The list of detection agents used in this study.

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.</div

MCF7-ras spheroid budding in co-cultures requires cell contractility.

(A, C) Photographs of spheroids on day 4 of dispersed co-culture with fibroblasts upon treatment with no inhibitor (mock), with blebbistatin (Bleb, A) or with Y27632 (C). Top gray and red bars indicate proportion of branched spheroids out of all spheroids per condition. Scale bar: 100 μm. (B, D) Quantification of number of branches/buds per branched spheroid in conditions from (A). The plot shows mean ± SD, each lined dot shows mean from each experiment, each faint dot shows single spheroid measurement, n = 4 (B) or 5 (D) biologically independent experiments, N = 20 spheroids per experiment. Statistical analysis: two-tailored t test. The data underlying the graphs shown in the figure can be found in S1 Data. (TIFF)</p

Fibroblasts organization around bifurcating TEB.

Z-stack scroll-through of mammary gland whole-organ imaging, showing a bifurcating TEB. DAPI in blue, vimentin in white, tdTomato in red. MIP and appropriate scale bar are depicted in Fig 6. (AVI)</p

Combination of fibroblasts and FGF2-STAB induces TEB-like phenotype of organoids.

(A) Time-lapse snap-shots of organoids grown in basal organoid medium with no exogenous growth factors (basal M), with FGF2-STAB, co-cultured with fibroblasts or co-cultured with fibroblasts with FGF2-STAB. Scale bar: 100 μm. (B) Quantification of organoid branching. The plot shows mean ± SD. n = 2 independent biological replicates, N = 20 organoids per experiment. (C) Quantification of number of branches per branched organoid. The plot shows mean ± SD. n = 2 independent biological replicates, N = 12–19 branching organoids per experiment. (D) Examples of luminized and full branch on bright-field imaging and quantification of the branch phenotypes. n = 2 independent biological replicates, N = 12–19 branching organoids per experiment. (E) Representative confocal images of organoids on day 5 of culture with FGF2-STAB or fibroblasts. Scale bar: 100 μm. (F) Quantification of maximum number of cell layers in a branch in confocal images. The plot shows mean ± SD. The dots represent averages from individual experiments. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 9–13 organoids per experiment. (G) Quantification of the percentage of organoids with KRT5+ cells present within the layers of KRT5-cells (basal-in-luminal, BIL cells) in confocal images. The plot shows mean ± SD. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 9–13 organoids per experiment. (H) A schematic representation of uncoupling fibroblast contraction and growth factor signaling in organoids. The data underlying the graphs shown in the figure can be found in S1 Data. FGF2, fibroblast growth factor 2; TEB, terminal end bud.</p

<i>Myh9</i> knock-out does not impede fibroblast motility.

(A) Detailed time-lapse snapshots of fibroblast-organoid contact establishment in dispersed co-cultures with control or Myh9-KO fibroblasts and tdTomato+ organoids. Scale bar: 50 μm. (B) Quantification of fibroblast-organoid contacts established in the first 3 days of co-culture, comparing GFP+ and GFP- fibroblasts (GFP is a marker of adenoviral transduction). The plot shows mean ± SD. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 20 organoids per experiment. The data underlying the graphs shown in the figure can be found in S1 Data. (TIFF)</p

The list of primers used for qPCR in this study.

Epithelial branching morphogenesis is an essential process in living organisms, through which organ-specific epithelial shapes are created. Interactions between epithelial cells and their stromal microenvironment instruct branching morphogenesis but remain incompletely understood. Here, we employed fibroblast-organoid or fibroblast-spheroid co-culture systems and time-lapse imaging to reveal that physical contact between fibroblasts and epithelial cells and fibroblast contractility are required to induce mammary epithelial branching. Pharmacological inhibition of ROCK or non-muscle myosin II, or fibroblast-specific knock-out of Myh9 abrogate fibroblast-induced epithelial branching. The process of fibroblast-induced branching requires epithelial proliferation and is associated with distinctive epithelial patterning of yes associated protein (YAP) activity along organoid branches, which is dependent on fibroblast contractility. Moreover, we provide evidence for the in vivo existence of contractile fibroblasts specifically surrounding terminal end buds (TEBs) of pubertal murine mammary glands, advocating for an important role of fibroblast contractility in branching in vivo. Together, we identify fibroblast contractility as a novel stromal factor driving mammary epithelial morphogenesis. Our study contributes to comprehensive understanding of overlapping but divergent employment of mechanically active fibroblasts in developmental versus tumorigenic programs.</div

<i>Myh9</i> knock-out in fibroblast decreases their morphogenetic potential.

Time-lapse video captures 4 days of epithelial morphogenesis in fibroblast (cyan)-organoid(red) co-culture with either control (Ad-GFP; left) or Myh9 knocked-out fibroblasts (adeno-Cre-mediated knock-out; Ad-Cre-GFP; right). Snapshots from the video are depicted in Fig 4. Scale bar: 100 μm. (AVI)</p

Fibroblast-induced branching of organoids does not reproduce FGF2-induced budding.

(A) Snapshots from time-lapse imaging of primary mammary organoids in basal organoid medium (basal M) without any FGF supplementation (top), basal M with FGF2 (middle), or co-cultured with primary mammary fibroblasts (fibro) in basal M with no FGF supplementation (bottom). Scale bar: 100 μm. Full videos are presented in S1 Movie. (B) Quantification of percentage of branched organoids per all organoids in the conditions from (A). The plot shows mean ± SD, each dot represents biologically independent experiment, n = 3. Statistical analysis: Two-tailored t test. (C) Quantification of branch thickness from experiments in (A). The plot shows mean ± SD, each dot represents a biologically independent experiment, n = 3. Statistical analysis: Two-tailored t test. (D) Quantification of number of branches per branched organoids in conditions from (A). The plot shows mean ± SD, each lined dot shows mean from each experiment, each faint dot shows single organoid measurement, n = 3 biologically independent experiments, N = 20 organoids per experiment. Statistical analysis: Two-tailored t test. (E) Quantification of organoid circularity in conditions from (A). The lines represent mean, the shadows and error bars represent ± SD, n = 3 biologically independent experiments, N = 20 organoids per experiment. The schemes show representative shape of indicated circularity. (F) Detailed images of branch development in co-culture with fibroblasts from (A). Scale bar: 20 μm. (G) MIP of F-actin (red), DAPI (blue), and PDGFRα (white) in organoid with exogenous FGF2 or with fibroblasts (fibro). Zoom-in area from the box is depicted as MIP and single z slices. The asterisks denote lumen. Scale bar: 100 μm. (H) A scheme depicting differences between organoid budding induced by exogenous FGF2 and organoid branching in a co-culture with fibroblasts. The data underlying the graphs shown in the figure can be found in S1 Data. FGF2, fibroblast growth factor 2; MIP, maximum intensity projection.</p

Endogenous paracrine signals are not sufficient to induce organoid branching.

(A) Representative organoids cultured with exogenous FGF2 or with fibroblasts (fibro), treated with inhibitors of FGFR (SU5402) and MEK (U0126). Scale bar: 100 μm. (B) Quantification of branched organoid per all organoids, relative to mock. The plots show mean ± SD, each dot represents biologically independent experiment, n = 3–5, N = 20 organoids per experiment. Statistical analysis: Two-tailored t test. (C) Schemes and images on day 0 and day 4 of different organoid-fibroblast co-culture set-ups. Scale bar: 100 μm. (D) Quantification of organoid branching in different co-culture set-ups. The plot shows mean ± SD, each dot represents biologically independent experiment, n = 16 independent experiments for “no fibro,” 5 for “cond. medium,” 5 for “transwell,” 12 for ”bottom,” 16 for “dispersed,” and 4 for “aggregate,” N = 20 organoids per experiment. Statistical analysis: Multiple t tests compared to control “no fibro,” or indicated by the line. (E) Dispersed and aggregated co-culture of LifeAct-GFP fibroblast (white) and tdTomato organoid (red) at the beginning of the culture. Scale bar: 100 μm. (F) A scheme of MCF7-ras spheroid co-culture setup. (G) Representative MCF7-ras spheroids cultured in basal organoid medium (basal M) or basal M with exogenous FGF2. Scale bar: 100 μm. (H) Schemes and images on day 0 and day 4 of different spheroid-fibroblast co-culture set-ups. Top gray and red bars indicate proportion of branched spheroids out of all spheroids per condition, n = 3–5 independent experiments, N = 20 spheroids per experiment. Scale bar: 100 μm. The data underlying the graphs shown in the figure can be found in S1 Data. FGF2, fibroblast growth factor 2; FGFR, FGF receptor.</p

Contractility inhibitors do not impede fibroblast motility.

(A) Representative endpoint images of organoids in dispersed co-cultures with contractility inhibitors. Scale bar: 100 μm. (B) Detailed time-lapse snapshots of fibroblast-organoid contact establishment in co-cultures with or without the inhibitors. Scale bar: 50 μm. White arrowhead indicates the fibroblast of interest. (C, D) Quantification of fibroblast-organoid contacts established in co-cultures with inhibitors (Y = 10 μM Y27632, C; Bleb = 10 μM Blebbistatin, D) within the first 2 days. The plots show mean ± SD. Statistical analysis: two-tailored t test; n = 3 independent biological replicates, N = 10 organoids per experiment. The data underlying the graphs shown in the figure can be found in S1 Data. (TIFF)</p