Panel A: The panel shows four bar graphs depicting the absolute number of different cell types in tumor tissue along with a diagram. The cell types are myeloid cells, granulocytes, monocyte-derived macrophages, and tumor-associated macrophages. The x-axis represents different experimental conditions (WT, WT plus TNF alpha, TNFRKO, TNFRKO plus TNF alpha), and the y-axis represents the absolute number of cells per milligram. Each dot represents data from an individual tumor, with statistical comparisons indicated by p-values. Panel B: Similar to Panel A, this panel shows four bar graphs for a different tumor model (EO771 variants). The x-axis and y-axis are the same as in Panel A. Panel C: A bar graph shows the density of CD11b plus myeloid cells in tumors, either injected or not with TNF. The x-axis represents different experimental conditions, and the y-axis represents the number of cells per square millimeter. Statistical comparisons are performed by the Mann-Whitney U test. Panel D: Representative microscopy images show the density of CD11b plus myeloid cells in tumors, with a scale bar of 50 micrometers. Panel E: A volcano plot represents the main differentially expressed genes in CD11b plus tumor-infiltrating myeloid cells from TNFR1KO or WT EO771 tumors. The x-axis represents the log fold change, and the y-axis represents the negative log of the p-value. Panel F: A box plot compares the myeloid suppressive signature in mRNA sequencing data sets from sorted CD11b plus cells. The x-axis represents TNFR1KO and TNFR1.WT conditions, and the y-axis represents the enrichment score. Statistical comparisons are evaluated by the Mann-Whitney U test.
TNFR1KO cells recruit fewer MDSCs into the tumor tissue microenvironment. (A) Schematic representation of the experimental workflow in mice bearing tumors derived from either MC38 or MC38 TNFR1KO variants. Tumors were intratumorally injected with rTNFα as indicated and excised on day 10. Cell suspensions derived from such tumors were analyzed by multicolor flow cytometry to quantify the density of neutrophils (Gr-MDSC) and macrophages (M-MDSC). Each dot represents data from an individual tumor (n = 12–14). A t test was performed for statistical comparisons. (B) Experiments as in A with the EO771 variants (n = 12–14). A t test was performed for statistical comparisons. (C) Analysis by multiplex tissue immunofluorescence of the density of CD11b+ myeloid cells in the tumors, either injected or not with TNFα (n = 6–8). Statistical comparisons were performed by the Mann–Whitney U test. (D) Representative microscopy images from C (Scale bar: 50 µm). (E) CD11b+ tumor-infiltrating myeloid cells from TNFR1KO or WT EO771 tumors that were FACS-sorted and mRNA sequenced in triplicate. The volcano plot represents the main differentially expressed genes. (F) Comparison of myeloid suppressive signature (Ido1, Cd274, Lgals9, Tgfb1, Il10, Entpd1, Arg1, Vegfa, Il4ra, and Pges) in the mRNA-seq datasets from the sorted CD11b+ cells in E. Differential statistics were evaluated by the Mann−Whitney U test. P < 0.001 (***).