Abstract P6-09-04: M2 macrophages induced by mammary carcinoma are switched to M1 macrophages by Escherichia coli maltose-binding protein

Abstract

Abstract Background: The maltose-binding protein (MBP) is a component of the maltose transport system in the Escherichia coli. It is used to increase the stability and solubility of proteins in bacterial protein expression systems and is increasingly being used to facilitate the production and delivery of subunit vaccine. Tumor-associated macrophages (TAMs) are alternatively activated macrophages (M2) within the tumor microenvironment, which directly promoting tumor cell growth, extracellular matrix remodeling, and angiogenesis. Our previous studies revealed that MBP could nonspecifically induce Th1 cells activation and classically activate macrophages (M1). In the present study, we investigated the effect of MBP on re-polarization of mammary carcinoma induced M2 macrophage to pro-inflammatory M1 macrophages, in order to develop a novel antitumor immune adjuvant. Methods: We mimicked a tumor microenvironment by in vitro coculturing RAW264.7 macrophages with conditioned media (CM) from two different kinds of mammary carcinoma cell lines EMT6 and 4T1. In the absence or presence of MBP, the mammary carcinoma CM-induced macrophages were tested for their polarization types. The production of IL-12 and IL-10 were assessed by ELISA, the mRNA expression of iNOS and Arg-1 were detected by RT-PCR, and the expression of CD206 and CD16/32 were determined by flow cytometry and immunofluorescence staining. All of these important factors were taken as criteria for M1 and M2 polarization. The expression levels of TLR2 and TLR4 on the surface of RAW264.7 cells were determined by flow cytometry. By the addition of TLR2 or TLR4 blocking antibody, the ratio of IL-12 and IL-10 production was used to distinguish M1 and M2 macrophages in the absence or presence of MBP. For the investigation of the signal transduction pathways, the expression level of MyD88 and the phosphorylation levels of IκB-α, NF-κB, PI3K, Akt, MAPKs, JNK were analyzed by western blotting. Results: The mammary carcinoma CM induced the RAW264.7 macrophage to polarize to M2 macrophages. In the mammary carcinoma CM-induced macrophages, the expression level of CD206 was increased, the ratio of IL-12/IL-10 production was decreased, and the gene expression of Arg-1 was increased. In the presence of MBP, M2 polarization was attenuated as shown by the increase of CD16/32 and iNOS, the decrease of CD206 and Arg-1, an altered ratio of IL-12/IL-10, and accompanied with the up-regulation of TLR2 and TLR4 expressions. The effect of MBP on mammary carcinoma CM-induced macrophages was blocked by anti-TLR2 antibody, but not anti-TLR4 antibody or TLR4 inhibitor polymyxin B. Further studies confirmed that the effects of MBP on switching mammary carcinoma induced M2 macrophages to M1 depended on the activation of NF-κB and MAPKs. Conclusion: Our results provide new insights into the modulation of TAM polarization. MBP can be used as a novel antitumor adjuvant, which may stimulate a tumor-rejecting environment by switching M2 macrophages to classical pro-inflammatory M1 macrophages through TLR2 signaling and a combination of transcription factors including NK-κB and MAPKs. Accordingly, MBP may have the therapeutic potential in mammary carcinoma. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-04.