Abstract 6480: Single-nucleus RNA sequencing reveals the immunological microenvironment of HPV+ and HPV- penile squamous cell carcinoma

Abstract Penile squamous cell carcinoma (PSCC) has high morbidity and mortality rates in developing countries. Puerto Rico has the fifth-highest incidence of PSCC globally. HPV-associated PSCC accounts for 20-63% of cases, depending on the geographical area. The tumor microenvironment (TME) plays a crucial role in modulating disease progression. In this study, we seek to characterize the immunological TME in HPV+ and HPV- PSCC using single-nucleus RNA sequencing. We sequenced (Illumina HiSeq, CA, USA) approximately 3, 000 nuclei from thirteen fresh PSCC tumor samples (8 HPV- and 5 HPV+) using the 10X Chromium System (10X Genomics, CA, USA). Quality control and filter of raw data was performed using Seurat and UMAP dimension reduction technique was used to cluster the nuclei. Cell types were predicted based on nuclei gene expression using two reference datasets from the Curated Cancer Cell Atlas: Ji 2020 (Skin Squamous Cell Carcinoma) and Kurten 2021 (Head and Neck Squamous Cell Carcinoma). Enrichment and functional analysis of genes that were significantly different between HPV+ and HPV- within each cell type were performed using Metascape. There were 19 distinct cell clusters identified among all samples. Six immune cell clusters were identified: T cells, macrophages, myeloid cells, B cells, dendritic cells, plasma cells. The relative abundance of each cell type was not significantly different between HPV+ and HPV- PSCC. In this study we focused on the T cell, macrophage, and myeloid cell clusters, which are immunologically relevant in cancer. These cell clusters contain differentially expressed genes that belong to pathways associated with protein synthesis (PS), extracellular matrix modulation (ECMM), blood vessel development (BVD), cytokine signaling (CS), and cell motility (CM). In all three immune cell clusters, PS pathway genes such as RPL7, RPL18A, RPL21 were overexpressed in the HPV + tumors. ECMM pathway genes (MMPs, COL1A1), BVD pathway genes (VEGFA), and CM pathway genes (CXCL1, CXCL8) were found to be overexpressed in HPV + tumors. For T cells and macrophages, CS pathways genes such as IL1A and CXCL8 were overexpressed in HPV + tumors. However, myeloid cells overexpressed different genes from the CS pathways in both HPV + (IL18, CCL3) and HPV - (CDKN1A, IFNAR2) tumors. In our analysis of the cancer cell clusters, the top differentially expressed genes among HPV+ tumors were: IL1A, IL24, ATP13A3, CCN1, AREG. Meanwhile the top differentially expressed genes among cancer cells of HPV - tumors were: ATP10B, FXYD3, CSRP2, CYP2C18, and KCTD1. Understanding how HPV infection affects the TME through changes in the molecular pathway interactions is critical for the identification of novel biomarkers and therapeutic targets that could improve treatment outcomes for penile cancer. Citation Format: Andres E. Hernandez-Gonzalez, Maria Sanchez-Vazquez, Yakshi Ortiz-Maldonado, Carlos Rivera-Lopez, Brandon Torres-Rivera, Hector L. Franco, Josue Perez-Santiago, Maria Marcos-Martinez, Antonio Puras-Baez, Magaly Martinez-Ferrer. Single-nucleus RNA sequencing reveals the immunological microenvironment of HPV+ and HPV- penile squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6480.