Fig 1: Schematic representation shows the mechanism through which PBRM1MUT ccRCC cells modulate tumor microenvironment and promote ccRCC progression. The PBRM1 mutant ccRCC cells secrete CCL5 cytokines that promote mast cell recruitment into the TME. The mast cells secrete several factors such as VEGFA, VCAM1, and PDGFA that stimulate angiogenesis. The mast cells also reduce the infiltration of CD8+ T cells and CD4+ T cells. Simultaneously, PBRM1 mutations facilitate tumor cell growth by activating intrinsic HIF signaling pathways. The complex interactions between the mast cells, epithelial cells, T cells, and ccRCC tumor cells in the TME are aided by several cytokines and chemokines that are secreted by these cells regulates tumor progression.
Fig 2: PBRM1-silenced ccRCC cells recruit significantly higher numbers of mast cells in vitro. (A) qRT-PCR and (B) Western blot analysis shows PBRM1 mRNA and protein levels in control and PBRM1-silenced 786-O and Caki-1 cells. (C, D) Transwell migration assay results show the total numbers of migrating HMC-1 cells when co-cultured with control and PBRM1-silenced 786-O and Caki-1 cells or the conditioned media from these cells. The migrating HMC-1 cells are stained with crystal violet and counted. The experiments were performed in triplicate and the results are shown as means±SD. Student’s t-test was used to determine statistical significance.
Fig 3: The relationship between PBRM1 protein expression and mast cell infiltration in ccRCC based on IHC analysis. (A) Representative immunohistochemical images show PBRM1- and tryptase-positive mast cells in ccRCC and adjacent normal kidney tissue samples. (B) Dot plot of PBRM1 IHC staining score in adjacent normal kidney tissues (n=83) and ccRCC tissues (n=83). (C) Overall survival of ccRCC patients with PBRM1 IHC staining negative group (n=65) or PBRM1 IHC staining positive group (n=20). (D) Pearson correlation analysis shows the association between PBRM1 expression and mast cell infiltration in 85 out of 90 ccRCC patient tumor tissue samples. Data for five tumor tissues is not included (missing the tissues in TMA). Note: Statistical significance was based on Student’s t-test.
Fig 4: Correlation analyses between gene cluster modules, PBRM1 mutations, and mast cell infiltration in ccRCC patients. (A) The clustering dendrogram shows different gene cluster modules that are color-coded. The dissimilarity of genes is based on the topological overlap. (B) Heatmap shows the correlation between module eigengenes and immune cell infiltration in ccRCC samples. The correlation table is color-coded. The modules in the blue box are associated with PBRM1 mutations and mast cell infiltration. (C) Analysis of the association between the 27 gene cluster modules and the 4 mutant genotypes (VHL, PBRM1, SETD2, and BAP1) in ccRCC patients. Each cell represents a module correlation co-efficient and its corresponding p-value. (D) Pathway enrichment analysis of dark orange module. Dark orange gene cluster was positive with PBRM1 mutant and mast cell infiltration. (E) Pathway enrichment analysis of the white module. White gene cluster was positive with PBRM1 mutant and mast cell infiltration. (F) Enrichment plots show upregulated FARDIN hypoxia signaling (red), MENSE hypoxia signaling (green), MIZUKAMI hypoxia signaling (green), PID-HIF1-THPATHWAY (purple), PID-HIF2-PATHWAY (blue), and other gene sets in the PBRM1mut group of ccRCC patients. FARDIN hypoxia signaling gene set including the genes in the hypoxia signature, based on analysis of 11 neuroblastoma cell lines in hypoxia and normal oxygen conditions; MENSE hypoxia signaling gene set including hypoxia response genes up-regulated in both astrocytes and HeLa cell line; MIZUKAMI hypoxia signaling gene set including the genes up-regulated in colon cancer cells in response to hypoxia, might not be direct targets of HIF 1α; PID-HIF1-THPATHWAY gene set including the gens in HIF 1α transcription factor network; PID-HIF2-PATHWAY gene set including the gens in HIF 2α transcription factor network.
Fig 5: PBRM1 silencing enhances tumor angiogenesis and promotes cell proliferation of RCC cells in vitro. (A) Matrigel tube formation assay results show the tube-like structures formed in the matrigel by CM from control and PBRM1-silenced 786-O and Caki-1 cells. (B) MTT assay results show viability of control and PBRM1-silenced 786-O and Caki-1 cells. (C) The colony formation assay results show the total numbers of colonies formed by control and PBRM1-silenced 786-O and Caki-1 cells based on crystal violet staining. (D) Flow cytometry analysis shows the percentage of G1, S, and G2-M cells in control and PBRM1-silenced 786-O and Caki-1 cells based on PI staining. Note: The experiments were performed in triplicate and data are represented as means±SD; the statistical analysis was performed using Student’s t-test.
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