Abstract
Introduction
Benign Prostatic Hyperplasia (BPH) is a multifactorial disease characterized by the non-malignant enlargement of the prostate gland, leading to urethral compression. This disease manifests as lower urinary tract symptoms, which can significantly impair quality of life. There are currently no treatment options for BPH that target underlying disease mechanisms; therefore, it is imperative to identify new therapeutic targets beyond symptom management. Previously our lab had identified the presence of CD68+ macrophages migrating into the prostatic lumen and differentiating into lipid laden foam cells. We believe that epithelial cytokines promote this translocation and provide a target for inhibiting foam cell formation (FCF). Therefore, this research aims to uncover new targets driving FCF as well as characterizing BPH tissue microenvironments using spatial transcriptomics.
Methods
BPH prostate tissue was obtained from individuals who underwent Holmium Laser Enucleation of the Prostate (HoLEP) surgery. The tissue was fixed in formalin, embedded in paraffin and sectioned at 8 µm for probe-based spatial transcriptomics using 10x Genomics Visium HD Spatial Gene Expression method. Sections were deparaffinized, permeabilized, and hybridized with gene specific probes. After amplification, spatial transcriptomic libraries were generated and sequenced. Data were processed using the 10x Genomics Space Ranger pipeline in Cloud Analysis, including spatial barcode and UMI processing, gene assignment, image registration, and tissue detection. Gene expression data were aggregated into 8-µm spatial bins. Osteopontin (Spp1) was used to identify foam cell rich (FCR) and foam cell sparse (FCS) regions of epithelium and adjacent stroma, followed by differential gene expression analysis.
Results
Differential gene analysis showed significant upregulation of inflammatory cytokines CXCL8 (p = 1.01E⁻²²), CXCL6 (p = 1.17E⁻²⁰), CXCL1 (p = 1.41E⁻⁹), immune cell activator IL32 (p = 4.29E⁻²²) and CCL2 (p = 7.62E⁻¹¹), and efferocytosis gene ANXA1(p = 3.07E⁻¹⁷) in FCR epithelium. FCR stromal regions had increased expression of inflammatory chemokines, CXCL8, (p = 2.89E⁻²⁷), CXCL6 (p = 1.77E⁻⁶), and CCL2 (p = 4.89E⁻⁵) compared to FCS stroma. Additionally, there was increased expression of extracellular matrix (ECM) remodeling genes, including COMP (p = 4.93E⁻²⁵), MMP10 (p = 1.31E⁻⁹), FN1 (p = 2.08E⁻¹⁴), ELN (p = 7.05E⁻¹⁵), POSTN (p = 2.95E⁻⁷), and LTBP2 (p = 2.86E⁻⁹ in FCR stromal areas.
Conclusion
Spatial transcriptomics identified several candidates for epithelial cytokines in the FCR regions that may promote macrophage translocation. In addition, elevated expression of ECM genes suggest that FCR regions undergo active remodeling. Feature studies will provide validation with fluorescent in situ hybridization and utilize transurethral installation of selected cytokine candidates to test their role in macrophage translocation.