Sains Malaysiana 48(8)(2019): 1697–1706

http://dx.doi.org/10.17576/jsm-2019-4808-15

An in vitro Three-Dimensional Co-Culture System for Ameloblastoma Modelling

(Sistem Ko-Kultur Tiga Dimensi secara in vitro untuk Pemodelan Ameloblastoma)

SOO LENG LEE¹*, ZAINAL ARIFF ABDUL RAHMAN¹, HIDETSUGU TSUJIGIWA², MEI HAMADA³, KIYOFUMI TAKABATAKE³, KEISUKE NAKANO³, HITOSHI NAGATSUKA³ & CHONG HUAT SIAR¹

¹Department of Oral and Maxillofacial Clinical Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Federal Territory, Malaysia

²Laboratory of Histopathology, Department of Life Science, Faculty of Science, Okayama University of Science, Okayama, Japan

³Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

Received: 18 September 2018/Accepted: 16 May 2019

ABSTRACT

Ameloblastoma, the most clinically significant odontogenic epithelial tumor, is a locally-invasive and destructive lesion in the jawbones. However, the nature of this infiltrativeness and destructive behavior remains ill-understood. To address this, we established an in vitro three-dimensional (3D) co-culture system to simulate an amelobastoma disease model aimed at investigating the interactions between tumor cells and osteoblasts. Osteoblastic cell lines (KUSA/A1 and MC3T3-E1) and one stromal cell line (ST2) were separately co-seeded with ameloblastoma-derived cell line (AM-1) in a collagen scaffold (representing the extracellular bone matrix) and incubated with mineralization medium. Immunohistochemistry, double immunofluorescence and mineralization assay were performed. Only AM-1/KUSA-A1 co-culture showed a significant increase in AM-1 cell count, suggesting that heterotypic cell-cell interaction promotes tumoral cell growth, while formation of visible AM-1 epithelial nest-like structures resembling ameloblastoma cells in their native state, suggest morphodifferentiation. A RANK-high, RANKL-low and osteoprotegerin-low immunoprofile in co-culture AM-1 cells implies deregulated osteoclastogenesis. Mineralization assays showed diminished calcification in AM-1/KUSA-A1 co-culture extracellular matrix suggesting an altered local bone metabolism. In contrast, KUSA/A1 monocultures showed abundant extracellular matrix calcification. Taken together, these results suggest that a 3D co-culture system as an amelobastoma disease model provides insights that bidirectional ameloblastoma-osteoblastic interactions might play a role in modulating tumor growth and osteoclastogenesis.

Keywords: Ameloblast; ameloblastoma modelling; co-culture system; pre-osteoblast

ABSTRAK

Ameloblastoma, tumor epitelium odontogenik yang paling umum secara klinikal, ialah lesi invasif setempat dan lesi memusnah yang didapati di tulang rahang. Walau bagaimanapun, sifat penyusupan dan perilaku yang merosakkan masih tidak difahami. Untuk menangani ini, kami menubuhkan satu sistem in vitro tiga dimensi (3D) untuk mensimulasikan model penyakit amelobastoma, bertujuan untuk mengkaji interaksi antara tumor dan sel osteoblastik. Sel-sel osteoblastik (KUSA/A1 dan MC3T3-E1) dan satu sel stromal (ST2) bersandarkan dengan sel-sel tumor ameloblastoma, AM-1 secara berasingan dalam gel kolagen (mewakili matriks tulang ekstraselul) dan diinkubasi dengan medium pemineralan. Imunohistokimia, imunopendarfluor ganda dua dan asai mineral dijalankan. Hanya kultivar AM-1/KUSA-A1 menunjukkan kenaikan ketara dalam jumlah sel AM-1, menunjukkan bahawa interaksi sel heterotip menggalakkan pertumbuhan sel tumor, manakala pembentukan struktur seperti sarang epitelium AM-1 kelihatan sebagai sel ameloblastoma dalam keadaan asalnya, mencadangkan pembezaan morfotip. RANK-Tinggi, RANKL-rendah dan osteoprotegerin-rendah imunoprofil dalam ko-kultur dengan sel AM-1 mencadangkan osteoklastogenesis yang tidak terkawal. Pemineralan asai juga menunjukkan bahawa kalsifikasi dalam matriks ekstrasel AM-1/KUSA-A1 telah mencadangkan perubahan dalam metabolisme tulang setempat. Sebaliknya, monokultur KUSA/A1 menunjukkan kalsifikasi matriks ekstrasel yang signifikan. Secara eksplisit, keputusan ini menunjukkan bahawa sistem ko-kultur 3D sebagai model penyakit amelobastoma memberikan pandangan terperinci terhadap interaksi antara ameloblastoma dan osteoblas dan keupayaannya untuk memainkan peranan penting dalam pengubahan pertumbuhan tumor dan osteoklastogenesis.

Kata kunci: Ameloblas; pemodelan ameloblastoma; pra-osteoblas; sistem ko-kultur

REFERENCES

Andrade, F.R., Sousa, D.P., Mendonca, E.F., Silva, T.A., Lara, V.S. & Batista, A.C. 2008. Expression of bone resorption regulators (RANK, RANKL, and OPG) in odontogenic tumors. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 106(4): 548-55.

Antoni, D., Burckel, H., Josset, E. & Noel, G. 2015. Three-dimensional cell culture: A breakthrough in vivo. Int. J. Mol. Sci. 16(3): 5517-5527.

Chantravekin, Y. & Koontongkaew, S. 2014. Effects of ameloblastoma-associated fibroblasts on the proliferation and invasion of tumor cells. J. Cancer Res. Ther. 10(4): 1082-1087.

da Silva, T.A., Batista, A.C., Mendonca, E.F., Leles, C.R., Fukada, S. & Cunha, F.Q. 2008. Comparative expression of RANK, RANKL, and OPG in keratocystic odontogenic tumors, ameloblastomas, and dentigerous cysts. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 105(3): 333-341.

de Matos, F.R., de Moraes, M., das Neves Silva, E.B., Galvao, H.C. & de Almeida Freitas, R. 2013. Immunohistochemical detection of receptor activator nuclear kappa B ligand and osteoprotegerin in odontogenic cysts and tumors. J. Oral Maxillofac. Surg. 71(11): 1886-1892.

Eriksson, T.M., Day, R.M., Fedele, S. & Salih, V.M. 2016. The regulation of bone turnover in ameloblastoma using an organotypic in vitro co-culture model. J. Tissue Eng. 7: 2041731416669629.

Fong, E.L., Wan, X., Yang, J., Morgado, M., Mikos, A.G., Harrington, D.A., Navone, N.M. & Farach-Carson, M.C. 2016. A 3D in vitro model of patient-derived prostate cancer xenograft for controlled interrogation of in vivo tumor-stromal interactions. Biomaterials 77: 164-172.

Fuchigami, T., Kibe, T., Koyama, H., Kishida, S., Iijima, M., Nishizawa, Y., Hijioka, H., Fujii, T., Ueda, M., Nakamura, N., Kiyono, T. & Kishida, M. 2014. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1alpha in ameloblastoma. Biochem. Biophys. Res. Commun. 451(4): 491-496.

Harada, H., Mitsuyasu, T., Nakamura, N., Higuchi, Y., Toyoshima, K., Taniguchi, A. & Yasumoto, S. 1998. Establishment of ameloblastoma cell line, AM-1. J. Oral. Pathol. Med. 27(5): 207-212.

Khosla, S. 2001. Minireview: The OPG/RANKL/RANK system. Endocrinology 142(12): 5050-5055.

Kim, Y. & Othmer, H.G. 2013. A hybrid model of tumor-stromal interactions in breast cancer. Bull. Math. Biol. 75(8): 1304- 1350.

Kumamoto, H. & Ooya, K. 2004. Expression of parathyroid hormone-related protein (PTHrP), osteoclast differentiation factor (ODF)/receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoclastogenesis inhibitory factor (OCIF)/osteoprotegerin (OPG) in ameloblastomas. J. Oral Pathol. Med. 33(1): 46-52.

Liu, X.Q., Kiefl, R., Roskopf, C., Tian, F. & Huber, R.M. 2016. Interactions among lung cancer cells, fibroblasts, and macrophages in 3D co-cultures and the impact on MMP-1 and VEGF expression. PLoS ONE 11(5): e0156268.

Luis-Ravelo, D., Antón, I., Vicent, S., Hernández, I., Valencia, K., Zandueta, C., Martínez-Canarias, Gúrpide, A. & Lecanda, F. 2011. Tumor-stromal interactions of the bone microenvironment: in vitro findings and potential in vivo relevance in metastatic lung cancer models. Clinical & Experimental Metastasis 28(8): 779-791.

Ottewell, P.D. 2016. The role of osteoblasts in bone metastasis. Journal of Bone Oncology 5(3): 124-127.

Proff, P. & Romer, P. 2009. The molecular mechanism behind bone remodelling: A review. Clin. Oral Investig. 13(4): 355-362.

Qian, Y. & Huang, H.Z. 2010. The role of RANKL and MMP-9 in the bone resorption caused by ameloblastoma. J. Oral Pathol. Med. 39(8): 592-598.

Ringer, E. & Kolokythas, A. 2017. Bone margin analysis for benign odontogenic tumors. Oral Maxillofac. Surg. Clin. North Am. 29(3): 293-300.

Sandra, F., Hendarmin, L., Kukita, T., Nakao, Y., Nakamura, N. & Nakamura, S. 2005. Ameloblastoma induces osteoclastogenesis: A possible role of ameloblastoma in expanding in the bone. Oral Oncol. 41(6): 637-644.

Sandra, F., Hendarmin, L. & Nakamura, S. 2006. Osteoprotegerin (OPG) binds with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): Suppression of TRAIL-induced apoptosis in ameloblastomas. Oral Oncol. 42(4): 415-420.

Sathi, G.A., Inoue, M., Harada, H., Rodriguez, A.P., Tamamura, R., Tsujigiwa, H., Borkosky, S.S., Gunduz, M. & Nagatsuka, H. 2009. Secreted frizzled related protein (sFRP)-2 inhibits bone formation and promotes cell proliferation in ameloblastoma. Oral Oncol. 45(10): 856-860.

Sathi, G.A., Tsujigiwa, H., Ito, S., Siar, C.H., Katase, N., Tamamura, R., Harada, H. & Nagatsuka, H. 2012. Osteogenic genes related to the canonic WNT pathway are down-regulated in ameloblastoma. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 114(6): 771-777.

Sathi, G.S., Nagatsuka, H., Tamamura, R., Fujii, M., Gunduz, M., Inoue, M., Rivera, R.S. & Nagai, N. 2008. Stromal cells promote bone invasion by suppressing bone formation in ameloblastoma. Histopathology 53(4): 458-467.

Siar, C.H. & Ng, K.H. 2014. Differential expression of transcription factors snail, slug, SIP1, and twist in ameloblastoma. J. Oral Pathol. Med. 43(1): 45-52.

Siar, C.H., Tsujigiwa, H., Ishak, I., Hussin, N.M., Nagatsuka, H. & Ng, K.H. 2015. RANK, RANKL, and OPG in recurrent solid/multicystic ameloblastoma: Their distribution patterns and biologic significance. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 119(1): 83-91.

Takebe, Y., Tsujigiwa, H., Katase, N., Siar, C.H., Takabatake, K., Fujii, M., Tamamura, R., Nakano, K. & Nagatsuka, H. 2017. Parenchyma-stromal interactions induce fibrosis by secreting CCN2 and promote osteoclastogenesis by stimulating RANKL and CD68 through activated TGF-beta/BMP4 in ameloblastoma. J. Oral Pathol. Med. 46(1): 67-75.

Tay, J.Y., Bay, B.H., Yeo, J.F., Harris, M., Meghji, S. & Dheen, S.T. 2004. Identification of RANKL in osteolytic lesions of the facial skeleton. J. Dent. Res. 83(4): 349-353.

Tekkesin, M.S., Mutlu, S. & Olgac, V. 2011. The role of RANK/ RANKL/OPG signalling pathways in osteoclastogenesis in odontogenic keratocysts, radicular cysts, and ameloblastomas. Head and Neck Pathology 5(3): 248-253.

Umezawa, A., Maruyama, T., Segawa, K., Shadduck, R.K., Waheed, A. & Hata, J.I. 1992. Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo. J. Cell Physio. 151(1): 197-205.

Vered, M., Muller, S. & Heikinheimo, K. 2017. Ameloblastoma. In World Health Organization Classification of Head and Neck Tumours, edited by El-Naggar, A.K., Chan, J.K.C., Grandis, J.R., Takata, T. & Slootweg, P.J. 4th. Lyon IARC. pp. 215-218.

Wendler, F., Stamp, G.W. & Giamas, G. 2016. Tumor-stromal cell communication: Small vesicles signal big changes. Trends Cancer 2: 326-329.

Wright, J.M. & Vered, M. 2017. Update from the 4th Edition of the World Health Organization classification of head and neck tumours: Odontogenic and maxillofacial bone tumors. Head Neck Pathol. 11(1): 68-77.

*Corresponding author; email: leng527@siswa.um.edu.my