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PLoS One. 2016 Aug 8;11(8):e0160615. doi: 10.1371/journal.pone.0160615. eCollection 2016.

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression.

Author information

1
Vaidya Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, Maharashtra, India.
2
Gade Laboratory for Pathology, Department of Clinical Medicine, and Center for International Health, Department of Global Public Health and Primary Care, University of Bergen, Bergen, Norway.
3
Department of Pathology, Haukeland University Hospital, Bergen, Norway.
4
Nups and Sumo Biology Group, Department of Biological Sciences, Indian Institute of Science, Education and Research, Bhopal, Madhya Pradesh, India.
5
Oral Surgery, Head and Neck Unit, Tata Memorial Hospital (TMH), Parel, Mumbai, India.

Abstract

To study multistep tumorigenesis process, there is a need of in-vitro 3D model simulating in-vivo tissue. Present study aimed to reconstitute in-vitro tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted in-vitro models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced in-vitro. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in in-vitro grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis.

PMID:
27501241
PMCID:
PMC4976883
DOI:
10.1371/journal.pone.0160615
[Indexed for MEDLINE]
Free PMC Article

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