Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 1. Dinaciclib and anti-PD1 combina- tion therapy inhibits tumor growth in syn- geneic mouse tumor models. Dinaciclib was tested alone and in combination with anti- PD1 mAb in (A and C) C57/BL6J, (B) BALB/c, and (D) Rag1–/– mice implanted with (A and D) MC38, (B) CT26, or (C) MB49 tumor cells. Tumor volume is represented as the mean ± SEM. The percentage of TGI on day 20 is pre- sented for each treatment group compared with the control group. Arrows indicate the treatment time points. Data represent at least 2 independent experiments (n = 10–12 mice/group). ***P < 0.001 and *P < 0.05, by 2-way ANOVA with Bonferroni post-test.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: Control

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 2. Dinaciclib and anti-PD1 combination therapy induces immune cell infiltration and activation in tumors. Mice with established CT26 tumors were treated with dinaciclib and anti-PD1 mAb as described in Figure 1. Tumors were isolated on day 14, and immune cells were analyzed by flow cytometry (n = 5 mice/group). Shown are the numbers of tumor-infiltrating (A) CD8+ T cells, (B) CD4+ T cells, and (E) CD11b+CD11c+ DCs in the different treatment groups. Also shown is the activation status of these cell populations as measured by the percentage of CD69+ CD4+ and CD8+ T cells (C and D) and MHCII, CD80, and CD86 mean fluorescence intensity (MFI) on DCs (F). For functional analysis, TILs were isolated from dissociated tumors using density-gradient centrifugation. For the detection of intracellular cytokines, harvested TILs were stimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours. Shown are the percentages of (G) IFN-γ+, (H) TNF-α+, and (I) GzB+ CD8+ T cells. Data represent at least 2 independent experiments. ***P < 0.001, **P < 0.01, and *P < 0.05, by 1-way ANOVA with Bonferroni post-test.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: Activation Assay, Isolation, Flow Cytometry, Fluorescence, Functional Assay, Gradient Centrifugation

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 3. Dinaciclib treatment induces a type I IFN signature within tumors. (A) Fluidigm qPCR analysis of MC38, CT26, and MB49 cells treated with dinaciclib in vitro for 24 hours either continuously or by washing and replacing medium after a 2-hour pulse. Heatmap indicates type I IFN signature genes with a greater-than 2-fold change (log10 scale) over the untreated control and a P value of less than 0.05. Genes with a FC of less than 2 and a P value of greater than 0.05 are blacked out. (B and C) Mice bearing 100 mm3 MC38 tumors were treated as described in Figure 1. Twenty-four hours after the first dose, tumors were isolated, and gene expression was analyzed by RNA sequencing (n = 5/group). (B) The top upregulated functional pathways in the dinaciclib and dinaciclib plus anti-PD1 groups as determined by GO analysis and IPA. (C) Expression of type I IFN response genes is depicted by a heatmap showing the log10 FC only of genes that were significantly upregulated (>2-fold and P < 0.01) compared with the isotype control group. Genes that were upregulated by less than 2-fold and that had a P value of greater than 0.01 are blacked out (represented as FC = 0).

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: In Vitro, Control, Isolation, Gene Expression, RNA Sequencing, Functional Assay, Expressing

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 4. Dinaciclib induces immunogenic cancer cell death. (A–D) CT26 cells were treated for 24 hours in vitro with dinaciclib at the indicated concentrations. Graphical data show (A) the percentage of tumor cell apoptosis, release of (B) HMGB1 and (C) ATP into the culture supernatants, and (D) surface expression of CRT on viable cells. Data represent the mean value ± SEM of 2 to 3 replicates from 1 representative experiment. ***P < 0.001 and *P < 0.05, for comparisons between individual dinaciclib-treated groups and the untreated group (0 μM). Statisti- cal data obtained via 1-way ANOVA with Bonferroni post-test. (E) CT26 cells either treated in vitro with dinaciclib or freeze- thawed were inoculated s.c. into BALB/c mice. After 10 days, mice were rechallenged with live CT26 cells. Shown is the percentage of tumor-free mice pooled from 2 indepen- dent experiments. ***P < 0.001, by log-rank (Mantel-Cox) test.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: In Vitro, Expressing

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 6. Dinaciclib induces PD1 expression and is efficacious in PD1-KO mice. (A) CT26, MB49, and MC38 tumor-bearing mice were treated as described in Figure 1. Tumors were isolated on day 14, and PD1 expression on CD8 + T cells was analyzed by flow cytometry (n = 5 mice per group). (B) WT or PD1-KO mice with established MB49 tumors (~100 mm3) were treated with dinaciclib or vehicle as described in Figure 1. Tumor growth is represented as the mean tumor volume ± SEM. Data represent at least 2 independent experiments. Arrows indicate the treatment time points. *P < 0.05 and ***P < 0.001, by unpaired t test (A) applied to calculate 2-tailed P value to estimate statistical difference between vehicle and dinaciclib treatment groups and 2-way ANOVA with Bonferroni post-test (B) applied to assess differences in tumor growth kinetics.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: Expressing, Isolation, Flow Cytometry

Journal: Molecular Therapy

Article Title: Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression

doi: 10.1016/j.ymthe.2019.07.018

Figure Lengend Snippet: Organelle Distribution in HCT116, H358, and CT26.WT Cells by Immunofluorescence

Article Snippet: Cells were implanted by subcutaneous injection of 1 × 10 7 HCT116 cells/mouse, 3 × 10 6 H358 cells/mouse with 50% Matrigel, or 5 × 10 5 CT26.WT cells/mouse into the left flank of female nude (HCT116 and H358, 18 g + ) or BALB/c mice (CT26.WT, 16 g + , all Envigo UK).

Techniques: Immunofluorescence

Journal: Molecular Therapy

Article Title: Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression

doi: 10.1016/j.ymthe.2019.07.018

Figure Lengend Snippet: Analysis of Endolysosomal pH in HCT116, H358, and CT26.WT Using Ratiometric Dextran Probes to Measure pH

Article Snippet: Cells were implanted by subcutaneous injection of 1 × 10 7 HCT116 cells/mouse, 3 × 10 6 H358 cells/mouse with 50% Matrigel, or 5 × 10 5 CT26.WT cells/mouse into the left flank of female nude (HCT116 and H358, 18 g + ) or BALB/c mice (CT26.WT, 16 g + , all Envigo UK).

Techniques: