Spatial and maturity heterogeneity of tertiary lymphoid structures shapes immune microenvironment and progression in prostate cancer.

Background: Tertiary lymphoid structure (TLS), ectopic lymphoid aggregates formed in response to chronic inflammation, have emerged as potential prognostic biomarkers and mediators of anti-tumor immunity in various cancers. However, the heterogeneity of TLS spatial distribution, maturity, and their prognostic and immunological significance in prostate cancer (PCa) remain poorly characterized. Methods: We utilized immunohistochemistry, multispectral fluorescence immunohistochemistry (mIHC) and spatial multi-omics analyses to evaluate the heterogeneity of TLS and its relationship with immune components in the tumor microenvironment (TME). Prognostic implications were assessed in 701 PCa patients from the TCGA and Fudan University Shanghai Cancer Center cohorts. The association between TLS heterogeneity and immunoreactivity was assessed through the quantification of immune cell infiltration. CellTreck and robust cell type decomposition deconvolution algorithms were used to decipher the colocalization features of each cell, cell-cell communication and ligand-receptor features within TLS regions. Results: In PCa, TLSs were detected in approximately 20 % of patients across both cohorts, with intratumoral TLS (intra-TLS) being twice as prevalent as peritumoral TLS (peri‑TLS). Patients harboring intra-TLS exhibited significantly longer disease-free and progression-free survival. Compared to peri‑TLS, intra-TLS were more mature, characterized by increased T-effector cell infiltration, activation of interferon pathways, and the presence of follicular dendritic cell centers and B cell aggregates. Notably, compared with immature TLS, mature TLS were markedly associated with reduced PD-L1 expression, lower regulatory T cells (Tregs) infiltration, and increased high endothelial venules (HEVs) density, indicative of an immunologically active microenvironment. Spatial multi-omics analysis revealed that mature TLS exhibited enriched immune cell diversity and HEVs density, suggesting enhanced anti-tumor immunity. Furthermore, cell-cell communication analysis identified significant interactions between CCL5 Conclusions: In conclusion, this study highlights the prognostic and immunological implications of TLS heterogeneity in PCa, demonstrating that the spatial distribution and maturity of TLSs are closely linked to TME activation and improved clinical outcomes. These findings provide novel insights into the immune landscape of PCa and establish a foundation for immune-based precision stratification and therapeutic development.