Presentations

Abstract

Background:

Benign prostatic hyperplasia (BPH) is an age-related disease associated with deteriorating urinary symptoms. The etiology of BPH involves an increase in estradiol-to-testosterone ratio and chronic inflammation but data is limited on how these processes are interconnected. In a mouse model reproducing steroid hormone imbalance (T+E2), we observed increased macrophage infiltration, accumulation in the prostate lumen and foam cell formation. However, the specific role of foam cells in the prostate is unclear. Therefore, to characterize the foam cell transcriptome and to gain a better understanding on macrophage populations and chemokine signaling in the prostate, we conducted single-cell RNA sequencing (scRNA-seq) on prostates from T+E2 mice.

Methods:

We implanted male C57BL/6J mice with pellets containing 25 mg testosterone and 2.5 mg estradiol and collected ventral prostates two weeks later. Cells were dissociated with cold protease and were loaded on Chromium Next GEM (7000 cells/sample). Samples were sequenced on a NextSeq2000 instrument at 100 million reads/sample. Confirmation of scRNA-seq results was performed using in situ hybridization (ISH) or immunohistochemistry (IHC) with specific probes for Cd209a, Cxcl17, Folr2 and Pmepa1 and an antibody against TGF-b1. Tissue samples were analyzed using Mantra II. Pathological Workstation and InForm software.

Results:

Our scRNAseq successfully identified five distinct macrophage clusters named after their corresponding marker genes: MacFolr2+, MacPmepa1+, MacEar2+, MacCd209a+, and MacSpp1+, the latter representing foam cells. Further marker genes of MacSpp1+ were Gpnmb, Trem2, Fabp5, Ctsl and Mmp12. Several cytokines and growth factors, including Tgfb1, Vegf, Cxcl16, and Ccl6, were significantly upregulated in MacSpp1+ (confirmed via ISH and IHC). We also identified that MacPmepa1+ cells were elevated in response to steroid hormone imbalance. We then conducted a chemokine screen in our scRNAseq data for all Cxcl and Ccl genes. This identified Cxcl17 to be upregulated in epithelial cells which was then confirmed using ISH (13-fold, p<0.001).

Conclusion:

The upregulation of cytokines and growth factors in MacSpp1+ foam cells suggest their potential pathological role in BPH. MacPmepa1+ cells were also elevated highlighting their significance within the stroma. Furthermore, our cytokine screen identified Cxcl17 as a potential driver of macrophage recruitment in the prostate. These results may promote the development of macrophage-targeting therapies for BPH.