Fig. 4.

The combination of CCL2/20 neutralization plus anti-PD-1 therapy blocked PIGL depletion-driven liver cancer progression. A CCL2/20 mRNA levels were detected in Hepa1-6 cells with or without PIGL depletion using qRT‒PCR. B After treatment with or without CCL2/20 neutralizing antibodies, the effect of conditioned medium (CM) from the indicated genetically manipulated Hepa1-6 cells on regulatory T-cell recruitment was detected by performing a transwell migration assay. Scale bar, 20 μm. C Subcutaneous transplantation of Hepa1-6 cells with the indicated genetic manipulation into C57BL/6 mice (n = 6). The treatment was performed as described in the supplementary methods. D, E In the solid tumors shown in (C), in vivo cell proliferation (D) and cell death (E) were measured by performing Ki67 and TUNEL staining, respectively. Scale bar, 200 μm. F, G In the solid tumors shown in (C), the proportions of the T-cell subsets (F) and F4/80⁺ macrophages (G) were analyzed using FCM. C One-way ANOVA; A, B, D–G Two-tailed Student’s t-test. (N.S., not significant; *p < 0.05; **p < 0.01; ***p < 0.001)