Fig. 6: ACOD1 deletion promoted anti-cancer cell activity of iMACs against solid tumors in vitro and in vivo. | Nature Communications

Fig. 6: ACOD1 deletion promoted anti-cancer cell activity of iMACs against solid tumors in vitro and in vivo.

From: Metabolic Reprogramming via ACOD1 depletion enhances function of human induced pluripotent stem cell-derived CAR-macrophages in solid tumors

Fig. 6

a, b The expression and quantification of CD80, CD86, CD163, and CD206 in MSLN-CAR-iMACs or ACOD1-/- MSLN-CAR-iMACs after co-cultured with HO-8910 cells (E:T = 5:1) for 24 h were measured by flow cytometry and displayed as histograms (b, n = 3 biologically independent samples). statistics by two-way ANOVA test. (CD80, P = 0.0035; CD86, P = 0.0324; CD206, P < 0.0001; CD163, P < 0.0001) c Luciferase assays for CAR-iMAC cytotoxicity activity against cancer cells when co-cultured with HO-8910 cells for 24 h (E:T = 5:1, 10:1, or 20:1) (5:1, n = 3; 10:1, n = 5; 20:1, n = 5 biologically independent samples). statistics by two-way ANOVA test. (5:1, P = 0.2969; 10:1, P < 0.0001; 20:1, P < 0.0001) d Luciferase assays for CAR-iMAC cytotoxicity activity against cancer cells with or without 4-Octyl Itaconate (4-OI) addition when co-cultured with HO-8910 cells for 24 h (n = 3 biologically independent samples) (E:T = 10:1). Statistics by one-way ANOVA test. (MSLN(-) vs MSLN(+), P < 0.0001; ACOD1-/- MSLN(-) vs ACOD1-/- MSLN(+), P < 0.0001; MSLN(-) vs ACOD1-/- MSLN(-), P = 0.0002; MSLN(+) vs ACOD1-/- MSLN(+), P = 0.00021) iMACs were pre-treated with 4-OI (250 μM) or DMSO control for 3 h before challenge with LPS plus IFN-γ (50 ng/mL each) for 24 h. e Luciferase assays for MSLN-CAR-iMAC cytotoxicity activity against cancer cells with or without sulforaphane (SFN) (10 μM) when co-cultured with HO-8910 cells for 24 h (E:T = 10:1) (n = 3 biologically independent samples). Statistics by one-way ANOVA test. f Luciferase assays for the cytotoxicity activity of the co-culture supernatant with IgG control, neutralizing antibody (10 μg/mL) of IFN-γ or TNF-α (n = 3 biologically independent samples). Statistics by two-way ANOVA test. The supernatant was collected after iMACs were co-cultured with HO-8910 cells for 24 h (E:T = 10:1). g The levels of the indicated cytokines/chemokines in the medium of iMAC-HO-8910 co-culture system were determined 24 h post IFN-γ and LPS challenge (n = 3 biologically independent samples). Statistics by unpaired t test. (IL-6, P < 0.0001; IL-1β, P = 0.0016; CXCL-10, P = 0.0003; TNF-α, P = 0.2075; IFN-γ, P = 0.2026) h Luciferase assays for MSLN-CAR-iMAC cytotoxicity activity against cancer cells with or without N-Acetyl-L-cysteine (NAC) (2.5 mM) when co-cultured with HO-8910 cells for 48 h (E:T = 10:1) (n = 3 biologically independent samples). Statistics by one-way ANOVA test. (MSLN(-) vs MSLN(+), P < 0.0001; ACOD1-/- MSLN(-) vs ACOD1-/- MSLN(+), P < 0.0001; MSLN(-) vs ACOD1-/- MSLN(-),P < 0.0001; MSLN(+) vs ACOD1-/- MSLN(+), P < 0.0001) (b-h) Data was shown as mean ± SD. i A diagram of the in vivo treatment scheme. j In Vivo Imaging system (IVIS) images showing progression of tumor in the above conditions (n = 5 per group). k Tumor burden on day −1, 7, 11, and 14 was quantified and displayed as mean ± SD. (n = 5 per group) statistics by two-way ANOVA test. (PBS vs MSLN-CAR, P < 0.0001; PBS vs ACOD1-/- MSLN-CAR, P < 0.0001; MSLN-CAR vs ACOD1-/- MSLN-CAR, P = 0.0051) l The Kaplan-Meier curve demonstrating survival of the mice. Statistics by two-tailed log-rank test. Source data are provided as a Source Data file.

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