Single-Cell Transcriptomic Analysis of Tumor-Derived Fibroblasts and Normal Tissue-Resident Fibroblasts Reveals Fibroblast Heterogeneity in Breast Cancer.
ABSTRACT: Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for ?-smooth muscle actin and several other contractile proteins; 2) 'inflammatory' CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.
Project description:Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium. Gene profiles for each CAF subtype correlate to distinctive functional programs and hold independent prognostic capability in clinical cohorts by association to metastatic disease. In conclusion, the improved resolution of the widely defined CAF population opens the possibility for biomarker-driven development of drugs for precision targeting of CAFs.
Project description:Triple-negative breast cancer (TNBC) has the characteristics of a complex molecular landscape, aggressive or high proliferation leading to poor prognosis, and behavioral heterogeneity. The purpose of the present study was to determine the spatiotemporal expression of ?-smooth muscle actin (?-SMA)-positive cancer-associated fibroblasts (CAFs) at histological level in 4T1 tumors and to predict the sensitivity to 138 drugs in patients with TNBC according to ?-SMA expression. The quantitative results of fibrosis showed that the volume of 4T1 tumors correlated positively with the area of tumor fibrosis. Furthermore, we divided 4T1 tumors according to the degree of fibrosis and characterized the molecular characteristics of the four regions. Finally, the difference in the signaling pathways and sensitivity to 138 drugs was analyzed in patients with TNBC according to ?-SMA expression combined with The Cancer Genome Atlas (TCGA) database. The myogenesis, TGF-?, and Notch signaling pathways were upregulated and the patients with TNBC were significantly differentially sensitive to 25 drugs. The results of in vivo experiments showed that the inhibitory effect of embelin on 4T1 tumors with high ?-SMA expression was greater than that on 4TO7 tumors with low ?-SMA expression. At the same time, embelin significantly decreased ?-SMA and PDGFRA expression in 4T1 tumors compared with the control group. Our findings add to understanding of CAF distribution in the 4T1 tumor microenvironment and its possible role in treating cancer.
Project description:Cancer-associated fibroblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofibroblastic CAFs and inflammatory CAFs and define their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population "antigen-presenting CAFs" and find that they activate CD4+ T cells in an antigen-specific fashion in a model system, confirming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. SIGNIFICANCE: Appreciating the full spectrum of fibroblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifically target tumor-promoting CAFs. This work identifies MHC class II-expressing CAFs with a capacity to present antigens to CD4+ T cells, and potentially to modulate the immune response in pancreatic tumors.See related commentary by Belle and DeNardo, p. 1001.This article is highlighted in the In This Issue feature, p. 983.
Project description:Cancer-associated fibroblasts (CAFs) are key components of the dense, proliferating stroma observed in pancreatic ductal adenocarcinoma (PDAC), and CAF subpopulations drive tumor heterogeneity and play a major role in PDAC progression and drug resistance. CAFs consist of heterogenous subpopulations such as myoblastic CAF (myCAF) and inflammatory CAF (iCAF), and each has distinct essential roles. However, it is not clear how CAF subpopulations are formed in PDAC. Adipose-derived MSCs (AD-MSCs), which possess a high multilineage potential and self-renewal capacity, are reported to be one of the in vivo CAF sources. Here, we aimed to investigate whether AD-MSCs can act as precursors for CAFs in vitro. We recorded morphological features and collected omics data from two in vitro co-culture models for recapitulating clinical PDAC. Additionally, we tested the advantages of the co-culture model in terms of accurately modeling morphology and CAF heterogeneity. We showed that AD-MSCs differentiate into two distinct CAF subpopulations: Direct contact co-culture with PDAC cell line Capan-1 induced differentiation into myCAFs and iCAFs, while indirect co-culture induced differentiation into only iCAFs. Using these co-culture systems, we also identified novel CAF markers that may be helpful for elucidating the mechanisms of CAFs in the tumor microenvironment (TME). In conclusion, AD-MSCs can differentiate into distinct CAF subtypes depending on the different co-culture conditions in vitro, and the identification of potential CAF markers may aid in future investigations of the mechanisms underlying the role of CAFs in the TME.
Project description:Cancer-associated fibroblasts (CAFs) are highly prominent in breast tumors, but their functional heterogeneity and origin are still largely unresolved. We report that bone marrow (BM)-derived mesenchymal stromal cells (MSCs) are recruited to primary breast tumors and to lung metastases and differentiate to a distinct subpopulation of CAFs. We show that BM-derived CAFs are functionally important for tumor growth and enhance angiogenesis via up-regulation of Clusterin. Using newly generated transgenic mice and adoptive BM transplantations, we demonstrate that BM-derived fibroblasts are a substantial source of CAFs in the tumor microenvironment. Unlike resident CAFs, BM-derived CAFs do not express PDGFR?, and their recruitment resulted in a decrease in the percentage of PDGFR?-expressing CAFs. Strikingly, decrease in PDGFR? in breast cancer patients was associated with worse prognosis, suggesting that BM-derived CAFs may have deleterious effects on survival. Therefore, PDGFR? expression distinguishes two functionally unique CAF populations in breast tumors and metastases and may have important implications for patient stratification and precision therapeutics.
Project description:Carcinoma-associated fibroblasts (CAFs) are abundant stromal cells in tumor microenvironment that are critically involved in cancer progression. Contrasting reports have shown that CAFs can have either pro- or antitumorigenic roles, indicating that CAFs are functionally heterogeneous. Therefore, to precisely target the cancer-promoting CAF subsets, it is necessary to identify specific markers to define these subpopulations and understand their functions. We characterized two CAFs subsets from 28 non-small cell lung cancer (NSCLC) patient tumors that were scored and classified based on desmoplasia [mainly characterized by proliferating CAFs; high desmoplastic CAFs (HD-CAF; n?=?15) and low desmoplastic CAFs (LD-CAF; n?=?13)], which is an independent prognostic factor. Here, for the first time, we demonstrate that HD-CAFs and LD-CAFs show different tumor-promoting abilities. HD-CAFs showed higher rate of collagen matrix remodeling, invasion, and tumor growth compared to LD-CAFs. Transcriptomic analysis identified 13 genes that were differentially significant (fold ?1.5; adjusted P value < .1) between HD-CAFs and LD-CAFs. The top upregulated differentially expressed gene, ST8SIA2 (11.3 fold; adjusted P value?=?.02), enhanced NSCLC tumor cell invasion in 3D culture compared to control when it was overexpressed in CAFs, suggesting an important role of ST8SIA2 in cancer cell invasion. We confirmed the protumorigenic role of ST8SIA2, showing that ST8SIA2 was significantly associated with the risk of relapse in three independent NSCLC clinical datasets. In summary, our studies show that functional heterogeneity in CAF plays key role in promoting cancer cell invasion in NSCLC.
Project description:<b>:</b> Cancer-associated fibroblasts (CAFs) in the tumor microenvironment contribute to all stages of tumorigenesis and are usually considered to be tumor-promoting cells. CAFs show a remarkable degree of heterogeneity, which is attributed to developmental origin or to local environmental niches, resulting in distinct CAF subsets within individual tumors. While CAF heterogeneity is frequently investigated in late-stage tumors, data on longitudinal CAF development in tumors are lacking. To this end, we used the transgenic polyoma middle T oncogene-induced mouse mammary carcinoma model and performed whole transcriptome analysis in FACS-sorted fibroblasts from early- and late-stage tumors. We observed a shift in fibroblast populations over time towards a subset previously shown to negatively correlate with patient survival, which was confirmed by multispectral immunofluorescence analysis. Moreover, we identified a transcriptomic signature distinguishing CAFs from early- and late-stage tumors. Importantly, the signature of early-stage CAFs correlated well with tumor stage and survival in human mammary carcinoma patients. A random forest analysis suggested predictive value of the complete set of differentially expressed genes between early- and late-stage CAFs on bulk tumor patient samples, supporting the clinical relevance of our findings. In conclusion, our data show transcriptome alterations in CAFs during tumorigenesis in the mammary gland, which suggest that CAFs are educated by the tumor over time to promote tumor development. Moreover, we show that murine CAF gene signatures can harbor predictive value for human cancer.
Project description:Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGF? pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.
Project description:Increased collagen expression in tumors is associated with increased risk of metastasis, and triple-negative breast cancer (TNBC) has the highest propensity to develop distant metastases when there is evidence of central fibrosis. Transforming growth factor-? (TGF-?) ligands regulated by cancer-associated fibroblasts (CAFs) promote accumulation of fibrosis and cancer progression. In the present study, we have evaluated TNBC tumors with enhanced collagen to determine whether we can reduce metastasis by targeting the CAFs with Pirfenidone (PFD), an anti-fibrotic agent as well as a TGF-? antagonist. In patient-derived xenograft models, TNBC tumors exhibited accumulated collagen and activated TGF-? signaling, and developed lung metastasis. Next, primary CAFs were established from 4T1 TNBC homograft tumors, TNBC xenograft tumors and tumor specimens of breast cancer patients. CAFs promoted primary tumor growth with more fibrosis and TGF-? activation and lung metastasis in 4T1 mouse model. We then examined the effects of PFD in vitro and in vivo. We found that PFD had inhibitory effects on cell viability and collagen production of CAFs in 2D culture. Furthermore, CAFs enhanced tumor growth and PFD inhibited the tumor growth induced by CAFs by causing apoptosis in the 3D co-culture assay of 4T1 tumor cells and CAFs. In vivo, PFD alone inhibited tumor fibrosis and TGF-? signaling but did not inhibit tumor growth and lung metastasis. However, PFD inhibited tumor growth and lung metastasis synergistically in combination with doxorubicin. Thus, PFD has great potential for a novel clinically applicable TNBC therapy that targets tumor-stromal interaction.