Journal: bioRxiv

Article Title: Fibroblast-derived Collagen VI shapes the structure and function of the tumor-immune microenvironment in clear cell renal cell carcinoma

doi: 10.64898/2026.03.19.712351

Figure Lengend Snippet: (A) Schematic depicting cancer cell reseeding on TK173-synthesized CDMs and further functional workup (Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y ). (B) Volcano plot of RNA sequencing analysis depicting differentially expressed genes of A498 cancer cells after reseeding on TK173-synthesized CDMs (COL6-containing CDM or depleted CDM; red dots indicate significantly regulated genes with adjusted p<0.05 and log 2 fold change (FC) > |0.5|). (C) Gene ontology (GO) gene set enrichment analysis of GO-terms of the molecular function category. (D) Volcano plot analysis for filtered matrisome genes. (E) IF staining for Nucleus (blue), F-actin (white), FN (green) and COL6 (violet) of A498 cells reseeded on shCTRL- and shCOL6-CDMs. (F) Scanning electron microscopy of A498 cells interacting with CDMs (yellow dotted boxes highlight areas of zoom in). (G&H) Morphometric analysis of reseeded cells (cell area and perimeter; Violin plots of individual cells of N=3 independent experiments, Mann-WhitneyLULtest). (I) Quantification of confluence of A498 cells growing on shCTRL- and shCOL6-CDMs (N=4 independent experiments, unpaired t test). (J&K) Representative phase-contrast images with DNA staining (blue) of A498 cells after cultivation for 3 days. Dot plot depicting quantification of A498 cells per cm² grown on CDMs (N=4 independent experiments, unpaired t test). (L&M) Representative IF staining of ccRCC patient samples for DNA (blue), PAX8 (red), KI-67 (green) and COL6 (violet) was used to classify and mask (bottom image) for proliferating tumor cells (orange), non-proliferative tumor cells (grey), as well as COL6 (violet). Violin plot depicting quantification of the distance of non-proliferative and proliferating tumor cells to COL6 (Mann-WhitneyLULtest). (N) Heatmap depicting Spearman’s correlation analysis of indicated proliferative cell state marker genes with COL6 , SERPINE1 and SERPINE2 genes in the ccRCC TCGA and CPTAC cohorts. (O&P) Spatial transcriptomics analysis of ccRCCs for spatial co-occurrence of COL6 , SERPINE1 , SERPINE2 and proliferative cell state marker genes. (O) Exemplary spatial mapping of the co-occurrence score on one sample (PD47512) and spatial mapping for the COL6, SERPINE and proliferative gene scores. (P) Correlation matrix of the spatial correlation between the COL6, SERPINE and proliferative gene sets (N=5 patients, Spearman’s correlation). Bars indicate mean and S.E.M in dot plots or median and quartiles in violin plots; ∗ – p < 0.05, ∗∗ – p < 0.01 and ∗∗∗∗ – p < 0.0001.

Article Snippet: Human renal cancer cell lines A498 (HTB-44, ATCC,) and 786-O (CRL-1932, ATCC) as well as human renal fibroblasts TK173 (RRID: CVCL_C8FA) and human renal proximal tubule epithelial cells (RPTEC/TERT1, ATCC, CRL-4031) were used and obtained as recently described .

Techniques: Synthesized, Functional Assay, RNA Sequencing, Staining, Electron Microscopy, Marker, Spatial Transcriptomics