One of the main reasons for cancer’s low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate the immunosuppression, which favors tumor progression. Extracellular vesicles (EVs) have shown great potential for targeted therapies as, depending on their biological origin, they can present different therapeutic properties, such as enhanced accumulation in the target tissue or modulation of the immune system. In the current study, EVs were isolated from M1-macrophages (M1-EVs) pre-treated with hyaluronic acid (HA) and the β-blocker carvedilol (CV). The resulting modulated-M1 EVs (MM1-EVs) were further loaded with doxorubicin (MM1-DOX) to assess their effect in a mouse model of metastatic tumor growth. The cell death and cell migration profile were evaluated in vitro in 4T1 cells. The polarization of the RAW 264.7 murine macrophage cell line was also analyzed to evaluate the effects on the TME. Tumors were investigated by qRT-PCR and immunohistochemistry. MM1-DOX reduced the primary tumor size and metastases. NF-κB was the major gene downregulated by MM1-DOX. Furthermore, MM1-DOX reduced the expression of M2-TAM (CD-163) in tumors, which resulted in increased apoptosis (FADD) as well as decreased expression of MMP-2 and TGF-β. These results suggest a direct effect in tumors and an upregulation in the TME immunomodulation, which corroborate with our in vitro data that showed increased apoptosis, modulation of macrophage polarization, and reduced cell migration after treatment with M1-EVs combined with HA and CV. Our results indicate that the M1-EVs enhanced the antitumor effects of DOX, especially if combined with HA and CV in an animal model of metastatic cancer.

中文翻译：

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来自 M1 极化巨噬细胞的细胞外囊泡与透明质酸和 β 受体阻滞剂联合通过下调乳腺癌中肿瘤相关巨噬细胞来增强阿霉素的抗肿瘤活性

癌症对化疗药物的临床反应低的主要原因之一是高度免疫抑制的肿瘤微环境 (TME)。肿瘤相关的 M2 巨噬细胞 (M2-TAM) 协调免疫抑制，这有利于肿瘤进展。细胞外囊泡 (EV) 已显示出靶向治疗的巨大潜力，因为根据其生物学来源，它们可以呈现不同的治疗特性，例如增强在靶组织中的积累或调节免疫系统。在目前的研究中，EV 是从用透明质酸 (HA) 和 β-受体阻滞剂卡维地洛 (CV) 预处理的 M1 巨噬细胞 (M1-EV) 中分离出来的。将所得调制的 M1 EV (MM1-EV) 进一步加载多柔比星 (MM1-DOX)，以评估它们在转移性肿瘤生长小鼠模型中的作用。在 4T1 细胞中体外评估细胞死亡和细胞迁移曲线。还分析了 RAW 264.7 鼠巨噬细胞系的极化以评估对 TME 的影响。通过qRT-PCR和免疫组织化学研究肿瘤。MM1-DOX 减少了原发肿瘤的大小和转移。NF-κB 是 MM1-DOX 下调的主要基因。此外，MM1-DOX 降低了肿瘤中 M2-TAM (CD-163) 的表达，从而导致细胞凋亡 (FADD) 增加以及 MMP-2 和 TGF-β 的表达降低。这些结果表明在肿瘤中的直接作用和 TME 免疫调节的上调，这与我们的体外数据证实，即在 M1-EV 与 HA 和 CV 联合治疗后细胞凋亡增加、巨噬细胞极化调节和细胞迁移减少。