Cancer research is one of today’s major research fields producing thousands of publications every year. Traditional therapeutic approaches aim to eliminate as much of the tumor mass as possible by means of surgery, irradiation, chemotherapy and biologics. However, evidence suggests that these techniques do not target the root of the disease: the cancer stem cell (CSC) subpopulation within the tumor.1 Cancer stem cells have been identified in various cancers including hematopoietic and many solid tumors.5 CSCs are defined as a small subset of malignant cells within the tumor with the exclusive ability to self-renew and maintain stem cell properties. They can differentiate into a heterogeneous mass of both non-tumorigenic and tumorigenic cell types and evade traditional chemotherapy and lead to cancer regression and metastasis.3
The gold standard functional in vivo assay for CSCs is the serial transplantation of isolated single cancer cells into immunocompromised mice (i.e. NOD SCID mouse). However, this method requires lengthy protocols and results are often difficult to interpret. The in vitro spheroid formation assay includes methods to generate colony-forming units (CFUs) in both semi-solid media and as 3D tumorsphere aggregates. The tumorsphere formation efficiency (TFE assay) indicates the percentage of cells within a cancer cell culture that are capable of forming a sphere from a single cell. TFE values are a quantitative measurement to measure the amount of cancer stem cells within a tumor/cancer cell line and is correlated with cancer metastasis and aggressiveness.
The PromoCell 3D Tumorsphere Media XF (C-28070, C-28075) is designed to serially passage cancer stem cells as undifferentiated 3D tumospheres with high cell proliferation rates. It’s defined and serum-free formualtion allows consistent and standardized expansion of sphereoids while maintaining the cancer stem cell characteristics such as self-renewal and chemoresistance. The media has been shown to support spheroid cell cultures from the following cell lines: U-87 MG, MCF-7, MDA-MB-231, HT-29, HT1080, HepG2, A549, Panc-1, LNCaP and A431.
Figure 1.The cancer stem cell hypothesis. The in vitro spheroid formation assay is a common assay used to measure the self-renewal and multipotent nature of the cancer stem cell subpopulations within a tumor or cancer cell line.
Figure 2.Overview of the spheroid formation assay protocol.
Note: It is recommended to use the mean diameter of the cell aggregate as a readout parameter using a microscopre grid or ruler. Ideally, the median sphere diameter of a specific cell line is allready known from previous experiments. For example, MCF-7 cells exhibit a mean diameter of > 150 µM after 10-12 days. Aggregates with < 80 µM should never be counted as positive. Some slowly growing cell lines require more than 7-12 day growth to achieve large enough spheroids to quantify.
Figure 3.Expected results from an in vitro spheroid formation assay. A) Positive tumorsphere derived from a single MCF-7 cell larger than the 150 µm cutoff size. B) Negative tumorsphere derived from a single MCF-7 cell smaller than the 150 µm cutoff size.
Figure 4.Tumorsphere culture of MCF-7 breast cancer cells in the PromoCell 3D Tumorsphere Media XF after 10 passages. The tumorsphere culture was subject to serial passage every 9 days by enzymatic dissociation according to the protocol. Robust tumorsphere formation was maintained during serial culture