Fig 1: Jagged-1 mediates signal from ECM-sensing basal cells to drive luminal cell differentiation.a JAG1 expression in basal cells from control, DDR1i, and DDR1i Rel tissues. P(DDR1iR vs Ctrl) < 2.5e-14, p(DDR1i vs Ctrl) < 7e-20, p(DDR1iR vs DDR1i) = 0.38. The likelihood ratio test (FDR) was used, not corrected for multiple testing. b JAG1 expression among epithelial clusters. c Immunofluorescent staining of Jagged-1 (green) and E-cadherin (red) in 3D-cultured patient-derived tissues. Results are representative of three independent repeats of this experiment. d, e Flow cytometry analysis of primary tissue in 3D hydrogels treated with DDR1i for 12 days. Basal cells were identified based on EpCAM and CD49f expression and further analyzed for Jagged-1 expression (d). The percentage of Jagged-1+ cells (e) was compared between control and treated groups. Data were derived from n = 3 independent patient samples. P-value = 0.035, two-sided Student’s t-test. * Indicates P = 0.05. Data are presented as mean values ± SD. f–g Development of MCF10A cells (WT, overexpressing LacZ or overexpressing JAG1) in 3D collagen with or without DDR1i. Representative images shown in (f), diameter of organoids shown in (g). Organoid diameter was measured for each treatment group. n = 206 and n = 145 organoids were measured in the WT group cultured under active or inactive DDR1, respectively. n = 166 and n = 92 organoids were measured in the LacZ group cultured under active or inactive DDR1, respectively. n = 183 and n = 92 organoids were measured in the JAG1 group cultured under active or inactive DDR1, respectively. Letters over graph bars indicate statistically different groups, P = 3 × 10-13 (One-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean values ± SD. h Quantification of secondary organoid development by cells isolated from structures formed by WT, LacZ-overexpressing, or JAG1-overexpressing MCF10A cells with or without DDR1i treatment. Data were derived from n = 10 fields analyzed for each treatment group. Letters over graph bars indicate statistically different groups, P = 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean values ± SD. i Immunofluorescent staining of Jagged-1 (green), Notch1 (red) and Hoechst (gray) in 3D-cultured patient-derived tissues. (I) 3D reconstruction of a Z-stack captured by confocal microscopy. (II) a single 2D plane across lobule framed by rectangle in (I). (III) high-power (×60) image of lobule framed by rectangle in (I) and (II). (IV) Enlargement of area framed by rectangle in (III). j Model for the role of DDR1, Jagged-1, and Notch1 within the mammary differentiation hierarchy (left) and visual depiction of how tissue regeneration and morphogenesis is controlled by these factors through spatially regulated cell fate decisions (right). Scale bars = 50 µm. * Indicates p < 0.05; ** indicates p < 0.01. Source data are provided as a Source data file.
Fig 2: scRNA-seq of primary tissues shows cell-state changes mediated by DDR1.a scRNA-seq data from all cells (n = 1467) from patient-derived hydrogel-grown tissues projected onto two dimensions using t-SNE on the top eight principal components across 7193 variable genes. b Bar chart of an embryonic stem cell gene set enrichment in each of the epithelial clusters. c, d Volcano plot visualizing differential gene expression between clusters 0 and 1 (c) and between clusters 4 and 6 (d). Cell cycle genes are highlighted in green. Ki67 (MKI67, www.ncbi.nlm.nih.gov/gene/4288) is labeled individually. The likelihood ratio test was used, p-values not corrected for multiple tests. e Inferred lineage relationships of all cells (black) were projected onto two dimensions as basal and luminal differentiation trajectories, using Monocle. Cluster 0 (red; left panel) and cluster 1 (yellow; right panel) cells are highlighted. f–g Stacked bar charts indicating the distribution of basal (f) and luminal (g) clusters in control, DDR1i, and DDR1i Rel tissues. h Heatmap of enriched gene sets using the Broad Institute’s MAigDB dataset with the hypergeometric test (FDRs were calculated) on DDR1i and DDR1i Rel tissues. i Cells treated with DDR1i, released from DDR1i and untreated controls projected onto basal and luminal differentiation trajectories, using Monocle. j Proposed model for the role of DDR1 in mammary epithelial cell differentiation. Source data are provided as a Source data file.
Fig 3: CRISPR screen in 3D mammary epithelial cell culture for regulators of stem cell self-renewal.a Development of primary breast cells (single MECs), primary organoids (Organoid MECs), and MCF10A cells in 3D hydrogels. Phalloidin-stained Type I and II lobules that developed from organoids are shown. Results are representative of at least 3 independent experiments. Scale bars = 100 µm (Single MECs and MCF10A), 200 µm (Phalloidin stained lobules) or 500 µm (Organoid MECs). b Schematic of pooled CRISPR screening strategy. c Phalloidin-stained secondary organoids from MCF10A cells with or without sgRNAs (top), and quantification of organoids (bottom). P = 0.0007 (two-sided Student’s t-test). Data were derived from n = 4 independently analyzed gels. Scale bar = 100 µm. d Screened kinases scored by significance relative to a null distribution using RIGER (y-axis) and by comparing the mean differential abundance of sgRNAs targeting the kinase (x-axis). Adjacent histograms indicate p-value distribution (right y-axis) and mean differential (top x-axis). RIGER’s p-values are adjusted for multiple tests. Significant genes previously implicated in cellular differentiation indicated in green. e Western blot for total DDR1 in parental MCF10A cells or after knocking out DDR1 with two independent sgRNAs. f Phalloidin-stained secondary organoids from parental or DDR1 knocked-out MCF10A cells. Scale bar = 100 µm. g Quantification of organoids from (f). P(Ctrl vs. sg1) = 2.28 × 10-6; P(Ctrl vs. sg2) = 7.56 × 10-7 (ordinary one-way ANOVA with Dunnett’s multiple comparisons test). Data were derived from n = 4 independently analyzed gels for each treatment group with n = 3 fields sampled per gel. h Quantification of secondary organoid development with or without DDR1 inhibition. P = 8.2 × 10-5 (two-sided Student’s t-test). Data were derived from n = 4 independently analyzed gels. For all bar graphs in this figure, data are presented as mean values ± SD. *** indicates P = 0.001; **** indicates p = 0.0001. Source data are provided as a Source data file.
Fig 4: DDR1 is required for breast tissue regeneration.a Percentage of patient-derived single primary breast epithelial cells able to give rise to a branched stricture in 3D hydrogel. Data for patients 1–3 was derived from n = 7 gels each, and n = 8 gels for patient 4. Data are presented as mean values ± SD. b Development of single primary breast cells in hydrogels with or without DDR1i treatment. Inset in D18 top panel depicts image directly below (DDR1i, day 18) scaled to match top image (Ctrl, day 18). Results are representative of two independent repeats of this experiment. Scale bar = 100 µm. c Development of breast tissue organoids in hydrogels with or without DDR1i treatment (left) and quantification of lobules (right). Red arrowheads indicate lobules, blue arrows indicate ducts that terminated without a lobule. P = 0.0167 (two-sided Student’s t-test). Data were derived from n = 11 and n = 14 gels for the Ctrl and DDR1i-treated groups, respectively. Data are presented as mean values ± SEM. Scale bars = 100 µm. d Schematic of DDR1-inhibition and withdrawal experiments (left) and representative bright-field images after withdrawing DDR1i (right). Scale bars = 100 µm. e Schematic of stem/progenitor assay in primary breast organoids with or without DDR1i treatment. Representative image of vehicle-treated development after 14 days is shown below. Scale bar = 100 µm. f Clone formation by cells isolated from primary breast organoids with or without DDR1i treatment, and 2 days following release from DDR1 inhibition. Data were derived from n = 12 gels for each treatment group. P(Ctrl vs. DDR1i) = 0.0003; P(Ctrl vs. DDR1i-Rel) = 0.02 (ordinary one-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean values ± SD. g Percentage of clones composed exclusively of cells stained with either of the lineage markers CK18 and p63, or mixed. Data was derived from n = 12 gels for each treatment group. Data are presented as mean values ± SD. h Mammosphere formation by cells isolated from primary breast organoids cultured in hydrogel with or without DDR1i treatment. P = 0.0006 (two-sided Student’s t-test). Data were derived from n = 24 gels for each treatment group. Data are presented as mean values ± SD. i Mammosphere formation by primary breast cells cultured in suspension culture with or without DDR1i treatment. P = 8.8 × 10-5, 0.001 and 3 × 10-6 comparing Ctrl and DDR1i groups, for patients A, B, and C, respectively (ordinary two-way ANOVA with Sidak’s multiple comparisons test). Data were derived from n = 3 gels per patient for each treatment group. Data are presented as mean values ± SD. j Flow-cytometry dot-plot analysis of EpCAM and CD49f expression on cells from primary breast organoids grown in hydrogels for 14 days without or with DDR1i treatment, or released from DDR1 inhibition for the last 2 days. k Graph depicts cell population percentages under each condition. Data were derived from n = 2, n = 4, and n = 3 gels for the Ctrl, DDR1i and DDR1i-Rel groups, respectively. Data are presented as mean values ± SD. * Indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001. Source data are provided as a Source data file.
Fig 5: DDR1 signaling activates Notch1 to drive luminal differentiation.a Violin plot showing the distribution of DDR1 expression in epithelial clusters. b Immunofluorescent staining of DDR1 (green), E-cadherin (red) and Hoechst nuclear staining (blue) in patient-derived hydrogel-grown tissues. Arrows: basal expression of DDR1. Arrowheads: luminal expression of DDR1. Right panels depict enlargement of region in white rectangle. Results are representative of three independent repeats of this experiment. c Flow cytometry analysis of primary breast tissue cultured in 3D hydrogels for 12 days. Basal and luminal cells were identified based on EpCAM and CD49f expression and further analyzed for DDR1 expression. d The percentage of DDR1+ cells was compared between the basal and luminal populations. P = 0.03 (two-sided Student’s t-test). e DDR1 signal intensity was compared between the basal and luminal populations. P = 0.0008 (two-tailed Student’s t-test). Data were derived from n = 3 independent patient samples. Data are presented as mean values ± SD. f GSEA plots depicting enrichment of a Notch1 target gene set among genes that are overexpressed in cells from DDR1i Rel tissues compared to DDR1i tissues. Plots depict enrichment across all clusters, luminal clusters only and basal clusters only. Normalized enrichment score (NES) and false discovery rate q-value (FDR-q) are indicated. g Western blot for phospho-DDR1, total-DDR1, and cleaved Notch1 (ICN1) under the indicated conditions. ß-tubulin was used as a loading control. h Graph depicts quantification of n = 3 biological repeats of western blot seen in (g), comparing DDR1i treated samples with untreated controls in the presence of collagen. P(ICN1 vs. Ctrl) = 0.0373, P(pDDR1 vs. Ctrl) = 0.0012 (two-sided Student’s t-tests). Data are presented as mean values ± SD. i Western blot for cleaved Notch1 (ICN1) and Jagged-1 in separately obtained MCF10A cell lines cultured in 3D collagen in the presence or absence of DDR1i. ß-tubulin was used as a loading control. j Graph depicts quantification of n = 3 biological repeats seen in (i), comparing DDR1i treated samples with untreated controls. P(Ctrl vs. ICN1) = 0.039, P(Ctrl vs. JAG1) = 0.028. (two-sided Student’s t-test)* indicates P < 0.05. ** indicates P < 0.01. *** indicates P < 0.001. Data are presented as mean values ± SD. * indicates P < 0.05. Source data are provided as a Source data file.
Supplier Page from Abcam for Anti-MCK10/NEP antibody [51D6] (PE)