Phenotypic and Spatial Characterization of Tumor-Associated Macrophages in Non-Metastatic Seminoma: Association with Local Tumor Progression.

Background: Seminoma is the most common subtype of testicular germ cell tumors in young men; however, the contribution of tumor-associated macrophages (TAMs) to disease progression remains insufficiently understood. This study aimed to quantitatively and phenotypically characterize CD68⁺ and CD163⁺ TAMs in non-metastatic seminomas (pT1N0M0 and pT2N0M0). Methods: This retrospective, multicenter, cohort, observational, analytical study was conducted from 1 January 2015 to 1 January 2025 at two branches of the National Medical Research Radiological Center of the Ministry of Health of the Russian Federation: the A. Tsyb Medical Radiological Research Center and the P. Hertsen Moscow Oncology Research Institute. Archived paraffin-embedded tumor samples from 96 patients and 21 samples of normal testicular tissue were analyzed using immunohistochemistry and digital morphometric analysis with QuPath software to assess macrophage density and spatial distribution. Results: Compared to normal testicular tissue, seminomas demonstrated more than a 10-fold increase in CD68⁺ TAMs and over a 100-fold increase in CD163⁺ TAMs. CD68⁺ cells predominantly localized to peripheral tumor regions, while CD163⁺ cells formed diffuse clusters in central tumor zones and around peripheral vessels. No statistically significant differences in CD68⁺ cell density were found between pT1 and pT2 stages. However, pT2 tumors showed a trend toward higher CD163⁺ TAMs density, suggesting increased M2 polarization with advancing tumor stage. Conclusions: These findings highlight the spatial and phenotypic heterogeneity of TAMs in seminoma and indicate a shift toward an immunosuppressive tumor microenvironment during local progression. Future studies should assess macrophage polarization and progression-free survival to evaluate their potential as prognostic biomarkers and therapeutic targets in seminoma.