巨噬细胞集落刺激因子对巨噬细胞极化及卵巢癌细胞侵袭、转移的影响

目的探讨卵巢癌微环境中巨噬细胞集落刺激因子(M-CSF)促进M2型巨噬细胞的极化和浸润,及其对于癌细胞侵袭、转移的影响。方法选取卵巢癌细胞系A2780和SKOV3与THP-1来源巨噬细胞,在体外构建共培养模型,ELISA和实时荧光定量PCR(qRT-PCR)检测培养基中M-CSF含量及其mRNA的表达水平。流式细胞术分析CD68^+CD163^+M2型巨噬细胞的极化比例。Transwell迁移试验检测2种卵巢癌细胞系与M2型巨噬细胞共培养后侵袭、转移能力的变化。免疫组化染色法检测卵巢癌(52例)及同期卵巢良性肿瘤(18例)石蜡组织切片中M-CSF、CD68^+、CD163^+和E-钙黏蛋白(E-cad)的表达及其相关性,并分析M-CSF与卵巢癌患者临床病理特征的关系。结果 A2780和SKOV3细胞与巨噬细胞共培养后,其上清中M-CSF浓度明显高于2种癌细胞单独培养组(t分别为14.315、12.338,P均<0.01)。荧光定量PCR结果显示,升高的M-CSF来源于共培养后卵巢癌细胞的分泌(t分别为29.915、36.826,P均<0.01)。CD68^+CD163^+M2型巨噬细胞在A2780和SKOV3共培养组的极化比例分别为(6.14±0.50)%和(7.32±0.67)%,明显高于单独培养组(1.82±0.34)%,差异有统计学意义(t分别为12.289、12.711,P均<0.01)。Transwell试验结果显示,2种卵巢癌细胞共培养后其侵袭能力增强(24.00±4.81 vs 75.20±6.42,t=11.058;18.40±2.31 vs 61.60±9.66,t=7.537,P均<0.01)。卵巢癌组织中M-CSF的表达与CD68^+、CD163^+阳性细胞数正相关且均高于对照组(r分别为0.690、0.596,P均<0.01),而E-cad阳性表达则与之呈负相关且低于对照组(r=-0.566,P<0.01)。M-CSF表达在不同肿瘤FIGO分期、分化程度和淋巴结转移情况组间差异有统计学意义(χ^2分别为6.240、6.612、4.544,P均<0.05)。结论卵巢癌微环境中M-CSF的表达升高诱导CD68^+CD163^+M2型巨噬细胞的极化和浸润,进而促进癌细胞的侵袭、转移。

Effects of macrophage colony-stimulating factor on macrophage polarization, invasion and metastasis of ovarian cancer

Objective To investigate the effects of macrophage colony-stimulating factor(M-CSF) on the polarization and infiltration of M2 macrophages and the invasion and metastasis of tumor cells in ovarian cancer microenvironment. Methods A co-culture system consisting of ovarian cancer cells(A2780 and SKOV3) and THP-1 derived macrophages was established in vitro. The M-CSF levels in culture medium and M-CSF mRNA levels in cancer cells and macrophages were detected by ELISA and qRT-PCR, respectively. The proportion of CD68^+CD163^+ M2 macrophages(polarization cells) was determined by flow cytometry. The invasive and metastatic ability of A2780 and SKOV3 cells after co-culturing with M2 macrophages were analyzed using Transwell assay. The expression levels of M-CSF, CD68^+, CD163^+ and E-cad in paraffin sections of 52 patients with ovarian cancer and 18 patients with benign ovarian tumor were detected by the immunohistochemistry staining, and their correlations and the relationship between M-CSF and clinicopathological features of ovarian cancer patients were analyzed. Results The M-CSF levels in culture medium of the co-culture group(A2780 and SKOV3 cells co-cultured with M2 macrophages) were significantly higher than that of A2780 and SKOV3 cells alone(t=14.315 and 12.338, P<0.01). Fluorescence quantitative PCR results showed that the increased M-CSF originated from the secretion of co-cultured ovarian cancer cells(t=29.915 and 36.826, P<0.01). The proportions of CD68^+CD163^+ M2 macrophages in the A2780 cells co-cultured with M2 macrophages group and SKOV3 cells co-cultured with M2 macrophages group were(6.14±0.50)% and(7.32±0.67)%, respectively, which were significantly higher than that in the M2 macrophages alone group([1.82±0.34]%, t=12.289 and 12.711, P<0.01). Transwell assay showed that the co-culture environment enhanced the invasion of A2780 and SKOV3 cells(24.00±4.81 vs 75.20±6.42, t=11.058;18.40±2.31 vs 61.60±9.66, t=7.537, P<0.01). The expression levels of M-CSF in ovarian cancer tissues were positively correlated with the number of CD68^+ cells and CD163^+ cells(r=0.690 and 0.596, P<0.01), and negatively with the expression levels of E-cad(r=-0.566, P<0.01). Moreover, the expression levels of M-CSF and the number of CD68^+ cells and CD163^+ cells in ovarian cancer tissues were significantly higher than that in benign ovarian tumor tissues, however, the expression levels of E-cad were on the contrary. The expression levels of M-CSF in ovarian cancer tissues were significantly correlated with tumor stage, differentiation and lymphatic node metastasis(χ^2=6.240, 6.612 and 4.544, respectively, P<0.05). Conclusion The increased expression of M-CSF in ovarian cancer microenvironment may induce the polarization and infiltration of CD68^+CD163^+ M2 macrophages, and then promote the invasion and metastasis of ovarian cancer cells.