4 Discussion
Despite recent progress in CRC treatment, patients’ quality of life has not significantly improved. Nearly 50% of patients experience tumor recurrence and drug resistance20. Therefore, it is necessary to explore CRC pathogenesis and develop new treatment alternatives. IFN-λ is a new type of interferon, recently discovered and initially recognized for its antiviral activity21, 22. Later, its anti-tumor and immunomodulatory activities have attracted researchers’ attention6-9, 23-25. Many groups have reported the anti-tumor effects of IFN-λ in tumor models—these include liver cancer14, lung cancer15, esophageal cancer26, breast cancer27, prostate cancer28, and fibrosarcoma29. We investigated the effects and mechanism of subcutaneous IL-28B injection on colon tumors. We confirmed that IL-28B inhibited MC38 mouse colon cancer models and regulated immune cell behavior. Furthermore, we found that M2 macrophages play an important role in IL-28B anti-tumor activity.
We found that IL-28B effectively inhibited colon cancer growth. Tumor volume and quality were significantly reduced compared with the control group. The necrosis of tumor tissue after IL-28B expression was significantly increased. IFN-III exerts direct anti-tumor effects by inhibiting tumor cell proliferation and promoting tumor cell apoptosis and cell cycle stagnation30. However, we found that even IL-28B concentrations of 1000 ng/mL did not affect MC38 apoptosis and cell viability in vitro . Interestingly, we confirmed the same result with IL-28B-treated RM-1 cells (mouse prostate cancer cell line)in vitro . We speculated that the role of IL-28B in colon cancer might be related to its immunomodulatory effects.
IFN-λ exerts indirect anti-tumor activity by regulating host immune cells such as natural killer cells31 and T cells32. Therefore, we detected immune cells in the peripheral blood, spleen, and tumor tissues. We found that IL-28B regulates immune cells, consistent with previous reports. Interestingly, the difference in M2 macrophages in the tumor tissue was the most significant. After IL-28B treatment, M2 macrophages in tumor tissues were significantly reduced. To the best of our knowledge, macrophages have two types of activation, M1 and M2. M2 macrophages exert a powerful effect on tumor growth and are involved in the proliferation, angiogenesis, tumor invasion, and metastasis of tumor cells33. However, the regulatory mechanism of IL-28B in M2 macrophages is not well understood.
Therefore, we cultivated BMDMs in vitro to explore the regulatory mechanism of IL-28B on M2 macrophages. CD11b, F4/80, and CD206 were labeled M2 in vitro . In the pre-experiment, we found IL-28B addition before differentiation into M2 led to no significant effects. Li et al.34 found that IFN-λ time-dependently influences cell behavior and biology. Therefore, we tuned the timing of IL-28B addition. Compared with that in the control group, IL-28B inhibited the polarization of BMDMs to M2 in the IL-28B-treated groups. Consistent with in vivo results, the relative abundance of M2 macrophages was significantly reduced. Furthermore, we analyzed TGF-β and Arg-1 expression and cytokine secretion in M2 macrophages. We found that TGF-β and Arg-1 expression was significantly reduced. These results indicated that IL-28B directly regulated M2 macrophages. Clinical studies show that tumor treatment and prognosis are associated with M2 macrophages in tumor tissue30. Therefore, we believe that IL-28B exerted anti-tumor effects by adjusting M2 macrophage abundance and behavior in the tumor microenvironment.
Signal transducer and activator of transcription (STAT) and MAPK pathways are closely related to the polarization of M2 macrophages35. Yu et al. found that in the tumor microenvironment, M2 polarization is related to STAT3 overexpression, and STAT3 activation can increase blood vessel production, transfer, and promote the expression of the proliferation-related genes36. JNK affects the polarization of M2 macrophages, and in turn, the induction of M2-IL-4 activates JNK37. Our results showed that IL-28B affected the polarization of M2 macrophages by downregulating the JNK and STAT3 pathways.