TITLE: Isolation of cancer stem cells by sphere formation assay
AUTHORS: Kwangok  P Nickel, Austin M. Maas, Randall Kimple

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cancer stem cells (CSCs) are a small subpopulation of tumor cells that are thought to be responsible for recurrence and metastasis of cancer due to their ability for self-renewal and differentiation into multiple cancer cell types. </div><div class = "text-block">CSCs are also known to play a key role in the development of metastases.  Cancer cells undergoing Epithelial to Mesenchymal Transition (EMT) have also been shown to attain CSC phenotypes and acquire invasive and migratory properties. Therefore, understanding the characteristics of CSCs may help to develop better treatment strategies. </div><div class = "text-block">The sphere formation assay has been widely used to isolate CSCs using anchorage-independent sphere culture.  CSCs can grow on ultra-low attachment plates that are coated with a layer to inhibit the attachment of cells. When cells are grown in serum-free and non-adherent conditions, CSCs can survive and clonally expand to form spheres, whereas differentiated tumor cells undergo apoptosis due to their anchorage dependence. </div></div>

[STEPS]
?. Prepare Sphere Formation Media () Base media is DMEM/F12   Penicillin and Streptomycin B-27  bFGF  hEGF  of DMEM/F12.
100 ml
1 ml
2 ml
20 µl
10 µl
96.97 ml
?. Dilute cells to a final concentration of 2000 cells/ml in Sphere Formation Media.Note that the final concentration of cells/ml should be optimized to your specific cell line or cancer type.
?. Add   of cell mix (from Step 3) to each well of a 24-well low attachement plate (final concentration of 500 cells/well).Note that the optimal number of cells is dependent on tumor type. Use low-attachment plates (see materials) NOT standard tissue culture plates.
250 µl
?. Place plates into humidified (95% relative humidity) incubator at  and 5% CO2.
37 °C
?. Check plate daily and add fresh media to keep the wells approximately 20% filled based on evaporation.
?. Prepare a single cell suspension from either cell culture or primary tumor tissue.  Count cells using a hemocytometer or an automated cell counter.Note:  In this example, a head and neck cancer cell line, UM-SCC47 , is analyzed. The experimental approach can be used to study cells isolated from primary human cancers or with established cancer cell lines.
?. [Results]
In this assay, the endpoint of interest is sphere formation efficiency (i.e., the percentage of plated cells that form a sphere). This is calculated using the following formula which is calculated for each well.Sphere formation efficiency (%) = (number of spheres)/(number of cells seeded) x 100.A minimum of 3 biologic replicates should be performed and the average and standard deviation reported.
?. Check spheres after 2 days and then daily by visualization under a microscope. We use a size of   to identify spheres. The time required for sphere formation can vary significantly based on the cell type used.  We typically expect approximately 10 spheres per well.  In this example, spheres were first identified 3-4 days after plating and a final count was made 7 days after plating the cells.