anti collagen type vi alpha 6  (Novus Biologicals)

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 the processing of a cohort of cases of 23 ccRCCs for proteomic analysis, histological tumor segmentation and spatial multiplex immunofluorescence (NAT – normal adjacent tissue). (B) Volcano plot analysis of the ccRCC proteome highlighting significantly regulated matrisome proteins (red dots indicate proteins with an adjusted p-value <0.05 and log 2 FC (fold change) > |0.5|; dashed boxed show unfiltered proteomic analysis). (C) Heatmap presentation of significant differentially expressed matrisome proteins in the proteomic analysis of ccRCC samples sorted by matrisome categories (yellow box highlights upregulated collagen-6 chains in ccRCC tissue). (D) GO enrichment analysis shows an increase in categories such as collagen fibril organization in ccRCC tumor tissue. (E) Compartment segmentation of ccRCC tumor tissue (top – Ca. = cancer (blue), TS = tumor stroma (red), Ery. = erythrocytes (green), Ø = no tissue (cysts, vessels) (grey)) and cells (bottom – CC = cancer cells (blue), MC = mesenchymal cells (red), IC = immune cells (violet)). (F) Sample wise correlation analysis of histological compartment composition by machine learning based segmentation shown in panel (E) and z-scores of COL6A1, COL6A2 and COL6A3 expression in the ccRCC proteome (N=23 patients, Pearson correlation; ∗ – p < 0.05, ∗∗ – p < 0.01 and non-significant (n. s.)). (G) Multiplex IF staining demonstrates predominant deposition of COL6 in the interstitial compartment (dashed boxes indicate regions of magnified areas, asterisk indicates perivascular mesenchymal cells (Fibroblasts, Pericytes), arrows indicate the ECM, arrowheads indicate the basement membrane). (H) Quantification of COL6A1 positive area in cancer and stroma tissue compartments of ccRCC (N=23 patients, ∗∗∗∗ – p<0.0001, paired t test). (I) Dot plot heatmap of gene expression in indicated cell populations derived from available scRNA sequencing analysis of ccRCCs (N=7 patients).

Article Snippet: For CDM supplementation experiments, pre-formed CDMs on coverslips were overlaid with neutralized COL1 (5005, Advanced Biomatrix) or COL6 (009-001-108, Rockland Immunochemicals) solutions (250 μg/ml in PBS), incubated for 24 h at 37°C, and subsequently processed for immunofluorescence staining.

Techniques: Multiplex Assay, Immunofluorescence, Expressing, Staining, Membrane, Gene Expression, Derivative Assay, Sequencing

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 representation of the generation of COL6 knockdown in the 786-O cell line. Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y . (B) Representative images of colony formation assays of 786-O shCTRL and shCOL6 cells; Dot plots depict the number of colonies and the relative area covered (N=3 independent experiments, unpaired t test). (C) Dot plot indicating cell proliferation analysis by BrdU in 786-O shCOL6 and shCTRL cells (N=9 replicates of 3 independent experiments, performed in triplicate and pooled for analysis, unpaired t test). (D) IF staining of focal adhesion (FA) markers paxillin (PXN, purple) and zyxin (ZYX, yellow), as well as Nucleus (blue) and F-actin (green) in 786-O shCTRL and shCOL6 cells (white dashed boxes highlight area of zoom-in; Violin plots depicting FA area (top) and FA number normalized to respective cell area (dots indicate cells from 3 independent experiments, Mann-WhitneyLULtest). (E) Volcano plot of differential gene expression of all genes (top left) and matrisome-filtered genes (bottom left) in 786-O COL6A2 knockdown and control cells; Right panel shows gene-ontology (GO) enrichment analysis represented in a bubble plot. (F) Schematic representation of the generation of COL6 knockdown in the TK173 cell line. Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y . (G) Representative images of colony formation assays of TK173 shCTRL and shCOL6 cells; Dot plots depict the number of colonies and the relative area covered (N=3 independent experiments, unpaired t test). (H) Dot plot indicating cell proliferation analysis by BrdU in TK173 shCOL6 and shCTRL cells (N=9 replicates of 3 independent experiments, performed in triplicate and pooled for analysis, unpaired t test). (I) IF staining of focal adhesion (FA) markers paxillin (PXN, violet) and zyxin (ZYX, yellow), as well as Nucleus (blue) and F-actin (green) in TK173 shCTRL and shCOL6 cells (white dashed boxes highlight areas of zoom-in; Violin plots depicting FA area (top) and FA number normalized to respective cell area (dots indicate cells from 3 independent experiments, Mann-WhitneyLULtest). (J) Volcano plot of differential gene expression of all genes (top left) and matrisome-filtered genes (bottom left) in TK173 COL6A2 knockdown and control cells; Right panel shows GO enrichment analysis represented in a bubble plot. Bars indicate mean and S.E.M in dot plots or median and quartiles in violin plots; ∗ – p < 0.05, ∗∗ – p < 0.01 and n.s – not significant.

Article Snippet: For CDM supplementation experiments, pre-formed CDMs on coverslips were overlaid with neutralized COL1 (5005, Advanced Biomatrix) or COL6 (009-001-108, Rockland Immunochemicals) solutions (250 μg/ml in PBS), incubated for 24 h at 37°C, and subsequently processed for immunofluorescence staining.

Techniques: Knockdown, Staining, Gene Expression, Control

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 the generation and imaging of cell-derived matrices (CDM). Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y . (B) IF of CDM samples generated from TK173 shCTRL and shCOL6 cells stained for Fibronectin (FN, green), Collagen VI (COL6, violet) and color-coded FN-fiber direction. (C) Density plot (left) showing quantification (mean and S.E.M.) of FN-fiber orientation in shCTRL (blue) and shCOL6 (red). Dot plot (right) of mean area under curve (AUC) within ±30° centralized peak (N=5 independent experiments, unpaired t-test). (D) Dot plot of mean CDM thickness of CDMs derived from shCTRL and shCOL6 cell lines (N=3 independent experiments, unpaired t-test). (E) High-resolution IF imaging of CDMs from shCTRL (top) and shCOL6 (bottom) for FN (green) and COL6 (violet). White arrows indicate patch-like COL6 structures. (F) SEM imaging of CDMs with an overview (left) of shCTRL and shCOL6 and zoom-ins (right). The white arrow in the shCTRL-CDM indicates a continuous sheath-like surface of interconnected fibers. The white arrow for the shCOL6 points to fragmented and singularized fibers. (G) Schematic illustration of AFM (left). Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y ; Violin plot shows Young’s modulus (log 2 scale) of shCTRL-and shCOL6-CDMs (dots indicate individual measurements of 3 independent experiments, Mann-WhitneyLULtest). (H) Representative IF image stained with FN (green) and COL6 (violet), as well as color-coded FN fiber direction of shCOL6 CDM after 24h supplementation of COL6. (I) Density plot (left) depicting quantification of FN fiber orientation of CDMs supplemented with COL6 (violet and brown) and COL1 (blue and red). Dot plot (left) of mean AUC within ±30° of centralized peak (N=3 independent experiments, one-way ANOVA with Tukey post-hoc test for multiple comparisons). Bars indicate mean and S.E.M in dot plots or median and quartiles in violin plots; ∗ – p < 0.05, ∗∗ – p < 0.01, ∗∗∗ – p < 0.001 and n.s – not significant.

Article Snippet: For CDM supplementation experiments, pre-formed CDMs on coverslips were overlaid with neutralized COL1 (5005, Advanced Biomatrix) or COL6 (009-001-108, Rockland Immunochemicals) solutions (250 μg/ml in PBS), incubated for 24 h at 37°C, and subsequently processed for immunofluorescence staining.

Techniques: Imaging, Derivative Assay, Generated, Staining

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: For CDM supplementation experiments, pre-formed CDMs on coverslips were overlaid with neutralized COL1 (5005, Advanced Biomatrix) or COL6 (009-001-108, Rockland Immunochemicals) solutions (250 μg/ml in PBS), incubated for 24 h at 37°C, and subsequently processed for immunofluorescence staining.

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

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) 17-plex SeqIF analysis for COL6 and the spatial distribution of immune cell populations in ccRCC was applied on 15 cores from 7 cancer cases. Representative images show IF stainings of indicated cell and compartment markers. Arrows indicate representative cell types: light green – CD3+CD8- (A-1), purple – CD3+CD8+ (A-1), yellow – CD3+CD8+PD1+ (A-1), light orange – KI67+ (A-2), red – CD4+ (A-4), brown – CD4+FOXP3+ (A-4), dark blue – CD20+ (A-4), dark green – PDL-1+ (A-5), dark red – CD56+ (A-5), orange – CD68+ (A-4). (B) UMAP of major segmented cell populations (53 102 cells, tumor cells – pink, myeloid cells – red, lymphocytes – blue and mesenchymal cells – green). (C) Log 2 density of indicated immune cell population in analyzed samples (dots indicate N=15 cores in graphs C- K). Percent numbers indicate the mean density of CD3+CD4+FOXP3+ cells of total CD3+CD4+ cells and CD3+CD8+PD1+ cells of total CD3+CD8+ cells. (D) Correlation analysis of whole tumor immune cell densities with COL6 expression (Pearson correlation). (E) Relative distribution of CD3+ cells to the COL6+ stromal compartment, peristromal boundary zone (COL6 boundary) and the remaining cancer cell compartment (cancer) in ccRCC. (F ) Absolute density of CD3+ cells in indicated compartments (N=15, RM one-way ANOVA with Geisser-Greenhouse correction and Tukey’s multiple comparison test is used in graphs F, G, I and J). (G) Absolute density of CD3+CD8+ cells in the indicated compartments. (H) Correlation analysis of absolute CD3+CD8+ cell density in the cancer compartment with tumor COL6 expression (Spearman correlation). (I) Absolute density of CD3+CD8+PD1+ cells in the indicated compartments. (J) Percentage of CD3+CD8+PD1+ cells of total CD3+CD8+ cells in indicated compartments. (K) Relative distribution of CD3+CD8+PD1+ cells to indicated compartments. (L) Scanning electron microscopy (SEM) images of peripheral blood mononuclear cells (PBMCs) seeded on shCTRL- and shCOL6-CDMs after incubation for 6 hours. (M) IF images of T cells incubated on shCTRL- and shCOL6-CDMs stained for DNA (blue), FN (green), and F-actin (white). T cells incubated on shCOL6-CDMs exhibit an increased formation of protrusions (white arrow). (N) Quantification of T cell morphology based on cell area and cell perimeter, after incubation with respective CDMs (Violin plots of analyzed cells of N=4 independent experiments, Mann-WhitneyLULtest). (O) Quantification of transwell migration of PBMCs through CDMs synthesized by shCOL6 and shCTRL fibroblasts (N=7 replicates of 4 independent experiments, paired t-test). (P) SeqIF analysis of an immune checkpoint inhibitor (ICI – Nivolumab & Ipilimumab) treated ccRCC patient sample. Representative images of regions with vital cancer, immune infiltration and cancer necrosis, and residual fibrosis are shown (markers as indicated). (Q) Quantification of COL6 positive area in the vital cancer compartment of whole tumor section of 3 ICI-treated patients and 10 control ccRCCs (dots indicate individual patients analyzed, Mann-WhitneyLULtest). Bars indicate mean and S.E.M in dot plots or median and quartiles in violin plots; ∗ – p < 0.05, ∗∗ – p < 0.01, ∗∗∗ – p < 0.001, ∗∗∗∗ – p < 0.0001 and non-significant (n. s.).

Article Snippet: For CDM supplementation experiments, pre-formed CDMs on coverslips were overlaid with neutralized COL1 (5005, Advanced Biomatrix) or COL6 (009-001-108, Rockland Immunochemicals) solutions (250 μg/ml in PBS), incubated for 24 h at 37°C, and subsequently processed for immunofluorescence staining.

Techniques: Expressing, Comparison, Electron Microscopy, Incubation, Staining, Migration, Synthesized, Control

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 ) Survival analysis for high or low expression of COL6A1 and COL6A2 in the CheckMate 025 study cohort of Nivolumab (anti-PD-1) or everolimus (mTOR inhibitor) treated patients . Graphs show Kaplan-Meier plots and Log-rank (Mantel-Cox) tests. (B-D) Analysis of COL6 deposition in CDMs treated with cabozantinib (Cabo.), sunitinib (Suni.), temsirolimus (Temsi.), or DMSO (Ctrl.) treated TK173 cells. (B) Dot plot depicting relative median fluorescence intensities (MFI) of COL6 in CDMs of respectively treated cells in the screening experiment (N=10 regions of interest, one-way ANOVA with Tukey’s multiple comparison test). (C) Dot plot depicting mean relative MFI of COL6 in CDMs of respectively treated TK173 cells (dots indicate mean of N=4 independent experiments, unpaired t test). (D) IF images stained for FN (green) and COL6 (violet) of synthesized CDMs of TK173 cells treated with the indicated drugs. (E) Schematic description of RNA sequencing analysis of cabozantinib or DMSO control-treated TK173 fibroblasts and acute organotypic slice cultures (OTSCs) of ccRCCs (Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y ). (F&G) Volcano plot of differential gene expression analysis of matrisome genes in cabozantinib-treated TK173 cells and ccRCC OTSCs. The inserts depict a volcano plot of all genes. Red dots indicate significantly regulated genes with adjusted p<0.01 and log 2 fold change (FC) > |1|). (H) Heatmap analysis of significantly regulated matrisome genes in cabozantinib-treated TK173 cells and ccRCC OTSCs (genes with adjusted p<0.0001 and fold change > |2| are depicted for TK173 cells or with adjusted p<0.01 and fold change > |1.5| for OTSCs). (I) Heatmap analysis of log 2 expression fold changes (FC) of immunomodulatory genes in TK173 cells and ccRCC OTSCs (N/A – not announced). (J&K) IF validation of COL6 regulation in cabozantinib-treated ccRCC OTSCs. Images show IF staining for Nuclei by Hoechst (blue), CK (yellow) and COL6A1 (violet) of OTSCs treated with the indicated drugs. Quantification of COL6A1-positive areas in OTSCs of 10 ccRCC patients (dots indicate individual patients analyzed, ratio paired t test). (L) Schematic summary illustrating the findings of the study, highlighting COL6 expression in tumor stroma as well as the remodeling of the ECM architecture in dependence of COL6 abundance and the subsequent implications for cancer and immune cells (Created in BioRender. Schell, C. (2026) https://BioRender.com/x5sqb3y ).

Article Snippet: For CDM supplementation experiments, pre-formed CDMs on coverslips were overlaid with neutralized COL1 (5005, Advanced Biomatrix) or COL6 (009-001-108, Rockland Immunochemicals) solutions (250 μg/ml in PBS), incubated for 24 h at 37°C, and subsequently processed for immunofluorescence staining.

Techniques: Expressing, Fluorescence, Comparison, Staining, Synthesized, RNA Sequencing, Control, Gene Expression, Biomarker Discovery