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. 2023 Nov 13;41(11):1911–1926.e8. doi: 10.1016/j.ccell.2023.09.001

Figure 2.

Figure 2

Analysis of the role of TIM-3 in the tumorigenesis of human and murine DIPG cell lines

(A) Western blot analysis of TIM-3 expression in DIPG human and murine cell lines, using GRB-2 as a housekeeping protein.

(B) Viability assay (MTS) of TP54 and DIPG007 cells 7 days after TIM-3-specific shRNA knockdown.

(C) Viability assay (MTS) of TP54 and DIPG007 cells 7 days after treatment with a human anti-TIM-3 antibody (provided by BMS, BMS-986258).

(D) Western blot analysis of TIM-3 expression in control guide NP53 and TIM-3KO (G2C1, G2C9, and G1C7) NP53 different cell lines.

(E) Clonogenicity assay with control guide NP53 cells versus three different TIM-3KO NP53 clones at 2 weeks.

(F) Evaluation of the proliferative capacity by a BrdU assay performed with TIM-3KO clones normalized to control guide cells (mean ± SEM) (G) Apoptosis analysis of TIM-3KO cells compared to control guide cells. Viable (Annexin V−7AAD−), early apoptosis (Annexin V+7AAD−), and late apoptosis (Annexin V+7AAD+).

(H) Viability assay (MTS) of NP53 control guide, G2C9 and G1C7 TIM-3KO cells 7 days after treatment with galectin-9 and CEACAM1 (TIM-3 main ligands).

(I) Kaplan-Meier survival curves of immunocompetent mice bearing control guide NP53 cells (n = 6), G2C9 TIM-3KO cells (n = 5, log rank; p = 0.0012), or G1C7 TIM-3KO cells (n = 5, log rank; p = 0.0012).

(J) Gene pathways enriched in the set of genes with differential expression between TIM-3+ and TIM-3KO cells. Results obtained from RNA-seq data.

(K) Heatmap of the differentially expressed genes between TIM-3+ and TIM-3KO cells related to the MAPK and PI3K-AKT pathways. Results were obtained from previous RNA-seq data.

(L) Western blot analysis of PTEN, MEK1, pMEK, ERK1/2, pERK1/2, and c-JUN expression in TIM-3+ and TIM-3KO murine DIPG cell lines. Standardized quantification against its own vinculin (housekeeping) and a Student’s t test is performed for TIM-3+ versus TIM-3KO statistical analysis.

(M) Kaplan-Meier survival curves of immunocompetent mice bearing HSJD-007 tumors and treated with an anti-human TIM-3 antibody (BMS-986258) (n = 9) or IgG1 (n = 9 log rank; p = 0.0001). Student’s t test (panel B, C), one-way ANOVA (panel E) and two-way ANOVA (panel F, H) were performed. Bar graphs indicate the mean ± SEM. (ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). See also Figures S2 and S3.