Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations
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Marketa Svobodova1,2,3, Martina Raudenska1,3, Jaromir Gumulec1,3, Jan Balvan1, Michaela Fojtu1, Monika Kratochvilova1,3, Hana Polanska1,2,3, Zuzana Horakova4, Rom Kostrica4, Petr Babula1, Zbynek Heger3,5 and Michal Masarik1,2
1Department of Physiology, Faculty of Medicine, Masaryk University, CZ-62500 Brno, Czech Republic
2Department of Pathological Physiology, Faculty of Medicine, Masaryk University, CZ-62500 Brno, Czech Republic
3Central European Institute of Technology, Brno University of Technology, CZ-61600 Brno, Czech Republic
4Department of Otorhinolaryngology and Head and Neck Surgery, St. Anne’s Faculty Hospital, CZ-65691 Brno, Czech Republic
5Department of Chemistry and Biochemistry, Mendel University in Brno, CZ-61300 Brno, Czech Republic
Michal Masarik, email: [email protected]
Keywords: head and neck neoplasms, coculture techniques, cell line, tumor, carcinoma
Received: May 24, 2017 Accepted: June 28, 2017 Published: August 03, 2017
In this study, we describe the establishment of the human papillomavirus 18-positive, stage II, grade 1, T2N0M0 head and neck tumor primary cell line derived from oral squamous cell carcinoma of a non-smoking patient by using two different protocols. Furthermore, a preparation of subpopulations derived from this primary cell line according to the cluster of differentiation molecules CD44/CD90 status using magnetic bead-based separation and their characterization was performed. Impedance-based real-time cell analysis, enzyme-linked immunsorbant assay (ELISA), wound-healing assay, flow-cytometry, gene expression analysis, and MTT assay were used to characterize these four subpopulations (CD44+/CD90−, CD44−/CD90−, CD44+/CD90+, CD44−/CD90−). We optimised methodics for establishement of primary cell lines derived from oral squamous cell carcinoma tissue samples and subsequent separation of mesenchymal (CD90+) and epithelial (CD90−) types of tumorous cells. Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase. According to our results, CD90 separation is a necessary step in preparation of permanent tumor-tissue derived cell lines. Based on the wound-healing assay, CD44+ cells exhibited stronger migratory capacity than CD44− subpopulations. CD44+ subpopulations had also significantly higher expression of BIRC5 and SOX2, lower expression of FLT1 and IL6, and higher levels of basal autophagy compared to CD44− subpopulations. Furthermore, co-cultivation experiments revealed that CD44−/CD90+ cells supported growth of epithelial tumor cells (CD44+/CD90−). On the contrary, factors released by CD44+/CD90+ type of cells seem to have rather inhibiting effect. The most cisplatin-resistant subpopulation with the shortest doubling time was CD44−/CD90+, but this subpopulation had a low migratory capacity.
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