, Bissell MJ. Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes. Breast Cancer Res. 2004;6:335. https://doi.org/10.1186/bcr634.

    Article  CAS  Google Scholar 

  91. Djonov V, Andres A, Ziemiecki A (2001) Vascular remodelling during the normal and malignant life cycle of the mammary gland. Microsc Res Techniq 52:182–189. https://doi.org/10.1002/1097-0029(20010115)52:2%3C182::aid-jemt1004%3E3.0.co;2-m

    Article  CAS  Google Scholar 

  92. Matsumoto M, Nishinakagawa H, Kurohmaru M, et al. Pregnancy and lactation affect the microvasculature of the mammary gland in mice. J Vet Med Sci. 1992;54:937–43. https://doi.org/10.1292/jvms.54.937.

    Article  CAS  PubMed  Google Scholar 

  93. Wang Y, Chaffee TS, LaRue RS, et al. Tissue-resident macrophages promote extracellular matrix homeostasis in the mammary gland stroma of nulliparous mice. Elife. 2020;9:e57438. https://doi.org/10.7554/elife.57438.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Chakarov S, Lim H, Tan L, et al. Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches. Science. 2019;363:eaau0964. https://doi.org/10.1126/science.aau0964.

    Article  CAS  PubMed  Google Scholar 

  95. Nagy D, Gillis CMC, Davies K, et al (2020) Developing mammary terminal duct lobular units have a dynamic mucosal and stromal immune microenvironment. bioRxiv. https://doi.org/10.1101/2020.11.05.369843.

  96. Rios AC, Fu N, Cursons J, et al. The complexities and caveats of lineage tracing in the mammary gland. Breast Cancer Res. 2016;18:116. https://doi.org/10.1186/s13058-016-0774-5.

    Article  PubMed  PubMed Central  Google Scholar 

  97. Plaks V, Brenot A, Lawson DA, et al. Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis. Cell Reports. 2013;3:70–8. https://doi.org/10.1016/j.celrep.2012.12.017.

    Article  CAS  PubMed  Google Scholar 

  98. Fernandez-Gonzalez R, Illa-Bochaca I, Welm BE, et al. Mapping mammary gland architecture using multi-scale in situ analysis. Integr Biol. 2008;1:80–9. https://doi.org/10.1039/b816933k.

    Article  CAS  Google Scholar 

  99. Lee HJ, Gallego-Ortega D, Ledger A, et al. Progesterone drives mammary secretory differentiation via RankL-mediated induction of Elf5 in luminal progenitor cells. Development. 2013;140:1397–401. https://doi.org/10.1242/dev.088948.

    Article  CAS  PubMed  Google Scholar 

  100. Rios AC, Fu NY, Jamieson PR, et al. Essential role for a novel population of binucleated mammary epithelial cells in lactation. Nat Commun. 2016;7:11400. https://doi.org/10.1038/ncomms11400.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Sun X, Robertson S, Ingman W. Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland. Cytokine. 2013;61:377–88. https://doi.org/10.1016/j.cyto.2012.12.002.

    Article  CAS  PubMed  Google Scholar 

  102. Degnim AC, Brahmbhatt RD, Radisky DC, et al. Immune cell quantitation in normal breast tissue lobules with and without lobulitis. Breast Cancer Res Tr. 2014;144:539–49. https://doi.org/10.1007/s10549-014-2896-8.

    Article  CAS  Google Scholar 

  103. Warburton MJ, Mitchell D, Ormerod EJ, Rudland P. Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating, and involuting rat mammary gland. J Hist Cytochem. 1982;30:667–76. https://doi.org/10.1177/30.7.6179984.

    Article  CAS  Google Scholar 

  104. Muschler J, Streuli CH. Cell–matrix interactions in mammary gland development and breast cancer. Csh Perspect Biol. 2010;2:a003202. https://doi.org/10.1101/cshperspect.a003202.

    Article  CAS  Google Scholar 

  105. Russell JS, McGee SO, Ip MM, et al. Conjugated linoleic acid induces mast cell recruitment during mouse mammary gland stromal remodeling. J Nutrition. 2007;137:1200–7. https://doi.org/10.1093/jn/137.5.1200.

    Article  CAS  Google Scholar 

  106. Ramirez RA, Lee A, Schedin P, et al. Alterations in mast cell frequency and relationship to angiogenesis in the rat mammary gland during windows of physiologic tissue remodeling. Dev Dynam. 2012;241:890–900. https://doi.org/10.1002/dvdy.23778.

    Article  CAS  Google Scholar 

  107. Hitchcock JR, Hughes K, Harris OB, Watson CJ. Dynamic architectural interplay between leucocytes and mammary epithelial cells. Febs J. 2019. https://doi.org/10.1111/febs.15126.

    Article  PubMed  PubMed Central  Google Scholar 

  108. Walker NI, Bennett RE, Kerr JFR. Cell death by apoptosis during involution of the lactating breast in mice and rats. Am J Anat. 1989;185:19–32. https://doi.org/10.1002/aja.1001850104.

    Article  CAS  PubMed  Google Scholar 

  109. Fadok VA. Clearance: The last and often forgotten stage of apoptosis. J Mammary Gland Biol. 1999;4:203–11. https://doi.org/10.1023/a:1011384009787.

    Article  CAS  Google Scholar 

  110. Monks J, Smith-Steinhart C, Kruk ER, et al. Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland. Biol Reprod. 2008;78:586–94. https://doi.org/10.1095/biolreprod.107.065045.

    Article  CAS  PubMed  Google Scholar 

  111. Teplova I, Lozy F, Price S, et al. ATG proteins mediate efferocytosis and suppress inflammation in mammary involution. Autophagy. 2013;9:459–75. https://doi.org/10.4161/auto.23164.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Sandahl M, Hunter DM, Strunk KE, et al. Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation. Bmc Dev Biol. 2010;10:122. https://doi.org/10.1186/1471-213x-10-122.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Martinez-Hernandez A, Fink LM, Pierce GB. Removal of basement membrane in the involuting breast. Lab Invest J Technical Meth Path. 1976;34:455–62.

    CAS  Google Scholar 

  114. O’Brien J, Lyons T, Monks J, et al. Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species. Am J Path. 2010;176:1241–55. https://doi.org/10.2353/ajpath.2010.090735.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Zwick RK, Rudolph MC, Shook BA, et al. Adipocyte hypertrophy and lipid dynamics underlie mammary gland remodeling after lactation. Nat Commun. 2018;9:3592. https://doi.org/10.1038/s41467-018-05911-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Wang QA, Song A, Chen W, et al. Reversible de-differentiation of mature white adipocytes into preadipocyte-like precursors during lactation. Cell Metab. 2018;28:282–8.e3. https://doi.org/10.1016/j.cmet.2018.05.022.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. YASUGI T, KAIDO T, UEHARA Y. Changes in density and architecture of microvessels of the rat mammary gland during pregnancy and lactation. Arch Histol Cytol. 1989;52:115–22. https://doi.org/10.1679/aohc.52.115.

    Article  CAS  PubMed  Google Scholar 

  118. Betts CB, Quackenbush A, Anderson W, et al (2020) Mucosal immunity and liver metabolism in the complex condition of lactation insufficiency. J Hum Lact 089033442094765. https://doi.org/10.1177/0890334420947656.

  119. Hughes K, Wickenden JA, Allen JE, Watson CJ. Conditional deletion of Stat3 in mammary epithelium impairs the acute phase response and modulates immune cell numbers during post-lactational regression. J Pathology. 2012;227:106–17. https://doi.org/10.1002/path.3961.

    Article  CAS  Google Scholar 

  120. Lilla JN, Joshi RV, Craik CS, Werb Z. Active plasma kallikrein localizes to mast cells and regulates epithelial cell apoptosis, adipocyte differentiation, and stromal remodeling during mammary gland involution. J Biol Chem. 2009;284:13792–803. https://doi.org/10.1074/jbc.m900508200.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Elder AM, Stoller AR, Black SA, Lyons TR. (2020) Macphatics and PoEMs in postpartum mammary development and tumor progression. J Mammary Gland Biol 1–11. https://doi.org/10.1007/s10911-020-09451-6.

  122. Elder AM, Tamburini BA, Crump LS, et al (2018) Semaphorin 7A promotes macrophage-mediated lymphatic remodeling during postpartum mammary gland involution and in breast cancer. Cancer Res.1642.2018. https://doi.org/10.1158/0008-5472.CAN-18-1642.

    Article  PubMed  PubMed Central  Google Scholar 

  123. Lyons TR, Borges VF, Betts CB, et al. Cyclooxygenase-2–dependent lymphangiogenesis promotes nodal metastasis of postpartum breast cancer. J Clin Invest. 2014;124:3901–12. https://doi.org/10.1172/jci73777.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Richardson D, Lichtman J. Clarifying tissue clearing. Cell. 2015;162:246–57.

    Article  CAS  Google Scholar 

  125. Chung K, Wallace J, Kim SY, et al. Structural and molecular interrogation of intact biological systems. Nature. 2013;497:332–7. https://doi.org/10.1038/nature12107.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Tainaka K, Kuno A, Kubota SI, et al. Chemical principles in tissue clearing and staining protocols for whole-body cell profiling. Annu Rev Cell Dev Bi. 2015;32:1–29. https://doi.org/10.1146/annurev-cellbio-111315-125001.

    Article  CAS  Google Scholar 

  127. Rios AC, Capaldo BD, Vaillant F, et al. Intraclonal plasticity in mammary tumors revealed through large-scale single-cell resolution 3D imaging. Cancer cell. 2019;35:618–32.e6. https://doi.org/10.1016/j.ccell.2019.02.010.

    Article  CAS  PubMed  Google Scholar 

  128. Li W, Germain RN, Gerner MY. Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D). Proc Natl Acad Sci. 2017;114:E7321–30. https://doi.org/10.1073/pnas.1708981114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Susaki EA, Tainaka K, Perrin D, et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 2014;157:726–39. https://doi.org/10.1016/j.cell.2014.03.042.

    Article  CAS  PubMed  Google Scholar 

  130. Tainaka K, Kubota SI, Suyama TQ, et al. Whole-body imaging with single-cell resolution by tissue decolorization. Cell. 2014;159:911–24. https://doi.org/10.1016/j.cell.2014.10.034.

    Article  CAS  PubMed  Google Scholar 

  131. Ke M-T, Fujimoto S, Imai T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci. 2013;16:1154–61. https://doi.org/10.1038/nn.3447.

    Article  CAS  PubMed  Google Scholar 

  132. Wang D, Cai C, Dong X, et al. Identification of multipotent mammary stem cells by protein C receptor expression. Nature. 2015;517:81–4. https://doi.org/10.1038/nature13851.

    Article  CAS  PubMed  Google Scholar 

  133. Wuidart A, Ousset M, Rulands S, et al. Quantitative lineage tracing strategies to resolve multipotency in tissue-specific stem cells. Genes Dev. 2016;30:1261–77. https://doi.org/10.1101/gad.280057.116.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Fu NY, Nolan E, Lindeman GJ, Visvader JE. Stem cells and the differentiation hierarchy in mammary gland development. Physiol Rev. 2020;100:489–523. https://doi.org/10.1152/physrev.00040.2018.

    Article  CAS  PubMed  Google Scholar 

  135. Watson CJ, Khaled WT. Mammary development in the embryo and adult: new insights into the journey of morphogenesis and commitment. Development. 2020;147:dev169862. https://doi.org/10.1242/dev.169862.

    Article  CAS  PubMed  Google Scholar 

  136. Huebner RJ, Lechler T, Ewald AJ. Developmental stratification of the mammary epithelium occurs through symmetry-breaking vertical divisions of apically positioned luminal cells. Development. 2014;141:1085–94. https://doi.org/10.1242/dev.103333.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Condeelis J, Segall JE. Intravital imaging of cell movement in tumours. Nat Rev Cancer. 2003;3:921–30. https://doi.org/10.1038/nrc1231.

    Article  CAS  PubMed  Google Scholar 

  138. Ellenbroek SIJ, Rheenen J van. Imaging hallmarks of cancer in living mice. Nat Rev Cancer. 2014;14:406–18. https://doi.org/10.1038/nrc3742.

    Article  CAS  PubMed  Google Scholar 

  139. Kedrin D, Gligorijevic B, Wyckoff J, et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat Meth. 2008;5:1019–21. https://doi.org/10.1038/nmeth.1269.

    Article  CAS  Google Scholar 

  140. Shan S, Sorg B, Dewhirst MW. A novel rodent mammary window of orthotopic breast cancer for intravital microscopy. Microvasc Rese. 2003;65:109–17.

    Article  Google Scholar 

  141. Sobolik T, Su YJ, Ashby W, et al. Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging. IntraVital. 2016;5:e1125562. https://doi.org/10.1080/21659087.2015.1125562.

    Article  PubMed  PubMed Central  Google Scholar 

  142. Entenberg D, Pastoriza JM, Oktay MH, et al. Time-lapsed, large-volume, high-resolution intravital imaging for tissue-wide analysis of single cell dynamics. Methods. 2017;128:65–77. https://doi.org/10.1016/j.ymeth.2017.07.019.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Ewald AJ, Werb Z, Egeblad M. Preparation of mice for long-term intravital imaging of the mammary gland. Cold Spring Harbor Protocols. 2011;2011:168–73. https://doi.org/10.1101/pdb.prot5562.

    Article  Google Scholar 

  144. Harney AS, Wang Y, Condeelis JS, Entenberg D. Extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment. J Vis Exp. 2016;112:e54042. https://doi.org/10.3791/54042.

    Article  Google Scholar 

  145. Kotsuma M, Parashurama N, Smith BR, et al. Nondestructive, serial in vivo imaging of a tissue-flap using a tissue adhesion barrier. IntraVital. 2012;1:69–76. https://doi.org/10.4161/intv.21769.

    Article  Google Scholar 

  146. Hor JL, Whitney PG, Zaid A, et al. Spatiotemporally distinct interactions with dendritic cell subsets facilitates CD4 + and CD8 + T cell activation to localized viral infection. Immunity. 2015;43:554–65. https://doi.org/10.1016/j.immuni.2015.07.020.

    Article  CAS  PubMed  Google Scholar 

  147. Qi H, Egen JG, Huang AYC, Germain RN. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells. Science. 2006;312:1672–6. https://doi.org/10.1126/science.1125703.

    Article  CAS  PubMed  Google Scholar 

  148. Harper KL, Sosa MS, Entenberg D, et al. Mechanism of early dissemination and metastasis in Her2 + mammary cancer. Nature. 2016;540:588–92. https://doi.org/10.1038/nature20609.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. Gyorki DE, Asselin-Labat ML, van Rooijen N, et al. Resident macrophages influence stem cell activity in the mammary gland. Breast Cancer Res. 2009;11:R62.

    Article  Google Scholar 

  150. Li Z, Liu S, Xu J, et al. Adult connective tissue-resident mast cells originate from late erythro-myeloid progenitors. Immunity. 2018;49:640–53.e5. https://doi.org/10.1016/j.immuni.2018.09.023.

    Article  CAS  PubMed  Google Scholar 

  151. Dawson CA, Mueller SN, Lindeman GJ, et al. Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue. Nat Protoc. 2021. https://doi.org/10.1038/s41596-020-00473-2.

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J.E.V. was supported by NHMRC Fellowships #1037230 and #1102742.

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Dawson, C.A., Visvader, J.E. The Cellular Organization of the Mammary Gland: Insights From Microscopy. J Mammary Gland Biol Neoplasia 26, 71–85 (2021). https://doi.org/10.1007/s10911-021-09483-6

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