Fibroblast Activation Protein Defines an Aggressive, Immunosuppressive EMT-associated Tumor Subtype in Highly Inflamed Localized Clear Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC) is among the most immune-infiltrated cancers, where high inflammation correlates with poor clinical outcomes. While epithelial-to-mesenchymal transition (EMT), immunosuppression, and cancer-associated fibroblasts (CAFs) have been linked with poor prognosis in metastatic ccRCC, their relevance and interplay in localized disease remain uncertain. To address this gap, we performed single-cell spatial profiling of highly inflamed localized tumors to characterize interactions between EMT-associated cancer cells and immune subsets, aiming to identify tumor microenvironment cell subsets and states responsible for the poor prognosis in highly inflamed ccRCC. Multiplexed immunofluorescence imaging with a 33-marker panel was applied to 1,728 tissue cores collected from tumor centers, invasive borders, and adjacent benign tissue from 435 localized ccRCC patients. Independent discovery (n=196) and validation (n=239) cohorts were used to confirm the significance of identified prognostic markers. Hierarchical clustering revealed TME subsets enriched for CD45⁺ immune cells, CD31⁺ endothelial cells, or fibroblasts. High CD45⁺ cell density significantly correlated with poor recurrence-free survival across tumor regions, including tumor center, invasive border, and adjacent benign tissue. Within tumors exhibiting high CD45⁺ infiltration, EMT-associated cancer cells strongly expressed fibroblast activation protein (FAP) at invasive borders, independently predicting worse prognosis and increased risk of liver metastasis. Furthermore, CD45^hightumors with elevated FAP expression featured immunosuppressive FAP⁺ CAFs, M2-like macrophages, exhausted T cells, and regulatory T cells. Tumor-cell-specific FAP expression was validated as an independent prognostic biomarker in both early-stage localized ccRCC (pT1-2) and patients subsequently developing metastases who received sunitinib, highlighting its potential for improved patient stratification.