Abstract 1163: Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment
Authors: Soupir Alex C. , Hayes Mitchell T. , Peak Taylor C. , Ospina Oscar E. , Chakiryan Nicholas H. , Berglund Anders E. , Stewart Paul A. , Nguyen Jonathan , Moran-Segura Carlos M. , Francis Natasha L. , Ramos-Echevarria Paola M. , Chahoud Jad , Li Roger , Tsai Kenneth Y. , Balasi Jodi A. , Caraballo-Perez Yamila , Dhillon Jasreman , Martinez Lindsey A. , Gloria Warren E. , Schurman Nathan , Kim Sean , Gregory Mark , Mule James J. , Fridley Brooke L. , Manley Brandon J. 
Published: March 2024

Abstract
Immunotherapy (IO) has improved the survival of patients with clear cell renal cell carcinoma (ccRCC); however, most patients will not have a durable complete response and will die from their disease. Our understanding of the development of IO resistance for ccRCC is very limited. We employed spatial transcriptomics (ST) with single cell resolution on patients with IO naïve tumors and IO exposed tumors to characterize spatial properties and differences between these two populations. Fourteen matched tumor and adjacent stromal fields of view (FOV) from surgically resected primary kidney specimens were analyzed using CosMx Spatial Molecular Imager (SMI). Non-tumor cell phenotyping was performed using the Kidney Cell Atlas. A generalized linear model was developed to identify malignant tumor cells in kidney tissues and validated by a pathologist. Differential cell abundance, clustering, and gene expression for each cell phenotype were compared between the two groups. Spatial gene set enrichment (GSEA) of modified hallmark gene sets were also compared. CD8+ T cells were more abundant in IO exposed vs IO naïve tumor FOVs. YES1 expression in the malignant tumor cells was significantly higher in IO exposed samples compared to IO naïve samples (false discovery rate [FDR] = 0.084). Spatial GSEA showed significant clustering of many gene sets (cells of high enrichment closer together than expected by chance), most notably epithelial-mesenchymal transition (EMT) pathway. Next, a post-hoc analysis of genes belonging to known ligand-receptor pairs in the EMT pathway was completed using bivariate Moran’s I to estimate spatial autocorrelation. We found significantly higher spatial autocorrelation of ITGAV-COL4A1 in the stroma of IO exposed FOVs than IO naïve FOVs (FDR = 0.008). Cell types with the highest expression of ITGAV and COL4A1 were found to be fibroblasts, endothelial, and tumor cells. Protein validation with multiplex immunofluorescence (mIF) confirmed cells positively stained for anti-integrin alpha-V were more abundance on stromal FOVs (p &lt; 0.001) in IO exposed samples. Fibroblast (SMA+) cells in stromal FOVs were more likely to be positively stained for anti-integrin alpha-V (p &lt; 0.001) in IO exposed than IO naïve FOVs. ITGAV protein is involved in migration from primary to metastatic disease in several cancers, and its inhibition increases T cell killing indicating ccRCC endothelial and tumor cells may be using this signaling for immune evasion and disease progression. Further, collagen and cancer associated fibroblasts (CAFs) play a role in tumor growth. To our knowledge, this is the first study to leverage single-cell resolution ST to identify potential ways by which ccRCC tumors become resistant to IO therapy.
Citation Format: Alex C. Soupir, Mitchell T. Hayes, Taylor C. Peak, Oscar E. Ospina, Nicholas H. Chakiryan, Anders E. Berglund, Paul A. Stewart, Jonathan Nguyen, Carlos M. Moran-Segura, Natasha L. Francis, Paola M. Ramos-Echevarria, Jad Chahoud, Roger Li, Kenneth Y. Tsai, Jodi A. Balasi, Yamila Caraballo-Perez, Jasreman Dhillon, Lindsey A. Martinez, Warren E. Gloria, Nathan Schurman, Sean Kim, Mark Gregory, James J. Mule, Brooke L. Fridley, Brandon J. Manley. Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1163.