Geospatial and Cell Density Analysis Using Multiplex Immunofluorescence Reveals an Important Role of Clustering Patterns of Immunosuppressive Macrophages in Survival Outcomes of Penile Squamous Cell Carcinoma

Background/Objectives: Penile squamous cell carcinoma (PSCC) is a rare malignancy with poor prognosis in advanced and recurrent disease, and therapeutic options remain limited. Increasing evidence suggests that the tumor immune microenvironment (TIME), including immune cell composition and spatial organization, plays a critical role in tumor progression and survival outcomes. This study aimed to characterize immune cell density and geospatial clustering patterns within the TIME of PSCC and to evaluate their associations with clinical outcomes. Methods: Multiplex immunofluorescence (mIF) was performed on tumor samples from 57 patients with PSCC using a panel of immune markers to identify lymphoid and myeloid cell populations. Immune cell densities were quantified within tumoral and stromal compartments. Spatial relationships among immune cells and between immune cells and tumor cells were analyzed using point pattern analysis. Survival outcomes, including overall survival (OS), recurrence-free survival (RFS), and cancer-specific survival (CSS), were assessed using Kaplan–Meier methods and Cox proportional hazards models, with analyses stratified by nodal and human papillomavirus (HPV) status. Results: Higher intratumoral and stromal densities of pro-immunogenic M1 macrophages were associated with improved OS. Increased densities of CD3+CD4+ helper T cells in both compartments were also associated with favorable survival outcomes. In contrast, close clustering of pro-tumorigenic M2 macrophages with tumor cells and with one another was associated with worse OS, RFS, and CSS. Bivariate clustering of helper T cells with tumor cells was associated with improved OS, including among patients with node-positive disease. Survival outcomes did not differ significantly by HPV status in patients with high helper T cell clustering. Conclusions: Immune cell density and spatial organization within the TIME are associated with survival outcomes in PSCC. Favorable patterns involving helper T cells and M1 macrophages correlate with improved survival, whereas clustering of M2 macrophages is associated with poorer outcomes, supporting the relevance of spatial immune profiling in this disease.