Proteomic characterization of the pseudocapsule of clear cell renal cell carcinoma in VHL disease reveals a distinct microenvironment at the tumor boundary zone

Von Hippel-Lindau (VHL) disease describes a hereditary tumor predisposition syndrome, caused by germline mutations in the VHL tumor suppressor gene, resulting in the functional loss of the VHL protein (pVHL). pVHL loss translates into a pseudo-hypoxic state that drives clear cell renal cell carcinoma (ccRCC) development. ccRCC tumors frequently form a pseudocapsule (PC) at the tumor boundary. This study describes the first comprehensive proteomic analysis of the PC in ccRCC patients with hereditary VHL inactivation, revealing a distinctive matrisomal signature. We conducted a deep, mass spectrometry-based proteomic analysis of 130 formalin-fixed paraffin-embedded (FFPE) ccRCC samples, comprising 54 tumor, 45 PC, and 31 non-malignant adjacent tissue (NAT) specimens from 34 patients. The PC exhibited unique matrisomal features, with pronounced enrichment of structural extracellular matrix (ECM) components, ECM processing enzymes, and secreted signaling proteins such as TGFβ2. Its proteome composition, including proteins involved in immune response, varied with tumor size and semi-tryptic peptide analysis indicated selective ECM processing in the PC and elevated levels of proteolysis within the tumor. Further, tumor proteomes reflected canonical VHL-driven metabolic reprogramming, including upregulated glycolysis and hypoxia markers, suppressed aerobic metabolism, and dysregulated fatty acid metabolism. Enriched immunoproteasome, MHC-I, and inflammasome proteins indicated an active immune response. Pro-angiogenic factors enriched in the tumor partially extended into the PC. Comparison of primary vs metachronous ccRCC cases uncovered proteomic tumor plasticity in VHL disease. Together, our study delineates the PC as an active, signaling-rich compartment at the ccRCC boundary with potential implications for tumor progression and clinical relevance beyond a mere structural scaffold.