Fibroblast heterogeneity in prostate carcinogenesis

Abstract

Our understanding of stromal components, specifically cancer-associated fibroblasts (CAF), in prostate cancer (PCa), has evolved from considering these cells as inert bystanders to acknowledging their significance as players in prostate tumorigenesis. CAF are multifaceted-they promote cancer cell growth, migration and remodel the tumor microenvironment. Although targeting CAF could be a promising strategy for PCa treatment, they incorporate a high but undefined degree of intrinsic cellular heterogeneity. The interaction between CAF subpopulations, with the normal and tumor epithelium and with other cell types is not yet characterized. Defining these interactions and the critical signaling nodes that support tumorigenesis will enable the development of novel strategies to control prostate cancer progression. Here we will discuss the origins, molecular and functional heterogeneity of CAF in PCa. We highlight the challenges associated with delineating CAF heterogeneity and discuss potential areas of research that would assist in expanding our knowledge of CAF and their role in PCa tumorigenesis.

Keywords: Cancer associated fibroblasts; Fibroblast heterogeneity; Myofibroblasts; Prostate cancer; Stromal heterogeneity.

Figure 1.. Fibroblast heterogeneity in normal prostate and prostate cancer tissues.

(A) The stromal tissue of normal prostate is predominantly composed of smooth muscle cells derived from the fetal urogenital sinus mesenchyme, a few resting fibroblasts and two distinct fibroblast subpopulations associated with tissue repair (Sca1+/CD90-) and cell survival (Sca1+/CD90+). Green arrow indicates interaction between epithelial and stromal compartments required for maintenance of organ homeostasis. (B) Stromal composition in prostate cancer (PCa) is dominated by fibroblasts with few smooth muscle cells. These activated fibroblastic cells are collectively known as cancer associated fibroblasts (CAF). CAF are in constant communication with each other, with cancer cells and with the other cells in the local tumor microenvironment (Red arrows). Bone marrow derived stem cells, adipose tissue, circulating fibroblasts, and resident fibroblasts can contribute to the heterogeneity of CAF. CAF in TME secrete growth factors and cytokines that impact cancer development and progression. CAF subpopulations with therapeutic potential are indicated in the box.

Figure 2:. Isolation and “functional” in vivo characterization of tumor-promoting CAF from human PCa tissue.

(A) Post-surgical removal of prostate tissue it is subjected to enzymatic and mechanical digestion followed by fibroblast enrichment in cell culture to isolate the stromal components. Isolated fibroblasts are maintained in culture followed by xenografting (under the kidney capsule) in mice with benign initiated epithelial cells (BPH1). (B) Fibroblasts that induce the malignant transformation of pre-malignant BPH1 cells and formation of invasive tumors (+growth/invasion) are characterized as cancer associated fibroblasts (CAF). Tumor growth and invasion was calculated as previously described. The degree of growth/invasion (small tumors vs large tumors) reflects inter-patient variability. Normal prostate fibroblasts (NPF) isolated from benign prostate tissue result in small to no tumor formation (-growth/invasion). Blue arrows indicate tumor (gross picture of the whole kidney) and asterisks the corresponding areas in histological images (H&E) surrounded by kidney parenchyma. (C) Analysis of the resultant tumors indicates a significant increase in graft size in CAF xenografts compared to that of NPF. Once tumors are formed under the influence of CAF, growth and invasion show a linear correlation.

Figure 3. scRNA-seq analysis of CAF freshly isolated from prostate cancer tissue versus cultured cells.

Uniform Manifold Approximation and Projection (UMAP) plot comparing freshly isolated fibroblasts from a prostate cancer patient versus cultured CAF from another prostate cancer patient (unpublished data). Fibroblasts were isolated from the prostatic peripheral zone, followed by enzymatic digestion and either fluorescence activated cell sorting (EpCAM-CD45-CD200-CalceinAM+ cells) or culturing/passaging of cells. Cells were prepared for scRNA-seq analysis using 10X Chromium (v2.0 for cultured CAF and v3.0 for freshly isolated CAF). Bioinformatics analysis was conducted using CellRanger, R, and Bioconductor, and visualization of naïve clustering was completed using Seurat.