[Gastric cancer-derived mesenchymal stem cells regulate the M2 polarization of macrophages within gastric cancer microenvironment via JAK2/STAT3 signaling pathway].

Objective: To investigate the role and mechanism of tumor-derived mesenchymal stem cells in regulating the M2 polarization of macrophages within gastric cancer microenvironment. Methods: Gastric cancer tissues and the adjacent non-cancerous tissues were collected from patients underwent gastric cancer resection in the First People's Hospital of Lianyungang during 2018. In our study, THP-1-differentiated macrophages were co-cultured with gastric cancer-derived mesenchymal stem cells (GC-MSCs). Then, the M2 subtype-related gene, the markers expressed on cell surface and the cytokine profile were analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry and Luminex liquid chip, respectively. The key cytokines mediating the inducing effect of GC-MSCs on macrophage polarization into the M2 subtype were detected and screened by Luminex liquid chip, which were further confirmed by the neutralizing antibody test. The expressions of macrophage proteins involved in M2 polarization-related signaling pathways under the different co-culture conditions of GC-MSCs were detected by western blot. Results: In Mac+ GC-MSC-culture medium (CM) group, the expression levels of Ym-1 and Fizz-1 (1.53±0.32 and 13.22±1.05, respectively), which are markers for M2 subtype, were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.60±0.41) was significantly lower than that of Mac group (1.06±0.38, P=0.023). In Mac+ GC-MSC-Transwell (TW) group, the expression levels of Ym-1 and Fizz-1 (1.47±0.09 and 13.16±2.77, respectively) were both significantly higher than those of Mac group (1.00±0.05 and 1.21±0.38, respectively, P<0.05). The level of iNOS in Mac+ GC-MSC-CM group (0.56±0.03) was significantly lower than that of Mac group (1.06±0.38, P=0.026). The ratios of CD163(+) /CD204(+) cells in Mac+ GC-MSC-CM and Mac+ GC-MSC-TW groups (3.80% and 4.40%, respectively) were both remarkably higher than that of Mac group (0.60%, P<0.05). The expression levels of IL-10, IL-6, MCP-1 and VEGF in Mac+ GC-MSC-CM group were (592.60±87.52), (1 346.80±64.70), (11 256.00±29.03) and (1 463.90±66.67) pg/ml, respectively, which were significantly higher than those of Mac group [(41.03±2.59), (17.35±1.79), (5 213.30±523.71) and (267.12±12.06) pg/ml, respectively, P<0.05]. The levels of TNF-α, IP-10, RANTES and MIP-1α were (95.57±9.34), (410.48±40.68), (6 967.30±1.29) and (1 538.70±283.04) pg/ml, which were significantly lower than those of Mac group [(138.01±24.31, (1 298.60±310.50), (14 631.00±4.21) and (6 633.20±1.47) pg/ml, respectively, P<0.05]. The levels of IL-6 and IL-8 in GC-MSCs [(11 185.02±2.82) and (12 718.03±370.17) pg/ml, respectively] were both strikingly higher than those of MSCs from adjacent non-cancerous gastric cancer tissues [(270.71±59.38) and (106.04±32.84) pg/ml, repectively, P<0.05]. The ratios of CD86(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (28.80% and 31.40%, respectively) were both higher than that of Mac+ GC-MSC-CM group (24.70%). Compared to Mac+ GC-MSC-CM group (13.70%), the ratios of CD204(+) cells in Mac+ IL-6-blocked-GC-MSC-CM and Mac+ IL-8-blocked-GC-MSC-CM groups (9.90% and 8.70%, separately) were reduced. The expression levels of p-JAK2 and p-STAT3, which are proteins of macrophage M2 polarization-related signaling pathway, in Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, respectively) were significantly higher than those of Mac group (0.50±0.01 and 0.82±0.01, respectively, P<0.05). The expression levels of p-JAK2 in Mac+ IL-6-blocked-GC-MSC-CM group (0.47±0.02) were significantly lower those that of Mac+ GC-MSC-CM group (0.86±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-8-blocked-GC-MSC-CM group (0.50±0.01 and 0.85±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). The expression levels of p-JAK2 and p-STAT3 in Mac+ IL-6/IL-8-blocked-GC-MSC-CM group (0.37±0.01 and 0.65±0.01, respectively) were both significantly lower than those of Mac+ GC-MSC-CM group (0.86±0.01 and 1.08±0.01, P<0.05). Conclusion: GC-MSCs promote the activation of JAK2/STAT3 signaling pathway in macrophages via high secretions of IL-6 and IL-8, which subsequently induce the macrophage polarization into a pro-tumor M2 subtype within gastric cancer microenvironment.目的： 探讨胃癌微环境中肿瘤来源间充质干细胞对巨噬细胞M2亚型极化的调节作用及机制。 方法： 胃癌及癌旁正常组织来源于2018年于连云港市第一人民医院行胃癌切除的患者(4例)。将THP-1诱导分化来源的巨噬细胞与胃癌来源间充质干细胞(GC-MSCs)进行体外共培养，细胞分为对照组(Mac组)、细胞上清处理组(Mac+GC-MSC-CM组)、Mac+GC-MSC-TW组、Mac+IL-6-blocked-GC-MSC-CM组、Mac+IL-8-blocked-GC-MSC-CM组和Mac+IL-6/IL-8-blocked-GC-MSC-CM组。采用实时荧光定量聚合酶链反应、流式细胞术和Luminex液态芯片技术，分别评价巨噬细胞中M2亚型相关基因、细胞表面标记蛋白以及细胞因子谱的表达情况。利用Luminex液态芯片技术检测并筛选GC-MSCs中可能发挥诱导巨噬细胞向M2亚型极化作用的关键细胞因子，并采用中和抗体试验进行验证。Western blot法检测GC-MSCs不同处理条件下，巨噬细胞中M2亚型极化相关信号通路蛋白表达情况。 结果： Mac+GC-MSC-CM组巨噬细胞中M2亚型相关基因Ym-1和Fizz-1表达水平分别为1.53±0.32和13.22±1.05，均高于Mac组(分别为1.00±0.05和1.21±0.38，均P<0.05)，iNOS基因表达水平(0.60±0.41)低于Mac组(1.06±0.38, P＝0.023)。Mac+GC-MSC-TW组巨噬细胞中Ym-1和Fizz-1基因表达水平分别为1.47±0.09和13.16±2.77，均高于Mac组(分别为1.00±0.05和1.21±0.38，均P<0.05)，iNOS基因表达水平(0.56±0.03)低于Mac组(1.06±0.38，P＝0.026)。Mac+GC-MSC-CM组和Mac+GC-MSC-TW组巨噬细胞中CD163(+)和CD204(+)细胞比例分别为3.80%和4.40%，均高于Mac组(0.60%)。Mac+GC-MSC-CM组巨噬细胞中白细胞介素10(IL-10)、IL-6、单核细胞趋化蛋白1和血管内皮生长因子的表达水平分别为(592.60±87.52)pg/ml、(1 346.80±64.70)pg/ml、(11 256.00±29.03)pg/ml和(1 463.90±66.67)pg/ml，均高于Mac组[分别为(41.03±2.59)pg/ml、(17.35±1.79)pg/ml、(5 213.30±523.71)pg/ml和(267.12±12.06)pg/ml，均P<0.05]，肿瘤坏死因子α、IP-10、RANTES和巨噬细胞炎症蛋白1α的表达水平分别为(95.57±9.34)pg/ml、(410.48±40.68)pg/ml、(6 967.30±1.29)pg/ml和(1 538.70±283.04)pg/ml，均低于Mac组[分别为(138.01±24.31)pg/ml、(1 298.60±310.50)pg/ml、(14 631.00±4.21)pg/ml和(6 633.20±1.47)pg/ml，均P<0.05]。IL-6、IL-8在GC-MSCs中的表达水平分别为(11 185.02±2.82)pg/ml和(12 718.03±370.17)pg/ml，均高于胃癌癌旁间充质干细胞[分别为(270.71±59.38)pg/ml和(106.04±32.84)pg/ml，均P<0.05]。Mac+IL-6-blocked-GC-MSC-CM和Mac+IL-8-blocked-GC-MSC-CM组巨噬细胞中CD86(+)细胞的比例(分别为28.80%和31.40%)均高于Mac+GC-MSC-CM组(24.70%)。Mac+IL-6-blocked-GC-MSC-CM和Mac+IL-8-blocked-GC-MSC-CM组中CD204(+)细胞比例(分别为9.90%和8.70%)均低于Mac+GC-MSC-CM组(13.70%)。Mac+GC-MSC-CM组巨噬细胞M2亚型极化相关信号通路蛋白p-JAK2和p-STAT3表达水平分别为0.86±0.01和1.08±0.01，均高于Mac组(分别为0.50±0.01和0.82±0.01，均P<0.05)，Mac+IL-6-blocked-GC-MSC-CM组巨噬细胞中p-JAK2蛋白表达水平(0.47±0.02)低于Mac+GC-MSC-CM组(0.86±0.01，P<0.05)。Mac+IL-8-blocked-GC-MSC-CM组巨噬细胞中p-JAK2和p-STAT3蛋白表达水平分别为0.50±0.01和0.85±0.01，均低于Mac+GC-MSC-CM组(分别为0.86±0.01和1.08±0.01，均P<0.05)。Mac+IL-6/IL-8-blocked-GC-MSC-CM组细胞中p-JAK2和p-STAT3蛋白表达水平分别为0.37±0.01和0.65±0.01，均低于Mac+GC-MSC-CM组(均P<0.05)。 结论： GC-MSCs通过分泌高水平IL-6和IL-8因子促进巨噬细胞中JAK2/STAT3信号通路活化，从而有效诱导胃癌微环境中巨噬细胞向具有促肿瘤作用的M2亚型发生极化。.