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Integrated single-cell spatial multi-omics of intratumor heterogeneity in renal cell carcinoma
Background: Clear cell renal cell carcinoma (ccRCC) exhibits conspicuous intratumor heterogeneity (ITH) - a driver of tumor evolution and metastasis. ITH in RCC has been studied extensively with bulk tumor DNA sequencing, which lacks the ability to integrate single cell resolution data, spatial architecture, and microenvironment composition. Therefore, we analyzed primary ccRCC tumors at multiple biopsy sites with CyTOF, multiplex immunofluorescence (MxIF), whole exome sequencing (WES), RNA sequencing (RNA-seq), single nuclei RNA-seq (snRNA-seq), and whole genome bisulfite sequencing (WGBS). Methods: Primary ccRCC tumors collected from 6 patients (pts) were biopsied at multiple locations and subjected to CyTOF (n = 21 sites, 6 pts), MxIF (20 markers, n = 8 sites, 3 pts), WES (n = 8 sites, 3 pts), RNA-seq (n = 8 sites, 3 pts), snRNA-seq (n = 8 sites, 3 pts), and WGBS (n = 8 sites, 3 pts), enabling integrated multi-omics analysis. MxIF, CyTOF, and genomic/transcriptomic analyses were performed by BostonGene. Results: Genomic intratumor (IT) evolution of ccRCC cells was tracked with WES, and subclonal distribution of SETD2, STAG2, TSC2 and PBRM1 mutations was observed in different IT regions. Different regions of the same tumor were similar, whereas individual patient tumors were distinct according to tumor microenvironment cellular composition measured by CyTOF or deconvoluted from RNA-seq. The cellular deconvolution of the ccRCC tumors reconstructed from RNA-seq correlated with CyTOF, snRNA-seq and WGBS, showing high concordance among the methods. The promoter CpG island methylation levels, averaged across all genes, positively correlated with ccRCC grade. MxIF revealed spatial IT heterogeneity in the distribution of immune infiltrate components. Macrophages and T cells dispersed among malignant cells; whereas, T cells formed clusters at unique tumor margins. Conclusions: The utilization of multi-omics methods produced a high-resolution portrait of the ccRCC tumor composition and identified differential ITH among regions within the primary tumors or among individual primary tumors. This study demonstrated strong concordance among the different technologies, suggesting that tumor deconvolution by bulk RNA-seq might be clinically applicable for ccRCC tumors. MxIF analysis enabled a fine elucidation of the spatial relationships among the tumor and the immune and stromal cells, missed by common omic platforms. Integrated single cell multi-omics could render specific pathobiological and therapeutic insights that impact treatment decisions.