Spatial and molecular determinants of response in penile squamous cell carcinoma.

12 Background: Penile squamous cell carcinoma (PSCC) is a rare, aggressive malignancy with limited treatment options and poor outcomes. The tumor microenvironment (TME) plays a key role in disease behavior and response to immunotherapy, but its immune composition and spatial organization in PSCC remain poorly defined. Methods: We conducted the first and largest longitudinal, multi-omics study of PSCC, integrating spatial transcriptomics (NanoString CosMx 6K panel) and single-cell RNA sequencing across 81 patients spanning all disease stages. Tissue Microarray 1 (TMA1) included 35 primary tumor samples from 31 patients, yielding > 390,000 spatially resolved cells. Patients were stratified by progression status, HPV, stage, and chemotherapy response (n = 12) per RECIST 1.1. Spatial cell–cell proximity and immune neighborhood analyses were used to define TME architecture linked to outcomes. Results: Spatial transcriptomic profiling of 35 primary PSCC samples identified two major TME patterns linked to outcomes. Progressors (n = 18) showed perivascular immunosuppressive hubs enriched with Tregs, SPP1⁺/M2-like macrophages, and CD14⁺ monocytes, along with reduced immune–endothelial cell distances (CD8, CD4, B, macrophages; p < 0.05). This vascular–myeloid suppressive axis was most evident in HPV-negative tumors (n = 17) and chemotherapy non-responders (n = 4). Conversely, patients that never progressed and chemotherapy responders displayed antigen-presenting, cytotoxic hubs enriched with CD8 effector and ISG⁺ T cells, cDC1s, and B/TLS features, with closer CD8–neutrophil interactions (p < 0.05), a pattern favoring HPV-positive disease. Early-stage (I–II) progressors showed higher Treg and fibroblast levels, suggesting stromal constraint while advanced-stage (III–IV) tumors demonstrated monocyte and CD4⁺ T-cell accumulation, indicating a shift toward myeloid-driven inflammation with interferon and exhaustion features. Across samples, reduced immune-endothelial proximity was linked to progression and non-response, whereas stronger CD8-neutrophil and cDC1-CD8 interactions correlated with treatment response (p < 0.05). Conclusions: This study establishes the first spatially resolved atlas of PSCC, integrating spatial and single-cell multi-omics to uncover distinct immune and stromal architectures linked to progression and therapy resistance. The discovery of vascular–myeloid immunosuppressive hubs versus cytotoxic immune neighborhoods provides a framework for precision immunotherapy development and biomarker stratification in this rare cancer.