'Cancer-Associated Fibroblasts' Are Present In Normal Bladder Tissue But Differentially Located In The Bladder Tumor Microenvironment Compared To Normal Controls
Benjamin T. Ristau, MD1, Dylan Baker, PhD2, Paul Robson, PhD2.
1University of Connecticut Health Center, Farmington, CT, USA, 2The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.
BACKGROUND: Cancer-associated fibroblasts (CAFs) are cells within the tumor microenvironment that have been implicated in tumor growth, progression, and treatment resistance by providing a supportive milieu. Recently, unique CAF subpopulations have been characterized by single cell RNA sequencing (scRNA-seq) supporting a role for fibroblasts in bladder cancer biology. Multiple bladder CAF's have been identified with markers such as SLC14A1, PDGFRA, and RGS5 and contribute to several aspects of cancer biology including inflammation and cancer stem maintenance. Here, we used scRNA-seq on normal human bladders and patient-derived bladder tumor specimens to demonstrate that previously identified CAF subpopulations exist in normal tissue, have corollaries in the mouse bladder, and are associated with other distinct cell types within the microenvironment.
METHODS: scRNA-seq was performed on transurethral resection of bladder tumor (TURBT) samples and normal bladder samples from cadavers utilizing 10X chromium gene expression kit. Preprocessing was performed with cellranger and downstream analysis performed via scanpy. After initial processing and clustering, fibroblasts were extracted from normal and tumor datasets for further subclustering analysis. Human fibroblasts were compared with mouse fibroblasts by Pearson correlation of marker genes identified in mouse and human fibroblast datasets. Cell type proportions associated with fibroblasts was confirmed in bulk RNA sequencing datasets from the UROMOL consortium by deconvolution of bulk RNA seq data with Scaden using in-house scRNA seq data as a reference. RESULTS: Four distinct fibroblasts populations were identified in scRNA-seq data from normal human bladders defined by DPT, SLC14A1, PI16 and CXCL14. The DPR and SLC14A1 populations were present in bladder tumors however the Pi16 and CXCL14 populations were absent in tumors. Comparison to mice showed that each of the fibroblasts present in humans had a fibroblast subtype corollary in mice with the DPT and SLC14A1 populations associated with detrusor resident fibroblasts and fully differentiated suburothelial myofibroblasts, respectively. Proportionally, DPT hi and SLC14A1 hi datasets were associated with other cell types and molecular signatures with DPT hi datasets associated with M2-like macrophages and SCL14A1 hi associated with ACKR1+ endothelial cells and PLN+ vascular smooth muscle. These cellular associations were confirmed in bulk RNA seq data where after deconvolution DPT hi datasets and SLC14A1 hi datasets had similar cellular proportion signatures originally identified in scRNA-seq. CONCLUSIONS: We identified distinct fibroblast populations in both normal human bladder and human bladder cancers defined by SLC14A1 and DPT. In normal bladder tissue, SLC14A1 is localized in the suburothelial location and DPT is present within the detrusor muscle. Despite these same fibroblasts being in a different location within bladder cancers compared to normal bladder, SLC14A1 and DPT fibroblasts within bladder cances remain associated with a distinctive microenvironment. SLC14A1 fibroblasts are associated with subtypes of endothelial and vascular smooth muscle and DPT fibroblasts are associated with anti-inflammatory macrophages and regulatory T cells. These findings suggest that tumors have the ability to co-opt normal cellular machinery to create a tumor microenvironment that supports carcinogenesis.
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