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 evaluate the effect of metformin.

- To facilitate the tumor sphere formation for cancer stem cells enrichment, dilute the cells in the appropriate volume of cancer stem cell medium to a final concentration of 2 times 10 to the 3 cells per milliliter, and chill them on ice for a few minutes. Then, after adding 500 microliters of PBS to the first and last rows of a 24 ultra-low attachment cell plate, resuspend the cells by pipette and seed 1 milliliter of cells per well into the empty wells of the plate.

Treat four wells of cells with the drug of interest and at least four negative control wells with vehicle. Then incubate the cells at 37 degrees Celsius with 5% carbon dioxide for one week. Every other day, add fresh treatment or vehicle to each of the wells.

On day 5 or 7, assess the number of formed tumor spheres with an automated cell counter that allows the identification of larger structures. For serial passaging of the tumor spheres, on day 7, use a 40-micrometer cell strainer to harvest the spheres. Next, spin down the spheres and incubate them in 1-milliliter of trypsin for 20 minutes at 37 degrees Celsius to dissociate the pellet into a single-cell suspension. Then expand the cells for another seven days, as just demonstrated.