Sphere Formation Assay: An In Vitro Method to Identify Pancreatic Cancer Stem Cells and Screen Therapies

- Pancreatic Ductal Adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic Cancer Stem Cells (CSC). CSCs are self-renewable, pluripotent, and capable of forming tumors. They express anti-apoptotic proteins and also contain multidrug-resistant genes, making them resistant to chemotherapy. To identify cancer stem cells and study the effect of drugs on them, we perform a sphere formation assay.

First, suspend the extracted cancer stem cells in the desired cell culture medium. Next, seed the cell suspension into a multi-well plate and incubate for the desired duration. Add a required amount of drug of interest into a few wells and placebo in other wells as a negative control. Incubate the cells for the desired duration. Cancer stem cells form sphere-shaped colonies under a non-adherent culture environment.

After incubation, pipette a small amount of cell suspension onto a hemocytometer and place it into an automated cell counter. Now, count the number of spheres formed. The drug-treated cells usually show a substantial reduction in the size and number of spheres. In the following protocol, we will perform a sphere formation assay to identify pancreatic cancer stem cells and