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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 4  |  Page : 1082-1087

Effects of ameloblastoma-associated fibroblasts on the proliferation and invasion of tumor cells


Faculty of Dentistry, Thammasat University, Khlong Luang, Pathum Thani, Thailand

Date of Web Publication9-Jan-2015

Correspondence Address:
Yosananda Chantravekin
Faculty of Dentistry, Thammasat University, Rangsit Campus, 99 Moo 18, Paholyothin Rd. T.Khlong Nueng, A.Khlong Luang, Pathum Thani, 12121
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.138005

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 > Abstract 

Context: Ameloblastoma is the most common odontogenic tumor, however, the molecular pathology, especially the role of the tumor stroma, is still unclear.
Aims: To investigate the effects of the ameloblatoma-associated fibroblast (AAFs) on the proliferation and invasion of tumor cells.
Settings and Design: Cell culture, ELISA, proliferation and invasion assays.
Methods and Material: The cocultivations and three-dimensional organotypic cultures of the AAFs and the tumor cells were performed. The gingival fibroblasts (GFs) were used as the control. The levels of TGF-β and HGF in the conditioned media were analyzed using ELISA technique. The MTT proliferation assays and Boyden chamber chemoinvasion assays were also performed.
Statistical Analysis Used: ANOVA.
Results: Both AAFs and GFs stimulated tumor cell growth. The TGF-β level in the AAF group was more than those of GF group, whereas the HGF levels were not different. The AAF conditioned media also stimulated tumor cell proliferation and invasion more than the GF conditioned media. However, no difference in the thickness and morphology between the AAF and GF groups could be demonstrated in the organotypic models.
Conclusions: Both AAFs and GFs support the proliferation of the tumor cells in cocultivation experiment and three-dimensional organotypic cultures. However, the AAFs have a tendency to stimulate the proliferation and induce the invasion more than GFs. Increased TGF-β levels in the AAF condition media suggested the possible role of this growth factor in the ameloblastoma biology.

 > Abstract in Chinese 

釉母细胞瘤相关的纤维母细胞对肿瘤细胞的浸润和增殖的影响

摘要

背景:釉母细胞瘤是最常见的牙源性肿瘤,然而,其分子病理学,尤其是肿瘤间质的作用,目前还不清楚。

目的:探讨了釉母细胞瘤相关的纤维母细胞(AAFS)对肿瘤细胞的浸润和增殖的影响。

设置和设计:采用细胞培养,酶联免疫吸附测定,细胞增殖和侵袭实验。

材料和方法:对AAFs和肿瘤细胞进行协同培养和三维器官型培养。牙龈成纤维细胞(GFS)作为对照。采用ELISA技术分析条件培养基的TGF-β和HGF水平。MTT增殖实验和Boyden小室趋化侵袭实验也被采用。

统计分析:方差分析。

结果:AAFs 和 GFs均刺激肿瘤细胞的生长。TGF-β水平在AAF组高于GF组,而HGF水平是一样的。AAF条件培养基刺激肿瘤细胞增殖和侵袭也超过GF条件培养基。然而,在器官模型上,AAF和GF组之间的厚度和形态无差异。

结论:AAFs 和 GFs支持协同培养实验和三维器官培养实验的肿瘤细胞的增殖。然而,AAFs有刺激增殖和诱导侵袭超过GFS的倾向。在AAF条件培养基中增加的TGF-β水平说明这种生长因子在釉母细胞瘤的生物学方面可能的作用。

关键词:釉母细胞瘤,细胞增殖,细胞生长因子,肿瘤,侵袭性,转化生长因子β

Keywords: Ameloblastoma, cell proliferation, hepatocyte growth factor, neoplasm, invasiveness, transforming growth factor beta


How to cite this article:
Chantravekin Y, Koontongkaew S. Effects of ameloblastoma-associated fibroblasts on the proliferation and invasion of tumor cells. J Can Res Ther 2014;10:1082-7

How to cite this URL:
Chantravekin Y, Koontongkaew S. Effects of ameloblastoma-associated fibroblasts on the proliferation and invasion of tumor cells. J Can Res Ther [serial online] 2014 [cited 2020 Apr 9];10:1082-7. Available from: http://www.cancerjournal.net/text.asp?2014/10/4/1082/138005


 > Introduction Top


Ameloblastoma is the most common odontogenic tumor. It is slow-growing, but locally aggressive with a high recurrent rate if it is not removed adequately. Because it seldom produces pain, most of the patients have delayed seeking treatment. Much more complicated surgery with residual deformities may be a result. From the large epidemiological study, this tumor is common within the Asian population. [1],[2],[3] Although many studies have identified the molecular alterations responsible for development and progression, its etiology and pathogenesis are still unclear.

In cancer biology, the roles of the stroma and tumor microenvironment are well-established. The normal stroma acts as a barrier to block tumor initiation and progression. [4],[5],[6],[7],[8] However, there is much evidence to suggest that the stroma itself undergoes a series of alterations, the stromagenesis. [4],[6],[9],[10],[11] The tumor stroma lacks the regulatory mechanisms, then it supports the tumor progression. Tumor-associated fibroblasts (TAFs) or cancer-associated fibroblasts (CAFs) promote the proliferation and invasion of the tumor cells via many autocrine and paracrine factors. [12],[13],[14],[15],[16],[17]

In ameloblastoma biology, most of the studies focus on the alteration of the tumor cells, not the tumor microenvironment. However, there are many studies that have revealed the abnormalities in the stromal part of ameloblastoma. These abnormal expressions could be classified into three groups: (1) The growth factors such as hepatocyte growth factor (HGF) and transformimg growth factor-beta (TGF-β), [18],[19] (2) the osteolytic cytokines such as parathyroid-related protein (PTHrP), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG), [20],[21] and (3) the matrix-degrading proteinases such as matrix metalloproteinases (MMPs) [22],[23] and heparanase. [24] Most of these studies used the immunohistochemistry technique which is proper for demonstrating the abnormal protein expression. To elucidate the role of stoma on tumor biology, other approaches are necessary.

In the present study, we used the cell culture model, as well as the cocultivation and the three-dimensional organotypic model, to investigate the effects of ameloblastoma-associated fibroblasts (AAFs) on the proliferation and invasion of tumor cells.


 > Subjects and Methods Top


Cells and cell cultures

Head and neck squamous cell carcinoma cell lines (HN4 and 12) were generously provided by Dr. J Silvio Gutkind (NIDCR, Bethesda, MD, USA). HN4 and HN12 were both derived from the same patient. HN4 was obtained from the primary tongue lesion, whereas, HN12 was obtained from lymph node metastasis. The immortalized keratinocyte cell line (OKF-6/TERT2) was provided by Dr. Dunyaporn Trachootham (Faculty of Dentistry, Thammasat University, Pathum Thani, Thailand). The HN4 and HN12 were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum ( FCS) and antibiotics, whereas the OKF-6/TERT2 was cultured in keratinocyte medium.

The AAFs were cultured from the central part of seven cases of ameloblastoma lesions. The control fibroblasts included four cases of gingival fibroblasts (GFs) cultured from biopsies of healthy gingiva of patients during impacted tooth removal. The biopsies were approved by Thammasat University Ethic Committee No. 030/2552. Informed consent was obtained from all subjects participating in this study. The specimens were cut into small pieces and transferred to culture dishes. Both AAFs and GFs were cultured on 35-mm plastic culture dishes in DMEM supplemented with 10% FCS and antibiotics. The media were changed twice weekly. The cells in the third to seventh passages were used for experiments.

Cocultures

In order to investigate the effects of AAFs on the proliferation and invasion of tumor cells, coculture experiments using Transwell® inserts were performed. HN and OKF-6/TERT2 cell lines were seeded in the 6-well culture plates at 1 × 10 5 cells per well. Two milliliter of DMEM was added to each well. On the next day, the AAFs and GFs were seeded in the HTS Transwell® -6 permeable support inserts at 2 × 10 5 cells per insert. The HN and OKF-6/TERT2 with the same cell number were cultured as the control group. The cocultivations were performed for 10 days. The conditioned media were collected and replaced on day 3, 7, and 10.

Determination of TGF-β and HGF levels in the conditioned media

The growth factor levels, including TGF-β and HGF, in the conditioned media were quantified using the enzyme-linked immunosorbent assay (ELISA) technique. The Quantikine® (R and D Systems, MN, USA) ELISA kits (DB100B for TGF-β and DHG00 for HGF) were used in the study. The conditioned media from the cocultivations were analyzed following the manufacturer's protocol. Briefly, the assay diluent was added to each well. Then the standard, control, or sample was added and incubated for 2 h. The aspiration and washing were performed four times. The conjugate was added and incubated for 1.75-2 h. Then the substrate was added and incubated for 30 min, followed by stop solution. The samples were read by microplate reader at 450 nm wavelength.

MTT proliferation assays

In order to measure the effect of AAFs on the proliferation of tumor cells, the MTT proliferation assays were performed. [25] HN and OKF-6/TERT2 cell lines were seeded in the 96-well culture plates at 5 × 10 3 cells per well. One day later, the medium was replaced by 90% DMEM + 10% conditioned media from the cocultivation experiments. The cells were incubated in the CO 2 incubator (5% CO 2 , 37°C) for 7 days, then MTT proliferation assays were performed. The 96-well plates were centrifuged at 200 g for 5 min. The media were discarded and replaced with the new media (DMEM + 10% FCS + antibiotics, 200 μl/well) plus MTT solution (5 mg/ml, 50 μl/well). The plates were wrapped with aluminum foil, and incubated in the CO 2 incubator for 4 h. The media and MTT solution were discarded. Then the MTT formazan crystal was detected using the dimethyl sulfoxide (DMSO, 200 μl/well) and glycine buffer (25 μl/well). The samples were read by microplate reader at 570 nm wavelength. Triplication was done for this experiment.

Three-dimensional organotypic cultures

The three-dimensional organotypic culture models in deep well plates were constructed. [26] Acellular layer of collagen was prepared in the insert using the Premix solution (DMEM + reconstitution medium + glutamine + antibiotics), FCS, and type 1 rat-tail collagen. The AAFs and GFs were trypsinized and counted. The cellular layer of the gel composed of the Premix, FCS, type 1 rat-tail collagen, and fibroblasts at 3 × 10 5 cells per insert. The fabricated gels were left to completely polymerized at 37°C for 2 h, then filled with DMEM. The gels were incubated in the CO 2 incubator (5% CO 2 , 37°C) for 7 days. Then the HN and OKF-6/TERT2 were seeded at 1 × 10 6 cells per well). Keratinocyte medium was filled inside the insert, whereas, the fibroblast medium was filled outside. The gels were continuously incubated in the CO 2 incubator (5% CO 2 , 37°C) for 7 days. The air-lift process was performed to let the keratinocytes get the nutrient only from the collagen. The incubation was continued for 14 days, and then the harvesting was performed. The specimen was fixed in formalin solution and prepared for histopathological examination.

Boyden chamber chemoinvasion assays

The blind-well Boyden chemotaxis chamber was used for chemoinvasion assay. [27] The upper and lower chambers were separated with the a 12 μm pore-size polycarbonate filter coated with 1 mg/ml basement membrane (Matrigel). The HN4 and HN12 (40,000 cells/chamber) were cultured in 200 μl DMEM in the upper chambers. The conditioned media from the cocultivation experiments were placed in lower chamber. The blind-well Boyden chambers were incubated in the CO 2 incubator (5% CO 2 , 37°C) for 5 h. The membranes were fixed and the cells were stained with 0.5% crystal violet in 25% methanol. The invaded cells were counted using a phase contrast microscope.


 > Results Top


Cocultures

Both AAFs and GFs stimulated the HN and OKF-6/TERT2 growths. After 7 days of cocultivation, the confluences of the keratinocytes in the 6-well plates were approximately 100% in both AAF and GF groups, whereas, the cell confluence in the control group (without cocultivation) was about 50% [Figure 1].
Figure 1: Photographs of HN12 on Day 7 of cocultivation. Approximately 100% cell confluences were found in both (a) Ameloblastoma-associated fibroblast (AAF) group and (b) Gingival fibroblast (GF) group, whereas, 50% cell confluence was found in the (c) Control group

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Determination of TGF-β and HGF levels in the conditioned media

The level of TGF-β in the conditioned media of the AAF group was 2,480 ± 173.6 pg/ml, whereas, the level of the GF group was 917.9 ± 60.25 pg/ml with statistical difference (P < 0.0001). When analyzed separately, for OKF-6/TERT2 cocultivated groups, the TGF-β level in the AAF group was 2,587 ± 343.2 pg/ml, whereas, the level of the GF group was 820.9 ± 138.2 pg/ml with statistical difference (P = 0.0008). For HN4 cocultivated groups, the TGF-β level in the AAF group was 2,237 ± 319.5 pg/ml, whereas, the level of the GF group was 1,106 ± 127.5 pg/ml with statistical difference (P = 0.0126). For HN12 cocultivated groups, the TGF-β level in the AAF group was 2,598 ± 343.1 pg/ml, whereas, the level of the GF group was 796.6 ± 166.4 pg/ml with statistical difference (P = 0.002).

The level of HGF in the conditioned media of the AAF group was 5,173 ± 513.6 pg/ml, whereas, the level of the GF group was 5,147 ± 281.2 pg/ml with no statistical difference (P = 0.9685). When analyzed separately, for OKF-6/TERT2 cocultivated groups, the HGF level in the AAF group was 6,833 ± 1,083 pg/ml, whereas, the level of the GF group was 6,567 ± 272.2 pg/ml with no statistical difference (P = 0.8328). For HN4 cocultivated groups, the HGF level in the AAF group was 4,990 ± 863.7 pg/ml, whereas, the level of the GF group was 4,692 ± 338.5 pg/ml with no statistical difference (P = 0.7729). For HN12 cocultivated groups, the HGF level in the AAF group was 3,941 ± 570.6 pg/ml, whereas the level of the GF group was 4,183 ± 374.9 pg/ml with no statistical difference (P = 0.7558).

MTT proliferation assays

For OKF-6/TERT2, the proliferation in the AAF group was 1.422 ± 0.061 folds of the control, whereas, GF group stimulated cell proliferation at 1.523 ± 0.062 folds, with no statistical difference (P = 0.2925). For HN4, the proliferation in the AAF group was 1.737 ± 0.078 folds, whereas, the GF group was 1.464 ± 0.057 folds, with statistical difference (P = 0.0213). For HN12, the proliferation in the AAF group was 2.194 ± 0.162 folds, whereas, the GF group was 1.317 ± 0.062 folds, with statistical difference (P = 0.0004).

Three-dimensional organotypic cultures

After 7 days of fabricated gel incubation, the gel had shrunk to about half original size because the action of fibroblasts on the collagen. Then the epithelial seeding was performed. The gel shrinkage continued, and the surface of the gel turned to white [Figure 2] because of multiplying of the epithelial cells. From histopathological examination, the proliferation of the epithelial cells could also be demonstrated [Figure 3]. However, no difference of thickness and morphology of the epithelial layers were found between the AAF and GF group.
Figure 2: (a) The fabricated gel appearance on day 7. The shrinkage of the gel was revealed. (b) The gel appearance on day 10. The gel shrinkage continued. The surface of the gel became white

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Figure 3: Proliferation of the epithelial cells could be demonstrated on the gel surface

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Boyden chamber chemoinvasion assays

From the chemoinvasion assays of HN4, the average invaded ratio in the AAF group was 56.63 ± 2.46%, whereas, the ratio in the GF group was 26.59 ± 2.32%, with statistical difference (P < 0.0001). For HN12, the average invaded ratio in the AAF group was 59.63 ± 2.61%, whereas, the ratio in the GF group was 45.49 ± 2.90% [Figure 4], with statistical difference (P = 0.0006).
Figure 4: Photographs of polycarbonate filters coated with Matrigel from Boyden chamber chemoinvasion assays. (a) Invaded HN12 in AAF group and (b) invaded HN12 in GF group

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 > Discussion Top


In cancer biology, the role of tumor stroma is well-established. There is much evidence to suggest that TAFs secrete the cytokines, which induce proliferation and invasion, and regulate apoptosis of keratinocytes. These cytokines include the MMPs, TGF-β, HGF, fibroblast growth factor (FGF), chemokine (C-X-C motif) ligand 12 (CXCL12), and Wnt. [12],[13],[14],[15],[16],[17],[28] There was no previous evidence about the role of the stroma in the ameloblastoma biology, but from this study, AAFs also have a tendency to regulate the proliferation and invasion of the tumor cells. From coculture experiment of this study, both AAFs and GFs stimulated the HN and OKF-6/TERT2 growths. After 7 days of cocultivation, the confluences of the keratinocytes in the 6-well plates were approximately 100% in both AAF and GF groups, whereas, the cell confluence in the control group (without cocultivation) was about 50%. These results conformed to the results of three-dimensional organotypic cultures. After 21 days of three-dimensional cocultivation, the tumor cell proliferation in both AAF and GF groups could be demonstrated. Difference between the AAF and GF groups was revealed in the MTT proliferation assays. The HN4 proliferation in the AAF group was 1.737 ± 0.078 folds of the control, whereas, the HN4 proliferation in the GF group was 1.464 ± 0.057 folds, with statistical difference (P = 0.0213). The HN12 proliferation in the AAF group was 2.194 ± 0.162 folds, whereas, the GF group was 1.317 ± 0.062 folds, with statistical difference (P = 0.0004). These results suggest that, both AAFs and GFs stimulate the growth of keratinocytes, but the level of upregulation in the AAF group is much higher, especially in the tumor cell groups.

The results in the invasion experiments have a similar tendency. The results of the three-dimensional organotypic cultures could not demonstrate the invasion in both AAF and GF groups. The difference between the AAF and GF groups was revealed in the Boyden chamber chemoinvasion assay. From these 5-h assays, the average invaded ratio in the AAF groups were 56.63 ± 2.46% for HN4 and 59.63 ± 2.61% for HN12, whereas, the ratio in the GF groups were 26.59 ± 2.32% for HN4 45.49 ± 2.90% for HN12 [Figure 4], with statistical difference (P < 0.0001 and = 0.0006, respectively), suggest that the AAFs have more tendency to stimulate the tumor invasion than the GFs. These characteristics of AAFs were similar to the characteristics of TAFs, which secrete many paracrines and involve in the carcinogenesis, tumor progression as well as tumor invasion. [12],[13],[14],[15],[16],[17]

In the study of growth factor levels in the coculture-conditioned media, there was no difference in the HGF level between the AAF and GF groups. However, the TGF-β levels in both groups had statistical difference. This increased TGF-β level in the AAF group may come from three sources: (1) The fibroblasts themselves, (2) the fibroblasts stimulate the tumor cells to secrete more TGF-β, and (3) the increased number of the tumor cells because the MTT proliferation assays suggest that AAFs upregulate the tumor cell growth much more than the GFs.

TGF-β is a pleiotropic growth factor expressed both by cancer and stromal cells. [29] It has both tumor suppressor and tumor promotion effects. [29],[30],[31],[32] As tumor suppressor, TGF-β inhibits epithelial cell growth, represses human telomerase reverse transcriptase (hTERT) as well as induces apoptosis. As tumor promoter; it induces angiogenesis; suppresses immune system; and promotes migration, invasion, and metastasis. Although it has both effects, the TGF-β expression in the cancer is often increased. [33] The sources of TGF-β include the TAFs, inflammatory cells, and the cancer cells themselves. In the experiment of Kupperwasser et al., the human immortalized mammary fibroblasts transduced to ectopically express HGF and/or TGF-β1 were recombined with nontransformed human mammary epithelial organoids, and were induced to form ductal carcinoma in situ, adenocarcinoma, and poorly differentiated carcinomas; whereas, the mammary organoid combined with nontransfected fibroblast did not show evidence of neoplastic transformation. [10]

Mazzocca et al., used the TGF- β receptor inhibitor to inhibit the synthesis and release of connective tissue growth factor (CTGF) in the hepatocellular carcinoma (HCC) and found that this substance could interrupt the cross-talk between cancer cells and TAFs, leading to reduction of tumor growth and dissemination, [34] whereas, Bhowmick et al., blocked the TGF-β receptor type II in the mouse fibroblasts, which resulted in neoplastic changes of the adjacent epithelia including intraepithelial neoplasia and invasive squamous cell carcinoma. [35] The promotion of cancer establishment and progression by inactivation of the TGF-β receptor type II was also reported by Biswas et al. [36] This evidence suggests the important role of TGF-β in the tumor initiation and progression.

There are a few studies on the expression of TGF-β in ameloblastoma. Kumamoto et al., detected TGF-β in both tumor and stromal cells as well, but could detect the TGF-β receptor only in the tumor cells. Expressions of the TGF-β receptor in the tumor parts of aggressive ameloblastoma and ameloblastic carcinoma were less than those of benign types of ameloblastoma. [18] Heikinheimo et al., could detect the TGF-β2 in different phases of tooth formation as well as odontogenic tumors including ameloblastoma. [19] These evidences support our finding that the TGF-β may have an important role in the ameloblastoma biology as well.

In summary, both AAFs and GFs support the proliferation of the tumor cells in cocultivation experiment and three-dimensional organotypic cultures. However, the AAFs have a tendency to stimulate the proliferation and induce the invasion more than GFs in the MTT proliferation and Boyden chamber chemoinvasion assays, respectively. Increased TGF-β levels in the AAF condition media was also detected suggesting the possible role of this growth factor in the ameloblastoma biology.


 > Acknowledgments Top


We would like to thank Dr. Dunyaporn Trachootham, for the OKF-6/TERT2 cell line, as well as her recommendations about the three-dimensional organotypic culture technique. Providing of the primary cell culture of ameloblastoma came from the university affiliating hospitals with many helps from Dr. Wiwat Chatwongwan, Dr. Thanasak Chengsuntisuk and Dr. Nakarin Kitkamthorn.

 
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]



 

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