Polyhema coated plates

A total of 500 single HCC cells were plated onto 24-well poly-HEMA-coated plates (Sigma-Aldrich, Shanghai, China). The cells were cultured for 3–4 weeks in DMEM/F12 medium (Invitrogen, Shanghai, China) supplemented with 4 mg/mL insulin (Sigma-Aldrich, Shanghai, China), B27 (1:50, GIBCO, Shanghai, China), 20 ng/mL EGF (Sigma-Aldrich, Shanghai, China) and 20 ng/mL basic FGF (Sigma-Aldrich, Shanghai, China). For serial passage of primary spheres, the primary spheres were collected, subsequently dissociated with trypsin and resuspended in DMEM/F12 medium with the above supplements. The surviving colonies were measured depending on their diameter, and the data are expressed as the mean ± SD of triplicate wells within the same experiment.

OSCC tumorspheres were prepared as previously reported [72 (link), 73 (link), 147 (link), 148 (link)]. In brief, OSCC cells grown overnight were challenged with T. denticola, P. gingivalis or F. nucleatum for 2 h, then tumorspheres were developed by maintaining cells under suspension conditions on poly-HEMA coated plates (7.5 mg/ml in 95% ethanol, Sigma-Aldrich, St.Louis, MO) in the presence or absence of nisin ZP for 24 h. In this assay, cells that survive anchorage withdrawal form multicellular aggregates or tumorsphere. A tumorsphere is defined as an aggregate of cells that is at least 50 μm in diameter. The total area occupied by tumorspheres in each well was quantified for each treatment condition using NIS-Elements BR4.13.04 imaging software.

Five hundred ccRCC cells were seeded onto 24-well polyHEMA-coated plates (Sigma–Aldrich, St Louis, MO, USA) and cultured for 2 weeks in DMEM/F12 medium (Invitrogen, Carlsbad, CA) supplemented with 4 μg/ml insulin (BIOIND, Kibbutz Beit Haemek, Israel), B27 (1:50, Gibco, Grand Island, NY, USA), 20ng/ml epidermal growth factor (Pepro Tech, Rocky Hill, NJ, USA), and 10ng/ml basic fibroblast growth factor (Pepro Tech, Rocky Hill, NJ, USA).