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[Preprint]. 2023 Nov 17:2023.11.16.567457.
doi: 10.1101/2023.11.16.567457.

Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment

Alex C Soupir  1 , Mitchell T Hayes  2 , Taylor C Peak  2 , Oscar Ospina  1 , Nicholas H Chakiryan  3 , Anders E Berglund  1 , Paul A Stewart  1 , Jonathan Nguyen  4 , Carlos Moran Segura  4 , Natasha L Francis  5 , Paola M Ramos Echevarria  6 , Jad Chahoud  2 , Roger Li  2 , Kenneth Y Tsai  4 , Jodi A Balasi  4 , Yamila Caraballo Peres  4 , Jasreman Dhillon  4 , Lindsey A Martinez  5 , Warren E Gloria  4 , Nathan Schurman  1   2   3   7   4   5   6   7 , Sean Kim  1   2   3   7   4   5   6   7 , Mark Gregory  1   2   3   7   4   5   6   7 , James Mulé  7 , Brooke L Fridley  1 , Brandon J Manley  2
Affiliations

Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment

Alex C Soupir et al. bioRxiv. .

Update in

Abstract

Background: Immunotherapy (IO) has improved survival for patients with advanced clear cell renal cell carcinoma (ccRCC), but resistance to therapy develops in most patients. We use cellular-resolution spatial transcriptomics in patients with IO naïve and IO exposed primary ccRCC tumors to better understand IO resistance. Spatial molecular imaging (SMI) was obtained for tumor and adjacent stroma samples. Spatial gene set enrichment analysis (GSEA) and autocorrelation (coupling with high expression) of ligand-receptor transcript pairs were assessed. Multiplex immunofluorescence (mIF) validation was used for significant autocorrelative findings and the cancer genome atlas (TCGA) and the clinical proteomic tumor analysis consortium (CPTAC) databases were queried to assess bulk RNA expression and proteomic correlates.

Results: 21 patient samples underwent SMI. Viable tumors following IO harbored more stromal CD8+ T cells and neutrophils than IO naïve tumors. YES1 was significantly upregulated in IO exposed tumor cells. The epithelial-mesenchymal transition pathway was enriched on spatial GSEA and the associated transcript pair COL4A1-ITGAV had significantly higher autocorrelation in the stroma. Fibroblasts, tumor cells, and endothelium had the relative highest expression. More integrin αV+ cells were seen in IO exposed stroma on mIF validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV. In CPTAC, collagen IV protein was more abundant in advanced stages of disease.

Conclusions: On spatial transcriptomics, COL4A1 and ITGAV were more autocorrelated in IO-exposed stroma compared to IO-naïve tumors, with high expression amongst fibroblasts, tumor cells, and endothelium. Integrin represents a potential therapeutic target in IO treated ccRCC.

Keywords: Single-cell RNA; immunotherapy resistance; ligand receptor; malignant cell typing; spatial transcriptomics.

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Conflict of interest statement

Competing interests: The corresponding author certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (ie. employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: ACS, MTH, TCP, OEO, NHC, AEB, PAS, JN, CMS, NLF, PMRE, KYT, JAB, YCP, JD, LAM, WEG, and BLF have no relevant disclosures; BJM is an NCCN Kidney Cancer Panel Member and an advisor for Merck; RL received research support from Predicine, Veracyte, CG Oncology, Valar Labs, and Merck, is on the clinical trials committee for CG Oncology, is scientific advisor for Bristol Myers Squibb, Merck, Fergene, Arquer Diagnostics, Urogen Pharma, Lucence, CG Oncology, and Janssen, and has received honoraria from SAI MedPartners, Solstice Health Communications, Putnam Associates, and UroToday; JJM is Associate Center Director at Moffitt Cancer Center, has ownership interest in Aleta Biotherapeutics, CG Oncology, Turnstone Biologics, Ankyra Therapeutics, and AffyImmune Therapeutics, and is a paid consultant/paid advisory board member for ONCoPEP, CG Oncology, Turnstone Biologics, Vault Pharma, Ankyra Therapeutics, AffyImmune Therapeutics, UbiVac, Vycellix, and Aleta Biotherapeutics; NS, SK, and MG are or formerly were employees of Nanostring.

Figures

Figure 1:
Figure 1:
Phenotyping of cells following assignment with ‘InSituType’. A) and C) show UMAPs of T cells and mononucleic phagocytes (MNP), respectively, calculated with all cells in all fields of view (FOVs). Refined phenotypes from calculating new PCA/UMAP, clustering the subset with Louvain, and identifying markers with ‘FindAllMarkers’ can be seen in B) and D). Final cell assignments of all cells, including tumor cells, are shown in E).
Figure 2:
Figure 2:
Examples of FOVs used for malignant cell identification with LASSO generalized linear models. UMAPs were created of kidney tissue specific cells (non-immune, non-fibroblast) for a tumor (A) and stroma (F) FOV. Gene expression was plotted over the UMAP for TP53, EGFR, MYC, and VEGFA with ‘FeaturePlot’ to aid in identifying malignant cells (B and G). VEGFA showed high expression in malignant proximal tubule cells (C) while expression in normal proximal tubule cells (H) was low. H&E images (D and I) who cores sent for CosMx SMI. After ‘InSituType’ and malignant cell classification, polygon plots were constructed with final cell assignments (E and J).
Figure 3:
Figure 3:
Spatial enrichment of cells with high (> mean + 1 standard deviation) Hallmark gene sets scores on tumor and stromal FOVs showing high spatial enrichment of gene sets in IO exposed tumor FOVs.
Figure 4:
Figure 4:
Expression of COL4A1 and ITGAV on RCC4 – FOV8. Figure A) shows overlap for myofibroblasts and tumor and high gene expression. Locations of all cells and their respective phenotypes can be seen in B). Pathway description of VEGF mediation neovascularization, of which integrin and YAP are involved.
Figure 5:
Figure 5:
Example core that subjected to different assays. A) displays multiplex immunofluorescence staining for pancytokeratin (endothelium, PCK), smooth muscle actin (fibroblasts, SMA), integrin subunit alpha (ITGAV), and type 4 collagen (COL4). Cell types derived from CosMx SMI gene expression (B) show structure identified in both the multiplex immunofluorescence image and H&E (C).
Figure 6:
Figure 6:
Exploration of COL4A1 and ITGAV in TCGA and CPTAC. Gene expression in tumor and normal for COL4A1 (A) and ITGAV (E). Protein abundance between low stage ccRCC (I, II, and III) and high stage ccRCC (IV) for COL4A1 (B) and ITGAV (F). C) and G) show the copy number change associated with the two genes. TCGA gene expression against methylation levels are shown in D) and H).
See this image and copyright information in PMC

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