Fig 2: Carcinogen-induced immunogenicity is dependent on reduced M-CSF and CD155 expression by cancer cells.(A) Cytokine array on supernatant from DMBA3-4 and DMSO3-1 cells. Red and blue boxes indicate the upregulated and downregulated proteins secreted by DMBA3-4 compared with DMSO3-1 cells, respectively. (B) Relative levels of the select upregulated and downregulated proteins from the DMBA3-4/DMSO3-1 cytokine array (n=2 per group). (C) M-CSF protein levels in DMBA3-4 compared with DMSO3-1 cell lysates (n = 9 per group). (D) Csf1 mRNA expression levels in DMBA3-4 compared with DMSO3-1 cells (n = 7 per group). (E) BMDM migration toward DMBA3-4 versus DMSO3-1 cells in the presence of anti-CSF1R or IgG control antibody. Fold change is determined as the ratio of BMDM migration in the absence of tumor cells at 96 hours after coculture (n=7 per group). (F) CD155, CD112, PD-L1 and PD-L2 expression on DMBA3-4 and DMSO3-1 cells. Numbers on the flow histograms represent the ligands’ MFI. (G) DMBA3-4 plus DMSO3-1 mixed tumor growth in WT mice treated with anti-TIGIT and/or anti-CSF1R antibody compared with IgG-treated controls (n = 10 per group). Mice received 450,000 DMBA3-4 plus 50,000 DMSO3-1 cells per injection site. (H) Survival rate of WT mice that received DMBA3-4 plus DMSO3-1 cells and treated with anti-TIGIT and/or anti-CSF1R antibody compared with IgG-treated controls (n = 10 per group). Unpaired t test (C–E), 2-way ANOVA (G) and log-rank test (H), bar graphs show mean ± SD.

Fig 3: Pro-lymphatic differentiation coincides with IL-10 dependent shift to M2 phenotype.(A) Representative histograms demonstrating expression of M2-specific myeloid markers CD163, CD209, and PD-L1 in freshly-isolated BM cells (Ex-vivo, blue line) and differentiated cells (CSF-1 + LPS, red line). Numbers in black font indicate the percentage of cells expressing an M2-specific marker. (B) The mean percentage of positive cells and (C) MFI (x103) of M2 marker expression in ex-vivo and differentiated cells. (D) Histograms demonstrating the expression of CD204 in CSF-1/LPS differentiated cells derived from wild-type C57BL/6 mice in the presence of rat isotype-matched control (left panel) or anti-IL-10R antibody (center panel). The right panel shows CD204 expression in identically differentiated cells from IL-10R knockout (KO) mice. Secondary controls are shown by the grey area outlined by black line. Transcriptional analysis determined by qPCR on day 6 of differentiation of anti-IL-10R antibody-treated BM cells compared with rat control antibody, and BM cells from IL-10R-/- compared with those from WT mice. Analyzed markers included those for (E) M1 phenotype, (F) immunosuppressive regulators, (G) Th2 regulators, and (H) M2 phenotype. Expression in WT cells in the absence or presence of control rat IgG was taken as 1. Stars represent statistical significance determined by Student’s t-test with P-values indicated by *<0.05, **<0.01, and ***<0.001. All experiments were performed in duplicate and reproduced at least twice.

Fig 4: CSF-1/TLR4 induced differentiation promotes endothelial progenitors with predominant lymphatic phenotype.(A) Schematic of the protocol used to differentiate BM cells by sequential activation of CSF-1 and TLR4 pathways. (B) Representative histograms demonstrating expression of lymphatic specific targets Vegfr-3, Colec12, Itga9, Lyve-1, and Pdpn in freshly-isolated BM cells (upper panel, Ex-vivo, blue line) and cells differentiated with CSF-1 and LPS (lower panel, red line). Numbers pointing to blue and red lines indicate the percentage of marker-positive cells. Secondary controls are shown by the grey area outlined by black line in each panel. (C) The mean percentage of positive cells and (D) MFI (x103) of BM cells expressing LEC markers. (E) Representative histograms demonstrating the percentage of Ex-vivo (blue line) and differentiated (red line) cells positive for blood vascular endothelial markers Vegfr-1, CD105, and Vegfr-2. Secondary controls are shown by the grey area outlined by black line in each panel. (F) The mean percentage of positive cells and (G) MFI (x103) of various endothelial markers in ex-vivo and differentiated cells. Black bars indicate statistical significance determined by Student’s t-test with P-values shown by *<0.05, **<0.01, and ***<0.001. All experiments were performed in duplicate and reproduced three times.

Fig 5: IL-10 pathway regulates transcription of specific markers of multiple lineages in BM myeloid precursors.Marker expression levels in CSF-1/LPS differentiated BM cells from WT mice in the presence of anti-IL-10R antibodies and from IL-10R KO mice were compared with the levels in WT cells treated with a control antibody. Cells harvested on the sixth day of differentiation were analyzed by RT-qPCR for mRNA expression of markers of (A) lymphatic-specific, (B) endothelial, (C) myeloid, (D) erythroid, (E) T-cell, and (F) B-cell lineages. All analyses were performed in triplicate for each target, normalized to β-actin, and independently reproduced twice. Data for each target are reported as the mean fold-change in treated cells compared with the WT cells treated with control rat IgG ± S.D. Statistical significance of expression changes compared with control group was determined by Student’s t-test with P-values indicated by *<0.05, **<0.01, and ***<0.001.