Single-Cell and Spatial Transcriptomic Analysis Reveals a High-Risk Malignant Epithelial Subtype and Collagen VI-Driven Immune Escape in Clear Cell Renal Cell Carcinoma

Background: Clear cell renal cell carcinoma (ccRCC) is characterized by substantial intratumoral heterogeneity and a highly remodeled tumor microenvironment. However, the mechanisms linking malignant epithelial states to extracellular matrix remodeling and immune escape remain insufficiently understood.Methods: We integrated five public single-cell RNA sequencing datasets comprising 32 ccRCC tumor samples to characterize malignant epithelial heterogeneity. Spatial transcriptomic data and TCGA-KIRC bulk transcriptomic data were further incorporated for spatial mapping and clinical validation. Non-negative matrix factorization, CytoTRACE, pseudotime trajectory analysis, cell–cell communication inference, immune infiltration estimation, and deconvolution analyses were performed to define tumor subtypes and their microenvironmental features. Clinical samples, renal cancer cell lines, co-immunoprecipitation, Transwell assays, and murine tumor models were used for experimental validation.Results: Four malignant epithelial subtypes were identified in ccRCC, among which Subtype1 and Subtype4 showed the most significant prognostic differences. Subtype1 was associated with favorable survival, whereas Subtype4 was linked to poor clinical outcome. SCGN and ANGPTL8 were identified as representative markers of Subtype1 and Subtype4, respectively, and both exhibited significant associations with tumor progression and prognosis. Cell–cell communication analysis revealed that collagen signaling was markedly enhanced in Subtype4, especially the COL6A1/COL6A2-CD44 axis. Immune infiltration analysis indicated that Subtype1 was more closely associated with macrophage M1 polarization, whereas Subtype4 was related to a functionally constrained macrophage state. Spatial transcriptomic analysis further demonstrated a stronger co-localization of Subtype4 with COL6A1- and COL6A2-enriched regions. Functional experiments confirmed that overexpression of COL6A1 or COL6A2 inhibited macrophage migration and promoted macrophage polarization toward a tumor-supportive phenotype, while CD44 blockade reversed these effects. In vivo, CD44 inhibition suppressed tumor growth, whereas PPAR activation partially rescued this phenotype.Conclusions: Our study reveals a clinically relevant malignant epithelial subtype associated with poor prognosis and collagen-rich microenvironmental remodeling in ccRCC. The COL6A1/COL6A2-CD44-PPAR axis may represent a critical mechanism underlying macrophage dysfunction and immune escape. These findings provide new insight into the interplay between tumor cell heterogeneity and the immune microenvironment and suggest a potential therapeutic target for high-risk ccRCC.