肿瘤相关巨噬细胞促进尤文肉瘤细胞增殖、侵袭及血管拟态形成
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摘要
目的:探究肿瘤相关巨噬细胞对尤文肉瘤细胞增殖、侵袭及血管拟态形成能力的影响及其可能的分子作用机制。方法:体外诱导人单核细胞U937向M2型巨噬细胞分化,实时荧光定量PCR法检测诱导前后单核细胞中M2型巨噬细胞相关因子CD68、CD163及CD206的表达差异。将诱导后的人单核细胞U937与尤文肉瘤细胞A673及SK-ES-1共培养后,分别采用CCK-8法、Transwell小室侵袭实验及血管拟态形成实验检测尤文肉瘤细胞增殖、侵袭及血管拟态形成能力的变化。蛋白质印迹法检测共培养后尤文肉瘤细胞中信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)及其下游的磷酸化STAT3(phospho-STAT3,p-STAT3)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)和周期蛋白D2(cyclin D2,CCND2)表达的变化。结果:体外诱导后的人单核细胞U937中CD68、CD163及CD206表达水平明显升高(P值均<0.01),提示单核细胞成功分化为M2型巨噬细胞。将诱导后的M2型U937细胞分别与尤文肉瘤细胞A673及SK-ES-1共培养48 h后,A673及SK-ES-1细胞增殖能力较未共培养的对照组明显增强(P值均<0.05),且穿透基质胶的细胞数较对照组明显增多(P值均<0.05),新生血管分支数也较对照组明显增多(P值均<0.05)。共培养后的尤文肉瘤细胞中,STAT3及其下游p-STAT3、MMP2和CCND2蛋白表达水平均较对照组明显升高(P值均<0.01)。结论:肿瘤相关巨噬细胞与尤文肉瘤细胞共培养能够明显促进尤文肉瘤细胞的增殖、侵袭及血管拟态形成,其作用可能是通过激活尤文肉瘤细胞中STAT3通路来实现的。
Objective :To investigate the effects of tumor-associated macrophages on the proliferation, invasion and vasculogenic mimicry of Ewing sarcoma cells, and to explore the involved molecular mechanism.Methods: Human monocytic U937 cells were cultured and induced to differentiate into M2-type macrophages in vitro. The expressions of M2-type macrophages-relative cytokines such as CD68, CD163 and CD206 in U937 cells before and after induction were detected by realtime fluorescent quantitative PCR. Ewing sarcoma A673 and SK-ES-1 cells were co-cultured with the induced U937 cells(macrophages). Then the effects of co-culture with macrophages on the proliferation, invasion and vasculogenic mimicry of Ewing sarcoma cells were performed using CCK-8, Transwell chamber invasion and matrigel tubule formation assays, respectively.The expressions of signal transducer and activator of transcription 3(STAT3) and its downstream phospho-STAT3(p-STAT3), matrix metalloproteinase 2(MMP2) and cyclin D2(CCND2)in Ewing sarcoma cells co-cultured with macrophages were detected by Western blotting.Results: The expression levels of CD68, CD163 and CD206 in monocytic U937 cells were significantly up-regulated after induction in vitro(all P < 0.01), suggesting that the human monocytic U937 cells were differentiated into M2-type macrophages. After the M2-type macrophages were co-cultured with Ewing sarcoma A673 and SK-ES-1 cells for 48 h, the proliferation ability of A673 and SK-ES-1 cells increased significantly as compared with the un-co-cultured control group(both P < 0.05), the number of A673 and SK-ES-1 cells passing through matrigel increased significantly as compared with the control group(both P <0.05), the branches of new vascular in co-cultured groups were more than those in control group(both P < 0.05), and the expression levels of STAT3 and its down-stream p-STAT3,CCND2 and MMP2 proteins were up-regulated in A673 and SK-ES-1 cells(all P < 0.01).Conclusion: Co-culturing with tumor-associated macrophages can induce the proliferation,invasion and vascular mimetic formation of Ewing sarcoma cells, which may be mediated by activating the STAT3 signaling pathway in Ewing sarcoma cells.
Objective :To investigate the effects of tumor-associated macrophages on the proliferation, invasion and vasculogenic mimicry of Ewing sarcoma cells, and to explore the involved molecular mechanism.Methods: Human monocytic U937 cells were cultured and induced to differentiate into M2-type macrophages in vitro. The expressions of M2-type macrophages-relative cytokines such as CD68, CD163 and CD206 in U937 cells before and after induction were detected by realtime fluorescent quantitative PCR. Ewing sarcoma A673 and SK-ES-1 cells were co-cultured with the induced U937 cells(macrophages). Then the effects of co-culture with macrophages on the proliferation, invasion and vasculogenic mimicry of Ewing sarcoma cells were performed using CCK-8, Transwell chamber invasion and matrigel tubule formation assays, respectively.The expressions of signal transducer and activator of transcription 3(STAT3) and its downstream phospho-STAT3(p-STAT3), matrix metalloproteinase 2(MMP2) and cyclin D2(CCND2)in Ewing sarcoma cells co-cultured with macrophages were detected by Western blotting.Results: The expression levels of CD68, CD163 and CD206 in monocytic U937 cells were significantly up-regulated after induction in vitro(all P < 0.01), suggesting that the human monocytic U937 cells were differentiated into M2-type macrophages. After the M2-type macrophages were co-cultured with Ewing sarcoma A673 and SK-ES-1 cells for 48 h, the proliferation ability of A673 and SK-ES-1 cells increased significantly as compared with the un-co-cultured control group(both P < 0.05), the number of A673 and SK-ES-1 cells passing through matrigel increased significantly as compared with the control group(both P <0.05), the branches of new vascular in co-cultured groups were more than those in control group(both P < 0.05), and the expression levels of STAT3 and its down-stream p-STAT3,CCND2 and MMP2 proteins were up-regulated in A673 and SK-ES-1 cells(all P < 0.01).Conclusion: Co-culturing with tumor-associated macrophages can induce the proliferation,invasion and vascular mimetic formation of Ewing sarcoma cells, which may be mediated by activating the STAT3 signaling pathway in Ewing sarcoma cells.
引文
[1]ZHU C,OLSON KA,ROTH M,et al.Provider views on the management of Ewing sarcoma of the spine and pelvis[J].J Surg Oncol,2018,117(3):417-424.
[2]ARAS S,ZAIDI MR.TAMeless traitors:macrophages in cancer progression and metastasis[J].Br J Cancer,2017,117(11):1583-1591.
[3]LI Y,HUANG G,ZHANG S.Associations between intratumoral and peritumoral M2 macrophage counts and cervical squamous cell carcinoma invasion patterns[J].Int J Gynaecol Obstet,2017,139(3):346-351.
[4]FUIJIWARA T,FUKUSHI J,YAMAMOTO S,et al.Macrophage infiltration predicts a poor prognosis for human ewing sarcoma[J].Am J Pathol,2011,179(3):1157-1170.
[5]HAM JS,PARK HY,RYU KJ,et al.Elevated serum interleukin-10 level and M2 macrophage infiltration are associated with poor survivalinangioimmun oblastic T-cell lymphoma[J].Oncotarget,2017,8(44):76231-76240.
[6]NA YR,JE S,SEOK SH.Metabolic features of macrophages in inflammatory diseases and cancer[J].Cancer Lett,2018,413:46-58.
[7]MIYAUCHI JT,CAPONEGRO MD,CHEN D,et al.Deletion of neuropilin1 from microglia or bone marrowderived macrophages slows glioma progression[J].Cancer Res,2017,78(3):685-694.
[8]TAVAZOIE S F,ALARCóN C,OSKARSSON T,et al.Endogenous human microRNAs that suppress breast cancer metastasis[J].Nature,2008,451(7175):147-152.
[9]TAYLOR R,KNOMLES HJ,ATHANASOU NA.Ewing sarcoma cells express RANKL and support osteoclastogenesis[J].J Pathol,2011,225(2):195-202.
[10]ZHANG Z,LI Y,HUANG L,et al.Let-7a suppresses macrophage infiltrations and malignant phenotype of Ewing sar coma via STAT3/NF-kappaBpositive regulatory circuit[J].Cancer Lett,2016,374(2):192-201.
[11]李亚,陈思成,王静,等.肿瘤相关M2型巨噬细胞通过上调VEGFR3的表达促进肺腺癌A549细胞的迁移和侵袭[J].肿瘤,2016,36(8):846-856.
[12]张伟娜.结直肠癌细胞EGFR信号通路调节巨噬细胞极性的机制研究[D].北京:北京协和医学院,2016.
[2]ARAS S,ZAIDI MR.TAMeless traitors:macrophages in cancer progression and metastasis[J].Br J Cancer,2017,117(11):1583-1591.
[3]LI Y,HUANG G,ZHANG S.Associations between intratumoral and peritumoral M2 macrophage counts and cervical squamous cell carcinoma invasion patterns[J].Int J Gynaecol Obstet,2017,139(3):346-351.
[4]FUIJIWARA T,FUKUSHI J,YAMAMOTO S,et al.Macrophage infiltration predicts a poor prognosis for human ewing sarcoma[J].Am J Pathol,2011,179(3):1157-1170.
[5]HAM JS,PARK HY,RYU KJ,et al.Elevated serum interleukin-10 level and M2 macrophage infiltration are associated with poor survivalinangioimmun oblastic T-cell lymphoma[J].Oncotarget,2017,8(44):76231-76240.
[6]NA YR,JE S,SEOK SH.Metabolic features of macrophages in inflammatory diseases and cancer[J].Cancer Lett,2018,413:46-58.
[7]MIYAUCHI JT,CAPONEGRO MD,CHEN D,et al.Deletion of neuropilin1 from microglia or bone marrowderived macrophages slows glioma progression[J].Cancer Res,2017,78(3):685-694.
[8]TAVAZOIE S F,ALARCóN C,OSKARSSON T,et al.Endogenous human microRNAs that suppress breast cancer metastasis[J].Nature,2008,451(7175):147-152.
[9]TAYLOR R,KNOMLES HJ,ATHANASOU NA.Ewing sarcoma cells express RANKL and support osteoclastogenesis[J].J Pathol,2011,225(2):195-202.
[10]ZHANG Z,LI Y,HUANG L,et al.Let-7a suppresses macrophage infiltrations and malignant phenotype of Ewing sar coma via STAT3/NF-kappaBpositive regulatory circuit[J].Cancer Lett,2016,374(2):192-201.
[11]李亚,陈思成,王静,等.肿瘤相关M2型巨噬细胞通过上调VEGFR3的表达促进肺腺癌A549细胞的迁移和侵袭[J].肿瘤,2016,36(8):846-856.
[12]张伟娜.结直肠癌细胞EGFR信号通路调节巨噬细胞极性的机制研究[D].北京:北京协和医学院,2016.