Curcumin analog GO-Y078 enhanced tumor immunity cooperate with anti-OX40 antibody (Raw data)

Fig. 1 GO-Y078 treatment induced apoptotic cell death in B16-F10 melanoma cells

(A) Live cell counting assay. B16-F10 melanoma cells were cultured with 0.007 μM DMSO (control) and 12.5–1.5625 μM GO-Y078 as indicated for 20 h, followed by the addition of the Cell Counting Kit 8 reagent (4 h). The circles indicate independent experiments. The horizontal bars represent the mean. (B, C) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). B16-F10 melanoma cells were cultured in the presence of 0.007 μM DMSO or 12.5–1.5625 μM GO-Y078 for 24 h. The data show representative density plots (B). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) and early apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO-treated cultured B16-F10 melanoma cells. Data show triplicate and are representative of three independent experiments (C). (D) The real-time quantitative analysis results of 0.007 μM DMSO- or 1.5625 μM GO-Y078-treated B16-F10 melanoma cells for 24 h. Data show triplicate and are representative of three independent experiments. (E) l-lactate production of cultured B16-F10 melanoma cells between DMSO and GO-Y078 treatments for 24 h. Data show triplicate and are representative of three independent experiments. (F, G) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). B16-F10 melanoma cells were cultured in the presence of 0.007 μM DMSO or 1.5625 μM GO-Y078 for 2 h. The data show representative density plots (F). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO-treated cultured B16-F10 melanoma cells. Data show triplicate and are representative of three independent experiments (G). (H) Glycolysis measured using the glycolytic rate test. Glycolysis was calculated after Rotenon/Antimycin A treatment based on the rate of OCR in gastric tumor SH-10-TC cells. White bar indicates 0.007 μM DMSO treatment; black bar indicates 1.5625 μM GO-Y022 treatment for 2 h. Student’s t-test (D, E, G, H) or one-way ANOVA with post hoc Dunnett’s test for multiple comparison (A and C) was used. Statistical significance was set at p < 0.05; *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 2 GO-Y078 treatment prevented tumor growth in vivo

(A) Representative picture of tumor size at the end of experiment (Day 13). (B) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (DMSO = 7, GO-Y078 = 8, mean ± standard deviation). (C, D) Representative intracellular staining of Foxp3+ Tregs (TCRβ+ CD4+ CD8α- Zombie-Foxp3+ population) in tumor-infiltrating lymphocytes. (E, F) Representative staining of exhausted CD8+ T cells (TCRβ+ CD4- CD8α+ Zombie- PD-1+ Tim3+ population) in tumor-infiltrating lymphocytes. Student’s t-test (B, D, F) was used. Statistical significance was set at p < 0.05; *p < 0.05, and **p < 0.01.

Fig. 3 GO-Y078 treatment augmented CD8+ T cell-induced tumor cytotoxicity in vitro

(A) Diagram of co-culture experiment. OVA-carrying B16-F10 melanoma cells (5 × 105 cells) labeled with CellTrace VioletTM and cultured with CD8+ T cells (1 × 105 cells) from OT-I mice. 25% culture supernatants from OVA-carrying B16-F10 melanoma cells were used and cultured in the presence of 0.007 μM DMSO or 0.25 μM GO-Y078 for 48 h at 37°C and 5% CO2. (B, C) Representative Annexin V and propidium iodide staining to check the percentage of apoptotic cell death (Annexin V+ propidium iodide+) in tumor cells (CellTrace Violet+ CD8-) or CD8+ T cells (CellTrace Violet- CD8+). (D) Absolute number of CD8+ T cells (CellTrace Violet- CD8+ Annexin V- propidium iodide-) in the co-culture experiment. (E) Scheme of tumor models using OVA-carried B16-F10 melanoma cells. 0.75 × 106 OVA-carried B16-F10 melanoma cells were subcutaneously injected at day 0. Seven days later, DMSO- or GO-Y078-PBS injected intraperitoneal in each days. In the end of experiments (day 13), collect the tumors and analyze. (F) Representative picture of tumor size at Day 13. (G) Tumors’ weight at Day 13. (H) Absolute numbers of CD8+ T cells per tumor volumes. (I, J) Representative OVA-tetramer and CD8α staining in tumor microenvironments. (K) Scheme of tumor models using Rag-1-deficient mice. Rag-1-deficient mice received B16-F10 melanoma cells (0.25 x105 cells: S.C. injection) or CD8+ T cells (1x106 cells: I.V. injection) at day 0. Seven days later, DMSO- or GO-Y078-PBS injected intraperitoneal in each days. In the end of experiments (day 13), collect the tumors and analyze. (L) Representative picture of tumor size at Day 13. (M) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (n = 10, mean ± standard deviation). Student’s t-test (C, D, G, H, J) or One-way ANOVA with post hoc Dunnett’s test for multiple comparison (M) were used. Statistical significance was set at p < 0.05; *p < 0.05., **p < 0.01.

Fig. 4 GO-Y078 treatment induced apoptotic cell death in CD8+ T cells even in the presence of the supernatant of tumor cells 

(A) Representative picture of tumor size at the end of experiment (Day 13). (B) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (n = 8, mean ± standard deviation). (A, B) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). Naïve CD8+ T cells were cultured in the presence of 0.007 μM DMSO or 0.1–0.25 μM GO-Y078 for 48 h. The data show representative density plots (A). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) and early apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO-treated. Data show triplicate and are representative of three independent experiments (B). (C) Live cell counting assay. Naïve CD8+ T cells were cultured with 0.007 μM DMSO (control), 0.1–0.25 μM GO-Y078 as indicated for 44 h, followed by the addition of the Cell Counting Kit 8 reagent (4 h). The circles indicate independent experiments. The horizontal bars represent the mean. (D) Absolute number of cultured naïve CD8+ T cells (48 h). 0.007 μM DMSO or 0.1–0.25 μM GO-Y078 treatment for 48 h. Data show triplicate and are representative of three independent experiments. (E, F) Flow cytometry analysis using live and dead staining (Annexin V and propidium iodide). Naïve CD8+ T cells were cultured in the presence of 0.007 μM DMSO or 0.25 μM GO-Y078 for 48 h in the presence or absence of the tumor supernatant. Data show representative density plots (E). Statistical analyses of apoptotic dead cells (Annexin V+propidium iodide+) and early apoptotic dead cells (Annexin V+propidium iodide+) compared with 0.007 μM DMSO treatment. Data show triplicate and are representative of three independent experiments (F). Student’s t-test (B, C, D, F) was used. Statistical significance was set at p < 0.05; *p < 0.05. Statistical significance was set at p < 0.05; *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 5 GO-Y078 treatment in cooperation with anti-OX40 induced strong antitumor effects in vivo

(A) Representative picture of tumor size at the end of experiment (Day 13). (B) Calculation of the tumor volume (mm3) for each day beginning 7 days after tumor injection (n = 8, mean ± standard deviation). (C) Representative picture of tumor-infiltrating CD8+ cells (green) and DAPI (blue). The original magnification is 20×.  (D)Absolute number of CD8+ T cells in tumor area.  (E) IFN-γ production from CD8+ T cells in TIL. (F) The real-time quantitative analysis results of Control antibody with DMSO-treated or anti-OX-40 antibody with GO-Y078-treated B16-F10 melanoma tissue at 13 days (n = 8, mean). One-way ANOVA with post hoc Dunnett’s test for multiple comparison (D) or Student’s t-test (E) was used. Statistical significance was set at p < 0.05; *p < 0.05.