Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A) Flow cytometry analysis of melanoma antigen panel for MHC-I, MHC-II and PD-L1 in control, responder, and treatment escape groups. Tumor cells were normalized to SOX10 + cells, n=3/group. (B) Immunohistochemistry (IHC) staining and quantification of MHC-I and PD-L1 expression in control, responder, and treatment escape groups, n=3/group. H-score was used to access MHC-I expression and the percentage of positive cells for PD-L1. (C) Flow cytometry analysis of T cell panel (number of infiltrated CD4 + and CD8 + T cells, activated CD4 + /CD69 + and CD8 + /CD69 + T cells, and memory CD44 + /CD62L − /CD4 + and CD44 + /CD62L − /CD8 + T cells) of control, responder, and treatment escape groups. Cells were normalized to the total number of live cells. (D) Flow cytometry analysis of Foxp3 + CD4 + T cells (Tregs) in control, responder, and treatment escape groups. Tregs were normalized to the total number of CD4 + cells. (E) Immunohistochemistry staining and quantification of CD8, CD69 and PD-1 expression in control, responder, and treatment escape groups. n=3/group. H-score was used to access CD8 and CD69 expressions and the percentage of positive cells for PD1, according to the best fit suggested by the software. (F) Flow cytometry analysis of exhaustion markers (PD-1, TIM-3, LAG-3 and CTLA-4) in control, responder, and treatment escape groups. T cells were normalized to the total number of live cells. Scale bar = 200 μm (G) Multiplex immunofluorescence (IF) staining of control, responder, and treatment escape tumors for DAPI, CD4, CD8, CD11b, CD11c, Ly6G, Ly6C, CD34 and SOX10 expression (H) Scoring of the multiplex IF images of the whole slides. (I) Degrees of clustering of CD4 + or CD8 + T cells versus M-MDSCs (CD11b + Ly6C + Ly6G − ) and PMN-MDSCs (CD11b + Ly6C − Ly6G + ) of control, responder, and treatment escape groups, n=3/group. Cells were normalized by the number of total cells. The graphs are represented as average ± SEM. One-way ANOVA was performed followed by Tukey’s multiple comparisons test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns: non-significant; C: control (not treated cells); R: responder; TE: treatment escape.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Flow Cytometry, Control, Immunohistochemistry, Expressing, Staining, Software, Multiplex Assay, Immunofluorescence

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A-B) Tumor growth and survival curves following pre-treatment with IgG control (n=5) or anti-CD4 + anti-CD8a +/− RMC-7977 (all n=15). Vehicle or RMC-7977 treatment was initiated when tumors reached around 100mm 3 . For T cell depletion, mice were treated 3 days before the injection of the tumor with the Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P.) and then every 4 days thereafter. The T cell depleted mice were treated with Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P., every 4 days), Anti-CD4 + Anti-CD8a + RMC-7977 was treated with Anti-CD4 and Anti-CD8a antibodies (1µg/µL, I.P., every 4 days) and RMC-7977 (25 mg/kg, P.O., q.d.); and RMC-7977 group was treated with only RMC-7977 (25 mg/kg, P.O., q.d.) for 7 weeks. Plots correspond to each individual mouse. Dotted lines indicate when treatment started and stopped.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Control, Injection

Journal: Cancer immunology research

Article Title: RAS(ON) multi-selective inhibition drives antitumor immunity in preclinical models of NRAS -mutant melanoma

doi: 10.1158/2326-6066.CIR-25-0744

Figure Lengend Snippet: (A-B) The effects of IgG control, anti-PD-1, RMC-7977 or anti-PD-1+RMC-7977 on the growth of OSUMMER.13 tumors. Treatment was initiated when tumors reached around 100mm 3 . The IgG group was treated with IgG control (2µg/µL, I.P., every 5 days: n=5), the vehicle group was treated with vehicle (P.O., q.d.: n=5), the vehicle + IgG group was treated with IgG control (2µg/µL, I.P., every 5 days: n=5) and vehicle (P.O., q.d.: n=5), total of n=15M for controls, the anti-PD-1 group was treated with Anti-PD-1 (2µg/µL, I.P., every 5 days: n=7), the RMC-7977 group was treated with RMC-7977 (25 mg/kg, P.O., q.d.: n=7), and the RMC-7977 + anti-PD-1 group (n=7) was treated with a combination of RMC-7977 and Anti-PD1 as indicated for 7 weeks. Plots correspond to each individual mouse. (C) Immunohistochemistry and quantification of pERK, Ki67, Melan-A, CD8, CD69, PD-1 and MHC-I of control (vehicle), anti-PD-1, RMC-7977 and RMC-7977 + anti-PD-1 group, n=3/group. H-score was used to access pERK, Ki67 and MHC-I expressions and the percentage of positive cells for melan-A and PD1, according to the best fit suggested by the software. Scale bar = 200 μm (D) Flow cytometry analysis of the T cell panel (number of infiltrated CD4 + and CD8 + T cells, activated CD4 + /CD69 + and CD8 + /CD69 + T cells, and memory CD44 + /CD62L − /CD4 + and CD44 + /CD62L − /CD8 + T cells) of vehicle, anti-PD1, RMC-7977 and the RMC-7977 + anti-PD-1 groups. The graphs are represented as average ± SEM. One-way ANOVA was performed followed by Tukey’s multiple comparisons test: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. ns: non-significant. Dotted lines indicate when treatment started, stopped and when rechallenge was carried out.

Article Snippet: For CD4 + and CD8 + T-cell depletion, specific antibodies (anti-CD4, clone YTS191; cat. #BE0003–1; BioXCell, RRID:AB_1107636 and anti-CD8a, clone YTS169.4, cat. #BE0117, BioXCell, RRID:AB_10950145) were used.

Techniques: Control, Immunohistochemistry, Software, Flow Cytometry