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.
Highlights
Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide
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
Primary cell line prepared by using trypsin proteolysis was more viable than the one prepared by using collagenase
Summary
Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide. Regardless of advances in diagnostic methods and therapy, survival of HNSCC remains almost unchanged with treatment resistance and metastases being the most important indicator of adverse outcome [1]. A recently disclosed www.impactjournals.com/oncotarget significant problem is the distinct cellular heterogeneity in the HNSCC tumor tissues that may contribute to formation of metastases or treatment resistance [2]. Recent data suggests that many potential cancer stem cell (CSC) markers are differentially expressed in different subpopulations of cells derived from a particular tumor [3]. One of the relevant characteristics of HNSCC subpopulations is the selective expression of surface receptors. In the context of HNSCC, CD44 and CD90 molecules are extensively discussed [4,5,6]
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