Isolation and Characterization of Ascites Tumor Cells

Ascites samples were processed within 24 hours of collection. Cells in the ascites were separated by the method developed in our laboratory as described previously (37 (link)). Briefly, approximately 100-500mL of ascites was centrifuged and the supernatant collected and stored at -80°C. Red blood cells from the cell pellet were removed by hypotonic shock. The remaining cells were suspended in a 1:1 ratio of MCDB131 (Life Technologies, CA, USA) and DMEM (Sigma-Aldrich), supplemented with 10% (v/v) heat inactivated FBS (Thermo Fisher Scientific, MA, USA), 2mM L-glutamine (Invitrogen Corporation, CA, USA), and an antibiotic combination of 1% (v/v) streptomycin and penicillin (Invitrogen Technologies, Vic, Australia). Approximately 1x10⁵-1x10⁶ tumor cells per well were seeded onto 6-well Corning^® Ultra-Low attachment plates (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37°C in 5% CO₂. Seeded cells grew into two distinct populations: the non-adherent epithelial cells and adherent mesenchymal population were collected and frozen in TRIzole (ThermoFisher Scientific, Melbourne, Australia) for RNA extraction.