Abstract 4013: Functional heterogeneity of cancer-associated stromal subtypes in the prostate cancer microenvironment

The stromal tumor microenvironment plays a central role in the development, progression and therapy resistance of prostate cancer (PCa), the second leading cause of cancer-related death in males in western nations. Functional and single cell RNA sequencing (scRNA-seq) studies unequivocally demonstrate the coexistence of functionally and spatially distinct fibroblast and mural cell states within the tumor microenvironment of various non-prostate tumors. Stromal cellular heterogeneity in PCa however remains poorly understood. This study therefore aimed to (1) comprehensively analyze stromal cell states within the PCa microenvironment and (2) to identify and characterize onco-supportive fibromuscular entities in respect to their potential clinical exploitation. Thus, we performed bulk tissue scRNA-seq on biopsy cores from benign and cancerous prostate tissue of 4 different patients. Analyses presented here focused on the fibroblast and mural cell clusters, which each delineated further into 2 subclusters. One fibroblast subcluster showed strong similarity to published signatures of inflammatory cancer-associated fibroblasts (iCAF). A closely related but distinct fibroblast subcluster exhibited hallmarks of benign interstitial fibroblasts with enrichment of GO terms such as antigen processing and presentation. Based on expression signatures and spatial localization via immunohistochemistry, the 2 mural cell subclusters (MCAM+) were identified as vascular smooth muscle cells (vSMCs) and dedifferentiated prostate SMCs/myofibroblasts. Notably, the gene signature of this latter subcluster correlated with increasing Gleason score and decreased disease-specific survival. On the basis of scRNA-seq data from this and independent datasets, panels of subcluster-specific markers were selected for validation studies via FACS, multiplex immunohistochemistry and quantitative real time PCR. Thus far, immunohistochemical studies validated not only the in vivo presence of the 4 distinct cellular states but also revealed their distinct spatial localization. We will present molecular and functional data from these ongoing analyses in (i) immortalized fibroblast and smooth muscle cell lines, (ii) heterogeneous ex vivo outgrowth cultures of primary PCa fibroblasts/myofibroblasts and (iii) primary cells from single cell suspensions of PCa biopsies enzymatically digested using an in-house approach optimized for maximal recovery of fibromuscular cells. These studies aim to evaluate t