Immune cells in the tumor microenvironment (TME) play a critical role in regulating tumor development and response to targeted immunotherapy. While it is well-accepted that clear cell renal carcinoma (ccRCC) is characterized by a T-cell infiltrate, differential activation states within the tumor and the contribution of other immunomodulatory cells remain relatively unexplored. We sequenced approximately 30,000 immune cells at the single-cell level from tumor and matched adjacent normal tissue in ccRCC patients. With a median of 3300 Unique Molecular Identifiers (UMIs)/cell, we were able to capture the expression of 1000 genes per cell on average and 21,000 genes across all cells from each patient. We used Multidimensional Scaling (MDS) -transformation to visualize the various clusters and found that the tumor and adjacent normal tissue were distinct, with each having two major sub-clusters - lymphoid and myeloid cells. The data demonstrated a higher fraction of lymphoid infiltrate(~70-75%) than myeloid in a relatively stable ratio across patients, with tumor containing a slightly higher fraction of lymphoid cells. The normal and tumor cell subtypes were distinguished by multiple iterations of PAM clustering (Partitioning Around Mediods) with feature selection using the iterClust algorithm. Top differentially expressed genes in each cluster were identified and cross-referenced with known immune cell markers along with pathway enrichment analysis for cell typing. Among the tumor lymphoid population, genes specific for T-cells, including markers of functional exhaustion were the most highly expressed. A separate sub-cluster of cells consistent with cytotoxic T and/or NK cells was also identified. We found a distinct population of monocytes within the myeloid cluster that was present exclusively in the tumor tissue. This population expressed high levels of complement factors C1QA, C1QB, C1QC, as well as ApoE. The latter has been implicated as a driver of myeloid cell immunosuppression, indicating a possible regulatory role in the TME of ccRCC that has not previously been described. The phenotype of the immune cell populations in tumor was further validated by spectral flow cytometry. This study demonstrates relative abundance of cell types in the immune microenvironment within ccRCC tumors and also identifies a novel C1Q-high myeloid sub-population specific to the tumor, suggesting its possible role in RCC tumorigenesis or progression.

Citation Format: Nivedita Chowdhury, Aleksandar Obradovic, David Aggen, Vinson Wang, Xinzheng Guo, Guarionex J. DeCastro, Hongxu Ding, Andrea Califano, Charles G. Drake. Decoding the immunologic landscape of human clear cell renal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1476.