In the last decade, organoids became a tremendously popular technique in developmental and cancer biology for their high pathophysiological relevance to in vivo models with the advantage of easier manipulation, real-time observation, potential for high-throughput studies, and reduced ethical issues. Among other fundamental biological questions, mammary organoids have helped to reveal mechanisms of mammary epithelial morphogenesis, mammary stem cell potential, regulation of lineage specification, mechanisms of breast cancer invasion or resistance to therapy, and their regulation by stromal microenvironment. To exploit the potential of organoid technology to the fullest, together with optimal organoid culture protocols, visualization of organoid architecture and composition in high resolution in three dimensions (3D) is required. Whole-mount imaging of immunolabeled organoids enables preservation of the 3D cellular context, but conventional confocal microscopy of organoid cultures struggles with the large organoid sample size and relatively long distance from the objective to the organoid due to the 3D extracellular matrix (ECM) that surrounds the organoid. We have overcome these issues by physical separation of single organoids with their immediate stroma from the bulk ECM. Here we provide a detail protocol for the procedure, which entails single organoid collection and droplet-based staining and clearing to allow visualization of organoids in the greatest detail.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Sumbal J, Budkova Z, Traustadóttir GA, Koledova Z (2020) Mammary organoids and 3D cell cultures: old dogs with new tricks. J Mammary Gland Biol Neoplasia 25(4):273–288 DOI

Ewald AJ, Brenot A, Duong M, Chan BS, Werb Z (2008) Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev Cell 14:570–581 DOI

Neumann NM, Perrone MC, Veldhuis JH, Huebner RJ, Zhan H, Devreotes PN et al (2018) Coordination of receptor tyrosine kinase signaling and interfacial tension dynamics drives radial intercalation and tube elongation. Dev Cell 45:67–82.e6 DOI

Zhang X, Martinez D, Koledova Z, Qiao G, Streuli CH, Lu P (2014) FGF ligands of the postnatal mammary stroma regulate distinct aspects of epithelial morphogenesis. Development 141:3352–3362 DOI

Simian M, Hirai Y, Navre M, Werb Z, Lochter A, Bissell MJ (2001) The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development 128:3117–3131 DOI

Centonze A, Lin S, Tika E, Sifrim A, Fioramonti M, Malfait M et al (2020) Heterotypic cell–cell communication regulates glandular stem cell multipotency. Nature 584:608–613 DOI

Sumbal J, Chiche A, Charifou E, Koledova Z, Li H (2020) Primary mammary organoid model of lactation and involution. Front Cell Dev Biol 8:68 DOI

Alladin A, Chaible L, Garcia del Valle L, Sabine R, Loeschinger M, Wachsmuth M et al (2020) Tracking cells in epithelial acini by light sheet microscopy reveals proximity effects in breast cancer initiation. elife 9:e54066 DOI

Sirka OK, Shamir ER, Ewald AJ (2018) Myoepithelial cells are a dynamic barrier to epithelial dissemination. J Cell Biol 217:3368–3381 DOI

Georgess D, Padmanaban V, Sirka OK, Coutinho K, Choi A, Frid G et al (2020) Twist1-induced epithelial dissemination requires Prkd1 signaling. Cancer Res 80:204–218 DOI

Cheung KJ, Gabrielson E, Werb Z, Ewald AJ (2013) Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155:1639–1651 DOI

Havas KM, Milchevskaya V, Radic K, Alladin A, Kafkia E, Garcia M et al (2017) Metabolic shifts in residual breast cancer drive tumor recurrence. J Clin Invest 127(6):2091–2105 DOI

Duarte AA, Gogola E, Sachs N, Barazas M, Annunziato S, de Ruiter JR et al (2018) BRCA-deficient mouse mammary tumor organoids to study cancer-drug resistance. Nat Methods 15:134–140 DOI

Sumbal J, Koledova Z (2019) FGF signaling in mammary gland fibroblasts regulates multiple fibroblast functions and mammary epithelial morphogenesis. Development 146(23):dev185306 PubMed

Koledova Z, Zhang X, Streuli C, Clarke RB, Klein OD, Werb Z et al (2016) SPRY1 regulates mammary epithelial morphogenesis by modulating EGFR-dependent stromal paracrine signaling and ECM remodeling. Proc Natl Acad Sci U S A 113(39):E5731–E5740 DOI

Sachs N, de Ligt J, Kopper O, Gogola E, Bounova G, Weeber F et al (2018) A living biobank of breast cancer organoids captures disease heterogeneity. Cell 172:373–386.e10 DOI

Padmanaban V, Grasset EM, Neumann NM, Fraser AK, Henriet E, Matsui W et al (2020) (2020) Organotypic culture assays for murine and human primary and metastatic-site tumors. Nat Protoc 15(8):2413–2442 DOI

Nguyen-Ngoc K-V, Shamir ER, Huebner RJ, Beck JN, Cheung KJ, Ewald AJ (2015) 3D culture assays of murine mammary branching morphogenesis and epithelial invasion. Methods Mol Biol 1189:135–162 DOI

Koledova Z, Lu P (2017) A 3D fibroblast-epithelium co-culture model for understanding microenvironmental role in branching morphogenesis of the mammary gland. Methods Mol Biol 1501:217–231 DOI

Miller DH, Sokol ES, Gupta PB (2017) 3D primary culture model to study human mammary development. Methods Mol Biol 1612:139–147 DOI

Linnemann JR, Miura H, Meixner LK, Irmler M, Kloos UJ, Hirschi B et al (2015) Quantification of regenerative potential in primary human mammary epithelial cells. Development 142:3239–3251 PubMed PMC

Koledova Z (2017) 3D coculture of mammary organoids with fibrospheres: a model for studying epithelial-stromal interactions during mammary branching morphogenesis. Methods Mol Biol 1612:107–124 DOI

Dekkers JF, Alieva M, Wellens LM, Ariese HCR, Jamieson PR, Vonk AM et al (2019) High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc 14:1756–1771 DOI

Fibroblast-induced mammary epithelial branching depends on fibroblast contractility