Identification of DDR1 as a regulator of tissue regeneration
When seeded as single cells in 3D matrix hydrogels, human breast epithelial cells from patient reduction mammoplasty specimens can regenerate an entire functional ductal-lobule unit7 (Fig. 1a). This occurs in a stepwise manner whereby single stem cells proliferate to form small spheroids before undergoing multilineage differentiation into organoids that initiate branching on day 10–13. Ducts then undergo budding and lobule formation by day 18 and continue to mature for an additional 10 days7 (Fig. 1a). When clusters of cells from partially-dissociated human breast tissue (organoids) are seeded in hydrogels, they regenerate complex ductal and lobular structures that closely resemble the functional TDLU present in the human breast7 (Fig. 1a). The kinetics of organoid regeneration is accelerated compared to single MECs, with initiation of branching typically occurring within the first 4 days and lobule maturation around days 10–127 (Fig. 1a). Similarly, single cells from the human breast MCF10A cell line can give rise to complex ductal, lobular, and ductal-lobular tissues in a stepwise manner. Cells first proliferate to form small spheroids that, through branching and budding, form complex ductal/lobule structures by day 14–158 (Fig. 1a). Importantly, when cultured in 3D collagen (in contrast to laminin-rich matrices such as Matrigel), MCF10A cells undergo multi-lineage differentiation toward the basal and luminal lineages8 (Supplementary Fig. 1a). Although generally developing similarly, MCF10A cells—unlike primary MECs—lose self-renewal activity during differentiation and maturation, and, consequently, are unable to regenerate secondary tissues. Inhibiting differentiation of MCF10A cells leads them to retain their self-renewal activity and the ability to reseed and regenerate secondary tissues8. Using this trait, we endeavored to identify potential regulators of tissue regeneration by screening for kinases that, when inhibited, would block differentiation. Kinases were selected due to their regulatory roles in cell differentiation, their interaction and cross-relationships with other signaling pathways implicated in cell fate decisions and importantly the existence of specific kinase inhibitors, enhancing the potential of findings being clinically relevant to breast cancer.
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.
A custom pooled CRISPR sgRNA library containing 5070 guides targeting 507 human kinases was generated for this purpose9. The experimental design is depicted in Fig. 1b. We transduced MCF10A cells with the pooled sgRNA library and seeded infected cells or uninfected control cells in 3D hydrogels. Both sgRNA library-infected and control cells formed primary organoids, which were then dissociated, re-seeded, and allowed to form secondary organoids (Fig. 1b). The number of secondary organoids, indicative of self-renewal capability, increased dramatically in sgRNA library-infected cells, compared to uninfected control cells (Fig. 1c).
To identify kinases that inhibited self-renewal, we sequenced the transduced sgRNAs from the secondary organoids. An enrichment score for each kinase was calculated by comparing the sgRNAs reads in the secondary organoids to that of sgRNAs reads in the original transduced cells grown in 2D. 32 kinases were enriched in secondary organoids relative to pre-screened cells (Supplementary Table 1). We noted that several kinases (FYN10 www.ncbi.nlm.nih.gov/gene/2534, MAPK711 www.ncbi.nlm.nih.gov/gene/5598, and MAP3K712 www.ncbi.nlm.nih.gov/gene/6885) have been implicated previously in cellular differentiation, validating the robustness of the approach.
The most significantly enriched kinase gene identified in the screen was DDR1 (www.ncbi.nlm.nih.gov/gene/780, Fig. 1d), encoding a collagen receptor. To validate the ability of DDR1 loss to enable self-renewal, MCF10A cells were transduced with two additional independent sgRNAs against DDR1 (Fig. 1e). Consistent with the screen results, cells lacking DDR1 expression showed a significant increase in secondary organoid formation in 3D, indicative of retaining self-renewal (Fig. 1f, g). Furthermore, we used an independent small-molecule chemical approach to inhibit DDR1 kinase activity by blocking its autophosphorylation (DDR1i13). MCF10A cells treated with DDR1i exhibited the same phenotype of increased secondary organoid formation as those transduced with sgRNAs against DDR1 (Fig. 1h).
To find out if the increase in secondary organoid formation was a result of increased proliferation induced by DDR1i treatment, we analyzed the effects of DDR1i on MCF10A cells cultured in 3D collagen gels. The results showed visible and measurable reduction in cell numbers following DDR1i (Supplementary Fig. 1b), confirmed by reduced dye dilution analysis of cell proliferation (Supplementary Fig. 1c). Cell cycle analysis indicated that DDR1i resulted in detectable increase in S and G2/M phase (Supplementary Fig. 1d). Given that this increase did not result in increase in cell numbers, we analyzed the effect of DDR1i on apoptosis using Annexin V assay and found no detectable effect (Supplementary Fig. 1e). Together, the results of these analyses indicate that increased proliferation is not a contributing factor to the increase in secondary structure formation. Thus, the increase in secondary organoid formation upon loss of DDR1 activity was confirmed both genetically and chemically, indicating that DDR1 is required for MCF10A stem cell differentiation.
To extend these findings to primary cells, primary MECs isolated from reduction mammoplasty patient tissue were seeded as single cells or as cell clusters (organoids) into 3D hydrogels and treated with DDR1i. The efficiency of branched structure formation from single cells seeded in hydrogels varies between donor samples and is around a frequency of ~1% of the total number of cells seeded (Fig. 2a). Based on this frequency, when seeding 4 gels with 1500 cells/gel, ~60 structures are expected to develop in total. However, when DDR1i was added to the media, no branched structures were identified in any of the gels seeded. Instead, single MECs formed small spheroids but failed to undergo multilineage differentiation and failed to initiate ductal elongation and lobule formation (Fig. 2b). When seeded as organoids in hydrogels in the presence of DDR1i, simple ducts formed but failed to initiate budding or form lobules, and TDLUs did not regenerate (Fig. 2c). To determine whether the block in TDLU regeneration could be reversed, organoids were cultured for 21 days: the first 12 days in the presence of DDR1i, and the last 9 days in the absence of DDR1i. Indeed, release from DDR1 inhibition led to full TDLUs formation (Fig. 2d) suggesting that DDR1 inhibition does not lead to an irreversible block in tissue regeneration. Together, these findings suggest that DDR1 activity is important for ductal elongation during early breast tissue regeneration, and for TDLU formation from the elongated ducts.
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.
DDR1 is required for bi-lineage differentiation
The block in tissue regeneration by inhibiting DDR1 could be due to defects in stem/progenitor cells and/or defects in differentiation. To assess this, primary tissues were cultured in 3D hydrogels in the presence or absence of DDR1i for 14 days or released from DDR1 inhibition for the last 2 days in culture (days 12–14, DDR1i-Rel). Cells were dissociated from the hydrogels and MECs were examined for stem/progenitor cell activity by colony-forming unit (CFU) assay and mammosphere assay (Fig. 2e–h). The CFU assay calculates the number of stem and progenitor cells by quantifying the number of unipotent (luminal or basal only) or bi-potent (mixed luminal and basal) colonies that form after seeding at low density. DDR1 inhibition led to >2-fold increase in the proportion of colony-forming cells (P ≤ 0.001, Fig. 2f). Immuno-staining of the clones that formed for the lineage markers CK18 and p63 revealed no significant changes in clone lineages between the control and DDR1i-treated groups. An increased percentage of luminal clones (CK18+) was noted two days following release from DDR1 inhibition, compared with control (P = 0.002, Fig. 2g). The mammosphere assay estimates the number of stem cells based on their ability to form colonies under non-adherent conditions (in suspension). Cells isolated from hydrogel structures that were under DDR1i treatment formed significantly more mammospheres compared with cells from untreated control hydrogels (3.6-fold, P = 0.0005, Fig. 2h). DDR1i treatment of primary human breast cells seeded directly in suspension culture (never cultured in hydrogels) resulted in dramatically diminished mammosphere formation compared with untreated controls, supporting inhibition of stem cell proliferation/differentiation (Fig. 2i). Together, these results indicate that the failure to form TDLUs was not due to a depletion in stem or progenitor numbers, but rather due to inhibition of their function.
The percentages of basal cells (CD49f+/EpCAM−/low) and luminal cells (CD49f−/low/EpCAM+) from 3D tissues grown in hydrogels in the presence or absence of DDR1i, or released from DDR1i for the last 2 days in culture (DDR1i-Rel), were analyzed by flow cytometry (Fig. 2j). Despite a trend toward increased percentage of luminal cells, DDR1i treatment did not result in statistically significant differences in the luminal/basal lineage ratio, similarly to the findings from the CFU assay. These results indicate that DDR1 may have a minor effect on the luminal/basal ratio of cells but does not significantly change the lineage balance.
To better understand the role of DDR1 in differentiation, we performed scRNA-seq using Seq-Well14 to generate a comparative cellular atlas at single-cell resolution on human breast tissues grown for 14 days in 3D hydrogels under three different conditions: 1) vehicle-treated control, 2) DDR1-inhibited (DDR1i), and 3) DDR1-inhibited for 12 days followed by release from inhibition for 2 days (DDR1i Rel). We first examined the molecular heterogeneity in 14-day human breast tissue outgrowths. Unsupervised analysis of 1467 cells, with greater than 500 genes detected per cell, revealed nine distinct clusters corresponding to different cell states in 3D human breast tissues (Fig. 3a).
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.
Using unbiased clustering analysis, spatial reconstruction of single cell data, and integrated analysis across all three conditions, we classified cells into 8 epithelial clusters based on a unique group of expressed genes (Fig. 3a). Table 1 summarizes the evidence for cell-state classification of each epithelial cluster. Analysis using genes encoding epithelial lineage-specific cytokeratins (Luminal: KRT8 www.ncbi.nlm.nih.gov/gene/3856, KRT18 www.ncbi.nlm.nih.gov/gene/3875, KRT19 www.ncbi.nlm.nih.gov/gene/3880; Basal: KRT14 www.ncbi.nlm.nih.gov/gene/3861, KRT5 www.ncbi.nlm.nih.gov/gene/3852) revealed six of the clusters were of the basal lineage (clusters 0, 1, 2, 3, 5, 7) while the remaining two clusters were of the luminal linage (clusters 4 and 6; Supplementary Fig. 2a, b). Gene set enrichment analysis (GSEA) for an embryonic stem cell geneset15 further defined clusters 0 and 1 as undifferentiated stem/progenitor cells within the basal lineage and cluster 6 as undifferentiated stem/progenitor cells within the luminal lineage (Fig. 3b). Analysis of differential expression of a cell cycle geneset16 between clusters 0 and 1 and clusters 4 and 6 revealed that clusters 1 and 6 displayed high scores for proliferation signature, indicating that these clusters were active progenitor cells (Fig. 3c, d). The remaining clusters (basal clusters 2, 3, 5, 7, and luminal cluster 4) consisted of more mature lineage-restricted differentiated cells. Luminal cluster 4 expressed high levels of LTF (encoding the protein Lactotransferrin, www.ncbi.nlm.nih.gov/gene/4057), consistent with mature lobular luminal cells (Supplementary Fig. 2c).
To understand the lineage relationships between cell clusters, we constructed a pseudotemporally ordered tree using Monocle. This analysis revealed that many cluster 0 cells were undifferentiated or along the basal differentiation trajectory with a minority along the luminal differentiation trajectory. Cluster 1 cells were farther along the basal differentiation trajectory and only a minority were undifferentiated. (Fig. 3e). Likewise, cluster 6 was restricted to the luminal lineage, giving rise to cluster 4, which scored further along the luminal differentiation dimension (Supplementary Fig. 2d).
Together, these results show that human breast tissues that grow in 3D hydrogels retain a cellular hierarchy including stem cells, lineage-restricted luminal and basal progenitors, and mature luminal and basal cells.
Having assessed the epithelial hierarchy and their linear relationship to differentiated cells in normal human breast tissue, we next asked how cell states were affected by DDR1 activity. Inhibition of DDR1 activity blocked the differentiation of stem cells, as evidenced by a dramatic increase in the proportion of bipotent stem cells, with a concomitant decrease in the number of mature basal cells (Fig. 3f). In addition, inhibition of DDR1 activity resulted in a significant increase in the fraction of proliferative luminal progenitors, indicating that in the absence of DDR1, luminal cells were trapped in a proliferative progenitor state but failed to differentiate into mature luminal cells (Fig. 3g). Upon release from DDR1 inhibition for the final two days of culture, expression levels of genes associated with mammary epithelial differentiation and early development were enriched (Fig. 3h). In addition, we observed a trend toward an increase in mature luminal cells with concomitant decrease in luminal progenitors, as well as a decrease in stem cell number concomitant with increase in the fraction of basal progenitors (Fig. 3f, g).
We analyzed the effect of DDR1i and release on the position of cells along the pseudotemporal trajectories of luminal and basal differentiation. DDR1 inhibition resulted in a block of cell progression along both trajectories, and accumulation of undifferentiated cells. Release of DDR1 inhibition released this block and allowed progression of cells toward both the luminal and basal differentiation axes (Fig. 3i).
Collectively, these results indicate that DDR1 is required for the differentiation of basal progenitors from bi-potent stem cells, as well as the differentiation of mature luminal cells from luminal progenitor cells (Fig. 3j).
DDR1 promotes luminal differentiation by activating Notch1
Recognizing that DDR1 activity promoted differentiation in more than one mammary lineage, we aimed to identify how DDR1 exerts its effect on different cell types. First, we looked to characterize DDR1 expression in the different cell types. Using scRNA-seq gene expression analysis and confocal imaging of immunofluorescence-stained 3D structures in hydrogel, we found that DDR1 is expressed in both luminal and basal cells (Fig. 4a, b). In luminal cells, DDR1 is localized to cell surfaces in contact with adjacent luminal cells, but not to basolateral surfaces in contact with basal cells (Fig. 4b, arrows). In contrast, in basal cells DDR1 is localized to the basolateral surfaces in contact with the ECM, but not on the apical surfaces in contact with luminal cells (Fig. 4b, arrowheads). Flow cytometry analysis of DDR1 expression in luminal and basal cells, as identified based on CD49f and EpCAM expression, showed that an average of 7% of basal cells are DDR1+ compared with only 2.5% of luminal cells (P < 0.05, Fig. 4c, d). Additionally, the signal intensity of DDR1 was significantly higher in basal compared with luminal cells (P < 0.001, Fig. 4e). DDR1 on basal cells can be activated by matrix collagen, but DDR1 sequestered to E-cadherin expressing cell–cell interfaces on luminal cells cannot17, raising the possibility that basally-expressed DDR1 acts indirectly on luminal cells to drive luminal cell maturation and lobular growth.
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. β-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. β-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.
As Notch signaling is required for luminal cell and lobular differentiation18,19,20 and DDR1 signaling has been shown in other contexts to activate Notch signaling21, we asked whether DDR1 activation results in upregulation of Notch signaling. GSEA analysis revealed that Notch1 target genes22 are significantly enriched among genes that are upregulated following release from DDR1 inhibition (DDR1i Rel tissues; Fig. 4f). Moreover, the enrichment of Notch1 target genes was higher in luminal cells compared with basal cells, making the luminal lineage the main contributor to the overall enrichment of Notch1 target genes (Fig. 4f). Expression of the Notch1 target genes MAP3K8 (www.ncbi.nlm.nih.gov/gene/1326), KRT23 (www.ncbi.nlm.nih.gov/gene/25984) and KLF6 (www.ncbi.nlm.nih.gov/gene/1316) was mapped to the clusters identified by the scRNA-seq, and was highly expressed in the mature luminal cluster 4, further supporting for the activation of Notch1 target genes in the luminal lineage (Supplementary Figs. 3a–c). Together, these findings suggest that DDR1 stimulation at the matrix-cell interface triggers Notch1 signaling in adjacent luminal cells.
To directly test whether DDR1 activation by collagen leads to Notch1 activation, we stimulated MCF10A cells in 2D culture with collagen and assessed DDR1 and Notch1 activity. DDR1 activation and phosphorylation was induced upon collagen stimulation as was activation of Notch1 cleavage (Fig. 4g). Densitometry quantification showed that when stimulated with collagen, DDR1 inhibition leads to significant reduction in Notch1 activation, as evidenced by decreased levels of cleaved Notch1 (Fig. 4h). Similarly, collagen failed to induce Notch1 cleavage in cells lacking DDR1 (DDR1KO, Fig. 4g). In addition, we analyzed the effect of DDR1 inhibition on Notch1 signaling in a 3D context (collagen gels) revealing reduced expression of both cleaved Notch1 (ICN1) and the Notch1 ligand Jagged-1 following DDR1 inhibition (Fig. 4i, j). Together, these results demonstrate that DDR1 activation by collagen activates Notch1 signaling.
Next, we examined whether the failure in TDLU formation upon DDR1 inhibition was due to the lack of Notch activation. We treated organoids seeded in 3D hydrogels with a γ-secretase inhibitor (GSI), which prevents Notch cleavage and nuclear translocation23. Indeed, the presence of GSI led to a failure of TDLU formation, phenocopying the defect due to inhibition of DDR1 (Supplementary Fig. 3d). Taken together, these findings indicate that breast tissue regeneration in 3D culture involves collagen stimulation of DDR1 in basal cells, leading to activation of Notch1 signaling in adjacent luminal cells, driving luminal differentiation and TDLU formation.
Jagged-1 mediates ECM-basal cell signals driving luminal differentiation
We next inquired how DDR1 stimulation on basal cells leads to Notch1 activation on luminal cells. Notch signaling is a conserved pathway that regulates cell fate decisions and is initiated by binding of a transmembrane ligand (Jagged (JAG) or Delta-like (DLL)) expressed on one cell to a Notch receptor expressed on an adjacent cell. We examined the expression of the Notch1 ligands JAG1 (www.ncbi.nlm.nih.gov/gene/182), JAG2 (www.ncbi.nlm.nih.gov/gene/3714), DLL1 (www.ncbi.nlm.nih.gov/gene/28514), DLL3 (www.ncbi.nlm.nih.gov/gene/10683) and DLL4 (www.ncbi.nlm.nih.gov/gene/54567) in basal cells following DDR1 inhibition and release and found that JAG1 expression is downregulated following DDR1 inhibition and upregulated following release (Fig. 5a). The other Notch1 ligands were minimally expressed (JAG2, DLL1, DLL3, and DLL4), and their expression did not change upon DDR1 inhibition and release (Supplementary Fig. 4). scRNA-seq revealed that JAG1 is expressed exclusively in basal clusters, including basal progenitors and mature basal cells, but not in MaSC (Fig. 5b). As expected, based on previous findings in-vivo24, immunofluorescent staining confirmed the basal location of Jagged-1 in primary breast tissue cultured in 3D hydrogels (Fig. 5c). To confirm the effect of DDR1 inhibition on Jagged-1 expression in basal cells, we treated primary breast tissue in 3D cultures with DDR1 inhibitor for 12 days and used flow cytometry to analyze Jagged-1 expression levels in the basal population, as defined by EpCAM and CD49f expression. The results confirmed the scRNA-seq finding that DDR1 inhibition leads to downregulation of Jagged-1 in the basal population (Fig. 5d, e).
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.
The finding that DDR1 regulates Jagged-1 expression in basal cells, while also regulating Notch1 signaling in adjacent luminal cells, suggested the possibility that Jagged-1 acts to transduce DDR1 signaling from the basal to the luminal layer, driving the differentiation of luminal progenitors to mature luminal cells via the Notch1 signaling pathway.
To establish whether Jagged-1 mediates the DDR1-induced differentiation signal, we tested whether JAG1 overexpression could rescue the block in differentiation induced by DDR1 inhibition. We created an MCF10A cell line that stably overexpresses JAG1 (MCF10A/JAG1) as well as a control cell line (MCF10A/LacZ, www.ncbi.nlm.nih.gov/gene/945006) (Supplementary Fig. 5a). 3D culture in collagen of the MCF10A/JAG1 in the presence or absence of DDR1i revealed that DDR1 inhibition resulted in downregulation of Jagged-1, which nevertheless remained 5-fold higher compared with DDR1i-treated MCF10A/LacZ control cells (Supplementary Figs. 5b, c). Wild-type (WT) MCF10A, MCF10A/LacZ, and MCF10A/JAG1 were cultured in collagen gels in the presence or absence of DDR1i and allowed to form 3D organoids. We noted that DDR1 inhibition resulted in significantly smaller organoids in WT MCF10A and MCF10A/LacZ, but not in MCF10A/JAG1, indicating that Jagged-1 overexpression enables normal organoid growth in the absence of active DDR1 (Fig. 5f, g). Despite their smaller size, DDR1i-treated organoids formed by WT MCF10A and MCF10A/LacZ, but not MCF10A/JAG1, had a higher percentage of DNA-replicating cells, as evidenced by EdU incorporation, compared with untreated organoids (Supplementary Fig. 5d, f). This demonstrates that active DDR1 enables cell differentiation, resulting in larger organoids comprised mainly of terminally differentiated, non-cycling cells. DDR1 inhibition arrested cell differentiation, resulting in smaller organoids with a higher percentage of DNA-replicating progenitor cells, an effect that was eliminated by JAG1 overexpression.
Finally, to assess their self-renewal potential, the organoids were dissociated into single cells and re-seeded in a second set of collagen gels. The second set of gels was not treated with DDR1i. As expected, DDR1i-treated WT MCF10A and MCF10A/LacZ cells formed significantly more secondary organoids compared with untreated controls, while MCF10A/JAG1 cells formed very few organoids regardless of DDR1i treatment (Fig. 5h), confirming that JAG1 overexpression rescued the DDR1i-induced differentiation arrest.
We were able to localize the region of potential Jagged-1-Notch1 interaction in 3D tissues by immunofluorescence staining. 3D reconstruction of regenerating TDLUs revealed the interface of Jagged-1-expressing basal cells and Notch1 expressing luminal cells, constituting the region in which the DDR1-Jagged-1-Notch1 signaling axis can take place to drive differentiation (Fig. 5i).
Taken together, these data show that DDR1 is required for TDLU regeneration through the differentiation of bipotent mammary stem cells into lineage-committed basal progenitors (Fig. 5j). Basal cells express the Notch1 ligand Jagged-1, which is required for activation of Notch1 signaling in luminal progenitor cells. This activation of luminal Notch1 signaling, in turn, induces the maturation of luminal progenitors into mature luminal cells, driving TDLU growth (Fig. 5j).
