Evaluating Colony and Spheroid Formation

To determine colony formation ability, 300–500 established CCA cells were plated in each well of a 6-well plate as described previously [41 (link)]. Drugs were added 24 h later and cells were allowed to grow until colonies appeared. Crystal violet (Sigma-Aldrich, St. Louis, MO, USA) was used to stain the colonies. The number of colonies was counted and the data were presented as a bar graph. For spheroid formation in PDXC, 1000 cells per well were plated, treated with the indicated drugs and allowed to grow for 6 days. Niraparib, olaparib and gemcitabine were purchased from Selleck chemicals (Houston, TX, USA).