Fig 2: PDAC cell–intrinsic GOT2 suppresses T cell–dependent immunologic control of tumor growth. A, Metascape pathway analysis depicting the top transcriptional programs inversely correlated with GOT2 expression in human PDAC. B and C, IHC staining of control and sgGot2 688M tumors for T-cell marker CD3 (B) and subtype markers CD4 and CD8 (C). Representative images are shown on the left (scale bars = 50 µm), with quantification on the right (ctrl: n = 5, sgGot2 a: n = 4, sgGot2 b: n = 4). Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA. D, IHC staining of control and shGot2 FC1245 tumors for T-cell markers CD3 and CD8. Representative images are shown on the left (scale bars = 50 µm), with quantification on the right (ctrl: n = 5, shGot2 a: n = 5, shGot2 b: n = 3). Data are presented as mean ± SEM. **, P < 0.01; ***, P < 0.001 by one-way ANOVA. E, IHC costaining of control and sgGot2 or shGot2 PDAC for macrophage marker F4/80 and immunosuppressive factor arginase-1 (Arg1). Representative images are from 688M tumors (scale bar on 20× images = 10 µm, scale bar on 63× images = 5 µm). Quantification of double-positive cells out of total F4/80+ cells in the 688M and FC1245 models is on the right; data are presented as mean ± SEM. **, P < 0.01; ****, P < 0.0001 by an unpaired t test. F, Multiplex IHC staining of control and sgGot2 FC1245 tumors for the indicated markers (large images, scale bar = 50 µm; insets, scale bar = 20 µm). G, Quantification of CD3 IHC on 688M PDAC at the indicated time points after transplantation (ctrl d11: n = 7, sgGot2 a d11: n = 6, ctrl d19: n = 3, sgGot2 a d19: n = 3, ctrl d27: n = 5, sgGot2 a d27: n = 4). *, P < 0.05; **, P < 0.01 by an unpaired t test. H, PDAC tumor weight at the experimental endpoint, 27 days after orthotopic transplantation of 688M cells and treatment with isotype control or T-cell depleting antibodies (details in Methods). Ctrl: n = 5 per cohort, sgGot2 a: n = 4 per cohort. Data are presented as mean ± SEM. ns = not significant. ****, P < 0.0001 by one-way ANOVA.

Fig 3: GOT2 promotes pancreatic tumor progression without impacting proliferation. A, Viable cell measurements in the indicated PDAC cell lines. Data are presented as mean ± SEM from biological triplicates. ****, P < 0.0001 by one-way ANOVA. Dox, doxycycline; RLU, relative light unit. Numbers in parentheses are the short hairpins (shRNAs) used for the GOT2 knockdowns. B, PDAC tumor weight at the experimental endpoint, 34 days after orthotopic transplantation of 688M cells into immune-competent hosts. Ctrl: n = 8, sgGot2 a: n = 9, sgGot2 b: n = 10. Data are presented as mean ± SEM. ns = not significant. ****, P < 0.0001 by one-way ANOVA. C, IHC staining of tumors in B for Ki-67 (proliferation) and pan-cytokeratin (panCK; tumor cells), with a DAPI counterstain (nuclei). Representative images are shown on the left (scale bars = 50 µm), with quantification on the right (ctrl: n = 6, sgGot2: n = 5). Data are presented as mean ± SEM. ns = not significant by an unpaired t test. D, PDAC tumor weight at the experimental endpoint, 22 days after orthotopic transplantation of FC1245 cells into immune-competent hosts. Ctrl: n = 5, shGot2 a: n = 5, shGot2 b: n = 3. Data are presented as mean ± SEM. ns = not significant. **, P < 0.01; ***, P < 0.001 by one-way ANOVA. E, PDAC tumor weight at the experimental endpoint, 18 days after orthotopic transplantation of FC1245 cells into immune-competent hosts. Ctrl: n = 5, sgGot2 a: n = 5. Data are presented as mean ± SEM. ****, P < 0.0001 by an unpaired t test.

Fig 4: PPARd activation restores tumor growth and T-cell exclusion in the absence of GOT2. A, Viable cell measurements in control or sgGot2 PDAC cells treated with vehicle or 100 nmol/L GW501516. RLU, relative light unit. B, PDAC tumor weight at the experimental endpoint, 30 days after orthotopic transplantation of the control or sgGot2 cells, with daily i.p. injection of vehicle or 4 mg/kg GW501516. Ctrl: n = 5 per cohort, sgGot2: n = 4 per cohort. Data are presented as mean ± SEM. ns = not significant. *, P < 0.05; **, P < 0.01 by one-way ANOVA. C, IHC staining of control and sgGot2 688M tumors treated with vehicle or GW501516 as in B for the T-cell marker CD3. Representative images are shown above (scale bars = 50 µm), with quantification below (ctrl: n = 5, ctrl + GW501516: n = 5, sgGot2: n = 3, sgGot2 + GW501516: n = 4). Data are presented as mean ± SEM. ns = not significant. **, P < 0.01 by one-way ANOVA. D, IHC staining for PTGS2/COX2 in control or sgGot2 PDAC treated with vehicle or GW501516 (representative of n = 3–5 per cohort). Scale bars = 50 µm. E, Viable cell measurements in control or sgGot2 PDAC cells stably transduced with empty vector or VP16–PPARd. Data are presented as mean ± SEM. F and G, PDAC tumor weight at the experimental endpoint in the indicated 688M (F) and FC1245 (G) lines. 688M: Ctrl: n = 5, sgGot2 a: n = 4, ctrl VP16–PPARd: n = 4, sgGot2 a VP16–PPARd: n = 4, endpoint = day 27. FC1245: Ctrl: n = 5, sgGot2 a: n = 5, ctrl VP16–PPARd: n = 5, sgGot2 a VP16–PPARd: n = 4, endpoint = day 18. Ctrl and sgGot2 FC1245 arms here are also depicted in Fig. 1E. ns = not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA. H, qPCR for PPARd-regulated genes in the indicated FC1245 stable cell lines, normalized to 36b4. Data are presented as mean ± SEM from biological triplicates. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 by one-way ANOVA. I, PDAC tumor weight at experimental endpoint (day 18) in ctrl (n = 4) and shPpard (n = 5 per hairpin) FC1245 tumors. **, P < 0.01 by one-way ANOVA. J, Quantification of CD3 IHC on the tumors from I (scale bars = 50 µm). ns = not significant. *, P < 0.05 by one-way ANOVA. K, Heat map depicting differentially expressed (DE) genes in control and sgGot2 FC1245 PDAC cells, untreated or treated with 500 nmol/L GW501516 for 24 hours (n = 3 per group), identified by RNA-seq using cutoff criteria Padj < 0.01 and logFC < -1 or > 1 in at least one comparison. L, Venn diagram showing RNA-seq results by the criteria in K, with sample gene identities at the overlap listed. Overlapping gene frequency: ***, P < 0.001 by permutation test. M, Molecular Signatures Database (MSigDB) pathway analysis showing the top 10 enriched pathways of genes at the overlap in L.