Blood-based single-cell and bulk RNA sequencing identifies CAD-driven CD4⁺ T cell dysfunction in aggressive small clear cell renal cell carcinoma

Background: Small (T1a) clear cell renal cell carcinoma (ccRCC) is typically indolent, yet a subset exhibits aggressive behavior with early metastasis or recurrence. Reliable preoperative biomarkers to distinguish aggressive from indolent tumors are lacking. This study aimed to develop a blood-based transcriptional signature predictive of aggressive behavior in small ccRCC using integrated bulk and single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs). Materials and Methods: Bulk and single-cell RNA sequencing were performed on PBMCs from nine patients with small ccRCC (five aggressive vs four non-aggressive) and two healthy controls. Aggressive tumors were defined by synchronous metastasis or postoperative recurrence. Differentially expressed genes were identified and validated using PBMC cytospin immunofluorescence and qPCR in matched tumor tissues. Cell type-specific expression and immunofluorescence co-localization studies defined the cellular origin. Pathway analysis elucidated functional relevance. Results: Aggressive small ccRCC was characterized by upregulation of CAD in CD4 + cytotoxic T lymphocytes (CTLs). Despite their expansion, these CD4 + CTLs exhibited reduced expression of cytotoxic genes ( GNLY, NKG7, CD247 ) and signs of exhaustion. Pathway analysis revealed dysregulated MHC class II and IL2-STAT5 signaling, suggesting chronic antigenic stimulation. We propose that persistent inflammatory cues from aggressive tumors lead to CD4 + CTL expansion followed by functional decline – a phenomenon akin to immune dysfunction. Conclusions: CAD expression in CD4⁺ CTLs may serve as a minimally invasive biomarker to identify aggressive small ccRCC. This immune exhaustion signature offers a potential tool for risk stratification and may support personalized treatment strategies including upfront systemic therapy or intensified surveillance. Further validation in larger cohorts is warranted.