Deep visual multi-omics profiling reveals mechanisms that underlie cancer cell differentiation and aggressiveness in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) exhibits significant intra-tumoral heterogeneity (ITH) at both morphological and genetic levels, complicating treatment and contributing to disease progression. Among these, ccRCCs with focal rhabdoid differentiation stand out as highly aggressive tumors distinguished by cells with unique morphological features. However, the correlation between distinct morphological phenotypes, specific molecular alterations, and their influence on tumor behavior remains poorly understood. In this study, we integrated advanced AI-based image analysis with single-cell isolation and multi-omics profiling to dissect the link between clinically relevant morphological and molecular features of ccRCC cells. Using a novel digital pathology workflow, we quantified low-grade, high-grade, and rhabdoid morphologies in ccRCC diagnostic images with unprecedented precision. Subsequently, isolation of two sets of 1,000 morphologically distinct cells for detailed mRNA and protein expression analyses, revealed significant increasing dysregulation associating with higher histopathological grades. Rhabdoid ccRCC cells (grade 4) demonstrated unique molecular profiles, including upregulated FOXM1-driven proliferation, disrupted cell-matrix interactions, and enhanced immune evasion pathways. Despite high T-cell infiltration in rhabdoid areas, we identified a rhabdoid-specific immunosuppressive network driven by cytokines, IFN-beta, and integrin signaling, likely contributing to T-cell exhaustion. Rhabdoid ccRCC cells develop a distinct immunosuppressive signaling network, involving PD-L1 and novel immunomodulatory factors such as CD38 and ITGB2. These findings provide a basis for novel therapeutic strategies targeting these pathways in combination with immunotherapy to improve outcomes for patients with aggressive rhabdoid ccRCC. Key PointsO_LIccRCC is characterized by well-established morphological heterogeneity but the correlation with the underlying molecular aberrations remained elusive. C_LIO_LIBy integrating AI-based image analysis with single cell isolation and deep multi-omics profiling, we dissect the molecular intricacies of ccRCC, from targeted collection of 1,000 morphologically distinct cells. C_LIO_LIOur results demonstrate significant dysregulation of gene and protein expression correlating with higher histopathological grades in ccRCC. C_LIO_LIAggressive ccRCC cells with rhabdoid differentiation (grade 4) display distinct molecular profiles, as they upregulate FOXM1-mediated proliferation, ECM remodeling and the immune evasion responses, suggesting new therapeutic avenues enhancing ICI efficacy in these patients. C_LI