Spheroid Viability Assay with Drugs

Monospheroids and heterospheroids were seeded as described before [16 (link),19 (link)], and after one day, treated with 5 µM TGF-β receptor type I/II (TGFBR1/2) kinase inhibitor (LY2109761, Sigma-Aldrich, #SML2051) or different therapeutic compounds, including 50 µM gemcitabine (Sigma-Aldrich, #G6423), 1 µM paclitaxel (Sigma-Aldrich, #580555), 1 µM SN38 (active metabolite of irinotecan; Sigma-Aldrich, #H0165) or 5 µM pitavastatin (a HMG-CoA reductase inhibitor; Selleckchem, #S1759). Three days later, spheroids were collected for RNA extraction or apoptosis detection. M30 Apoptosense® CK18 Kit (Diapharma #P10011) was used according to the manufacturer's instructions to quantitatively detect apoptosis in epithelial cells and differences between treatment groups were assessed by paired t-test.