The expression of prostate-specific membrane antigen (PSMA) increases with the progression and metastasis of prostate cancer, and PSMA PET has recently been used for prostate cancer diagnosis. Recently, we found that the intensity of PSMA expression in peritumoral vessels of renal cell carcinoma (RCC) correlates with recurrence rates. Furthermore, growth factors encapsulated within microvesicles secreted by RCC cells induce PSMA expression in surrounding blood vessels, thereby enhancing angiogenic potential. Therefore, this study aimed to evaluate the relationship between PSMA expression and angiogenic potential in the tumor microenvironment of RCC using high-resolution spatial gene expression analysis.

Formalin-fixed paraffin-embedded (FFPE) samples that passed RNA quality control (DV200 > 30%) were used for spatial transcriptome construction and sequencing. Tissue preparation was performed according to the Visium HD Spatial Gene Expression for FFPE tissue preparation guide (10x Genomics). Resected kidney tissue specimens were thinly sliced (10 µm thick), and Visium HD analysis was conducted on a designated area of 6.5 × 6.5 mm², including the peripheral tumor and normal vessel sites. After tissue permeabilization, mRNA was reverse-transcribed into cDNA, incorporating barcode information for spatial localization. The cDNA was amplified and sequenced to generate spatially resolved gene expression data. Each spot was assigned a barcode to enable mRNA analysis while preserving positional information. Sequencing was performed at the Center for Gene Research at Yamaguchi University using the MGI DNBSEQ-G400RS (MGI Tech Co., Shenzhen, China). The Space Ranger pipeline v2022.0705.1 (10x Genomics, CA, USA) and the GRCh38-2020-A reference genome were used to process FASTQ files.

Tumor vessels and normal vessels in RCC were identified as distinct clusters. The peritumoral vessel cluster exhibited significantly higher expression of FOLH1, the gene encoding PSMA. Pathway analysis of the RCC tumor vessel cluster using Gene Set Enrichment Analysis (GSEA) revealed the activation of multiple pathways involved in angiogenesis. Specifically, increased dimethylation at lysine 4 (H3K4me2) and trimethylation at lysine 27 (H3K27me3) of histone H3 were identified as potential regulators of angiogenesis-inducing transcription. Furthermore, increased expression of gene sets induced by tretinoin, which promotes angiogenesis, was also observed.

High-resolution spatial gene expression analysis has provided insights into the mechanisms underlying PSMA-mediated angiogenesis in peritumoral vessels. Future studies will further elucidate the regulatory mechanisms of PSMA expression in normal vascular endothelial cells, potentially contributing to the development of novel anti-angiogenic therapies.