Facilitation of Tumorsphere Formation Assay by Isolation of Cancer Stem Cell Markers and Enhanced Single Cell Sorting
Introduction
According to the cancer stem cell hypothesis, tumors are driven and maintained by a small population of cells that display stem-like properties. Researchers have relied on tumorsphereforming assays to try to identify and propagate these cancer stem cells.1 A tumorsphere is a solid, spherical cell formation that develops from a cancer stem cell and is not composed of aggregated cells of other origins. The cells grow in serum-free, non-adherent conditions and can be useful to characterize a cancer stem cell population in vitro.2 This assay can be used with various cell lines such as B16-F10, HT-29, MDA-MB-231, HCT-116, SKBR3, and MCF-7.
MCF-7 is a model breast cancer research cell line used for mammosphere assays that have cancer stem cell properties detected with markers such as CD44 and CD24. A key step for mammosphere development is to isolate single cells and then calculate the sphere forming efficiency (SFE).3
% SFE = Number of Spheres Counted per Well / Number of Seeded Cells *100
Traditional methods consist of using cell seeding densities ranging from 1,000 to 100,000 cells/mL leading to aggregation of cells, as opposed to only outgrowth from stem cells.4,5 By increasing the number of seeded cells, the sphere forming efficiency decreases. Because mammospheres need to arise from a single cell, plating a single cell per well would be ideal.
As a reflection of this, spheroids formed with lower cell densities produce consistent and reliable results when performing drug toxicity studies.6
By using the WOLF G2 Cell Sorter and the N1 Single Cell Dispenser, users can obtain cancer stem cells efficiently by isolating cells with markers of interest to increase sphere forming efficiency by depositing a relevant single cell per well.
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