Abstract
Most epithelial tumors have been shown to contain cancer stem cells that are potentially the driving force in tumor progression and metastasis (Kreso and Dick, 2014; Nassar and Blanpain, 2016). To study these cells in depth, cell isolation strategies relying on cell surface markers or fluorescent reporters are essential, and the isolation strategies must preserve their viability. The ability to isolate different populations of cells from the bulk of the tumor will continue to deepen our understanding of the biology of cancer stem cells. Here, we report the strategy combining mechanical tumor dissociation, enzymatic treatment and flow cytometry to isolate a pure population of epithelial cancer stem cells from their native microenvironment. This technique can be useful to further functionally profile the cancer stem cells (RNA sequencing and epigenetic analysis), grow them in culture or use them directly in transplantation assays.
Keywords: Cancer stem cells, Flow cytometry, Cell isolation, Squamous cell carcinoma
Background
Tumor recurrence and metastasis is the leading cause of most deaths related to cancer. Malignant tumors may be initiated and maintained by a stem cell population (Nassar and Blanpain, 2016; Bonnet and Dick, 1997), and these cells represent an important therapeutic target to prevent relapse (Baumann et al., 2008). Studies suggest that squamous cell carcinomas are maintained by a subpopulation of tumor cancer stem cells that are resistant to therapy and can initiate tumor recurrence by undergoing self-renewal and differentiation, like normal stem cells, giving rise to proliferating progenitor cells that differentiate and form the bulk of the tumor (Locke et al., 2005; Prince et al., 2007; Malanchi et al., 2008; de Sousa e Melo et al., 2017). In this scenario, tumor cell fate and behavior are determined by the specific combination of changes in genes or their expression that have occurred during tumor development (Wang, 2010). Alternatively, progenitor cells can also acquire mutations that give them the potential to self-renew or can acquire some plasticity that give them cancer stem cell properties (Shimokawa et al., 2017). Regardless of the origin of cancer stem cells, efficient techniques to isolate these cells while maintaining their viability is essential. We have extensively characterized cancer stem cells from anorectal transition zone squamous cell carcinoma which arise spontaneously in the absence of epithelial TGFβ signaling (Keratin14Cre; Tgfbr2flox/flox mice) (Guasch et al., 2007; McCauley and Guasch, 2013; McCauley et al., 2017 ). In this protocol, we describe a method to isolate cancer stem cells from these anorectal transition zone squamous cell carcinomas.
Materials and Reagents
Equipment
Procedure
Data analysis
Notes
Recipes
Acknowledgments
The protocol to dissociate the tumor is based on our original protocol published in Methods Mol Biol (McCauley and Guasch, 2013), with minor modifications. The protocol to make chelexed FBS and E media are abbreviated here, but made exactly as described in (Nowak and Fuchs, 2009). An abbreviated description of the flow cytometry protocol appeared in eLife (McCauley et al., 2017). We would like to acknowledge the assistance of the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center (supported in part by P30 DK07839) and the Flow Cytometry Core at the CRCM, FRANCE. The protocol related to this work was supported by grants from the V Foundation, the Sidney Kimmel Foundation and in part from the Foundation ARC pour la recherche sur le cancer (GG).
References
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