Fibroblasts are an integral cell type of mammary gland stroma, which plays crucial roles in development, homeostasis, and tumorigenesis of mammary epithelium. Fibroblasts produce and remodel extracellular matrix proteins and secrete a plethora of paracrine signals, which instruct both epithelial and other stromal cells of the mammary gland through mechanisms, which have not been fully understood. To enable deciphering of the intricate fibroblast-epithelial interactions, we developed several 3D co-culture methods. In this chapter, we describe methods for establishment of various types of embedded 3D co-cultures of mammary fibroblasts with mammary epithelial organoids, mammary tumor organoids, or breast cancer spheroids to investigate the role of fibroblasts in mammary epithelial development, morphogenesis, and tumorigenesis. The co-culture types include dispersed, aggregated, and transwell cultures.

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

Laboratory of Genetics and Developmental Biology Institut Curie INSERM U934 CNRS UMR3215 Paris France

Laboratory of Tissue Morphogenesis and Cancer Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Sorbonne Université Collège Doctoral Paris France

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Wiseman BS, Werb Z (2002) Stromal effects on mammary gland development and breast cancer. Science 296:1046–1049. https://doi.org/10.1126/science.1067431 PubMed DOI PMC

Sumbal J, Belisova D, Koledova Z (2020) Fibroblasts: the grey eminence of mammary gland development. Semin Cell Dev Biol 114:134–142. https://doi.org/10.1016/j.semcdb.2020.10.012 PubMed DOI

Koledova Z, Zhang X, Streuli C 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:E5731–E5740. https://doi.org/10.1073/pnas.1611532113 PubMed DOI PMC

Kouros-Mehr H, Werb Z (2006) Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis. Dev Dyn 235:3404–3412. https://doi.org/10.1002/dvdy.20978 PubMed DOI PMC

Sumbal J, Koledova Z (2019) FGF signaling in mammary gland fibroblasts regulates multiple fibroblast functions and mammary epithelial morphogenesis. Development 146:dev185306. https://doi.org/10.1242/dev.185306 PubMed DOI

Zhao C, Cai S, Shin K et al (2017) Stromal Gli2 activity coordinates a niche signaling program for mammary epithelial stem cells. Science 356:eaal3485. https://doi.org/10.1126/science.aal3485 PubMed DOI

Brownfield DG, Venugopalan G, Lo A et al (2013) Patterned collagen fibers orient branching mammary epithelium through distinct signaling modules. Curr Biol 23:703–709. https://doi.org/10.1016/j.cub.2013.03.032 PubMed DOI PMC

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

Jones CE, Hammer AM, Cho Y et al (2019) Stromal PTEN regulates extracellular matrix organization in the mammary gland. Neoplasia 21:132–145. https://doi.org/10.1016/j.neo.2018.10.010 PubMed DOI

Nerger BA, Jaslove JM, Elashal HE et al (2021) Local accumulation of extracellular matrix regulates global morphogenetic patterning in the developing mammary gland. Curr Biol 31:1903–1917.e6. https://doi.org/10.1016/j.cub.2021.02.015 PubMed DOI PMC

Peuhu E, Kaukonen R, Lerche M et al (2017) SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland. EMBO J 36:165–182. https://doi.org/10.15252/embj.201694387 PubMed DOI

Houthuijzen JM, de Bruijn R, van der Burg E et al (2023) CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer. Nat Commun 14:183. https://doi.org/10.1038/s41467-023-35793-w PubMed DOI PMC

Sahai E, Astsaturov I, Cukierman E et al (2020) A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer 20:174–186. https://doi.org/10.1038/s41568-019-0238-1 PubMed DOI PMC

Calvo F, Ege N, Grande-Garcia A et al (2013) Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts. Nat Cell Biol 15:637–646. https://doi.org/10.1038/ncb2756 PubMed DOI

Avery D, Govindaraju P, Jacob M et al (2018) Extracellular matrix directs phenotypic heterogeneity of activated fibroblasts. Matrix Biol 67:90–106. https://doi.org/10.1016/j.matbio.2017.12.003 PubMed DOI

Strell C, Paulsson J, Jin S-B et al (2019) Impact of epithelial-stromal interactions on peritumoral fibroblasts in ductal carcinoma in situ. J Natl Cancer Inst 111:983–995. https://doi.org/10.1093/jnci/djy234 PubMed DOI PMC

Costa A, Kieffer Y, Scholer-Dahirel A et al (2018) Fibroblast heterogeneity and immunosuppressive environment in human breast cancer. Cancer Cell 33:463–479.e10. https://doi.org/10.1016/j.ccell.2018.01.011 PubMed DOI

Bartoschek M, Oskolkov N, Bocci M et al (2018) Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing. Nat Commun 9:1–13. https://doi.org/10.1038/s41467-018-07582-3 DOI

Li CM-C, Shapiro H, Tsiobikas C et al (2020) Aging-associated alterations in mammary epithelia and stroma revealed by single-cell RNA sequencing. Cell Rep 33:108566. https://doi.org/10.1016/j.celrep.2020.108566 PubMed DOI PMC

Kanaya N, Chang G, Wu X et al (2019) Single-cell RNA-sequencing analysis of estrogen- and endocrine-disrupting chemical-induced reorganization of mouse mammary gland. Commun Biol 2:406. https://doi.org/10.1038/s42003-019-0618-9 PubMed DOI PMC

Yoshitake R, Chang G, Saeki K et al (2022) Single-cell transcriptomics identifies heterogeneity of mouse mammary gland fibroblasts with distinct functions, estrogen responses, differentiation processes, and crosstalks with epithelium. Front Cell Dev Biol 10:850568. https://doi.org/10.3389/fcell.2022.850568 PubMed DOI PMC

Koledova Z (2017) 3D Coculture of mammary organoids with fibrospheres: a model for studying epithelial-stromal interactions during mammary branching morphogenesis. In: Koledova Z (ed) 3D cell culture: methods and protocols, Methods in molecular biology, vol 1612. Springer, New York, pp 107–124. https://doi.org/10.1007/978-1-4939-7021-6_8 DOI

Koledova Z, Lu P (2017) A 3D fibroblast-epithelium co-culture model for understanding micro-environmental role in branching morphogenesis of the mammary gland. In: Martin F, Stein T, Howlin J (eds) Mammary gland development: methods and protocols, Methods in molecular biology, vol 1501. Springer, New York, pp 217–231. https://doi.org/10.1007/978-1-4939-6475-8_10 DOI

Sumbal J, Fre S, Sumbalova Koledova Z (2024) Fibroblast-induced mammary epithelial branching depends on fibroblast contractility. PLoS Biol 22(1): e3002093. https://doi.org/10.1371/journal.pbio.3002093

Kasid A, Lippman ME, Papageorge AG et al (1985) Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity. Science 228:725–728. https://doi.org/10.1126/science.4039465 PubMed DOI

Sumbal J, Koledova Z (2022) Single organoids droplet-based staining method for high-end 3D imaging of mammary organoids. In: del Mar Vivanco M (ed) Mammary stem cells: methods and protocols, Methods in molecular biology, vol 2471. Springer, New York, pp 259–269. https://doi.org/10.1007/978-1-0716-2193-6_14 DOI

Joshi PA, Waterhouse PD, Kasaian K et al (2019) PDGFRα + stromal adipocyte progenitors transition into epithelial cells during lobulo-alveologenesis in the murine mammary gland. Nat Commun 10:1760. https://doi.org/10.1038/s41467-019-09748-z PubMed DOI PMC

Guo Q, Minnier J, Burchard J et al (2017) Physiologically activated mammary fibroblasts promote postpartum mammary cancer. JCI Insight 2:e89206. https://doi.org/10.1172/jci.insight.89206 PubMed DOI PMC

Hamilton TG, Klinghoffer RA, Corrin PD et al (2003) Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms. Mol Cell Biol 23:4013–4025. https://doi.org/10.1128/MCB.23.11.4013-4025.2003 PubMed DOI PMC