Cancer Biology


Protocols in Current Issue
Protocols in Past Issues
0 Q&A 9138 Views Sep 5, 2018
Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in vitro. We further show how ELDA can be used on cells, before and after forced differentiation to amplify inherent differences in sphere-forming potential between mutant and control NSCs. Ultimately, ELDA exploits a difference in the ability of a single or a few seeded stem cells to self-renew, divide and form neurospheres. Importantly, the assay also allows a comparison between genetically distinct cells or between the same cells under different conditions, where the impact of target-specific drugs or other novel cancer stem cell therapies can be tested.
0 Q&A 13536 Views Apr 20, 2017
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.
3 Q&A 54100 Views Feb 5, 2013
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.
0 Q&A 21075 Views Dec 20, 2012
Self-renewing cells from adult tissue (such as bone) that represent a progenitor population can be grown in suspension cultures in the presence of defined serum-free medium. Progenitor cells can be identified by this property of anchorage-independent growth in suspension cultures. These spherical clusters of progenitor bone cells growing under non-adherent conditions are called osteospheres. Such progenitor populations often possess characteristics of multipotency and can differentiate into multiple mesenchymal lineages. Cancer cells capable of growing in suspension have also been reported in osteosarcomas, tumors of the bone tissue. These spherical colonies formed from single cells (clonal) in non-adherent conditions are generally considered to represent self-renewing, stem-like cells and can be employed for other assays such as multipotency and limiting dilution analysis (LDA).

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