Establishment of oral squamous cell carcinoma cell line and magnetic bead-based isolation and characterization of its CD90/CD44 subpopulations

// Marketa Svobodova 1, 2, 3 , Martina Raudenska 1, 3 , Jaromir Gumulec 1, 3 , Jan Balvan 1 , Michaela Fojtu 1 , Monika Kratochvilova 1, 3 , Hana Polanska 1, 2, 3 , Zuzana Horakova 4 , Rom Kostrica 4 , Petr Babula 1 , Zbynek Heger 3, 5 and Michal Masarik 1, 2 1 Department of Physiology, Faculty of Medicine, Masaryk University, CZ-62500 Brno, Czech Republic 2 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, CZ-62500 Brno, Czech Republic 3 Central European Institute of Technology, Brno University of Technology, CZ-61600 Brno, Czech Republic 4 Department of Otorhinolaryngology and Head and Neck Surgery, St. Anne’s Faculty Hospital, CZ-65691 Brno, Czech Republic 5 Department of Chemistry and Biochemistry, Mendel University in Brno, CZ-61300 Brno, Czech Republic Correspondence to: Michal Masarik, email: masarik@med.muni.cz Keywords: head and neck neoplasms, coculture techniques, cell line, tumor, carcinoma Received: May 24, 2017 Accepted: June 28, 2017 Published: August 03, 2017 ABSTRACT 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.