Extended Data Fig. 6: GABA suppresses CCL4/5 production in tumor cells to create a non-T cell-inflamed microenvironment.
From: Cancer-cell-derived GABA promotes β-catenin-mediated tumour growth and immunosuppression
a, Lymphocytes were isolated from lymph nodes of OTI mice, and then treated for 3 days with peptide antigen and the indicated concentrations of GABA. Cells were then collected and analyzed for IFNγ and TNFα expression by flow cytometry (left, n = 3 per group). Representative examples of IFNγ and TNFα staining with or without 0.1 μM GABA (gated live, TCRβ, CD8; right). b, MC38 cells stably expressing Gad1 shRNAs or NTC were subcutaneously injected into C57BL/6 mice. Tumor-bearing mice were given anti-mouse CD8β antibody or IgG1 isotype control by intraperitoneal injection on days -1, 0, 7, 14 and 21 (n = 5 per group). c, Lymphocytes were isolated from lymph nodes of OTII or OTI mice, and then treated for 3 days with peptide antigen and the indicated concentrations of GABA. CFSE staining was used for analyzing proliferation of OTII CD4+ T cells and OTI CD8+ T cells. d, Quantitative real-time PCR analysis of CCL4 and CCL5 expression in H520 (left) and HT29 (right) cells stably expressing GAD1 shRNAs (sh1 and sh2) or NTC, as indicated (n = 3 per group). e, Western blot analysis of GAD1 and β-catenin expression in tumor samples of LLC-ova, LG1233, and MC38 tumors stably expressing Gad1 shRNAs (sh1 and sh2) or NTC, as indicated. f, LLC-ova cells stably expressing Gad1 shRNAs (sh1 and sh2) or NTC were further infected with virus expressing β-catenin (∆GSK) mutant or empty vector (pRRLSIN). Ccl4 and Ccl5 mRNA levels were analyzed by quantitative real-time PCR (n = 3 per group). g, Quantitative real-time PCR analysis of CCL4 and CCL5 expression in H520 cells stably expressing GAD1 shRNAs or NTC were further infected with virus expressing mutant β-catenin (S33Y) or EV (n = 3 per group). h, LLC-ova cells stably expressing Gad1 shRNAs (sh1 and sh2) or NTC were further infected with virus expressing β-catenin (∆GSK) mutant or empty vector (pRRLSIN). GAD1, β-catenin, cyclin D1, and ATF3 protein levels were analyzed by western blot. i, Quantitative real-time PCR analysis of Ccl4 and Ccl5 expression in MC38 cells expressing Ccl4 and Ccl5 shRNAs or NTC (n = 3 per group). β-actin served as loading control in western blot analysis. Western blot data shown in (e, h) are repeated independently at least two times with similar results. n indicates the number of biological (a, b) or technical (d, f, g, i) replicates. Data are presented as the mean ± SEM (a) or mean ± SD (b, d, f, g, i). P-values were calculated by two-tailed Student’s t test (a, d, f, g, i) and two-way ANOVA (b). *P < 0.05, **P < 0.01, ***P < 0.001. Exact P values can be found in the Source Data.
