Fig 1: Gene expression and somatic mutations (deleterious, slice, missense/inframe or silent) in the genes BRAF, NRAS, NF1, PTEN, IDO1 and HLA-DRA in skin melanoma. The crystal structures (3D) of the genes’ encoded proteins along with their somatic mutations detected in the TCGA-SKCM dataset (purple color), were calculated using MuPIT (hg38 coding) and are depicted on the right of each plot. Hotspot BRAF and NRAS mutations are highlighted in red color in the corresponding crystal structures. Apart from BRAF, NRAS, NF1 and PTEN, all of which are well-known to be recurrently mutated in skin melanoma, IDO1 and HLA-DRA were also significantly mutated, but the somatic mutations did not seem to affect their protein expression.
Fig 2: The expression of CD8, PD-1, CTLA-4, IDO1, LAG3, HAVCR2, TIGIT, ILT2 and ILT4 was significantly higher in skin melanomas; whereas, C10orf54 (VISTA) and VTCN1, were expressed at markedly lower levels in the tumor samples compared to normal skin samples. Red asterisks (*) denote significant differences (|log2FC>1| and p<0.01) between skin melanomas from the TCGA-SKCM dataset and matched normal samples from TCGA and GTEx. One-way ANOVA, using disease state (skin melanoma or normal sample) was used to calculate differential expression. The expression data were first log2(TPM+1) transformed for differential analysis and the log2FC was defined as median (skin melanoma) - median (normal skin).
Fig 3: OXA treatment induces upregulation of IDO1 in patient-derived colorectal cancer cells(A) Gene expression in patient-derived colorectal cancer cells post-OXA treatment. Ten colorectal cancer samples obtained from patients underwent an enzymatic digestion, and digested single cells were cultured in complete DMEM medium containing 50 µM OXA. 2 days later, cells were harvested for the examination of gene expression. The mRNA expressions of genes associated with immunosuppression (including PD-1, PD-L1, CTLA-4, STAT3, CD39, CD73, IDO1, and CSF-1R) were examined by real-time PCR. GAPDH was detected as a control. (B) The protein level of IDO1 in three patient-derived colorectal cancer cells post-OXA treatment was examined by western blot. ß-actin was used as a loading control. (C) IDO1 expression in the CT26 cell after OXA treatment. Data represent mean ± SD. ***p < 0.001.
Fig 4: (A) Indicative immunohistochemistry (IHC) staining for the inhibitory receptors IDO1, PD-L1, PD-1, LAG3, CD8A/B (marker for cytotoxic T-cells) and FOXP3 (marker for Tregs) in an independent cohort of 11 cutaneous melanomas. H&E, hematoxylin and eosin staining. (B) Overall, the protein expression of these markers was either not detected (ND) or low and probably did not differ between TMBhigh and TMBlow tumors. (C) Immune-cell fractions across TMBhigh, TMBint and TMBlow skin melanomas, using extracted data (quanTIseq) from The Cancer Immunome Database. (D) The scatterplots depict the percentage of lymphocytes (%), average number of TIL patches and clusters (with standard deviation, SD) in TMBhigh (>30 mut/Mb) and TMBlow (<7.4 mut/Mb) skin melanomas. Neither of these differed significantly between the two subgroups of tumors. (E) The expression of CD8A (log2(TPM+1)) did not correlate with the neoantigen load in either TMB subgroup. Expression of CD8A, PDCD1 (PD-1), CD274 (PD-L1), PDCD1LG2 (PD-L2), IDO1 and CTLA-4 across different immune (F) and molecular (G) subtypes in skin melanoma. Immune subtypes: C1, wound healing (n=41); C2, IFN-gamma dominant (n=27); C3, inflammatory (n=14); C4, lymphocyte depleted (n=19); C5, immunologically quiet (n=0); C6, TGF-b dominant (n=2). Molecular subtypes: BRAFmut (n=150), NF1mut (n=27), RASmut (n=92), tripleWT (n=46).
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