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, CD45high tumors 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. SignificanceFAP expression identifies an aggressive, immunosuppressive EMT-associated subtype of localized ccRCC, uncovering critical interactions between cancer cells and the TME and revealing novel biological insights and therapeutic vulnerabilities driving early-stage ccRCC progression. Graphical AbstractO_ST_ABSTumor-cell FAP Expression Defines an Aggressive, Immunosuppressive EMT Subtype in Highly Inflamed Localized ccRCCC_ST_ABS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/620479v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@d25831org.highwire.dtl.DTLVardef@19c0de7org.highwire.dtl.DTLVardef@54bd9eorg.highwire.dtl.DTLVardef@16d0387_HPS_FORMAT_FIGEXP M_FIG C_FIG Localized ccRCC tumors were stratified by CD45 immune infiltration, revealing that CD45high tumors were associated with significantly poorer recurrence-free survival (5-year RFS rate: 39%). Among these, tumor cell-specific expression of fibroblast activation protein (FAP) identified a particularly aggressive subtype marked by epithelial-to-mesenchymal transition (EMT), reduced endothelial content, and an immunosuppressive tumor microenvironment enriched with M2-like macrophages, regulatory T cells, exhausted T cells, and FAP cancer-associated fibroblasts (CAFs). FAP tumors had an even lower 5-year RFS rate of 26%, compared to 50% in FAP- CD45high tumors. Tumor-cell FAP thus provides an additional layer of risk stratification within the CD45high subgroup and reflects a biologically distinct, immune-evasive tumor state. These findings highlight FAP as both a potential therapeutic target and imaging biomarker in localized ccRCC.