# 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

## TL;DR

This study finds that the spatial arrangement of immune cells, especially M2 macrophages, in penile cancer affects patient survival, suggesting new ways to improve treatment.

## Contribution

The study introduces spatial immune profiling as a novel prognostic tool in penile squamous cell carcinoma using multiplex immunofluorescence.

## Key findings

- Higher densities of M1 macrophages and helper T cells correlate with improved survival in penile cancer.
- Clustering of M2 macrophages with tumor cells is linked to worse survival outcomes.
- Helper T cell clustering with tumor cells is associated with better survival, even in node-positive patients.

## Abstract

Penile cancer is a rare malignancy with poor prognosis in advanced and recurrent stages. The currently limited treatment options and poor survival outcomes represent an unmet need for patients with advanced or recurrent penile cancer. Innovation in therapeutic options for patients with penile cancer relies on insight of underlying tumor biology and tumor–immune system interactions. In this paper, we use multiplex immunofluorescence to investigate the immune microenvironment of penile cancer, focusing specifically on geospatial clustering patterns of various immune effector cells within various stages of penile cancer and human papillomavirus status. We identified spatial and clustering patterns of pro-immunogenic macrophages, tumor-associated macrophages, and helper T cells as potential prognosticators of survival in 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.