Objective: To investigate the key cytokine which polarizes M2 macrophages and promotes invasion and metastasis in non-small cell lung cancer (NSCLC). Methods: After co-culture with A549 cells in vitro, the proportion of CD14(+) CD163(+) M2 macrophages in monocytes and macrophage colony stimulating factor (M-CSF) levels in culture supernatant were detected by flow cytometry, ELISA assay and real-time qPCR, respectively. The effects of CD14(+) CD163(+) M2 macrophages on invasion of A549 cells and angiogenesis of HUVEC cells were measured by transwell assay and tubule formation assay, respectively. The clinical and prognostic significance of M-CSF expression in NSCLC was further analyzed. Results: The percentage of CD14(+) CD163(+) M2 macrophages in monocytes and the concentration of M-CSF in the supernatant followed by co-culture was (12.03±0.46)% and (299.80±73.76)pg/ml, respectively, which were significantly higher than those in control group [(2.80±1.04)% and (43.07±11.22)pg/ml, respectively, P< 0.05]. Human recombinant M-CSF promoted M2 polarization of macrophages in vitro. M2 macrophages enhanced the invasion of A549 cells (66 cells/field vs. 26 cells/field) and the angiogenesis of HUVEC cells (22 tubes/field vs. 8 tubes/field). The mRNA expression of M-CSF in stage Ⅰ-Ⅱ patients (16.23±4.83) was significantly lower than that in stage Ⅲ-Ⅳ (53.84±16.08; P<0.05). M-CSF levels were associated with poorer overall survival and disease-free survival in NSCLC patients (P<0.05). Conclusions: Tumor-derived M-CSF can induce CD14(+) CD163(+) M2 polarization of macrophages, which can further promote the metastasis and angiogenesis of NSCLC. M-CSF could be used as a potential therapeutic target of NSCLC.
目的： 探讨非小细胞肺癌(NSCLC)微环境中巨噬细胞极化的关键因子及其促进NSCLC进展的作用机制，寻求NSCLC治疗的潜在靶点。 方法： 将单核细胞与A549细胞体外共培养，采用流式细胞术、酶联免疫吸附实验和实时荧光定量PCR法检测CD14(＋)CD163(＋)M2型巨噬细胞的比例，以及培养上清中巨噬细胞集落刺激因子(M-CSF)的含量和M-CSF mRNA的表达水平。采用Transwell迁移实验和血管生成实验分别检测CD14(＋)CD163(＋)M2型巨噬细胞对A549细胞侵袭转移和血管生成的影响。采用实时荧光定量PCR法检测NSCLC组织中M-CSF mRNA的表达，并分析其与NSCLC患者TNM分期和预后的关系。 结果： 单核细胞与A549细胞共培养后，CD14(＋)CD163(＋)M2型巨噬细胞的比例和培养上清中M-CSF的含量分别为(12.03±0.46)%和(299.80±73.76)pg/ml，均高于单独培养组的单核细胞[分别为(2.80±1.04)%和(43.07±11.22)pg/ml，均P<0.05]。人重组M-CSF能够促使单核细胞向CD14(＋)CD163(＋)M2型巨噬细胞极化。Transwell迁移实验的结果显示，加培养基组A549细胞和加CD14(＋)CD163(＋)M2型巨噬细胞培养上清组A549细胞的迁移细胞数分别为26个/视野和66个/视野，差异有统计学意义(P<0.01)。加培养基组人脐静脉内皮细胞系(HUVEC)细胞和加CD14(＋)CD163(＋)M2型巨噬细胞培养上清组HUVEC细胞的微血管密度分别为8个/视野和22个/视野，差异有统计学意义(P<0.01)。Ⅰ～Ⅱ期和Ⅲ～Ⅳ期NSCLC组织中M-CSF mRNA的表达水平分别为16.23±4.83和53.84±16.08，Ⅲ～Ⅳ期NSCLC患者组织中M-CSF mRNA的表达水平明显高于Ⅰ～Ⅱ期(P<0.05)。M-CSF低表达组(26例)和高表达组(27例)患者的中位总生存时间分别为26.3个月和21.4个月，差异有统计学意义(P<0.01)；中位无进展生存时间分别为25.3个月和16.6个月，差异亦有统计学意义(P<0.01)。 结论： NSCLC通过分泌M-CSF诱导单核细胞向CD14(＋)CD163(＋)M2型巨噬细胞极化，被极化的M2型巨噬细胞能进一步促进NSCLC的转移和血管形成。M-CSF可能可以作为NSCLC潜在的治疗靶点。.
Keywords: Angiogenesis; Carcinoma, non-small cell lung; Invasion; M2 macrophage; Macrophage colony stimulating factor; Tumor-associated macrophage.