Fig 2: Cyclin G2 knockout in macrophages attenuates the inhibitory effects of IFN-γ on colon cancer cell growth. A–C MC38 cells were mixed with BMDMs from WT and Ccng2−/− C57BL/6 mice at a ratio of 5:1 and injected subcutaneously into C57BL/6 mice, which were then treated with IFN-γ at specific times. Gross tumors (A), tumor weights (B), and tumor volumes (C) were measured at the endpoint. Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SEM (n = 5). D A schematic model depicting the role of cyclin G2 in macrophages after IFN-γ treatment. Upregulated cyclin G2 after IFN-γ treatment inhibited the interaction between PP2Ac and STAT1, thereby increasing the nuclear import of STAT1 and promoting CXCL9 transcription. Increased CXCL9 secretion can promote CTL chemotaxis and inhibit vascular endothelial cell angiogenesis, ultimately inhibiting tumor progression. **p < 0.01; ****p < 0.0001

Fig 3: Cyclin G2 in macrophages regulates CTL chemotaxis and vascular endothelial cell tube formation via CXCL9. A CXCL9 levels in the supernatants of BMDMs from WT and Ccng2−/− C57BL/6 mice treated with IFN-γ were determined by ELISA (representing 3 independent experiments). B, C CXCL9 levels in the supernatants of THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2 and Vector and Flag-cyclin G2) treated with IFN-γ were determined by ELISA (representing 3 independent experiments). D, E CTL chemotaxis analyzed by treating conditioned medium from BMDMs isolated from WT and Ccng2−/− C57BL/6 mice treated with or without recombinant CXCL9. Scale bar = 200 μm (representing 3 independent experiments). F Tube formation experiments showed the tube formation ability of SVEC4–10 cells treated with conditioned medium from BMDMs isolated from Ccng2−/− C57BL/6 mice. The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 500 μm (representing 3 independent experiments). G Tube formation experiments showed the tube formation ability of HUVECs treated with conditioned medium from a THP-1 stable cell line (shcyclin G2#1). The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 200 μm (representing 3 independent experiments). (A–C, E–G) Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SD **p < 0.01; ***p < 0.001; ns, not significant

Fig 4: CXCL9 enhances anti-tumor activity of CXCR3+ CD8+ T cells(A and B) Hepatic CD8+ T cells from WT mice were stimulated with serum from Sed or PEx WT mice for 24 h (A). ELISA analysis of Granzyme B, IFN-γ, and TNF-α levels in the medium (B) (n = 5/group).(C and D) Hepatic CD8+ T cells from WT mice were stimulated with serum from Sed and PEx Cxcl9 KO mice for 24 h (C). ELISA analysis of Granzyme B, IFN-γ, and TNF-α levels in the medium (D) (n = 5/group).(E and F) Cxcr3 KO hepatic CD8+ T cells were stimulated with serum from Sed and PEx WT mice for 24 h (E). ELISA analysis of Granzyme B, IFN-γ, and TNF-α levels in the medium (F) (n = 5/group).(G and H) Tumor CD8+ T cells from WT mice were stimulated with serum from Sed and PEx WT mice for 24 h (G). ELISA analysis of Granzyme B, IFN-γ, and TNF-α levels in the medium (H) (n = 5/group).(I and J) Tumor CD8+ T cells from WT mice were stimulated with serum from Sed and PEx Cxcl9 KO mice for 24 h (I). ELISA analysis of Granzyme B, IFN-γ, and TNF-α levels in the medium (J) (n = 5/group).(K and L) Tumor Cxcr3 KO CD8+ T cells were stimulated with serum from Sed and PEx WT mice for 24 h (K). ELISA analysis of Granzyme B, IFN-γ, and TNF-α levels in the medium (L) (n = 5/group).(M and N) Peripheral CD8+ T cells from healthy volunteers were stimulated with serum from the same CRLM patient before and after exercise for 24 h (M). ELISA analysis of Granzyme B, IFN-γ, and TNF-α levels in the medium (N) (n = 5/group). All experiments: error bars represent mean ± SEM, statistical analysis by Student’s t test. ns, not significant, ∗p < 0.05, ∗∗p < 0.01. See also Figure S7.

Fig 5: PEx increases CXCL9 release from KCs and promotes CXCR3+ CD8+ T cells recruitment(A) Heatmap showing the number of potential ligand-receptor pairs among liver immune cells from Sed and PEx mice 3 weeks after establishing the surgical stress-promoted CRLM model. Color intensity (red, high; blue, low) represents interaction strength.(B) Dot plot displaying CXCL9 expression levels in liver immune cell populations from Sed and PEx mice 3 weeks after establishing the surgical stress-promoted CRLM model.(C) Representative flow cytometry plots and quantification of CXCL9+ Clec4f+ KCs in the liver of Sed and PEx WT mice 3 weeks after establishing the surgical stress-promoted CRLM model (n = 6/group).(D) ELISA quantification of CXCL9 levels in the serum from Sed and PEx mice 3 weeks after establishing the surgical stress-promoted CRLM model (n = 6/group).(E) ELISA quantification of CXCL9 levels in the liver of Sed and PEx mice 3 weeks after establishing the surgical stress-promoted CRLM model (n = 6/group).(F) ELISA quantification of serum CXCL9 levels in CRLM patients before (Pre) and after (Post) the exercise intervention (n = 6/group).(G) Western blot showing CXCL9 levels in KCs from Non-PEx and PEx CRLM patients.(H) Dot plot showing CXCR3 expression across liver immune cell populations from Sed and PEx mice 3 weeks after establishing the surgical stress-promoted CRLM model.(I) Representative flow cytometry plots and quantification of CXCR3+ CD8+ T cells in the liver of Sed and PEx WT mice 3 weeks after establishing the surgical stress-promoted CRLM model (n = 6/group).(J) Western blot showing CXCR3 levels in peripheral CD8+ T cells from Non-PEx and PEx CRLM patients.(K) Representative flow cytometry plots and quantification of CD8+ T cells expressing Granzyme B, IFN-γ, and TNF-α in the liver of Sed and PEx WT mice 3 weeks after tumor injection (n = 6 per group). All experiments: error bars represent mean ± SEM, statistical analysis by Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S3.