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

• Basic Study • Previous Articles     Next Articles

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. 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 E-mail:2012181643016@stu.scu.edu.cn;wufei1122_qi@163.com;chengran@scu.edu.cn;zhouhm@scu.edu.cn

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

CLC Number: 

  • R78

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 components1) 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"

[1] Chen XM, Song EW. Turning foes to friends: targeting cancer-associated fibroblasts[J]. Nat Rev Drug Discov, 2019,18(2):99-115.
pmid: 30470818
[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: 10.1016/j.cell.2019.05.012 pmid: 31155233
[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: 10.1038/s41388-019-0765-y pmid: 30816343
[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: 10.3892/ijo.2018.4515 pmid: 30085337
[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: 10.3892/or.2017.5479
[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: 10.1016/j.yexcr.2018.04.029
[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.
pmid: 28742795
[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: 10.18632/oncotarget.22922 pmid: 29416611
[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: 10.1186/1471-2407-11-88 pmid: 21352603
[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: 10.1016/j.cmet.2018.09.012 pmid: 30293773
[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: 10.1111/j.1601-0825.2005.01207.x pmid: 16792722
[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: 10.3390/cells8010074
[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: 10.1007/s10529-017-2363-9 pmid: 28536939
[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: 10.1002/bit.26297 pmid: 28369747
[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: 10.1016/j.lungcan.2019.04.007
[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: 10.1111/brv.12293 pmid: 27545872
[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: 10.1002/ijc.31316 pmid: 29441570
[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: 10.1186/s12943-018-0758-4 pmid: 29325547
[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: 10.1093/carcin/bgw113 pmid: 27803052
[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: 10.1016/j.molonc.2013.10.008
[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: 10.3390/ijms17091458
[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: 10.1016/j.yexcr.2016.12.002 pmid: 27919745
[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: 10.1016/j.neo.2016.08.002 pmid: 27690238
[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: 10.1186/s13046-019-1225-9 pmid: 31200779
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