Isolation and Separation of Epithelial CD34 + Cancer Stem Cells from Tgfbr2 -deficient Squamous Cell Carcinoma

[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.


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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.  2. Our tumor-bearing mice contain the eYFP reporter allele ( Figure 1A). Because of this, we also sacrifice a healthy, wild-type mouse which does not contain a fluorescent reporter and dissociate keratinocytes from the wild-type anorectal transition zone to obtain cells for unstained and single-stained controls. Dissociate cells from the anorectal transition zone of this healthy, wildtype mouse in parallel to dissociation of tumor cells from the tumor-bearing mouse.

Materials and Reagents
3. Carefully dissect the tumor, ensuring that it is separated from skin, fat, muscle and other contaminating tissue ( Figure 1A). Remove any necrotic tissue.
4. Place the tumor, approximately 0.5 cm 2 , in 8 ml sterile 1x HBSS in a 10 cm Petri dish and mince using a scalpel until all pieces are uniform and small ( Figure 1B).

5.
Add 100 µl 20% collagenase (see Recipes) to dissociate the minced tumor. Incubate for 45 min while shaking at 37 °C. We place a rocking platform within a dry incubator set at speed 3 rpm.
6. After 45 min, the tumor mixture should be homogeneous and may appear viscous ( Figure 1C). 13. Bring the 7AAD with the sample to the flow cytometer. We prefer to add the 7AAD to the sample immediately before sorting to minimize cell death due to toxicity. We found 20 µl per million cells to be sufficient to label dead cells.
14. Record at least 10,000 events from the unstained and single-color controls to enable compensation.
15. See Figure 2 for an example of our gating strategy to isolate epithelial CD34 + cancer stem cells. b. For return to culture, collect as many cells as possible in 500 µl epithelial cell culture media in a screw-top tube. Centrifuge tubes at 200 x g for 5 min and very carefully aspirate supernatant. Resuspend in epithelial cell culture media and plate. We found that cells did not survive when plating directly on plastic. We prefer to plate cells on irradiated mouse fibroblasts to improve colony formation and survival. We found that plating less than 1,000 cells did not allow for cell survival. Transplanting larger numbers of cells will accelerate tumor formation.

Because the isolation of rare cells requires inclusion and exclusion of a number of markers, it is
imperative to have unstained and single-stained controls for effective compensation of spectral overlap.
2. The frequency of epithelial CD34 + cells ranged from 7% to 22%. This is due to the heterogeneity of the squamous cell carcinoma from mouse to mouse. It is therefore recommended to perform at least six biological repeats within each experimental group.

All experiments were approved by the Cincinnati Children's Hospital Research Foundation
Institutional Animal Care and Use Committee (protocol number 1D10087) and in agreement with European and national regulation (protocol number 4572), and carried out using standard procedures.
2. Trypsinizing the filter is a crucial step that can drastically impact the efficiency of CD34 + cancer stem cells isolated, as the cells may remain in clumps on top of the filter and be unintentionally discarded. Be sure to use pre-warmed 0.25% trypsin-EDTA, to incubate for the full 10:00 at 37 °C, and to vigorously pipet the trypsinized cells to ensure proper dissociation and maximal number of cells obtained.
3. It is possible to store the tumor samples in media containing serum overnight at 4 °C and perform the cell isolation the next day. Cell mortality will be increased but the overall percentage of cancer stem cells will not be altered.