3.2 M2-like macrophages play a pivot in IL-28B-mediated anti-tumor effects
We hypothesized that IL-28B impacted tumor growth by directly inhibiting MC38 cell proliferation. We found that IL-28B did not significantly inhibit MC38 cell proliferation (Fig. S1A). We also detected IL-28B effects on the proliferation of the RM-1 tumor cell line in mice. We found that IL-28B had no significant effect on RM-1 proliferation (Fig. S1B), which suggested that IL-28B anti-tumor effect is not mediated by the inhibition of MC38 tumor cell proliferation.
H&E staining revealed that IL-28B significantly increased necrosis in tumor tissues. We suspect that IL-28B inhibits tumor growth by directly promoting MC38 cell apoptosis. We used flow analysis techniques to detect the effects of IL-28B on the apoptosis of MC38 tumor cells. However, results showed that IL-28B did not affect apoptosis of MC38 tumor cells (Fig. S2A). We also found that IL-28B did not affect apoptosis in RM-1 tumor cells in mice (data not shown). These results show that IL-28B does not inhibit tumor growth by directly promoting the apoptosis of MC38 cells. We hypothesized that IL-28B might inhibit tumor growth by regulating immune responses in mice. To verify this hypothesis, we used flow cytometry to detect the proportion of macrophages and myeloid-derived suppressor cells (MDSCs) in peripheral blood, and the ratio of T cells, macrophages, and MDSCs in the spleen. However, there was no significant difference in the proportion of these immune cells (Fig. S3A-B). Next, we detected changes in T cells, macrophages, and MDSCs in tumors. The results showed that the relative abundance of M2 macrophages in the IL-28B tumor treatment group was significantly reduced compared to that in the control group, and the proportion of CD8+ T cells increased (Fig. 2A, B, G, I). However, the abundances of M1 macrophages, MDSCs, and CD4+ T cells were not significantly changed compared to those in the control group (Fig. 2C-H). Immunofluorescence staining of M2 macrophages (F4/80+CD206+) in the tumor tissues showed that M2 macrophages were significantly inhibited by IL-28B treatment, consistent with flow cytometry results (Fig. 2J). Overall, these data indicate that IL-28B decreased the proportion of M2 macrophages in the MC38 tumor microenvironment.