Fig 2: SOX10 expression is correlated with CD8+ T-cell infiltration and CEACAM1 levels(A) In vivo growth comparison between the pBABE control (n = 5) and the pBABE-SOX10 overexpressing (n = 5) YUMM1.7 cell lines in C57 immune-competent mice.(B) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE and pBABE-SOX10 tumors at endpoint.(C) Comparison of tumor size 24 days post injection between pBABE control (n = 4) and the pBABE-SOX10 overexpressing (n = 5) YUMM1.7 cell lines in RAG1 mice.(D) Flow analysis showing the proportion of CEACAM1 positive cells in various YUMM cell lines compared to isotype control.(E) Flow cytometric staining of SOX10 and CEACAM1 in OHRI-13 and SK-MEL28 human melanoma cell lines. Percentage of each group is indicated within their quadrant.(F) Flow cytometric staining of SOX10 and CEACAM1 in the YUMM1.1, YUMM1.7 and YUMM2.1 murine cell lines. Percentage of each group is indicated within their quadrant. Graphs show the mean ± SEM. **p < 0.005 by two-tailed t test (B).

Fig 3: SOX10 directly activates Ceacam1 on a distal enhancer region(A) YUMM1.1 cells were treated with siRNA at 200nM targeting Sox10 or a non-targeting control for 72 h and Sox10 knockdown was assessed by Immunoblot.(B) 72 h post siRNA transfection cells were isolated and stained for CEACAM1. Representative flow plots were generated with indicated percent CEACAM1+ cells.(C) qPCR was used to compare Sox10 knockdown efficiency with Ceacam1 isoform levels of the cells in (A).(D) YUMM1.7 pBABE and YUMM1.7 pBABE-SOX10 cells were collected and analyzed by qPCR for Sox10 and Ceacam1 isoforms.(E) YUMM1.7 pBABE and YUMM1.7 pBABE-SOX10 cells were isolated and stained for CEACAM1. Representative flow plots were generated with indicated percent CEACAM1+ cells.(F) Luciferase activity measurements from various Ceacam1 promoter fragments in YUMM1.1 and 2.1 cells.(G) RT-PCR of SOX10 ChIP in YUMM1.1 cells from various regions within the CEACAM1 -4135/-2785 fragment showing the fold enrichment over IgG. The red line represents the IgG control set to one. Data is representative of three independent experiments (C, D, F, and G). Graphs show the mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (C and D) or by two-tailed t test (F).

Fig 4: CEACAM1 decreases CD8+ T-Cell infiltration and promotes tumor growth(A) Overlaid flow profiles representing the YUMM1.7 cells stably expressing CEACAM1 Isoform 1 or 4.(B) Quantitation of primary and secondary sphere formation in SOX10-deficient YUMM1.7 cells stably expressing CEACAM1 and controls.(C) Representative images of both primary and secondary spheres from the assays in (B) (Scale bar = 100µM).(D and E) In vivo growth comparison between the pBABE controls (n = 5), the CEACAM1 iso1 (n = 5) and iso4 (n = 4) expressing YUMM1.7 cell lines in NCG immune compromised mice (D) and C57 immune-competent mice (E).(F) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE and pBABE-CEACAM1 tumors at endpoint. Data is representative of three independent experiments (B). Graphs show the mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001 by one-way ANOVA (B and F).