Intratumoral Microbiota and Immune Microenvironment in Prostate Cancer

Prostate cancer is a common malignancy in men, with rising incidence worldwide. Current clinical risk assessment tools-including PSA, Gleason score/ISUP grade, pathological stage, margin status, perineural invasion, and postoperative PSA kinetics-cannot fully explain the marked heterogeneity in disease progression, recurrence, and treatment response, highlighting the need for novel microenvironment-based biomarkers. Emerging evidence has identified intratumoral microbiota as a component of the tumor microenvironment in various solid tumors (e.g., breast, lung, ovarian, pancreatic, and melanoma), where microbial signals correlate with immune infiltration, inflammation, drug metabolism, and patient outcomes. However, the composition, spatial distribution, and immune-related roles of intratumoral microbiota in prostate cancer remain poorly characterized. Given that prostatic tissue resides at the urogenital junction and is continuously exposed to urine, prostatic fluid, and local inflammation, it is plausible that microbial components influence local immunity and tumor behavior. Importantly, intratumoral microbiota represent low-biomass samples, highly susceptible to contamination from reagents, environment, and laboratory procedures. Therefore, this study incorporates rigorous quality controls-including negative controls, process blanks, batch records, contaminant identification, spatial localization validation, and cross-platform verification-to ensure data reliability. Using residual tissue specimens, archived pathological slides, and comprehensive clinicopathological data from prostate cancer patients, we will employ 16S rRNA sequencing, metagenomic/metatranscriptomic sequencing, spatial transcriptomics, spatial proteomics, immunofluorescence, immunohistochemistry, and bioinformatic analyses to profile intratumoral microbiota. Our specific objectives are: (1) to compare microbial composition, abundance, and diversity among tumor, adjacent-normal, and benign tissues; (2) to validate spatial localization of candidate microbial signals via in situ hybridization, immunohistochemistry, and spatial omics; (3) to assess differences in immune cell infiltration, macrophage polarization, T-cell exhaustion markers, and inflammatory pathways between microbe-high and microbe-cold regions; (4) to explore associations between microbial features and Gleason score/ISUP grade, stage, perineural invasion, margin status, postoperative PSA changes, biochemical recurrence, and other clinical outcomes; and (5) to establish a standardized workflow for low-biomass intratumoral microbiome research in prostate cancer. This study aims to provide new insights into microenvironmental heterogeneity and to lay a foundation for identifying prognostic biomarkers and potential therapeutic targets.