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

Abstract

Background: Immunotherapy 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 immunotherapy naïve and exposed primary ccRCC tumors to better understand immunotherapy resistance.

Results: Spatial molecular imaging of tumor and adjacent stroma samples from 21 tumors suggests that viable tumors following immunotherapy harbor more stromal CD8 + T cells and neutrophils than immunotherapy naïve tumors. YES1 is significantly upregulated in immunotherapy exposed tumor cells. Spatial GSEA shows that the epithelial-mesenchymal transition pathway is spatially enriched and the associated ligand-receptor transcript pair COL4A1-ITGAV has significantly higher autocorrelation in the stroma after exposure to immunotherapy. More integrin αV + cells are observed in immunotherapy exposed stroma on multiplex immunofluorescence validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV. Assessing bulk RNA expression and proteomic correlates in CPTAC databases reveals that collagen IV protein is more abundant in advanced stages of disease.

Conclusions: Spatial transcriptomics of samples of 3 patient cohorts with cRCC tumors indicates that COL4A1 and ITGAV are more autocorrelated in immunotherapy-exposed stroma compared to immunotherapy-naïve tumors, with high expression among fibroblasts, tumor cells, and endothelium. Further research is needed to understand changes in the ccRCC tumor immune microenvironment and explore potential therapeutic role of integrin after immunotherapy treatment.

Keywords: Immunotherapy resistance; Ligand receptor; Malignant-cell typing; Single-cell RNA; Spatial transcriptomics.

Fig. 1

Phenotyping of cells following assignment with InSituType. A and C show UMAPs of T cells and MNPs, respectively, calculated with all cells in all FOV. 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. Abbreviations: FOV, fields of view; MNPs, mononucleic phagocytes; PCA, principal component analysis; UMAP, uniform manifold approximation and projection

Fig. 2

Examples of FOV used for malignant-cell identification with LASSO generalized linear models after cell typing with InSituType and the Kidney Cell Atlas. UMAPs were created of kidney tissue specific cells (nonimmune, nonfibroblast) for a tumor (A) and stroma (B) FOV. Gene expression was plotted over the UMAP for TP53, EGFR, MYC, and VEGFA with “FeaturePlot” to aid in identifying malignant cells (C and D). VEGFA showed high expression in malignant-proximal tubule cells (E), while expression in normal proximal tubule cells (F) was low. H&E images (G and H) who cores sent for CosMx SMI. After InSituType and malignant-cell classification, polygon plots were constructed with final cell assignments (I and J). Abbreviations: FOV, fields of view; LASSO, least absolute shrinkage and selection operator; UMAP, uniform manifold approximation and projection

Fig. 3

Expression of YES1 in malignant melanoma cells before and after treatment with immune checkpoint inhibitor anti-PD-1 (GSE115978)

Fig. 4

Spatial enrichment of cells with high (> mean + 1 standard deviation) hallmark gene sets scores on tumor and stromal FOV showing high spatial enrichment of gene sets in IO-exposed tumor FOV. Abbreviations: FOV, fields of view; IO, immunotherapy

Fig. 5

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 are shown in B. Pathway description of VEGF mediation neovascularization, of which integrin and YAP are involved (C). Expression of COL4A1 and ITGAV for cell types on RCC4 – FOV8 (D)

Fig. 6

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). Abbreviations: H&E, hematoxylin and eosin; PCK, pancytokeratin; SMA, smooth muscle actin; SMI, spatial molecular imager. D and E show results for comparisons that were made between cohorts at the ligand/receptor assignment level and the fibroblast/tumor + ligand/receptor assignment levels, respectively

Fig. 7

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 2 genes. TCGA gene expression against methylation levels are shown in D and H. Abbreviations: CPTAC, Clinical Proteomic Tumor Analysis Consortium; TGCA, The Cancer Genome Atlas