Published: Vol 7, Iss 8, Apr 20, 2017 DOI: 10.21769/BioProtoc.2233 Views: 14092
Reviewed by: Anonymous reviewer(s)
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Abstract
Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells (or cancer stem cells) self-renewal potential in vitro. When single cells are plated at low density in stem cell culture medium, only the cells endowed with self-renewal are able to grow in tridimensional clusters usually named spheres. In the recent years, SFA has also been used also to test the effect of several drugs, chemical and natural compounds or microenviromental components on stem cells self-renewal capacity. Here we will illustrate a detailed protocol to assess self-renewal of human melanoma stem cells, growing as melanospheres.
Keywords: MelanomaBackground
Cancer stem cells (CSCs) were first found in acute myeloma leukemia (Lapidot et al., 1994) and then were identified in many solid tumors including melanoma. CSCs are defined as cells strongly endowed with self-renewal and tumor initiating capacity, being able to regenerate the whole tumor heterogeneity in vivo. CSCs can be isolated from the tumor mass with different approaches based on phenotypic characteristic or biological properties, then their properties have to be tested in vitro (self-renewal) and in vivo (tumorigenic potential). Melanoma CSCs were isolated using a combination of cell surface markers, (Fang et al., 2005; Monzani et al., 2007; Schatton et al., 2008; Boiko et al., 2010; Boonyaratanakornkit et al., 2010) or through culture in specific stem cell media (Perego et al., 2010; Santini et al., 2012). To validate melanoma CSC self-renewal, and to study the effect of tumor microenvironmental factors on it (Tuccitto et al., 2016), we used the sphere formation assay (SFA) in vitro. Melanoma CSCs are plated at low density in stem cell culture medium and they grow in anchorage-independent, three-dimensional spherical structures, called melanospheres (tumorspheres, in general). Spheres forming efficiency is directly proportional to the number of melanoma CSCs present in the culture (one CSC corresponds to one melanosphere), thus giving a direct quantification of CSC amount in culture. This relatively simple method is useful to study the ability of any exogenous factors (growth factors, cytokines and chemokines, drugs) in perturbing CSC self-renewal (Tsuyada et al., 2012; Tuccitto et al., 2016). Here we provide detailed information about the SFA protocol we optimized in our laboratory for melanoma SFA.
Materials and Reagents
Equipment
Procedure
Notes:
Data analysis
Sphere efficiency can be reported as percentage (%) of spheres formed dividing by the original number of cells seeded or number of spheres counted considering the total number of single cells seeded. We usually presented the results as mean ± SD (or SEM) of biological replicates. Each cell line was tested multiple times to be sure that the sphere forming efficiency was maintained after extensive cell culture.
Notes
Recipes
Acknowledgments
This protocol was used in Perego et al., 2010 and Tuccitto et al., 2016. These works were supported by the Associazione Italiana Ricerca sul Cancro (CC IG-10615).
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Tuccitto, A., Beretta, V., Rini, F., Castelli, C. and Perego, M. (2017). Melanoma Stem Cell Sphere Formation Assay. Bio-protocol 7(8): e2233. DOI: 10.21769/BioProtoc.2233.
Category
Cancer Biology > Cancer stem cell > Cell biology assays
Stem Cell > Adult stem cell > Cancer stem cell
Cell Biology > Cell-based analysis > Non-adherent culture
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