Panel A: A scatter plot shows the fold change in neighborhood MDSC recruitment in TNFR1-high versus TNFR1-low tumor niches for colon and breast cancer. The x-axis represents different cancer types, and the y-axis represents the fold change. Panel B: A scatter plot displays the odds ratio of TNFRSF1A detection in MDSC-rich tumor neighborhoods for individual patient samples from colon and breast cancer. The x-axis lists patient samples, and the y-axis shows the odds ratio. Panel C: A heatmap illustrates the upregulation of various chemokines in TNFR1-high tumor niches compared to TNFR1-low niches. The x-axis lists different chemokines, and the color scale indicates the level of upregulation. Panel D: A bar graph shows the mRNA expression levels of TNFR1 in wild-type HT29 cells and four TNFR1 knockout clones. The x-axis lists the cell types, and the y-axis shows the expression levels. Panel E: A bar graph depicts IL-8 concentration levels after TNF stimulation in wild-type and TNFR1 knockout HT29 cells. The x-axis lists the cell types, and the y-axis shows the IL-8 concentration. Panel F: A bar graph shows TNF gene expression levels relative to RPLO mRNA levels in wild-type and TNFR1 knockout cultured variants. The x-axis lists the cell types, and the y-axis shows the relative expression levels. Panel G: A schematic diagram and bar graphs illustrate an experiment where NSG immunodeficient mice were xenografted with either HT29 wild-type or TNFR1 knockout variants, and tumors were injected with rTNF. The bar graphs show IL-8 concentrations in tumor tissue homogenate fluid and plasma. The x-axis lists the experimental groups, and the y-axis shows the IL-8 concentration. Panel H: A schematic diagram represents an experiment where immunodeficient Rag2-/-IL-2R-/- mice were xenografted with HT29 tumors, either wild-type or TNFR1 knockout. Panel I: A bar graph shows IL-8 concentrations in the culture supernatants of tumor fragments set in culture with or without rhTNF. The x-axis lists the experimental groups, and the y-axis shows the IL-8 concentration. Panel J: A bar graph shows TNF transcript levels assessed by quantitative RT-PCR in the cell pellets of the cultured tumor fragments. The x-axis lists the experimental groups, and the y-axis shows the relative expression levels.
Human evidence for a proinflammatory immunosuppressive role of TNFR1 expressed by malignant cells. (A) Bioinformatic analyses of digital spatial transcriptomics (Visium) of tumor niches in a series of colon and breast cancer analyses showing that tumor TNFR1-enriched areas express more prominently transcripts associated with MDSCs. (B) Patient-by-patient sample analyses show that MDSC-enriched tumor niches show more TNFR1 expression in malignant cells. A one-sided Mann–Whitney U test was performed. (C) Correlations of the transcriptional expression of the indicated inflammatory mediators with respect to TNFR1 expression in the tumor niches (Wilcoxon test). (D) mRNA expression of TNFR1 quantified by quantitative RT-PCR in WT HT29 cells and four CRISPR/Cas9 TNFR1KO clones. (E) IL-8 concentration levels after TNFα stimulation in WT and TNFR1KO HT29 cells, showing that the induction of IL-8 is lost in the TNFR1KO variants (n = 6–12). (F) TNFα gene expression levels relative to RPLO mRNA levels quantified by quantitative RT-PCR in WT and TNFR1KO cultured variants, showing that the induction of TNFα transcripts by human rTNFα is lost in the TNFR1KO variants (n = 6). (G) NSG immunodeficient mice were subcutaneously xenografted with either HT29 WT or TNFR1KO variants, and tumors were injected with or without rTNFα, as indicated. 9 days after tumor-cell inoculation, tumors were excised, and IL-8 concentrations (mean + SEM and t test for statistical comparisons) were assessed in the tumor tissue homogenate fluid (left) and in plasma (right) (n = 10). (H) Schematic representation of experiments in which immunodeficient Rag2−/−IL-2Rγ−/−mice were xenografted with HT29 tumors, either HT29 WT or HT29 TNFR1KO. 10 days after engraftment, tumors were excised, and small tumor fragments (approx. 1 mm × 1 mm) were set in culture as shown in the scheme, in the presence of either medium alone or rhTNFα. (I and J) IL-8 concentrations were monitored in the culture supernatants (I), and TNFα transcripts were assessed by quantitative RT-PCR in the cell pellets (J) (n = 8–9). P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).