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

Overview

This video demonstrates the protocol for developing pancreatic cancer stem cell spheroids in vitro. The assay can serve as a screening tool to identify compounds that potentially target cancer stem cells.

Protocol

1.Sphere Forming Assay and Analysis

1. Sphere Formation Assay and Metformin Treatment
   1. Take the required number of cells and add the appropriate volume of CSCs medium to prepare a cell concentration of 2,000 cells/ml. Do not keep the cell suspension on ice for no longer than 1h and mixed well prior to plating.
   2. Add 500 µl of 1X PBS to the first and last row of a 24-well plate for humidification in order to help minimize medium evaporation. Seed the cells into ultra-low attachment cells plates at a density of 2,000 cells per well in 1 ml of tumor sphere medium (2,000 cells/ml).
      ​NOTE: The number of cells used for tumor sphere formation assays may vary between tumor types.
   3. Treat at least 4 wells with vehicle (negative control) and 4 wells with each allocated treatment, e.g., 3 mM of metformin. Place the cells in an incubator set to 37 °C and supply the cells with 5% CO₂ for one week.
      NOTE: The medium should not be changed in order to allow the undisturbed formation of tumor spheres, but can be topped up with growth factors on a daily basis as they are not stable in culture medium.
   4. Every other day add treatment, e.g., 3 mM of metformin or vehicle to each of the wells. Assess the number of formed tumor spheres after 5 and/or 7 days by the use of an automated cell counter that allows for counting of larger structures (Figure 1).
      NOTE: Only tumor spheres with at least 40 µm should be considered and can be categorized as small (40 - 80 µm) or large (80 - 120 µm), respectively. The results can be illustrated as the percentage of tumor spheres divided by the initial number of cells seeded (e.g., 2,000 cells).
2. Serial Passaging of First Generation Tumor Spheres
   1. After 7 days of incubation harvest the tumor spheres using a 40 µm cell strainer and centrifuge them for 5 min at 900 x g at RT.
   2. Dissociate the pellet of tumor spheres to single cells using trypsin, and then expand the obtained single cell cells suspension again for another 7 days.

Representative Results

Figure 1. Metformin Treatment Inhibits Oxygen Consumption and Induces ROS Production. (A) Metformin addition inhibits oxygen consumption (OCR) of sphere-derived cells. Two different PDAC secondary spheres were treated with metformin and OCR changes were measured by extracellular flux analysis. Rotenone injection was used as a control for total mitochondrial OCR consumption. X-axis represents the oxygen consumption rate in pmol of oxygen/hr normalized by the protein content in each well. (B) PDAC spheres used in A were treated for 8 hr with metformin and ROS production was assessed by flow cytometry using carboxy-DCFDA. Left, representative flow cytometry plots. Right, summary of data from three independent experiments. Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
24-well Ultra-low Attachment Plates	Corning	3474
Sterile 1x Dulbecco’s Phosphate Buffered Saline	Sigma	D8537
Metformin	Sigma	D150959-5G
Trypsin solution, 0.05%	Life technologies	25300054
Incubator with CO2 input
CASY Cell Counter and Analyzer for proliferation and viability measurement	Roche Innovatis	AG CASY Model TTC 45,60,150 μm
Counting Chamber/ Hemocytometer	Hausser Scientific Co	3200
50 ml centrifuge tubes	Corning	430828
15-ml polypropylene conical tube	BD Falcon	352097
50-ml polypropylene conical tube	BD Falcon	352070