Abstract
The tumor immune microenvironment (TIME) shows significant heterogeneity in primary clear cell renal cell carcinoma (ccRCC). As TIME heterogeneity between primary and paired metastatic tumors of ccRCC is less understood, we characterized and compared the TIME of primary ccRCC with paired asynchronous metastases. We analyzed patients who developed ccRCC recurrence post radical nephrectomy and had both primary and metastatic treatment-naïve tissue available. Capture whole-transcriptome sequencing was performed on formalin-fixed paraffin-embedded (FFPE) specimens using the Illumina platform. Differential gene expression (DGE) analysis and gene set enrichment analysis (GSE) was performed using R packages limma and fgsea respectively. TIME deconvolution was quantified using CIBERSORT, an in-silico flow cytometry tool. In aggregate, 42 tumor samples from 19 patients (19 primary tumors with 23 matched metastases) were analyzed. Metastatic sites included lung (n = 6), bone (n = 6), adrenal (n = 4), liver (n = 2), lymph node (n = 2), and soft tissue (n = 3). In unsupervised hierarchical clustering, primary tumors clustered together and not with their matched metastatic tumor. Of the immune cells assayed, primary tumors displayed greater Tregs than their matched (and unmatched) metastases (p < 0.001). Among metastatic sites, bone had high levels of EMT activity compared to their matched primary tumors and lung metastatic tumors were enriched in E2F targets. We demonstrate differences in pathway enrichment and immune cell populations in primary ccRCC and their matched metastases, including a higher infiltration of immunosuppressive T regulatory cells in the tumor immune microenvironment of primary ccRCC. Metastatic tumors not only differed from their paired primary tumors but also differed in gene expression, gene set enrichment, and immune cell composition between metastatic tissue sites.
Data availability
Data used for downstream analyses such as differentially expressed genes, pathway enrichment scores, Cibersort and Estimate immune deconvolution results are deposited in Gene Expression Omnibus (GEO) under the accession code: GSE278174. R code will be provided by the corresponding author upon reasonable request.
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Funding
Funded by National Comprehensive Cancer Network (T.M.M.). S.S.S., T.M.M. are supported by the University of Michigan Health System-Peking University Health Science Center (UMHS-PUHSC) Joint Institute (JI). S.S.S. is supported by the Robert Wood Johnson Foundation as part of the Harold Amos Medical Faculty Development Program (AMFDP). S.S.S. is supported in part by the Urology Care Foundation Rising Stars in Urology Research Award Program and Astellas, Inc. R.M., S.S.S., T.M.M. are supported by P30 CA046592. B.H.C was supported by 5T32CA180984-06 Ruth L. Kirschstein NRSA Institutional Research Training Grant S.M.D supported in part by U24CA271037.
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B.C. interpreted the data, formulated conclusions, and wrote the main manuscript text, S.N. analyzed data and prepared figures S.M analyzed data and prepared figure supplement 2. S.S. provided guidance on experimental design and data interpretation, oversaw the overall research progress, and contributed to the critical review and editing of the manuscript. All authors reviewed the manuscript.
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Cotta, B., Nallandhighal, S., Monda, S. et al. Molecular profiling of primary versus paired asynchronous metastatic clear cell renal cell carcinoma reveals heterogeneity in tumor immune microenvironment. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35021-7
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DOI: https://doi.org/10.1038/s41598-026-35021-7
