Authors: Shyam Patel & Pranela Rameshwar
The tumorsphere assay allows one to assess whether a single cell harbors the potential to both initiate and maintain tumors in the absence of cellular interaction and adhesion. Stem cells are capable of unlimited self-renewal ability, and unique subsets of cancer cells that acquire stem cell properties have the theoretical ability to form de novo tumors when grown under low-attachment conditions within minimal growth factor supplementation. Primary stem cells and progenitor cells from the breast can be enriched within mammospheres, a concept that is analogous to neural stem cell enrichment in neurospheres. Tumorigenic efficiency of a subset of cancer cells can be determined based on number of spheres that emerge from single cells. On the contrary, cancer cells that lack stem cell properties have limited sphere-forming potential due to telomere loss and cellular senescence.
This assay will take approximately 3 hours for initial set-up. Total incubation time is approximately 10 days per passage. Timing varies depending on the doubling time of cancer cell population of interest.
Step 1: Be sure to use phenol red-free DMEM if fluorescence is required, as phenol red will interfere for excitation/emission and may create false positive results.
Step 1: Note that serum-free media can sometimes lead to cellular death. If this occurs, it may be beneficial to supplement with 1% FBS or FCS to prevent death. Alternatively, one can use low-serum media in the absence of additional growth factor supplementation.
Step 5: Be sure to allow enough time for spheres to form. This may take up to two weeks, based on the doubling time of different tissue types. Note that some cellular subsets may be more inherently quiescent and therefore require more time to initiate cell division and tumorsphere formation.
Step 5: Note that morphology of spheres may vary depending on the cell line or primary tissue used. Some spheres may be more compact while others may be loose.
The most primitive and stem-like cancer cells are expected to have the highest sphere-forming efficiency. Spheres that arise from stem-like cancer cells may have intratumoral heterogeneity. This may be evident by most primitive cells located centrally and the most differentiated cells located peripherally. This assessment can be made by fluorescent microscopy if the putative stem-like cells are designated by a fluorescent reporter. Progenitors may have intermediate efficiency in forming spheres. Cancer cells that lack stem-like properties may initially form cell clusters, but these clusters will regress due to lack of self-renewal ability. The majority of the non-stem cells are expected to die by anoikis. Normal epithelial cells (MCF-10 or MCF-12A) are not expected to form any spheres.
We would like to thank Dr. Gabriela Dontu from King’s College London School of Medicine for her advice on this assay.
Delineation of breast cancer cell hierarchy identifies the subset responsible for dormancy. Shyam A. Patel, Shakti H. Ramkissoon, Margarette Bryan, Lillian F. Pliner, Gabriela Dontu, Prem S. Patel, Sohrab Amiri, Sharon R. Pine, and Pranela Rameshwar. Scientific Reports 2 () 30/11/2012 doi:10.1038/srep00906
Shyam Patel & Pranela Rameshwar, Rameshwar Lab
Correspondence to: Pranela Rameshwar ([email protected])
Source: Protocol Exchange (2013) doi:10.1038/protex.2013.023. Originally published online 11 February 2013.