A nanopore-based sequencing of mitochondrial DNA of oral squamous cell carcinoma cell lines with differing responses to cisplatin

Mitochondrial DNA (mtDNA) alterations, including copy-number variation, point mutations, and methylation changes, have been reported in various cancers, suggesting their significant role on cancer development, survival and therapeutic resistance. In this work, mtDNA sequencing of two human oral squamous cell carcinoma (OSCC) cell lines, SAS and H103, and SAS-derived cancer stem-like cells (CSCs) with different sensitivity to cisplatin treatment was performed to examine the influence of mtDNA alterations on cisplatin responsiveness in OSCC. The SAS-derived CSCs were generated via a sphere-forming assay. Cisplatin sensitivity was determined by measurements of cell viability after 72 h treatments with cisplatin using CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS assay; Promega Inc., USA). Briefly, H103 and SAS tumour spheres had reduced sensitivity towards cisplatin, yielding higher IC50 values of cisplatin compared to SAS. MtDNA was extracted and processed for both native DNA and amplicon sequencing. Sequencing-ready native DNA and amplicons were analyzed using a MinION sequencer (Oxford Nanopore Technologies, UK). The sequencing data were acquired by the MinKNOW software (Oxford Nanopore Technologies, UK).