Effects of TGF-β1 on the migration of oral cancer-associated fibroblasts in two and three dimensional co-culture models

doi:10.12016/j.issn.2096-1456.2020.09.003

Journal of Prevention and Treatment for Stomatological Diseases ›› 2020, Vol. 28 ›› Issue (9): 562-568.DOI: 10.12016/j.issn.2096-1456.2020.09.003

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Effects of TGF-β1 on the migration of oral cancer-associated fibroblasts in two and three dimensional co-culture models

YANG Jin¹(),WU Feifei¹(),GAO Qinghong²,LI Xiaoyu³,MANABU Kato⁴,CHENG Ran⁵(),ZHOU Hongmei¹()

1. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Department of Oral Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
2. Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
3. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China
4. Urology, Mie University Hospital, Mie 514-8507, Japan
5. State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Department of Preventive Dentistry, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China

Received:2019-12-09 Revised:2020-01-22 Online:2020-09-20 Published:2020-08-24
Contact: Ran CHENG,Hongmei ZHOU

TGF-β1对口腔癌相关成纤维细胞在二维和三维共培养条件下迁移的影响

杨津¹(),吴飞飞¹(),高庆红²,李小玉³,MANABUKato⁴,程然⁵(),周红梅¹()

1.口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院口腔黏膜病科,四川成都（610041）
2.四川大学华西口腔医院口腔颌面外科,四川成都（610041）
3.口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院,四川成都（610041）
4.三重大学医院肾内科,日本三重县（514-8507）
5.口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心四川大学华西口腔医院预防科,四川成都（610041）

通讯作者: 程然,周红梅
作者简介:杨津,医师,博士,Email:2012181643016@stu.scu.edu.cn|吴飞飞,医师,硕士,Email:wufei1122_qi@163.com
杨津和吴飞飞为共同第一作者
基金资助:
国家自然科学基金资助项目(81772898)

Abstract

Abstract:

Objective To observe the effect of transforming growth factor-β1 (TGF-β1) on the migration of oral carcinoma associated fibroblasts (CAFs) with two-dimensional culture model and three-dimensional model.Methods Under two-dimensional culture conditions, CAFs stimulated by TGF-β1 with the addition of 10 ng/mL medium were used as the experimental group, and untreated CAFs were used as the control group. The migration of CAFs with the stimulation of TGF-β1 was measured by cell scratch assay and transwell assay. CAFs positive for green fluorescent protein (GFP) were cultured by retrovirus transfection. Human tongue squamous cell carcinoma cells SCC25, GFP(+) CAFs and CAFs with three-dimensional cell co-culture models were established. The three-dimensional model cultured under the stimulation of TGF-β1 with 10 ng/mL medium was used as the experimental group, and the three-dimensional model without TGF-β1 was used as the control group. The migration of CAFs with the stimulation of TGF-β1 was also measured by the three-dimensional models.Results It was verified that 10 ng/mL TGF-β1 promoted the migration of CAFs in the two-dimensional culture model. The three-dimensional co-culture models of SCC25, GFP(+) CAFs and CAFs were successfully established. The migration of SCC25 and CAFs was detected in the three-dimensional model. However, 10 ng/mL TGF-β1 had little effect on their migration.Conclusion The effect of TGF-β1 in vitro on the migration of oral CAFs was associated with different culture models in two and three dimensions.

Key words: carcinoma-associated fibroblasts, migration, three-dimensional cell culture model, transforming growth factor-β1, oral carcinoma

摘要：

目的通过二维和三维条件下的细胞共培养模型观察转化生长因子-β1（transforming growth factor-β1,TGF-β1）对口腔癌相关成纤维细胞（carcinoma associated fibroblasts,CAFs）迁移的影响。方法二维培养条件下,以培养基中加入10 ng/mL TGF-β1刺激的CAFs为实验组,以未经处理的CAFs细胞为对照组,通过划痕实验、Transwell实验观察CAFs的迁移现象;通过逆转录病毒转染,培养绿色荧光蛋白（green fluorescent protein,GFP）阳性的CAFs细胞,建立人舌鳞癌细胞SCC25、GFP(+) CAFs和CAFs三维细胞共培养模型,以培养基中加入10 ng/mL TGF-β1刺激下培养的三维模型为实验组,以未加的TGF-β1的三维模型为对照组,观察在三维模型的细胞迁移现象。结果在二维培养条件下验证了10 ng/mL TGF-β1可促进口腔CAFs的迁移;成功建立SCC25、GFP(+) CAFs和CAFs三维细胞共培养模型,观察到CAFs和口腔癌细胞的迁移现象,但10 ng/mL的TGF-β1对上述细胞的迁移在三维条件下无明显促进作用。结论体外TGF-β1对口腔CAFs迁移的影响与二维和三维不同培养模式有关。

CLC Number:

YANG Jin,WU Feifei,GAO Qinghong,LI Xiaoyu,MANABU Kato,CHENG Ran,ZHOU Hongmei. Effects of TGF-β1 on the migration of oral cancer-associated fibroblasts in two and three dimensional co-culture models[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2020, 28(9): 562-568.

杨津,吴飞飞,高庆红,李小玉,MANABUKato,程然,周红梅. TGF-β1对口腔癌相关成纤维细胞在二维和三维共培养条件下迁移的影响[J]. 口腔疾病防治, 2020, 28(9): 562-568.

Figures/Tables 6

Figure 1 Establishment of a three-dimensional culture model for the migration of CAFs and squamous carcinoma cells. Created with BioRender.com

Table 1 Proportion of gel components

Gel components^1）	Proportion
Collagen type I （4 mg/mL）	50%^2）
Matrigel	20%^2）
10× basal DMEM medium	10%
Fetal bovine serum（FBS）	10%
DMEM+10% FBS	10%

Figure 2 Immunocytochemistry staining to identify CAFs × 40 a： α-SMA （+）; b： FAP （+）; c： vimentin（+）; d： cytokeratin （-）

Figure 3 Comparison of the two-dimensional cell culture model formigration between the two groups a： TGF-β1 （-） CAFs and TGF-β1 （+） CAFs cell migration were observed by scratch test（× 40）; b： TGF-β1 （+） CAFs show a significantly greater average width on the scratch test; c&d： TGF-β1（-）CAFs （c） and TGF-β1（+）CAFs (d) cell migration was observed by transwell assay （× 100）; e： TGF-β1 （+） CAFs show significantly greater average number of migrated cells; *： P < 0.05, **： P < 0.01; ****： P < 0.000 1

Figure 4 Observation of the three-dimensional culture model for migration a： H&E staining observation and determination of the three-dimensional culture tissue （× 100）. The red solid lines indicate the depth of tumor invasion; b： observation and determination under fluorescence microscopy of the three-dimensional cell culture tissue （× 200）. The red arrow indicates GFP （+） CAFs, and the red dotted line indicates the initial boundary between GFP （+） CAFs and CAFs.

Figure 5 Comparison of the three-dimensional cell culture model for migration between two groups a： H&E staining of the three-dimensional cell culture model for migration between the TGF-β1 （-） and TGF-β1 （+） groups （× 100, ×200）. The red solid lines indicate the depth of tumor invasion; b： the TGF-β1 （+） group shows the same average depth of tumor invasion as the TGF-β1 （-） group, P > 0.05; c： GFP（+） CAF cell migration was observed with a fluorescence microscope （× 200）. The red dotted line indicates the initial boundary between GFP （+） CAFs and CAFs

References 27

[1]	Chen XM, Song EW. Turning foes to friends: targeting cancer-associated fibroblasts[J]. Nat Rev Drug Discov, 2019,18(2):99-115. URL PMID
[2]	Ligorio M, Sil S, Malagon-Lopez J, et al. Stromal microenvironment shapes the intratumoral architecture of pancreatic cancer[J]. Cell, 2019,178(1):160-175. DOI URL PMID
[3]	Bu L, Baba H, Yoshida N, et al. Biological heterogeneity and versatility of cancer-associated fibroblasts in the tumor microenvironment[J]. Oncogene, 2019,38(25):4887-4901. DOI URL PMID
[4]	Okuyama K, Suzuki K, Yanamoto S, et al. Anaplastic transition within the cancer microenvironment in early-stage oral tongue squamous cell carcinoma is associated with local recurrence[J]. Int J Oncol, 2018,53(4):1713-1720. DOI URL PMID
[5]	Shan T, Chen S, Chen X, et al. Cancer-associated fibroblasts enhance pancreatic cancer cell invasion by remodeling the metabolic conversion mechanism[J]. Oncol Rep, 2017,37(4):1971-1979. DOI URL
[6]	Li YY, Zhou CX, Gao Y. Interaction between oral squamous cell carcinoma cells and fibroblasts through TGF-beta1 mediated by podoplanin[J]. Exp Cell Res, 2018,369(1):43-53. DOI URL
[7]	Elmusrati AA, Pilborough AE, Khurram SA, et al. Cancer-associated fibroblasts promote bone invasion in oral squamous cell carcinoma[J]. Br J Cancer, 2017,117(6):867-875. URL PMID
[8]	Meng W, Liu C, Gao Q, et al. Effect of TGF-β1 on the migration of oral carcinoma-associated fibroblasts[J]. J Oral Sci Res, 2013,29(8):707-709.
[9]	Ham SL, Thakuri PS, Plaster M, et al. Three-dimensional tumor model mimics stromal--breast cancer cells signaling[J]. Oncotarget, 2018,9(1):249-267. DOI URL PMID
[10]	Meng W, Xia Q, Wu L, et al. Downregulation of TGF-beta receptor types Ⅱ and Ⅲ in oral squamous cell carcinoma and oral carcinoma-associated fibroblasts[J]. BMC Cancer, 2011,11:88. DOI URL PMID
[11]	Bertero T, Oldham WM, Grasset EM, et al. Tumor-stroma mechanics coordinate amino acid availability to sustain tumor growth and malignancy[J]. Cell Metab, 2019,29(1):124-140. DOI URL PMID
[12]	Liu Y, Hu T, Shen J, et al. Separation, cultivation and biological characteristics of oral carcinoma-associated fibroblasts[J]. Oral Dis, 2006,12(4):375-380. DOI URL PMID
[13]	Cheng R, Li D, Shi X, et al. Reduced CX3CL1 secretion contributes to the susceptibility of oral leukoplakia-associated fibroblasts to candida albicans[J]. Front Cell Infect Mi, 2016,6(1):150.
[14]	Namekawa T, Ikeda K, Horie-Inoue K, et al. Application of prostate cancer models for preclinical study: advantages and limitations of cell lines, patient-derived xenografts, and three-dimensional culture of patient-derived cells[J]. Cells, 2019,8(1):74. DOI URL
[15]	Zhao Y, Yan X, Li B, et al. Three-dimensional co-culture microfluidic model and its application for research on cancer stem-like cells inducing migration of endothelial cells[J]. Biotechnol Lett, 2017,39(9):1425-1432. DOI URL PMID
[16]	Chiew GGY, Wei N, Sultania S, et al. Bioengineered three-dimensional co-culture of cancer cells and endothelial cells: a model system for dual analysis of tumor growth and angiogenesis[J]. Biotechnol Bioeng, 2017,114(8):1865-1877. DOI URL PMID
[17]	Horie M, Saito A, Yamaguchi Y, et al. Three-dimensional co-culture model for tumor-stromal interaction[J]. J Vis Exp, 2015, (96):52469.
[18]	Nakamura H, Sugano M, Miyashita T, et al. Organoid culture containing cancer cells and stromal cells reveals that podoplanin-positive cancer-associated fibroblasts enhance proliferation of lung cancer cells[J]. Lung Cancer, 2019,134(1):100-107. DOI URL
[19]	Bielecka ZF, Maliszewska-Olejniczak K, Safir IJ, et al. Three-dimensional cell culture model utilization in cancer stem cell research[J]. Biol Rev Camb Philos Soc, 2017,92(3):1505-1520. DOI URL PMID
[20]	Sampson N, Brunner E, Weber A, et al. Inhibition of Nox4-dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal-mediated protumorigenic interactions[J]. Int J Cancer, 2018,143(2):383-395. DOI URL PMID
[21]	Ren Y, Jia HH, Xu YQ, et al. Paracrine and epigenetic control of CAF-induced metastasis: the role of HOTAIR stimulated by TGF-ss1 secretion[J]. Mol Cancer, 2018,17(1):5. DOI URL PMID
[22]	Cirillo N, Hassona Y, Celentano A, et al. Cancer-associated fibroblasts regulate keratinocyte cell-cell adhesion via TGF-beta-dependent pathways in genotype-specific oral cancer[J]. Carcinogenesis, 2017,38(1):76-85. DOI URL PMID
[23]	Karagiannis GS, Schaeffer DF, Cho CK, et al. Collective migration of cancer-associated fibroblasts is enhanced by overexpression of tight junction-associated proteins claudin-11 and occludin[J]. Mol Oncol, 2014,8(2):178-195. DOI URL
[24]	Eder T, Weber A, Neuwirt H, et al. Cancer-associated fibroblasts modify the response of prostate cancer cells to androgen and anti-androgens in three-dimensional spheroid culture[J]. Int J Mol Sci, 2016,17(9):1458. DOI URL
[25]	Yang L, Carrington LJ, Erdogan B, et al. Biomechanics of cell reorientation in a three-dimensional matrix under compression[J]. Exp Cell Res, 2017,350(1):253-266. DOI URL PMID
[26]	Miyake M, Hori S, Morizawa Y, et al. CXCL1-mediated interaction of cancer cells with tumor-associated macrophages and cancer-associated fibroblasts promotes tumor progression in human bladder cancer[J]. Neoplasia, 2016,18(10):636-646. DOI URL PMID
[27]	Hwang HJ, Oh MS, Lee DW, et al. Multiplex quantitative analysis of stroma-mediated cancer cell invasion, matrix remodeling, and drug response in a 3D co-culture model of pancreatic tumor spheroids and stellate cells[J]. J Exp Clin Cancer Res, 2019,38(1):258. DOI URL PMID

Effects of TGF-β1 on the migration of oral cancer-associated fibroblasts in two and three dimensional co-culture models

TGF-β1对口腔癌相关成纤维细胞在二维和三维共培养条件下迁移的影响

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