Progressive T cell exhaustion and predominance of aging tissue associated macrophages with advancing disease stage in penile squamous cell carcinoma.

11 Background: Penile squamous cell carcinoma (PSCC) is a rare malignancy with limited understanding of the tumor immune microenvironment (TIME). The interplay between PSCC and the immune system across disease progression and HPV infection status remains poorly characterized. This study aims to assess the TIME changes from localized to advanced disease and between HPV-positive versus negative tumors, we hope to identify potential immune evasion mechanisms for advanced PSCC. Methods: scRNA-seq was performed on ten PSCC tissue samples from penile, lymph node and distant metastatic sites with four matched penile and lymph node samples to understand the cellular heterogeneity within PSCC tumors. Analysis of immune cell populations and transcriptional hallmarks were performed stratified by localized (pT1-3, N0) versus advanced (N1-3, M0 or any N, M1) disease states and HPV infection status. Results: Patients with advanced stage disease were found to have a TIME enriched with terminally exhausted C8+ T cells. The myeloid compartment showed an increase in aging and immunosuppressive M2-like macrophages. Additionally, hypoxia-induced response patterns were more pronounced in advanced stages. HPV-negative tumors exhibited a quiescent TIME characterized by low immune cell infiltration. Conclusions: We observed significant differences in immune cell infiltration between localized and advanced PSCC disease states. Advanced disease states demonstrated an exhausted immune phenotype, characterized by terminally exhausted CD8 + T cells, M2-like macrophages and hypoxic signature. HPV-negative tumors displayed low immune cell infiltration. These findings offer valuable insights into the evolving PSCC immune landscape, paving the way for the development of potential novel macrophage directed therapies for PSCC.