Fig 1: GPR55 deficiency in the TME of KPCY55 tumors influences anti-PD-1 treatment. (A) RNA-seq-derived normalized counts (transcripts per million with p-adjusted values (p.adj)) and (B) RT-qPCR data (normalized to Hprt) for a select number of immune checkpoint and costimulatory molecules. n=6. (C) GPR55 KO and WT mice were subcutaneously (s.c.) injected on day 0 with 5x105 KPCY55 cells. Starting on day 14, six rounds of anti-PD1 antibodies or isotype control were injected i.p. into mice. On day 28, tumors were measured ex vivo and used for analysis. (D, E) Ex vivo measurement of tumor mass and volume in isotype control (Isotype) and anti-PD1 antibody (PD-1) treated mice. n=8-10 per group. Statistical differences were evaluated using unpaired Student’s t-test. (F) Flow cytometric analysis of monocytes and macrophages positive for CXCL9 expression in KPCY55 tumors from WT or GPR55KO mice. Statistical differences were evaluated using two-way ANOVA with Šídák’s multiple comparisons test. In all datasets, data indicate medians, 25th and 75th percentiles, and min-max values. *p<0.05, **p<0.01, ***p<0.001. n=7-10. WT, wildtype; KO, knockout; p.adj, adjusted p-value; PD-1, anti-PD-1 antibody; Isotype, isotype control; TME, tumor microenvironment.
Fig 2: Role of the CXCR3/CXCL9 axis in the migration of T cells. (A) Differential expression of Cxcl9, Cxcl10, Cxcr3, and Ifnγ in the TME of KPCY55 tumors from WT and GPR55 KO mice, presented by normalized counts (transcripts per million) with p-adjusted values (p.adj) from bulk RNA-seq. n=6. (B) Relative gene expression (2^-dCT) of Cxcl9, Cxcl10, and Ifnγ normalized to Hprt. Statistical differences were evaluated by unpaired Student’s t-test, **p<0.01, *p<0.05. n=6. (C) Chemotaxis assay of pan-T cells isolated from healthy spleens of GPR55 KO and WT mice, migrating towards different concentrations of CXCL9 in the bottom wells. Statistical differences were evaluated using two-way ANOVA and Tukey’s post hoc test. *p<0.05. n=4-5 (D) Geometric means of CXCR3 expression on lymphocytes in KPCY55 tumors from WT and GPR55 KO mice. Statistical differences were evaluated using two-way ANOVA with Šídák’s multiple comparisons test. ***p<0.001, ****p<0.0001. n=4-5. In all datasets, data indicate medians, 25th and 75th percentiles, and min-max values. (E) Schematic showing the comparison between the GPR55 WT and KO TME. p.adj, adjusted p-value; ns, not significant; WT, wildtype; KO, knockout; TME, tumor microenvironment. Created in BioRender. Ristic, (D) (2024) https://BioRender.com/j25e259.
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