GDF15 Promotes Colon Cancer Escape By Affecting Macrophages Polarization

The incidence of cancer remains a growing trend in the world. In recent years, tumor immunotherapy obtains more and more attention and recognition for its good clinical effect. However, tumor escape seriously limits the effectiveness of tumor immunotherapy. Studies have shown that dysfunction of immune cells under tumor microenvironment plays an important role in the process of tumor escape.Macrophages play important roles in anti-infection, presenting antigen to initiate immune response, killing tumor cells and immune regulation. Under different microenvironment, macrophages differentiate into different phenotypes and show different functions, namely polarization. Currently macrophages are referred to as classical activated(M1) or alternatively activated(M2) macrophages. M1 macrophages can secret pro-inflammatory cytokines to elicit Th1 response to kill intracellular bacterium and tumor cells. M2 macrophages can secret anti-inflammatory cytokines to promote tissue repair and support Th2 response to inhibit killing tumor cells.A large number of clinical and experimental evidences suggest that tumor infiltrated macrophages, namely tumor associated macrophages, generally have M2-like phenotypes in the majority of tumors. These TAMs can secrete various growth factors to promote tumor angiogenesis, secrete matrix degradation related enzymes to promote tumor invasion and metastasis, inhibit the proliferation and killing function of CTLs and NKs, suppress DCs activation and maturation, induces proliferation of Tregs, thus involves in tumor immune escape and promote the development of malignant tumors.GDF15 was first identified in cDNA library of macrophage activation related genes in U937 cell line. GDF15 is a member of the TGF-β superfamily, involves in many physiological and pathological processes. The dysregulation of GDF15 expression is also closely related with a variety of tumor development, and directly affect the prognosis and living quality of patients. Although GDF15 has received many attentions in the development of cancer as a tumor-derived factor, there are few exact reports of effects of GDF15 on the immune cells. Our previous studies for the first time confirmed that GDF15 can inhibit DCs maturation and function and the ability to stimulate anti-tumor specific immune response, and promote naive CD4+ T cells differentiate into Tregs by up-regulating Foxp3 expression.Whether GDF15 can affect macrophages polarization is still unknown. To clarify this question, we conducted the following work: 1) First, CD14+ monocytes were isolated from human peripheral blood and induced to differentiate into macrophages. By Real-time PCR, ELISA and FCM methods, we studied the effects of GDF15 on CD14+ monocytes and monocyte-derived macrophages at polarization phase, including membrane molecules expression, cytokines secretion, phagocytic capacity and the ability to induce Foxp3 expression in naive CD4+ T cells; 2) By lentivirus infection, we constructed GDF15 over-expressing murine colon carcinoma CT26 stably transfected cell line; 3) By Real-time PCR, FCM and MLR methods, we studied the effects of GDF15 on murine bone marrow-derived macrophages, including membrane molecules expression, cytokines secretion, NO secretion and the ability to stimulate T cells proliferation; 4) We constructed tumor-bearing mice to study the tumor promoting effects of GDF15 in vivo by ELISA, Real-time PCR, FCM and IHC methods; 5) THP-1 cells were selected as human macrophages model cells. We validated the effects of GDF15 on THP-1 cells polarization by Real-time PCR, ELISA and FCM methods. As TGF-β1 was the most significantly influenced cytokine by GDF15, we further studied the molecular mechanisms how GDF15 regulated TGF-β1 expression by dual luciferase reporter gene system, Real-time PCR, ELISA, Western blot, transcription factor activity chip, confocal, Ch IP and RNA interference methods.Through the above research work, we obtained the following results: 1) GDF15 directly induced CD14+ monocytes to differentiate into M2-like macrophages, and GDF15 inhibited M1-type polarization while promoted M2-like polarization of monocyte-derived macrophages; 2) GDF15 stimulated M2-like macrophages could induce naive CD4+ T cells to differentiate into Tregs; 3) GDF15 significantly induced TGF-β1 expression in CD14+ monocytes and monocyte-derived macrophages at polarization phase; 4) GDF15 induced murine bone marrow-derived macrophages to differentiated into M2-like macrophage and inhibited their ability to stimulate T cells proliferation; 5) GDF15 over-expressing promoted tumor growth in vivo, increased peritoneal M2 macrophages ratio and M2-like TAM infiltration; 6) Positive correlations existed among plasma GDF15 levels, CD206+ TAM infiltration ratio and TAM TGF-β1 expression; 7) GDF15 induced TGF-β1 expression in THP-1 cells; 8) GDF15 regulated TGF-β1 expression by K-Ras-JNK/ERK-c-Myc axis in THP-1 cells.We first identified that GDF15 could inhibit M1-type polarization and promote M2-like polarization, further studied the molecular mechanisms how GDF15 regulated TGF-β1 expression in THP-1 cells. Thus, we gave a new mechanism for the occurrence of tumor immune tolerance and provided new ideas and targets for blocking tumor escape and improving tumor immunotherapy effects.