Heterogeneity of human prostate carcinoma‐associated fibroblasts implicates a role for subpopulations in myeloid cell recruitment

Abstract

BackgroundCarcinoma–associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment (TME) that can promote tumorigenesis in the prostate. By understanding the mechanism(s) by which CAF contributes to tumor growth, new therapeutic targets for the management of this disease may be identified. These studies determined whether unique sub‐populations of human prostate CAF can be identified and functionally characterized.MethodsSingle‐cell RNA‐seq of primary human prostate CAF followed by unsupervised clustering was utilized to generate cell clusters based on differentially expressed (DE) gene profiles. Potential communication between CAF and immune cells was analyzed using in vivo tissue recombination by combining CAF or normal prostate fibroblasts (NPF) with non‐tumorigenic, initiated prostate epithelial BPH‐1 cells. Resultant grafts were assessed for inflammatory cell recruitment.ResultsClustering of 3321 CAF allows for visualization of six subpopulations, demonstrating heterogeneity within CAF. Sub‐renal capsule recombination assays show that the presence of CAF significantly increases myeloid cell recruitment to resultant tumors. This is supported by significantly increased expression of chemotactic chemokines CCL2 and CXCL12 in large clusters compared to other subpopulations. Bayesian analysis topologies also support differential communication signals between chemokine‐related genes of individual clusters. Migration of THP‐1 monocyte cells in vitro is stimulated in the presence of CAF conditioned medium (CM) compared with NPF CM. Further in vitro analyses suggest that CAF‐derived chemokine CCL2 may be responsible for CAF‐stimulated migration of THP‐1 cells, since neutralization of this chemokine abrogates migration capacity.ConclusionsCAF clustering based on DE gene expression supports the concept that clusters have unique functions within the TME, including a role in immune/inflammatory cell recruitment. These data suggest that CCL2 produced by CAF may be involved in the recruitment of inflammatory cells, but may also directly regulate the growth of the tumor. Further studies aimed at characterizing the subpopulation(s) of CAF which promote immune cell recruitment to the TME and/or stimulate prostate cancer growth and progression will be pursued.

The Role of Cancer-Associated Fibroblasts in Prostate Cancer Tumorigenesis

“…In addition, laminin expression is reduced in the reactive stroma indicating disruption of the basement membrane [106]. Subsequent studies have given evidence of the pro-angiogenic and immunomodulatory properties of CAFs in prostate carcinogenesis [58,107]. Overall, the properties of CAFs and ECM components in prostate reactive stroma support the aforementioned idea that tumors behave as 'wounds that do not heal'.…”

“…Another example of CAF heterogeneity in PCa is the co-existence of CAF subpopulations that are positive/negative for TGFβ receptor II (TGFβRII), a feature that seems to promote prostate tumorigenesis [56,57]. Recently, single-cell RNA sequencing of human prostate CAFs has revealed six main CAF subpopulations that secrete different arrays of cytokines with various immunomodulatory properties [58]. Among these CAF clusters, the chemokines (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 12 (CXCL12) were found to be differentially expressed-information that could not be revealed by sequencing the RNA of the bulk population.…”

“…Among these CAF clusters, the chemokines (C-C motif) ligand 2 (CCL2) and (C-X-C motif) ligand 12 (CXCL12) were found to be differentially expressed-information that could not be revealed by sequencing the RNA of the bulk population. These cytokines have different functions, but they both contribute to the generation of an immunosuppressive microenvironment [58]. Specifically, CCL2-secreting CAFs recruit tumor-associated macrophages, while CXCL12-secreting CAFs activate other immune components as mast cells, eosinophils, innate lymphoid cells, and helper T cells [58].…”

“…These cytokines have different functions, but they both contribute to the generation of an immunosuppressive microenvironment [58]. Specifically, CCL2-secreting CAFs recruit tumor-associated macrophages, while CXCL12-secreting CAFs activate other immune components as mast cells, eosinophils, innate lymphoid cells, and helper T cells [58].…”

Cancer-Associated Fibroblasts: Accomplices in the Tumor Immune Evasion

“…Other studies described additional mechanisms of the recruitment of MDSCs by CAFs involving the CXCL1 chemokine, followed by differentiation by CXCL12, IL-6, vascular endothelial growth factor (VEGF) and macrophage colony-stimulating factor (M-CSF) [ 28 , 29 ]. A single-cell RNA sequencing study conducted in prostate cancer demonstrated that different clusters of CAFs secreted various levels of proinflammatory cytokines such as CXCL12 and CCL2, highlighting that several CAF subpopulations may interact with MDSCs [ 51 ].…”