Abstract
A tumorsphere is a solid, spherical formation developed from the proliferation of one cancer stem/progenitor cell. These tumorspheres (Figure 1a) are easily distinguishable from single or aggregated cells (Figure 1b) as the cells appear to become fused together and individual cells cannot be identified. Cells are grown in serum-free, non-adherent conditions in order to enrich the cancer stem/progenitor cell population as only cancer stem/progenitor cells can survive and proliferate in this environment. This assay can be used to estimate the percentage of cancer stem/progenitor cells present in a population of tumor cells. The size, which can vary from less than 50 micrometers to 250 micrometers, and number of tumorspheres formed can be used to characterize the cancer stem/progenitor cell population within a population of in vitro cultured cancer cells and within in vivo tumors (Lo et al., 2012; Liu et al., 2009). While several cell lines can be used for tumorsphere formation assay (e.g. primary mammary tumor cells from Her2/neu-transgenic mice, MCF7, BT474 and HCC1954), some cell lines may not form typical tumorsphere structures and may be difficult to count or classify definitively as tumorspheres.
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was first described in Lo et al. (2012). This work was supported by the Elsa U. Pardee Cancer Foundation grant (B94AFFAA), the American Cancer Society Research Award (RSG-10-067-01-TBE) to HC and NIH grant (3P20RR017698-08) to HC and QW.
References
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Hello, I have had this happen once before with tumorspheres kept in culture for over one week. It is possible that the tumorspheres have remained in culture too long. We generally counted spheres after one week, so a lengthier culture can lead to attachment. From my understanding, some cells respond better to different types of non-tissue culture petri dishes and low attachment plates as well.I hope this helps.Sara
Dear Tara,When counting and photographing the spheres, we use the imaging software that accompanied our Olympus inverted scope (DP2-BSW software). This program has a 100 micrometer scale bar that allows us to estimate the size of the tumorspheres.I hope that this helps to answer your question,Sara
Dear Sara, How to identify the range of diameter for tumorspheres? 50-250 micrometers? Thanks! Ting Yu
Dear Melanine, We are using the flat-bottom plate. The cells are likely to be attacked to the edge but they should be able to form sphere well. Best, Hexin