癌相关成纤维细胞对人舌鳞状细胞癌上皮-间质转化的影响

摘要/Abstract

摘要： 目的:探讨癌相关成纤维细胞(CAFs)对舌鳞癌细胞侵袭、转移的作用及可能机制。方法:应用胶原酶消化法原代分离舌癌相关成纤维细胞及对应正常成纤维细胞(NFs),采用Western免疫印迹、ELISA、激光共聚焦和实时定量PCR分别检测vimentin、cytokeratin、α-SAM和SDF1蛋白质及mRNA水平表达差异。以舌鳞状细胞癌SCC9为研究对象,建立共培养体系,采用Western免疫印迹、激光共聚焦和实时定量PCR检测SCC9中上皮-间质转化标志蛋白E-cadherin、vimentin和fibronectin及其mRNA水平的变化,Transwell实验检测细胞侵袭、迁移能力。采用SPSS13.0软件包对数据进行统计学分析。结果:胶原酶消化法成功获取CAFs及对应NFs,CAFS和NFs均表达vimentin,不表达cytokeratin。其中,CAFs高表达α-SAM并分泌SDF1(P<0.01)。与SCC9共培养2周后,SCC9呈梭形改变,E-cadherin表达降低,vimentin和fibronectin升高(P<0.01),同时其迁移能力增强。结论:胶原酶消化法能成功获取原代CAFs。CAFs可通过诱导SCC9上皮-间质转化进而促进其侵袭、转移。

Abstract: PURPOSE: To examine the role of cancer-associated fibroblasts (CAFs) in promoting invasion of tongue squamous cell carcinoma (TSCC) and the possible mechanism. METHODS: A series of matched pairs of CAFs and normal fibroblasts (NFs) were primarily cultured in collagenase digestion. Identified markers of alpha smooth muscle actin (α-SMA), stromal cell-derived factor-1(SDF1), vimentin and cytokeratin (CK) were observed. Transwell assay, Western blot, reverse transcription-PCR and immunofluorescence staining were carried out to assess the role of CAFs in promoting migration, invasion and inducing epithelial to mesenchymal transition (EMT) of SCC9, compared with NFs. RESULTS: Both primarily cultured CAFs and NFs expressed vimentin, but not CK. CAFs showed significantly higher α-SMA expression and SDF1 secretion, as well as mRNA level of α-SMA and SDF1. Co-culture with CAFs induced up-regulation of EMT markers (fibronectin and vimentin) and down-regulation of E-cadherin of SCC9, as well as enhanced invasion. CONCLUSION: Collagenase digestion is a reliable way to primary culture of CAFs. CAFs may induce EMT markers and functional changes in SCC9. Supported by National Natural Science Foundation of China (81172563).

Key words: Cancer-associated fibroblast, Tumor microenvironment, Tongue squamous cell carcinoma, Epithelial-mesenchymal transition

中图分类号:

R739.8

周斌,陈伟良,王友元,林钊宇,张大明,范松. 癌相关成纤维细胞对人舌鳞状细胞癌上皮-间质转化的影响[J]. 中国口腔颌面外科杂志, 2014, 12(3): 198-204.

ZHOU Bin, CHEN Wei-liang, WANG You-yuan, LIN Zhao-yu, ZHANG Da-ming, FAN Song. Cancer-associated fibroblasts induce epithelial-to-mesenchymal transition in tongue squamous cell carcinoma[J]. China J Oral Maxillofac Surg, 2014, 12(3): 198-204.

参考文献

【1】Sun L, Yao Y, Liu B, et al. MiR-200b and miR-15b regulate chemotherapy-induced epithelial-mesenchymal transition in human tongue cancer cells by targeting BMI1 . Oncogene, 2012, 31(4): 432-445.
【2】Joyce JA, Pollard JW. Microenvironmental regulation of metastasis . Nat Rev Cancer, 2009, 9(4): 239-252.
【3】Zhang J, Liu J. Tumor stroma as targets for cancer therapy . Pharmacol Ther, 2013, 137(2): 200-215.
【4】Potenta S, Zeisberg E, Kalluri R. The role of endothelial-to-mesenchymal transition in cancer progression . Br J Cancer, 2008, 99(9): 1375-1379.
【5】Liu Y, Hu T, Shen J, et al. Separation, cultivation and biological characteristics of oral carcinoma-associated fibroblasts . Oral Dis, 2006, 12(4): 375-380.
【6】Hanna E, Quick J, Libutti SK. The tumour microenvironment: a novel target for cancer therapy . Oral Dis, 2009, 15(1): 8-17.
【7】Fujii N, Shomori K, Shiomi T, et al. Cancer-associated fibroblasts and CD163-positive macrophages in oral squamous cell carcinoma: their clinicopathological and prognostic significance . J Oral Pathol Med, 2012, 41(6): 444-451.
【8】Bello IO, Vered M, Dayan D, et al. Cancer-associated fibroblasts, a parameter of the tumor microenvironment, overcomes carcinoma-associated parameters in the prognosis of patients with mobile tongue cancer . Oral Oncol, 2011, 47(1): 33-38.
【9】Vered M, Dobriyan A, Dayan D, et al. Tumor-host histopathologic variables, stromal myofibroblasts and risk score,are significantly associated with recurrent disease in tongue cancer . Cancer Sci, 2010, 101(1): 274-280.
【10】Schoppmann SF, Berghoff A, Dinhof C, et al. Podoplanin-expressing cancer-associated fibroblasts are associated with poor prognosis in invasive breast cancer . Breast Cancer Res Treat, 2012, 134(1): 237-244.【11】Herrera M, Herrera A, Dominguez G, et al. Cancer-associated fibroblast and M2 macrophage markers together predict outcome in colorectal cancer patients . Cancer Sci, 2013, 104(4): 437-444.
【12】Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion . Cell, 2005, 121(3): 335-348.
【13】Lin J, Liu C, Ge L, et al. Carcinoma-associated fibroblasts promotes the proliferation of a lingual carcinoma cell line by secreting keratinocyte growth factor . Tumour Biol, 2011, 32(3): 597-602.
【14】Sobral LM, Bufalino A, Lopes MA, et al. Myofibroblasts in the stroma of oral cancer promote tumorigenesis via secretion of activin A . Oral Oncol, 2011, 47(9): 840-846.
【15】Wu MH, Hong HC, Hong TM, et al. Targeting galectin-1 in carcinoma-associated fibroblasts inhibits oral squamous cell carcinoma metastasis by downregulating MCP-1/CCL2 expression . Clin Cancer Res, 2011, 17(6): 1306-1316.
【16】Hinsley EE, Hunt S, Hunter KD, et al. Endothelin-1 stimulates motility of head and neck squamous carcinoma cells by promoting stromal-epithelial interactions. Int J Cancer, 2012, 130(1): 40-47.
【17】Xu J, Lu Y, Qiu S, et al. A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells . Cancer Lett, 2013, 335(2): 380-386.
【18】Kellermann MG, Sobral LM, da Silva SD, et al. Mutual paracrine effects of oral squamous cell carcinoma cells and normal oral fibroblasts: induction of fibroblast to myofibroblast transdifferentiation and modulation of tumor cell proliferation . Oral Oncol, 2008, 44(5): 509-517.
【19】Thiery JP, Acloque H, Huang RY, et al. Epithelial-mesenchymal transitions in development and disease . Cell, 2009, 139(5): 871-890.
【20】Vered M, Dayan D, Yahalom R, et al. Cancer-associated fibroblasts and epithelial-mesenchymal transition in metastatic oral tongue squamous cell carcinoma. Int J Cancer,2010,127(6): 1356-1362.
【21】Lebret SC, Newgreen DF, Thompson EW, et al. Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors . Breast Cancer Res, 2007, 9(1): R19.
【22】Soon P, Kim E, Pon CK, et al. Breast cancer-associated fibroblasts induce epithelial-to-mesenchymal transition in breast cancer cells . Endocr Relat Cancer, 2013, 20(1): 1-12.
【23】Giannoni E, Bianchini F, Masieri L, et al. Reciprocal activation of prostate cancer cells and cancer-associated fibroblasts stimulates epithelial-mesenchymal transition and cancer stemness. Cancer Res, 2010, 70(17): 6945-6956.
【24】DudÁs J, Bitsche M, Schartinger V, et al. Fibroblasts produce brain-derived neurotrophic factor and induce mesenchymal transition of oral tumor cells . Oral Oncol, 2011, 47(2): 98-103.
【25】Daly AJ, McIlreavey L, Irwin CR. Regulation of HGF and SDF-1 expression by oral fibroblasts-implications for invasion of oral cancer . Oral Oncol, 2008, 44(7): 646-651.
【26】Onoue T, Uchida D, Begum NM, et al. Epithelial-mesenchymal transition induced by the stromal cell-derived factor-1/CXCR4 system in oral squamous cell carcinoma cells . Int J Oncol, 2006, 29(5): 1133-1138.

[1]	鲁煜, 刘清海, 梁海胜, 邓刚. 3D-ASL对舌肿物的诊断价值及与病理学特征的相关性分析[J]. 中国口腔颌面外科杂志, 2024, 22(1): 42-48.
[2]	韩林孜, 周建华, 董磊, 赵璐, 袁荣涛. 肿瘤相关成纤维细胞对口腔鳞状细胞癌生物学行为影响的研究进展[J]. 中国口腔颌面外科杂志, 2024, 22(1): 84-91.
[3]	慕家宁, 袁文清, 陆洲, 赵阳, 王奇民, 童磊, 陈正岗. Cdc42在舌鳞状细胞癌中的表达及其对细胞增殖、迁移、侵袭的影响[J]. 中国口腔颌面外科杂志, 2023, 21(3): 209-217.
[4]	王博, 沈月洪, 杨宏宇. 鼻咽癌放化疗后继发舌鳞癌、口咽癌及牙龈癌1例并文献复习[J]. 中国口腔颌面外科杂志, 2023, 21(3): 301-305.
[5]	水文军, 高金星, 任蕾. 基质金属蛋白酶9抑制剂对人舌鳞癌SCC-25细胞侵袭、迁移能力的影响及机制探讨[J]. 中国口腔颌面外科杂志, 2021, 19(5): 424-428.
[6]	陈正岗, 王奇民, 童磊, 王云英, 徐晓娜, 王莹, 韩红钰, 盛善桂, 王少如. 沉默Icmt基因对舌鳞癌CAL-27和SCC-4细胞增殖、凋亡及细胞周期的影响[J]. 中国口腔颌面外科杂志, 2021, 19(2): 97-104.
[7]	孔宪琛, 姜晓东, 芦昕, 禹雯怡, 薛娇, 张鹏, 赵璐, 袁荣涛. 核糖体蛋白L29在舌鳞状细胞癌中的表达及意义[J]. 中国口腔颌面外科杂志, 2021, 19(2): 110-115.
[8]	李明彧, 马海龙, 杨溪, 张志愿. 癌相关成纤维细胞在肿瘤葡萄糖代谢中的作用[J]. 中国口腔颌面外科杂志, 2021, 19(1): 70-75.
[9]	王子昱, 丁宇洁, 陈冰, 沈昊翀, 孟箭, 仝伟凤. α桐酸对舌鳞状细胞癌CAL-27细胞的体外抑制作用[J]. 中国口腔颌面外科杂志, 2020, 18(6): 490-494.
[10]	周光明, 梁衍灿, 黄子贤, 张彬. 长链非编码RNA FOXD2-AS1对舌鳞癌细胞迁移、侵袭的影响[J]. 中国口腔颌面外科杂志, 2020, 18(6): 495-500.
[11]	陈永菊, 陈伟良, 周斌, 钟江龙, 汪延, 陈睿. 面-颏下动脉岛状皮瓣修复年轻与老年舌癌患者的效果比较[J]. 中国口腔颌面外科杂志, 2020, 18(2): 117-121.
[12]	林志立, 梁建平, 周斌. lncRNA-HOTTIP在低氧诱导舌鳞癌细胞上皮-间质转化过程中的作用探讨[J]. 中国口腔颌面外科杂志, 2019, 17(1): 26-31.
[13]	罗丹, 孙巧珍, 刘少华, 孙俊伟, 王升志. 热疗联合Id-1基因沉默对舌鳞癌CAL-27细胞增殖及侵袭的影响[J]. 中国口腔颌面外科杂志, 2018, 16(6): 488-492.
[14]	刘墨, 李劲松, 阮毅, 黄洪章. miR-181a抑制舌鳞癌细胞上皮-间充质转化及化疗耐药机制的研究[J]. 中国口腔颌面外科杂志, 2018, 16(4): 289-295.
[15]	孙俊伟, 罗丹, 刘浩, 王升志. 热疗对Cal27细胞Id-1、HSF1表达的影响[J]. 中国口腔颌面外科杂志, 2018, 16(2): 144-148.