Stem Cell

Collagen signaling and matrix stiffness regulate multipotency in glandular epithelial stem cells in mice

Time Stamp: December 17, 2024 7:00 PM
Source Node: 812351

Collagen expression is associated with BaSC multipotency in the MG and the prostate

To assess whether a common mechanism is controlling BaSC multipotency in MG and prostate, we determined the genes that are upregulated in MG BaSC when they are multipotent. We have previously established the 246 gene signatures of MG SC (embryonic multipotent progenitors, EMPs) when they are multipotent during their initial stage of embryonic development (E15)⁶ (Fig. 1a, Supplementary Fig. 1a and Supplementary Table 1). To identify the genes associated with multipotency during BaSC transplantation, we transplanted BaSC alone or together with LCs in the mammary fat pad, recovered them 3 days following transplantation, FACS isolated, and performed transcriptional profiling of these cells in these two conditions. By comparing the transcriptional profile of BaSC in these two conditions, we found 1713 genes that were associated with multipotency during BC transplantation alone (Fig. 1a, Supplementary Fig. 1b, and Supplementary Table 1). We and others have previously shown that the expression of an oncogenic mutant of PIK3CA, the most frequently mutated oncogene in human breast cancers^10,18 activates multipotency in both BaSC and LC in adult mice. We have previously established the multipotent signature of BaSCs and LCs upon oncogenic PIK3CA expression¹⁰, that are composed of 399 genes (Fig. 1a, Supplementary Fig. 1c, and Supplementary Table 1). We have also previously established the BaSC multipotency signature upon LC ablation of the MG in adult mice¹¹ (Fig. 1a, Supplementary Fig. 1d, and Supplementary Table 1).

**Fig. 1: Collagen 1 and ECM stiffness promotes BaSC multipotency in MG and the prostate.**

In the prostate, we previously demonstrated that during the early stage of prostate postnatal development, the BaSC at the tips of the ductal tree is multipotent, whereas the BaSC along the main ducts was already unipotent⁹. To define the multipotent gene signature of prostate BaSC, we microdissected the distal tip regions (tip—100 μm) from the main ducts of the developing prostate at postnatal day 10–12 under the stereoscope, isolated these two populations of BaSC by FACS, transcriptionally profile the unipotent and multipotent BaSC and found that 174 genes were associated with multipotency in prostate BaSC during postnatal development²⁷ (Fig. 1a, Supplementary Fig. 1e and Supplementary Table 1).

The overlap between all the multipotent signatures revealed a common upregulation of Col1a1, Col1a2, Col3a1 from BaSC of the MG and prostate across these different conditions (transplantation, oncogenic mutation, LC ablation, and development) (Fig. 1a, Supplementary Fig. 1a–e and Supplementary Table 1). Immunohistochemistry staining confirmed that Col1a1 was expressed more abundantly within the MG stroma following the induction of an oncogenic PIK3CA mutant in BCs and after LC ablation in adult mice (Fig. 1b, c).

Collagen 1 and ECM stiffness promote BaSC multipotency in MG and the prostate

To assess whether Col1 in addition to be expressed at higher level in BaSC in multipotent conditions is directly promoting multipotency, we embedded MG and prostate organoids derived from K5CreER/Rosa-tdTomato or K5CreER/Rosa-YFP mice in collagen I gel at varying concentrations (2 mg/ml, 4 mg/ml and 8 mg/ml). The organoids were growing well within the collagen I gel. TAM was added to the culture media after embedding the organoid either with Matrigel or with collagen I to lineage trace BCs (TOM+ or YFP+). Following TAM administration, TOM expression were mainly found in BC (TOM + K8−) and very few LCs (TOM + K8+) were TOM+ in MG organoid embedded in Matrigel (Fig. 1d, e). In contrast, there was an increase in the proportion of TOM+ in LCs in organoid embedded in collagen I in a concentration-dependent manner, showing that high level of collagen promotes the differentiation of BCs into LCs by promoting BaSC multipotency in the MG and prostate organoids (Fig. 1d–g).

As Matrigel contained approximately 60% laminin and 30% collagen IV, we assessed the effect of Matrigel concentration on MG BC multipotency. To this end, organoids were embedded in 50%, 70%, and 100% Matrigel, treated with TAM for 48 h, and then cultured for an additional 5 days after TAM and the proportion of BC and LC Tomato labeled cells were quantified by FACS. Our results show a higher proportion of TOM+ LCs in 100% Matrigel compared to 50% Matrigel, showing that increasing the concentration of Matrigel promotes BC multipotency (Supplementary Fig. 2 and Supplementary Fig. 3a–c).

To assess the impact of other ECM components on BC multipotency, we embedded organoids in 70% Matrigel supplemented with either additional laminin or fibronectin. Our results showed that increasing laminin concentrations minimally enhanced BC multipotency, while fibronectin did not change BC multipotency at all (Supplementary Fig. 3d–f).

Collagen can bind to different classes of receptors, including the integrin family (integrin α1β1, α2β1, α10β1, α11β1), the discoidin domain receptors (DDRs), glycoprotein VI (GPVI), and the leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1)²⁸. Particularly, Col1 binding to the integrin α2β1 activates a signaling cascade which involves the focal adhesion kinase (FAK) activation and the related downstream pathway to promote different cellular responses, including adhesion, survival, and migration^29,30,31,32. In addition, increased Col1 concentration increases ECM stiffness that can signal to the cells and activate the mechanotransduction signaling pathways^33,34.

To assess whether rising Col1 concentration promotes BaSC multipotency by increasing ECM stiffness, we performed lineage tracing of BCs in MG organoids and prostate organoids embedded in polyethylene glycol gels (PEGs) at different concentrations presenting increasing stiffness. Unlike the Matrigel, PEGs are inert gels in which different components of the ECM can be added and the degree of stiffness can be modulated by changing the concentration of the polymers^35,36,37. We embedded MG organoids and prostate organoids in PEG gels at different elastic modulus, including Matrigel (0.1 kPa stiffness), soft gel (2.5% PEG, 0.3 kPa stiffness), medium gel (3.5–4% PEG, 1–1.3 kPa stiffness) and stiff gel (7–9% PEG, stiffness 3.3–4 kPa stiffness), in the presence or absence of Arg-Gly-Asp (RGD) peptides, which are the minimal sequence motif on collagen bound by integrin³⁸. These stiffness values approximate the mechanical properties of normal mammary gland stroma (0.1 kPa) and tumor (4 kPa)³⁴, as well as collagen I gel at varying concentrations (2 mg/ml, 0.5 kPa; 4 mg/ml, 0.9 kPa; 8 mg/ml, 3.3 kPa, Advanced Biomatrix). We observed a dose-dependent enrichment of K8+ fluorescent labeled LCs arising from BaSC in the presence of increased gel stiffness in both MG and prostate (Fig. 1h–k). In addition, we found that the presence of RGD peptide was necessary for the activation of multipotency (Fig. 1h–k and Supplementary Fig. 4a–d). We used intracellular flow cytometry analysis of K5 and K8 expression to quantify MG BC multipotency mediated by increasing collagen concentration and stiffness using an orthogonal method. FACS quantification showed that similarly to what was found using immunostaining of K8 on organoid sections, high collagen concentration, and stiffness increased the proportion of TOM+ LCs (K5^low K8^high) coming from MG BaSCs (Fig. 1l, m), further supporting the notion that collagen I and ECM stiffness promote BaSC multipotency.

To assess whether collagen I and ECM stiffness promote LC multipotency, we embedded MG organoids derived from K8CreER/Rosa-YFP mice in four conditions: Mat, Soft (2.5% PEG + RGD), Col1 (4 mg/ml), and Stiff (7% PEG + RGD). One week following TAM administration, we observed that 15–20% LCs expressed YFP+, with only a small proportion YFP+ BCs (1–2%). Higher levels of collagen I and ECM stiffness did not lead to a further increase in YFP+ BCs (Fig. 1n and Supplementary Fig. 4e, f), suggesting that in contrast to BaSC, collagen I and ECM stiffness do not induce LC multipotency.

Altogether, these data demonstrate that Col1 and ECM stiffness promote the activation of BC multipotency in the MG and in the prostate.

Collagen 1 and ECM stiffness promote hybrid states in MG organoids

To understand the mechanisms by which collagen and stiffness promote multipotency, we performed 10x single-cell RNA sequencing (scRNA-seq) to profile MG organoids embedded in four different conditions promoting or not multipotency: MATR (Matrigel, stiffness 0.1 kPa), SOFT (2.5% PEG + RGD, stiffness 0.3 kPa), COL1 (Col1 gel, stiffness 0.9 kPa), and STIFF (7% PEG + RGD, stiffness 3.3 kPa). Following demultiplexing of the samples tagged with different antibodies and filtering out doublets/negative droplets, mitochondrial genes, and non-epithelial cells in the MG samples, we identified a total of 1390 epithelial cells in the MATR, 2511 cells in the SOFT, 718 cells in the COL1, and 2620 cells in the STIFF conditions.

Dimensionality reduction analysis using Uniform Manifold Approximation and Projection (UMAP) and Principal Component Analysis (PCA) in the various conditions showed the presence of three main clusters. Through the expression of well-known markers for BCs (Krt5), pan-LCs (Krt8), LC ER+ (Foxa1), and LC ER- (Elf5) we identified that the three main clusters were corresponding to BCs, LCs ER+, and LCs ER- found in MG in vivo^6,11,39,40 (Supplementary Fig. 5a–d). In addition to these three main clusters, there were additional clusters of cells expressing both BC and LC genes, particularly in COL1 and STIFF conditions, forming non-homogeneous clusters in the UMAP plot that could represent hybrid cells between BC and LC previously identified in previous single-cell analysis studies of the MG in conditions associated with multipotency^40,41,42, including embryonic MG and MG following LC ablation^6,11 (Supplementary Fig. 5a–d).

To study the differences and similarities between the four samples, and to identify the changes in cell states following embedding MG organoids in different ECM gels with increased concentration of collagen and stiffness, we have integrated the four samples using a set of anchor genes calculated by Seurat integration pipeline⁴³. After integration, we had a total of 1383 cells for MATR, 2508 cells for SOFT, 716 cells for COL1, and 2619 for STIFF. The UMAP dimensionality reduction of the integrated sample showed the presence of all expected cell types, including BC, LC ER+, and LC ER− clusters, as well as an increased enrichment of hybrid cells (HY ER+/ER−, HY BC/ER−, and a proliferative HY BC/ER−) specifically in the STIFF and COL1 conditions (Fig. 2a, b, Supplementary Fig. 5e). After data integration and plotting UMAPs split by sample (Fig. 2b), the cell type distribution mirrored what was observed in the individual datasets (Supplementary Fig. 5a–d). These observations suggest that the integration did not lead to an over-correction of the data and the disappearance of the clusters found before data integration. Additionally, we identified a new cluster called BC primed to LC differentiation (BC primed LC), which expressed BC, LC ER+, and LC ER− markers that are highly enriched in COL1 and STIFF conditions (Fig. 2a, b, Supplementary Fig. 5e).

**Fig. 2: Single-cell RNA sequencing shows an increase of hybrid state in MG organoid embedded in Collagen 1 and stiff matrix.**

To determine the proportion of the cells presenting a hybrid state, we quantified in the different conditions the fraction of cells expressing high proportion of LC and BC genes (more than 65% of the genes). In the MATR and the SOFT conditions, about 1% of cells present this hybrid signature whereas in COL1 and STIFF, this proportion increased to 4% and 3% of cells respectively (Fig. 2c–g).

To determine the differentiation path taken by the BCs when they differentiate into LC, we perform a lineage trajectory analysis on the integrated dataset using slingshot⁴⁴ and set the BC cluster as the starting point of the trajectory. We excluded the BC/LC ER- Pr cluster from the analysis because the genes associated with proliferation disrupt the lineage trajectories. Slingshot analysis suggested the existence of three trajectories that start from the BCs. One trajectory indicated the direction from BC toward LC ER+, passing through the BC presenting the LC priming (Fig. 2h), another trajectory from BC to LC ER− passing through the hybrid BC/ER- (Fig. 2i) and finally, a last trajectory originating from BC going toward LC ER− and then to the LC ER+ (passing through the hybrid ER+/ER−) (Fig. 2j). During the differentiation from BC to LC, there was a downregulation of BC markers following the trajectories, including Krt5, Krt14, Trp63 and Acta2. Expression levels of LC ER+ (Foxa1, Areg, Prlr, Ly6a) and LC ER- (Elf5, Ehf, Cd14) marker genes were gradually increased following the different trajectories (Fig. 2k–m).

To further define the genes associated with hybrid state, we performed analysis of the differentially expressed genes (DEG) comparing the BC cluster and the hybrid BC/ER- in Collagen and STIFF conditions. We found that 490 genes and 621 genes were significantly upregulated in HY BC/ER- under COL1 and STIFF conditions (Supplementary Fig. 6a, b). Among these differentially regulated genes, 379 genes were commonly upregulated in HY BC/ER- including many classical LC genes such as Epcam, Krt8, Krt18, Cldn3, 4 and 7, CD24a, or Elf5 (Supplementary Fig. 6a, b). This analysis also revealed that 283 genes were commonly downregulated in HY BC/ER- including canonical BC marker genes such Krt5, Krt14, Trp63, genes of the basal membrane (Col4a1, Col4a2, Col17a1, Itga6, Lama3, Lamb3), and myoepithelial markers (Acta2, Myh11, Myl9, Mylk) (Supplementary Fig. 6a, b). Additionally, three collagen genes (Col1a1, Col1a2, Col3a1) that are commonly upregulated across the different multipotent conditions (Fig. 1a) were exclusively expressed in BC primed LC differentiation during COL1 and STIFF conditions (Supplementary Fig. 6c, d).

To determine whether the hybrid state induced by high collagen and stiffness resembles to the hybrid state found in the other conditions associated with multipotency, we compared the hybrid gene signatures from EMP⁶ and LC ablation¹¹ (Fig. 1a, Supplementary Table 1) with the marker genes from the clusters BC-primed LC differentiation and HY BC/ER- state found in high collagen and stiffness (Supplementary Fig. 6e, f). Three genes (Col1a1, Col1a2, Col3a1) were also shared between BC primed LC differentiation and the EMP and LC ablation signatures. Five genes (Col8a1, Emp3, Sostdc1, Timp1, Tnc) overlapped with the LC ablation condition, while 6 genes (Col18a1, Vim, Serpinh1, Bcl11b, Lgals7, Ppic) overlapped with the EMP state, mainly linked to ECM composition. For the HY BC/ER- population, two genes (Fn1, Fscn1) were common across all conditions. Compared to the EMP hybrid state, 10 genes, including those related to cytoskeleton and ECM (Col18a1, Serpinh1, Vim) and tubulins (Tuba1a, Tubb2b, Tubb5), were shared. The LC ablation comparison showed an overlap of 29 genes, including those involved in cell proliferation (Cdk6, Cdkn2b, Nupr1, Nme1), ECM composition (Col8a1, Tnc, Thbs2, Spon1), and cytoskeleton regulation (Clu, Krt7). These findings suggest that the collagen-induced hybrid state is more similar to the hybrid state found in adult mice following LC ablation.

ATAC-seq and RNA-seq performed during MG development uncover luminal genes for which the chromatin regions surrounding the promoter are also accessible in basal cells, suggesting that some luminal genes are primed in BCs⁴⁵. We then assess whether these primed genes correlate with the hybrid and primed clusters induced by stiffness. Our data show that while primed genes were expressed in the BC primed to LC differentiation population, most showed higher expression in the HY ER+/ER− and LC ER+ populations (Supplementary Fig. 6g).

In situ characterization of hybrid cells

To further characterize these novel hybrid cells in situ, we performed immunostaining of basal and luminal markers following lineage tracing of BCs in MG and prostate organoids in the MATR, SOFT, COL1, and STIFF conditions. Our results revealed that both TOM+ and YFP+ cells, for the MG and the prostate organoids respectively, were restricted to K14+ basal cells under Matrigel and Soft conditions, whereas hybrid cells that co-expressed K14, K8, and TOM/YFP were found in COL1 and STIFF conditions (Fig. 3a–c and Supplementary Fig. 7a), showing that collagen and ECM stiffness promote the appearance of hybrid cells during activation BaSC multipotency. Interestingly, the appearance of hybrid cells was observed as early as two days after TAM administration in COL1 condition (Fig. 3d–f). These hybrid cells induced by Collagen and ECM stiffness fully differentiated into LCs over time as shown by the loss of K14/K5 and the gain of K8 expression at Day 7 (Figs. 3a–c and 1l, m). Taken together, these results indicate that the activation of BaSC multipotency induced by Collagen and ECM stiffness is a progressive process associated with the emergence of transient hybrid states that eventually differentiate into LCs after losing their BC identity.

Activation of AP-1 TFs during the promotion of BC multipotency by collagen and ECM stiffness

To understand which genes are promoting BC multipotency in different conditions, and whether common or different pathways are involved in the activation of multipotency in the stiff matrix and COL1 conditions, we used two different approaches. In the first approach, we performed DEG analysis between the BCs in Matrigel versus the other conditions (SOFT vs MATR, COL1 vs MATR, and STIFF vs MATR). Comparison of SOFT versus MATR conditions showed that only 6 genes were significantly differentially expressed, showing that these two conditions are very similar. In contrast, 26 and 40 genes were significantly different in COL1 and STIFF conditions compared to MATR (Supplementary Fig. 8a). Among those, 13 genes were commonly upregulated in the BCs in COL1 and STIFF conditions promoting BC multipotency, including AP-1-related genes such as Jun, Fos, Fosb, and Atf3 (Fig. 4a), as well as ribosomal genes like Rps29, Rsp28, and Rpl37 (Supplementary Fig. 8b). We also found upregulation of Spp1 (Osteopontin) and Dusp1 (Supplementary Fig. 8b).

**Fig. 4: Identification of genes and TFs associated with BC multipotency in MG organoids embedded in collagen 1 and stiff matrix.**

To enhance the statistical power of our analysis, we assessed using a linear model which genes are correlated with both increased stiffness and the presence of COL1. When we applied this model to the BC cluster, we identified the same set of genes previously found in the DEG analysis including the AP-1 family of TFs such as Jun, Fosb, Jund, Fos and Atf3 (Fig. 4b, Supplementary Fig. 8c). Furthermore, Jun and Fosb were also upregulated in BC primed LC and in hybrid BC/ER- cells in the stiff condition (Fig. 4c–f). These results reinforce the notion that AP-1 activation is associated with BC multipotency in both the COL1 and STIFF conditions. Additionally, ribosomal genes (Uba52, Rpl38, Rps29) which could be involved in translational regulation of stem cell function⁴⁶ and transcription factors such as Klf2 and Klf6 are also showed significance on the modeling (Supplementary Fig. 8d, e).

To further strengthen the identification of the transcription factors (TFs) that mediate BaSC multipotency and BC-to-LC differentiation in response to increased collagen concentration and ECM stiffness, we used SCENIC analysis, a bioinformatics tool that identifies regulatory regions through the inference of co-expression between TFs and their target genes⁴⁷ and compared TFs active in the different conditions using Fisher’s combined test. As previously found in scRNA-seq from adult MG in vivo⁶, we found that Trp63, Egr2, Foxp1, and Mef2c were enriched in organoid BCs. Foxa1 and Tbx3 were enriched in LC ER+, while well-known LC ER- TFs such as Elf5 and Hey1 were enriched in LC ER−. BC primed LC population showed a significant activation of the AP-1 family (Jun, Jund, and Atf3), and Klf2 as well as TFs known to promote either LC ER+ or LC ER− fate, such as Foxa1 and Creb5, respectively. Moreover, the HY BC/ER- state was enriched in regulons activated by Ets1, as well as Sox10 and Sox11, which are reported as upregulated in hybrid population of mammary epithelial cells in previous studies^{39,45,48,49,50} (Fig. 4g, Supplementary Fig. 8f). Also, AP-1 family of TFs (Fos, Fosb and Atf3) were predicted as activated in BC in conditions associated with multipotency (COL1 and STIFF) compared to the conditions associated with unipotency (MATR and SOFT) (Fig. 4h). Jun immunostaining in MG organoids showed that Jun was expressed in LCs, with minimal expression in BCs in Mat and Soft conditions (Fig. 4i, j), whereas in high collagen concentration and ECM stiffness promoted Jun expression in BC (Fig. 4i, j).

Altogether, these data show that AP-1 TFs and their target genes were activated during the activation of multipotency in COL1 and stiff conditions, further showing that AP-1 TFs are associated with BC multipotency in both COL1 and stiff conditions.

Integrin-β1/FAK signaling controls collagen and stiffness induced multipotency in glandular epithelia

Col1 can bind different integrin receptors and activate different intracellular signaling involved in developmental processes. Among the collagen-binding integrins, integrin β1 is the most common receptor⁵¹. To investigate the role of Col1-β1-integrin signaling in BC multipotency activation, we treated MG and prostate organoids with β1-integrin blocking antibodies under different conditions: MATR, SOFT, COL1, and STIFF. Interestingly, the addition of β1-integrin blocking antibody⁵² for 5–7 days after TAM administration significantly reduced the proportion of K8+ Tomato+ LC in the MG organoids from K5CreER/Rosa-tdTomato mice and K8 + YFP+ cells in the prostate organoids from K5CreER/Rosa-YFP mice compared to control IgG treated group under conditions promoting multipotency (COL1 and STIFF) (Fig. 5a, b). To further substantiate these findings, we pharmacologically inhibited α2β1 signaling by treating organoids with α2β1-inhibitor (BT 3033)⁵³. Consistent with results of β1-integrin blocking antibody, treatment with BT 3033 for 5-7 days after TAM administration decreases the proportion of K8+ Tomato+ LCs in the MG organoids and K8 + YFP+ LCs in the prostate organoids under conditions promoting multipotency, further showing the important role of β1-integrin signaling induced by high concentration of collagen and a stiff ECM (Fig. 5c, d).

Next, we assessed whether β1-integrin signaling promotes BC multipotency through its canonical downstream signaling focal adhesion kinase (FAK). To this end, we treated MG and prostate organoids with PF573228, an inhibitor of FAK⁵⁴, for 5-7 days following TAM administration. Treatment with the FAK inhibitor decreased the proportion of K8+ Tomato+ LCs in the MG organoids and K8 + YFP+ LCs in the prostate organoids induced by COL1 and STIFF conditions without inducing apoptosis (Fig. 5c, d and Supplementary Fig. 9a, b), indicating that the activation of FAK is required for the promotion of BaSC multipotency downstream of β1-integrin in response to high level of Col1 or stiff ECM.

FAK inhibition in vivo prevents BaSC multipotency and MG outgrowth during BC transplantation

To assess whether FAK signaling regulates BC multipotency in vivo, we inhibited FAK signaling following the transplantation of BC into the mammary fat pad. To this end, we FACS isolated YFP+ BCs from the MG of K14Cre/YFP mice (Supplementary Fig. 10) and transplanted them into the cleared mammary fat pads of NOD-SCID mice, followed by 8 weeks of FAK inhibitor treatment (10 mg/kg, i.p., twice a week)^55,56 (Fig. 5e). Interestingly, while the control mice developed well-branched mammary glands following transplantation of BCs, mice treated with FAK inhibitor showed only YFP+ cell clumps with poorly branched structures, suggesting FAK inhibition led to a decrease in MG outgrowth from BC (Fig. 5f, g). Immunostaining revealed that FAK inhibition disrupted the differentiation of BCs into LCs, resulting in smaller, less organized structures co-expressing basal and luminal markers. In contrast, the control mice showed clear segregation of BCs and LCs, forming well-developed glandular architecture (Fig. 5h). These results provide in vivo evidence that FAK signaling can regulate multipotency following BC transplantation.

Integrin-β1/FAK/AP-1 axis controls collagen and stiffness-induced multipotency in glandular epithelia

Next, we assessed whether the AP-1 transcriptional factors are promoting collagen and ECM stiffness-induced multipotency as suggested by our bioinformatic analyses (Fig. 4a–h, Supplementary Fig. 8c). Inhibition of β1-integrin and FAK signaling impaired collagen- and ECM stiffness-induced AP-1 activation, suggesting that AP-1 is activated by high collagen concentration and ECM stiffness downstream of β1-integrin and FAK signaling (Fig. 6a). To functionally investigate the role of AP-1 signaling in the activation of BC multipotency, we assessed whether T-5224⁵⁷, a pharmacological inhibitor of AP-1 reduced BaSC multipotency in response to high concentration of Col1 and stiff ECM in the MG and prostate organoids. Interestingly, AP-1 inhibitor treatment for 5-7 days following TAM administration decreased the proportion of K8+ Tomato+ LCs in the MG organoids as shown by immunostaining on organoid sections and FACS analysis and K8 + YFP+ LCs in the prostate organoids induced by COL1 and STIFF conditions and did not increase apoptosis (Fig. 6b–d, Supplementary Fig. 9a, b). Given that AP-1 plays a critical role in regulating cell proliferation⁵⁸, and our data showed that increased collagen and ECM stiffness promote cell proliferation (Supplementary Fig. 9c, d), we investigated whether proliferation regulates BC multipotency. To this end, we inhibited cell proliferation in MG organoids embedded in different ECM by using RO-3306, a CDK inhibitor¹¹. We found that decreasing proliferation reduced collagen- and ECM-stiffness-induced BC multipotency (Fig. 6e and Supplementary Fig. 9c, d).

To further substantiate our findings, we performed genetic deletion of Junb/Junc and assessed the importance of these TFs in regulating BC multipotency mediated by Col1 and ECM stiffness (Fig. 6f). To this end, we generated MG organoids from Junb^fl/fl/Junc^fl/fl /mTmG mouse model. To delete Junb/Junc and lineage-traced BCs, we infected organoids with adenoviruses expressing Cre under the control of K5 promoter⁵⁹ and embedded them in different ECM substrates. One week following Jun deletion, immunostaining showed that Jun could not be observed in BCs, and the proportion of K8 + YFP+ LCs compared to control organoids infected with CRE adenovirus was reduced (Fig. 6g-j), further demonstrating the importance of AP-1 TFs in regulating BaSC multipotency mediated by Col1 and ECM stiffness.

Taken together, these results show that collagen and ECM stiffness-induced multipotency in MG and the prostate is mediated by integrin β1/FAK/AP-1 axis.

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Source: https://www.nature.com/articles/s41467-024-54843-5

Immune checkpoint inhibitors therapy for solid organ malignancies after allogeneic hematopoietic stem cell transplantation: a retrospective study from the EBMT Transplant Complications Working Party

December 12, 2024 7:00 pm

Collagen signaling and matrix stiffness regulate multipotency in glandular epithelial stem cells in mice

Collagen expression is associated with BaSC multipotency in the MG and the prostate

Collagen 1 and ECM stiffness promote BaSC multipotency in MG and the prostate

Collagen 1 and ECM stiffness promote hybrid states in MG organoids

In situ characterization of hybrid cells

Activation of AP-1 TFs during the promotion of BC multipotency by collagen and ECM stiffness

Integrin-β1/FAK signaling controls collagen and stiffness induced multipotency in glandular epithelia

FAK inhibition in vivo prevents BaSC multipotency and MG outgrowth during BC transplantation

Integrin-β1/FAK/AP-1 axis controls collagen and stiffness-induced multipotency in glandular epithelia

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