Af 100 15 1mg

The HNSCC single cells were resuspended in a sphere-forming medium (DMEM/F12 + N2 supplement (1%, R&D Systems, Minneapolis, MN, USA, AR009) + B27 (1%, Gibco, A3582801) +bFGF (20 ng/mL, Invitrogen, RP-8627) + EGF (20 ng/mL, Gibco, AF-100-15-1MG)) and inoculated in ultra-low attachment 6-well plates (1000 cells/well, Corning, Corning, NY, USA). After 10 days of culture, cancer spheres larger than 100 μm in diameter were counted. The sphere formation assay was used to assess the sphere formation ability of the HNSCC cells.

ReNcell VM neural progenitor cells (Merck Millipore SCC008) were used in this study as an in vitro model of hNSC. They were grown either as neurospheres or as adherent monolayer on laminin coated plasticware in the following culture medium: 50% DMEM:F12 (GIBCO 11554546), 50% Neurobasal (GIBCO 21103049), 0.5% Penicillin-Streptomycin (Sigma-Aldrich P4333-100ML), 1% N2 supplement (GIBCO 17502048), 2% B27 NeuroMix (GIBCO 17504044), heparin (5μg/ml, Sigma-Aldrich 9041-08-1). Human Epidermal Growth Factor (hEGF, GIBCO AF-100-15-1MG) and human Fibroblast Growth Factor (hFGF, GIBCO 100-18B-1MG) were added to the cell culture medium just before use, both at a concentration of 20 ng/ml. Growth medium was replaced with fresh medium every 2 days.
Transfection of hNSC was performed by electroporation using a Nucleofector^TM 2b Device (Lonza) with Nucleofector^TM Kits for Mouse Neural Stem Cells as per manufacturer instructions (Lonza VPG-1004).