通过人参皂苷 Rh2 调节肿瘤相关巨噬细胞与非小细胞肺癌之间的串扰，抑制肿瘤迁移和侵袭。

BACKGROUND: Tumor-associated macrophages (TAMs) play a critical role in modulating the tumor microenvironment and promote tumor metastases. Our studies have demonstrated that ginsenoside Rh2 (G-Rh2), a monomeric compound extracted from ginseng, is a promising anti-tumor agent in lung cancer cells. However, it remains unclear whetherG-Rh2 can modulate the differentiation of TAMs and its interaction with tumor microenvironment. In this study, we investigated how G-Rh2 regulates the phenotype of macrophages and affects the migration of non-small cell lung cancer (NSCLC) cells. METHODS: Murine macrophage-like RAW264.7 cells and human THP-1 monocyte were differentiated into M1 and M2 subsets of macrophages with different cytokines combination, which were further identified by flow cytometry with specific biomarkers. M2 macrophages were sorted out to co-culture with NSCLC cell lines, A549 and H1299. Wound healing assay was performed to examine the cell migration. Expression levels of matrix metalloproteinases 2 and 9 (MMP-2, - 9) and vascular endothelial growth factor-C (VEGF-C) were measured by RT-qPCR and western blot, and the release of VEGF in the supernatant was measured by a VEGF ELISA kit. Finally, modulation of TAMs phenotype and VEGF expression by G-Rh2 was examined in vivo. RESULTS: We demonstrated that M2 subset of macrophages alternatively differentiated from RAW264.7 or THP-1cells promote migration of NSCLC cells. Further examinations revealed that NSCLC significantly increased the release of VEGF to the media and elevated the expression levels of VEGF at mRNA and protein levels after being co-cultured with M2 macrophages. Similar alterations in MMP-2 and MMP-9 were observed in NSCLC after being co-cultured. Of note,G-Rh2 had a potential to effectively convert M2 phenotype to M1 subset of macrophages. Importantly, G-Rh2 had a preference to decrease the expression levels of VEGF, MMP2, and MMP9 in co-cultured lung cancer cells, over than those in lung cancer cells without co-culturing. Consistently, G-Rh2 reduced M2 macrophage marker CD206 and VEGF expression levels in vivo. CONCLUSIONS: All of these results suggested that M2 subset macrophages drive lung cancer cells with more aggressive phenotypes. G-Rh2 has a potential to convert TAMs from M2 subset to M1 in the microenvironment and prevents lung cancer cell migration, suggesting the therapeutic effects of G-Rh2onlung cancer.

背景：肿瘤相关巨噬细胞（TAMs）在调节肿瘤微环境和促进肿瘤转移方面发挥着关键作用。我们的研究表明，从人参中提取的单体化合物人参皂苷 Rh2（G-Rh2）是肺癌细胞中一种很有前途的抗肿瘤药物。然而，目前尚不清楚 G-Rh2 是否可以调节 TAMs 的分化及其与肿瘤微环境的相互作用。在这项研究中，我们研究了 G-Rh2 如何调节巨噬细胞的表型以及如何影响非小细胞肺癌（NSCLC）细胞的迁移。

方法：用不同细胞因子组合将鼠源巨噬细胞样 RAW264.7 细胞和人源 THP-1 单核细胞分化为 M1 和 M2 亚群的巨噬细胞，并用特异性标志物通过流式细胞术进一步鉴定。将 M2 巨噬细胞分选出来与 NSCLC 细胞系 A549 和 H1299 共培养。通过划痕实验检测细胞迁移。通过 RT-qPCR 和 Western blot 检测基质金属蛋白酶 2 和 9（MMP-2、-9）和血管内皮生长因子-C（VEGF-C）的表达水平，并通过 VEGF ELISA 试剂盒测量上清液中 VEGF 的释放量。最后，在体内研究 G-Rh2 对 TAMs 表型和 VEGF 表达的调节作用。

结果：我们证明了从 RAW264.7 或 THP-1 细胞分化而来的 M2 亚群巨噬细胞可促进 NSCLC 细胞的迁移。进一步的研究表明，与 M2 巨噬细胞共培养后，NSCLC 细胞显著增加了 VEGF 向培养基中的释放，并在 mRNA 和蛋白水平上升高了 VEGF 的表达水平。与共培养的 NSCLC 细胞相比，MMP-2 和 MMP-9 也观察到类似的变化。值得注意的是，G-Rh2 具有将 M2 表型有效转化为 M1 亚群巨噬细胞的潜力。重要的是，G-Rh2 更倾向于降低共培养的肺癌细胞中 VEGF、MMP2 和 MMP9 的表达水平，而不是不与肺癌细胞共培养的细胞。同样，G-Rh2 降低了体内 M2 巨噬细胞标志物 CD206 和 VEGF 的表达水平。

结论：所有这些结果表明，M2 亚群巨噬细胞驱动具有更侵袭性表型的肺癌细胞。G-Rh2 具有在微环境中将 TAMs 从 M2 亚群转化为 M1 的潜力，并阻止肺癌细胞迁移，这表明 G-Rh2 对肺癌具有治疗作用。