Mouse 4T1 Breast Cancer Cell-Derived Exosomes Induce Proinflammatory Cytokine Production in Macrophages via miR-183.

Tumor-associated macrophages (TAMs) play a critical role in the tumor inflammatory microenvironment and facilitate tumor growth and metastasis. Most types of tumors aberrantly express microRNAs (miRNAs), which can be transferred between cells by exosomes and can regulate gene expression in recipient cells, but it remains unclear whether tumor-derived miRNAs are transferred by exosomes and regulate the TAM phenotype. We report that mouse 4T1 breast cancer cell-derived exosomes enhanced TAM expression of IL-1β, IL-6, and TNF-α and that inhibition of 4T1-cell exosome secretion through short hairpin RNA-mediated Rab27a/b depletion repressed tumor growth and metastasis and markedly downregulated IL-1β, IL-6, and TNF-α in a 4T1 breast tumor model. Furthermore, miRNA expression profiling revealed that three miRNAs (miR-100-5p, miR-183-5p, and miR-125b-1-3p) were considerably more abundant in 4T1 cell exosomes than in mouse bone marrow-derived macrophages, indicating potential exosome-mediated transfer of the miRNAs, and, notably, miR-183-5p was found to be transferred from 4T1 cells to macrophages through exosomes. Moreover, PPP2CA was verified as an miR-183-5p target gene, and PPP2CA downregulation enhanced NF-κB signaling and promoted macrophage expression of IL-1β, IL-6, and TNF-α. Lastly, when miR-183-5p was downregulated in exosomes through miR-183-5p sponge expression in 4T1 cells, these 4T1-derived exosomes triggered diminished p65 phosphorylation and IL-1β, IL-6, and TNF-α secretion, and the miRNA downregulation also led to repression of tumor growth and metastasis in the 4T1 breast tumor model in vivo. Thus, miR-183-5p expressed in tumor cells was transferred to macrophages by exosomes and promoted the secretion of proinflammatory cytokines by inhibiting PPP2CA expression, which contributed to tumor progression in a breast cancer model.

Guo J ，Duan Z ，Zhang C ，Wang W ，He H ，Liu Y ，Wu P ，Wang S ，Song M ，Chen H ，Chen C ，Si Q ，Xiang R ，Luo Y ... -  《-》

Exosome-mediated miR-33 transfer induces M1 polarization in mouse macrophages and exerts antitumor effect in 4T1 breast cancer cell line.

Macrophages are the most abundant tumor-infiltrating immune cells. Macrophages are conventionally classified as M1 or M2 types. M2 type is the dominant phenotype of macrophages in the tumor microenvironment. M2 macrophages support different aspects of tumor development, including tumor formation, growth, and metastasis. MicroRNAs (miRNAs) have been demonstrated to regulate numerous cellular processes, including macrophage polarization. To determine whether miR-33 containing exosomes can alter macrophage polarization, we used the exosomes isolated from 4T1 breast cancer cells to deliver miR-33 mimic into IL-4 induced M2 macrophages and treated macrophages with 4T1-conditioned media. Then, we assayed the expression of M1 specific markers and the production of cytokines using real-time PCR and ELISA, respectively. Additionally, we performed MTT, migration, and invasion assays to detect the effect of miRNA-mediated macrophage repolarization on cancer cell proliferation, migration, and invasion. The results of this study showed that miR-33 containing exosomes could convert M2 to M1 phenotype as indicated by an increase in expression of M1 markers, including Irf5, Nos2, and CD86, and a decrease in M2 markers including Arg, Ym1, and CD206. Furthermore, the secretion of TNF-α and IL-1β as M1 specific cytokines increased, while the secretion of IL-10 and TGF-β as M2 specific cytokines decreased. Incubation of 4T1 cells with conditioned media of treated macrophages showed reduced proliferation, invasion, and migration of these cells. So, our data suggests that exosomes can be used as an efficient nanocarrier for miR-33 delivery into macrophages. Also, miR-33 is capable of inducing M1 polarization in macrophages, which is essential for suppressing tumor growth and metastasis.

Moradi-Chaleshtori M ，Bandehpour M ，Heidari N ，Mohammadi-Yeganeh S ，Mahmoud Hashemi S ... -  《-》

Exosome derived from epigallocatechin gallate treated breast cancer cells suppresses tumor growth by inhibiting tumor-associated macrophage infiltration and M2 polarization.

Jang JY ，Lee JK ，Jeon YK ，Kim CW ... -  《BMC CANCER》

Cancer-derived exosomal miR-138-5p modulates polarization of tumor-associated macrophages through inhibition of KDM6B.

Rationale: Differential activation of macrophages correlates closely with tumor progression, and the epigenetic factor lysine demethylase 6B (KDM6B, previously named JMJD3) mediates the regulation of macrophage polarization through an unknown mechanism. Methods: We developed a suspension coculture system comprising breast cancer cells and macrophages and used RT-qPCR and western blotting to measure KDM6B expression. Bioinformatics and luciferase reporter assays were used to identify candidate microRNAs of cancer cells responsible for the downregulation of KDM6B. To determine if exosomes mediated the transfer of miR-138-5p between cancer cells to macrophages, we treated macrophages with exosomes collected from the conditioned medium of cancer cells. The effects of exosomal miR-138-5p on macrophage polarization were measured using RT-qPCR, flow cytometry, and chromatin immunoprecipitation assays. We employed a mouse model of breast cancer, metastatic to the lung, to evaluate the effects on tumor metastasis of macrophages treated with miR-138-5p-enriched exosomes. To develop a diagnostic evaluation index, the levels of exosomal miR-138-5p in samples from patients with breast cancer were compared to those of controls. Results: Coculture of breast cancer cells led to downregulation of KDM6B expression in macrophages. Cancer cell-derived exosomal miR-138-5p inhibited M1 polarization and promoted M2 polarization through inhibition of KDM6B expression in macrophages. Macrophages treated with exosomal miR-138-5p promoted lung metastasis, and the level of circulating exosomal miR-138-5p positively correlated with the progression of breast cancer. Conclusion: Our data suggest that miR-138-5p was delivered from breast cancer cells to tumor-associated macrophages via exosomes to downregulate KDM6B expression, inhibit M1 polarization, and stimulate M2 polarization. Therefore, exosomal miR-138-5p represents a promising prognostic marker and target for the treatment of breast cancer.

Xun J ，Du L ，Gao R ，Shen L ，Wang D ，Kang L ，Chen C ，Zhang Z ，Zhang Y ，Yue S ，Feng S ，Xiang R ，Mi X ，Tan X ... -  《Theranostics》

In vitro and in vivo evaluation of anti-tumoral effect of M1 phenotype induction in macrophages by miR-130 and miR-33 containing exosomes.

Moradi-Chaleshtori M ，Bandehpour M ，Soudi S ，Mohammadi-Yeganeh S ，Hashemi SM ... -  《-》