Name: Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples (Video) | JoVE Journal
Uploaded: 2017-05-10T15:00:00.000+00:00
Duration: 9 min 57 s
Description: Learn to detect and isolate cancer stem cells using a violet dye-based side population assay with flow cytometry. This functional method enables downstream analyses and therapeutic target discovery in cancer research.

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Maximilian Boesch is supported by an Erwin Schr&#246;dinger Fellowship of the Austrian Science Fund FWF (grant number J-3807).

This protocol is in full compliance with standard Institutional Ethical Review Board guidelines. If human or animal tissue is investigated using the protocol depicted here, researchers are obliged to have approval from the relevant review board of their institution or country.
Caution: Standard precautions for the safe handling of biological samples apply to this protocol. This includes wearing gloves and a lab coat, and performing the work under a biosafety cabinet whenever possible.
1. Preparation of Cells
<ol>
	<li>Cancer cell lines
	<ol>
		<li>Culture a respective human or murine cancer cell line at 37 &#176;C in 10-30 mL of appropriate medium (e.g., RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 1x penicillin/streptomycin) and harvest the cells at sub-confluency (i.e.&#160;70-90%) using digestion with 0.05% trypsin-EDTA or some other de-attachment procedure. Determine the cell count and check for viability using a counting chamber and trypan blue staining. The fraction of viable cells should exceed 85%. 
		NOTE: Examples of human cancer cell lines harboring SP&#160;compartments include MCF7/HTB-22 and SKBR3/HTB-30 (breast cancer), A2780 and SKOV-3/HTB-77 (ovarian cancer), and A549 (lung cancer)26.</li>
	</ol>
	</li>
	<li>Ex vivo tumor tissue
	<ol>
		<li>Take a fresh surgical tumor specimen or alternatively, harvest tumor tissue from a transplantable or genetically-engineered mouse tumor model. Take 200-1,000 mg of tissue and generate a single cell suspension by mechanical disaggregation (e.g., using scissors or a scalpel) and enzymatic digestion (e.g., using a collagenase/dispase/DNase cocktail).</li>
		<li>Filter the sample through a 70 &#181;m cut-off strainer. Determine the cell count and check for viability using a counting chamber and trypan blue staining. 
		NOTE: Digestion cocktails frequently contain 200 &#181;g/mL collagenase P, 0.8 U/mL dispase and 25 &#181;g/mL DNase I, and incubation times typically range from 20 - 50 min. Ideally, tissue disaggregation is performed according to a protocol optimized for the tissue under investigation 27,28,29.</li>
	</ol>
	</li>
</ol>
2. Test Samples
<ol>
	<li>Adjust cell concentration to 1 x 106 cells/mL in fresh culture medium (a nutrient-containing vehicle is important as dye extrusion is an active energy-consuming process). Finally, transfer 1 mL of sample to an ordinary flow cytometry/FACS round-bottom tube (labelled &#39;test&#39;). 
	NOTE: For optimal results, it can be necessary that the cell concentration is titrated (for instance 5 x 105-1 x 106-2.5 x 106 cells/mL), but a lower cell concentration generally yields a higher resolution19. Only for sorting applications where a maximum number of SP cells shall be recovered, it is recommended to increase the cell concentration to up to 1 x 107 cells/mL.</li>
	<li>Add 10 &#181;M of DCV to the sample and mix well, either by gentle vortexing or resuspending for 3-5x. Proceed to inhibition controls. 
	NOTE: Optimal results might depend on titration of the triggering dye (for instance 2.5; 5; 10; 20 &#181;M), but a higher dye concentration generally yields a higher resolution19.</li>
</ol>
3. Inhibition Controls
<ol>
	<li>Transfer 250 &#181;L of the dye-containing cell suspension to a new flow cytometry/FACS round-bottom tube (labelled &#39;control&#39;). Add 50 &#181;M of verapamil or 20 &#181;M of FTC and mix well by pipetting. 
	NOTE: Verapamil and FTC are the most well-established inhibitors for SP analysis but have different efflux pump specificity: Verapamil inhibits several ABC drug transporters including ABCB1 and ABCB5, whereas FTC is specific for ABCG26,19. If the SP status of an individual sample is unknown, it is thus recommended to use both verapamil and FTC in separate tubes.</li>
</ol>
4. Staining
<ol>
	<li>Cap both the &#39;test&#39; and &#39;control&#39; tubes and incubate them at 37 &#176;C in the dark for 90 min, with gentle agitation every 15 min. Most of the dye extrusion in SP cells will occur within the first 45 min, but dye accumulation in non-SP cells (contributing to resolution) is only maximal after 75-90 min19.</li>
	<li>After staining is complete, wash the cells in 3-4 mL ice-cold PBS and resuspend the pellet in a volume of 100 &#181;L (PBS as well). Keep the cells in the dark and chilled on ice, and proceed to the staining of additional markers. Alternatively, directly perform flow cytometric analysis.</li>
</ol>
5. Costaining for Other Markers
<ol>
	<li>Add a desired panel of fluorochrome-conjugated antibodies to the DCV-stained cells, mix well and incubate the cells at 4 &#176;C in the dark for 20-30 min. Wash the cells in 3 - 4 mL ice-cold PBS and proceed to dead cell discrimination. 
	NOTE: Antibody formats that are easily compatible with DCV and virtually do not require compensation include PerCP or PerCP-Cy5.5, PE-Cy7, APC, Alexa Fluor 647, APC-Cy7, APC-H7, BV711, Alexa Fluor 750 and BV786. Formats that are compatible with DCV but may require compensation include FITC, Alexa Fluor 488, PE and BV650. Formats that are virtually not compatible with DCV include BV421, V450 and V500.</li>
	<li>Add 7-AAD (or PI) to the DCV-stained cells at the concentration recommended by the particular manufacturer and incubate the samples for 5 - 10 min in the dark. Filter the samples through 70 &#181;m cut-off strainers and proceed to flow cytometric analysis.</li>
</ol>
6. Flow Cytometric Analysis
NOTE: It is recommended to switch on the respective flow cytometric instrument during the staining process already so that everything is set as soon as the samples are ready. This includes also standard maintenance procedures such as system performance tracking and tank refilling.
<ol>
	<li>Acquire the cells on the flow cytometer and gate them on a bivariate FSC/SSC dot plot (Figure 1B). Exclude doublets and aggregates by comparing the different signals of FSC (i.e.&#160;height, width, area) (Figure 1C).
	<ol>
		<li>If a dead cell marker has been included, gate on the viable cell fraction (e.g., 7-AAD-negative) (Figure 1D). Visualize the viable singlet cells on a bivariate dot plot for &#39;blue&#39; and &#39;red&#39; DCV emission. To this end, display the &#39;blue&#39; fluorescence channel on the y-axis (e.g., 450/50) and the &#39;red&#39; fluorescence channel on the x-axis (e.g., 525/50).</li>
		<li>Switch both channels to linear mode and adjust the detector voltages correspondingly so that the SP locates to the side of the plot in the lower left quadrant. Conversely, the non-SP locates to the upper right quadrant of the plot reflecting the cell cycle distribution (G1 and G2). Gate the SP and stop the acquisition (Figure 1E). 
		NOTE: DCV-defined SP cells can be detected using flow cytometric instruments equipped with a violet laser excitation source (i.e.&#160;a 405 nm laser line). It is one specific of SP analysis that the emitted fluorescence is measured in linear mode and in two separate channels displayed on a bivariate dot plot. The &#39;blue&#39; emission of DCV is measured at approximately 450 nm (e.g., with a 450/40 standard filter) and the &#39;red&#39; emission of DCV is measured at approximately 510 - 585 nm (e.g., with a 510/50, a 525/50 or a 585/15 filter)19. Due to the particular chemistry of the assay, the separation between SP and non-SP cells is generally higher in the &#39;red&#39; fluorescence channel19.</li>
	</ol>
	</li>
	<li>Load the inhibition control(s) and acquire the data. The SP cells have now disappeared or are reduced substantially (Figure 1F). If necessary, refine the SP gate based on these data.</li>
	<li>To investigate surface marker expression by SP cells, leave the &#39;red&#39; fluorescence of DCV on the x-axis and display a respective fluorescence channel on the y-axis in logarithmic mode (e.g., APC). Adjust the detector voltages correspondingly and gate the fraction of SP cells staining positive for the investigated marker(s) (Figure 1G). Co-expression of a particular marker by SP cells yields a respective signal in the upper left quadrant of the plot30.</li>
	<li>Record the data and/or sort the SP cells.</li>
</ol>

<ol><li>Pattabiraman, D. R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tackling+the+cancer+stem+cells+-+what+challenges+do+they+pose%3F%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Tackling the cancer stem cells - what challenges do they pose?</a> Nat Rev Drug Discov. 13 (7), 497-512 (2014).</li>
<li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterogeneity+of+Cancer+Stem+Cells%3A+Rationale+for+Targeting+the+Stem+Cell+Niche%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Heterogeneity of Cancer Stem Cells: Rationale for Targeting the Stem Cell Niche.</a> Biochim Biophys Acta. 1866 (2), 276-289 (2016).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+cancer+relapse+and+metastasis+by+inhibiting+cancer+stemness%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Suppression of cancer relapse and metastasis by inhibiting cancer stemness.</a> Proc Natl Acad Sci U S A. 112 (6), 1839-1844 (2015).</li>
<li>Greve, B., Kelsch, R., Spaniol, K., Eich, H. T., Götte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+in+cancer+stem+cell+analysis+and+separation%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Flow cytometry in cancer stem cell analysis and separation.</a> Cytometry A. 81 (4), 284-293 (2012).</li>
<li>Marcato, P., Dean, C. A., Giacomantonio, C. A., Lee, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aldehyde+dehydrogenase%3A+its+role+as+a+cancer+stem+cell+marker+comes+down+to+the+specific+isoform%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform.</a> Cell Cycle. 10 (9), 1378-1384 (2011).</li>
<li>Golebiewska, A., Brons, N. H., Bjerkvig, R., Niclou, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+appraisal+of+the+side+population+assay+in+stem+cell+and+cancer+stem+cell+research%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Critical appraisal of the side population assay in stem cell and cancer stem cell research.</a> Cell Stem Cell. 8 (2), 136-147 (2011).</li>
<li>Hirschmann-Jax, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+distinct+%26%2334%3Bside+population%26%2334%3B+of+cells+with+high+drug+efflux+capacity+in+human+tumor+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">A distinct &#34;side population&#34; of cells with high drug efflux capacity in human tumor cells.</a> Proc Natl Acad Sci U S A. 101 (39), 14228-14233 (2004).</li>
<li>Zhou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ABC+transporter+Bcrp1%2FABCG2+is+expressed+in+a+wide+variety+of+stem+cells+and+is+a+molecular+determinant+of+the+side-population+phenotype%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.</a> Nat Med. 7 (9), 1028-1034 (2001).</li>
<li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+side+population+of+ovarian+cancer+cells+defines+a+heterogeneous+compartment+exhibiting+stem+cell+characteristics%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics.</a> Oncotarget. 5 (16), 7027-7039 (2014).</li>
<li>Szotek, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ovarian+cancer+side+population+defines+cells+with+stem+cell-like+characteristics+and+Mullerian+Inhibiting+Substance+responsiveness%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.</a> Proc Natl Acad Sci U S A. 103 (30), 11154-11159 (2006).</li>
<li>Brown, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+benign+and+malignant+prostate+epithelial+Hoechst+33342+side+populations%5BTitle%5D)+AND+%22Prostate%22%5BJournal%5D)">Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.</a> Prostate. 67 (13), 1384-1396 (2007).</li>
<li>Mathew, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ABCG2-mediated+DyeCycle+Violet+efflux+defined+side+population+in+benign+and+malignant+prostate%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">ABCG2-mediated DyeCycle Violet efflux defined side population in benign and malignant prostate.</a> Cell Cycle. 8 (7), 1053-1061 (2009).</li>
<li>Engelmann, K., Shen, H., Finn, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MCF7+side+population+cells+with+characteristics+of+cancer+stem%2Fprogenitor+cells+express+the+tumor+antigen+MUC1%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">MCF7 side population cells with characteristics of cancer stem/progenitor cells express the tumor antigen MUC1.</a> Cancer Res. 68 (7), 2419-2426 (2008).</li>
<li>Ho, M. M., Ng, A. V., Lam, S., Hung, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Side+population+in+human+lung+cancer+cell+lines+and+tumors+is+enriched+with+stem-like+cancer+cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells.</a> Cancer Res. 67 (10), 4827-4833 (2007).</li>
<li>Kato, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endometrial+cancer+side-population+cells+show+prominent+migration+and+have+a+potential+to+differentiate+into+the+mesenchymal+cell+lineage%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Endometrial cancer side-population cells show prominent migration and have a potential to differentiate into the mesenchymal cell lineage.</a> Am J Pathol. 176 (1), 381-392 (2010).</li>
<li>Bleau, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PTEN%2FPI3K%2FAkt+pathway+regulates+the+side+population+phenotype+and+ABCG2+activity+in+glioma+tumor+stem-like+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">PTEN/PI3K/Akt pathway regulates the side population phenotype and ABCG2 activity in glioma tumor stem-like cells.</a> Cell Stem Cell. 4 (3), 226-235 (2009).</li>
<li>Harris, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+stem+cells+are+enriched+in+the+side+population+cells+in+a+mouse+model+of+glioma%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Cancer stem cells are enriched in the side population cells in a mouse model of glioma.</a> Cancer Res. 68 (24), 10051-10059 (2008).</li>
<li>Murase, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Side+population+cells+have+the+characteristics+of+cancer+stem-like+cells%2Fcancer-initiating+cells+in+bone+sarcomas%5BTitle%5D)+AND+%22Br+J+Cancer%22%5BJournal%5D)">Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas.</a> Br J Cancer. 101 (8), 1425-1432 (2009).</li>
<li>Boesch, M., Wolf, D., Sopper, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+Stem+Cell+Detection+Using+the+DyeCycle-Triggered+Side+Population+Phenotype%5BTitle%5D)+AND+%22Stem+Cells+Int%22%5BJournal%5D)">Optimized Stem Cell Detection Using the DyeCycle-Triggered Side Population Phenotype.</a> Stem Cells Int. 2016, 1652389(2016).</li>
<li>Golebiewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Side+population+in+human+glioblastoma+is+non-tumorigenic+and+characterizes+brain+endothelial+cells%5BTitle%5D)+AND+%22Brain%22%5BJournal%5D)">Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells.</a> Brain. 136 (PT 5), 1462-1475 (2013).</li>
<li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+transporter-mediated+protection+of+cancer+stem+cells+from+ionophore+antibiotics%5BTitle%5D)+AND+%22Stem+Cells+Transl+Med%22%5BJournal%5D)">Drug transporter-mediated protection of cancer stem cells from ionophore antibiotics.</a> Stem Cells Transl Med. 4 (9), 1028-1032 (2015).</li>
<li>Fukunaga-Kalabis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tenascin-C+promotes+melanoma+progression+by+maintaining+the+ABCB5-positive+side+population%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">Tenascin-C promotes melanoma progression by maintaining the ABCB5-positive side population.</a> Oncogene. 29 (46), 6115-6124 (2010).</li>
<li>Dean, M., Fojo, T., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumour+stem+cells+and+drug+resistance%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Tumour stem cells and drug resistance.</a> Nat Rev Cancer. 5 (4), 275-284 (2005).</li>
<li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183 (4), 1797-1806 (1996).</li>
<li>Telford, W. G., Bradford, J., Godfrey, W., Robey, R. W., Bates, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Side+population+analysis+using+a+violet-excited+cell-permeable+DNA+binding+dye%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Side population analysis using a violet-excited cell-permeable DNA binding dye.</a> Stem Cells. 25 (4), 1029-1036 (2007).</li>
<li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+prevalence+of+side+population+in+human+cancer+cell+lines%5BTitle%5D)+AND+%22Oncoscience%22%5BJournal%5D)">High prevalence of side population in human cancer cell lines.</a> Oncoscience. 3 (3-4), 85-87 (2016).</li>
<li>Ali, M. Y., Anand, S. V., Tangella, K., Ramkumar, D., Saif, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+Primary+Human+Colon+Tumor+Cells+from+Surgical+Tissues+and+Culturing+Them+Directly+on+Soft+Elastic+Substrates+for+Traction+Cytometry%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.</a> J Vis Exp. (100), e52532(2015).</li>
<li>Azari, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+expansion+of+human+glioblastoma+multiforme+tumor+cells+using+the+neurosphere+assay%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and expansion of human glioblastoma multiforme tumor cells using the neurosphere assay.</a> J Vis Exp. (56), e3633(2011).</li>
<li>Hasselbach, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+high+grade+glioma+cell+culture+from+surgical+specimens+for+use+in+clinically+relevant+animal+models+and+3D+immunochemistry%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Optimization of high grade glioma cell culture from surgical specimens for use in clinically relevant animal models and 3D immunochemistry.</a> J Vis Exp. (83), e51088(2014).</li>
<li>Boesch, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DyeCycle+Violet+used+for+side+population+detection+is+a+substrate+of+P-glycoprotein%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">DyeCycle Violet used for side population detection is a substrate of P-glycoprotein.</a> Cytometry A. 81 (6), 517-522 (2012).</li>
<li>Patrawala, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Side+population+is+enriched+in+tumorigenic%2C+stem-like+cancer+cells%2C+whereas+ABCG2%2B+and+ABCG2-+cancer+cells+are+similarly+tumorigenic%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2- cancer cells are similarly tumorigenic.</a> Cancer Res. 65 (14), 6207-6219 (2005).</li>
<li>Guha, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nicotine+promotes+apoptosis+resistance+of+breast+cancer+cells+and+enrichment+of+side+population+cells+with+cancer+stem+cell-like+properties+via+a+signaling+cascade+involving+galectin-3%2C+alpha9+nicotinic+acetylcholine+receptor+and+STAT3%5BTitle%5D)+AND+%22Breast+Cancer+Res+Treat%22%5BJournal%5D)">Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, alpha9 nicotinic acetylcholine receptor and STAT3.</a> Breast Cancer Res Treat. 145 (1), 5-22 (2014).</li>
</ol>

The authors have no conflicts of interest to declare. There is also no non-author involvement in the preparation of the manuscript.

Progress in the clinical management of cancer also depends on the development of treatment modalities that target CSCs. Methods allowing the reliable isolation of CSCs from biological samples will accelerate the identification of suitable targets and are therefore of critical importance in this endeavor. SP analysis is an established and valuable technique to identify CSC populations that express ABC drug transporters and the implementation of DCV has extended its applicability to standard flow cytometric instruments. Me...

The efficacy of primary cancer therapies has improved substantially over the last decade due to compelling advances in the genetic and molecular understanding of the disease and the advent of targeted drugs such as therapeutic antibodies and small molecule inhibitors. In contrast, metastatic and recurrent disease is still typically incurable, and morbidity and mortality remain high in these clinical settings. CSCs&#160;represent a distinct subpopulation within tumors and are endowed with canonical stem cell properties such as clonogenicity/tumorigenicity, multi-drug resistance and asymmetric cell division1,2. Thus, CSCs not only drive metastatic progression and tumor heterogeneity, but also persist during treatment to predispose the patient to relapse. Therapeutic CSC elimination is therefore an important medical need to prevent disease recurrence and allow long-term cure of cancer3.
Identification of vulnerabilities and elucidation of strategies to eradicate CSCs heavily depends on methods that allow their purification from biological samples for subsequent expression profiling and/or functional investigation. In turn, such methods rely on surface, intracellular or functional markers that are specific for these cells. CSC-specific surface markers include, but are not limited to, CD44, CD133, CD24 and CD90, and have been used to identify CSC populations in a variety of tumor entities including breast cancer and colon cancer4. Another marker, aldehyde dehydrogenase (ALDH), shows intracellular localization and can be functionally detected providing a respective substrate whose enzymatic conversion produces light. Using this test, CSC populations have been identified in diverse tumor entities as well5. A complementary method, commonly referred to as SP&#160;analysis and portrayed here in methodological detail, harnesses active dye efflux by ABC drug transporters to identify small populations of fluorescence-dim stem-like cells6,7,8. To achieve this, a given sample is incubated in the presence of a lipophilic DNA-binding fluorophore which enters all cells through passive diffusion and targets nuclear and mitochondrial DNA for binding. Non-CSCs devoid of ABC drug transporter expression accumulate the dye resulting in bright fluorescence, whereas CSCs actively extrude the dye which reduces fluorescence. Pharmacological inhibition of drug transporter activity abrogates and functionally confirms the SP phenotype and should be used for control purpose. CSC populations exhibiting SP characteristics have been disclosed, amongst others, in ovarian cancer9,10, prostate cancer11,12, breast cancer13, lung cancer14, endometrial cancer15, glioma16,17 and bone sarcoma18. Importantly, the SP assay is compatible with both cancer cell lines and primary tumor tissue, even though primary material poses additional challenges such as the requirement for a specific tumor cell discrimination strategy (certain host cell populations can exhibit SP characteristics as well)19,20.
The two most established SP-conferring drug transporters are ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD3388,9,21; however, other drug transporters can be a molecular determinant of the SP phenotype too (e.g., ABCB5)22. ABCB1 can be efficiently blocked with verapamil whereas the activity of ABCG2 can be specifically abrogated with fumitremorgin C (FTC)6,19. A particular strength of SP analysis is that it can be combined with other stainings (e.g., surface markers and ALDH) and that it allows live cell recovery thus being compatible with downstream functional investigations. Moreover, SP detection is broadly applicable owing to the high conservation of ABC drug transporters among CSC populations9,23.
Originally, SP detection has been performed using Hoechst 33342 as a triggering dye24. This dye achieves excellent resolution but requires ultraviolet laser excitation; hence its applicability is naturally limited to high-end flow cytometric instruments. The advent of DyeCycle Violet (DCV)25 has opened new avenues for SP analysis and extended the applicability of this method to standard flow cytometric instruments lacking an ultraviolet laser source (a violet laser source suffices to resolve DCV-SP cells). Importantly, Hoechst 33342 and DCV share common pump specificities, indicating that either dye should identify the same cell populations.
Here, we provide a detailed experimental protocol of DCV-based SP analysis for quick and easy reproduction in independent labs. We thus perceive our article as a resource for CSC researchers that should contribute to the optimization and standardization of this useful cell-biological method.

<table><tbody><tr><td>Vybrant DyeCycle Violet</td><td>Thermo Fisher Scientific</td><td>V35003</td><td>Ready to use</td></tr><tr><td>Verapamil hydrochloride</td><td>Sigma-Aldrich</td><td>V4629</td><td>Dissolve in ethanol</td></tr><tr><td>Fumitremorgin C</td><td>Sigma-Aldrich</td><td>F9054</td><td>Dissolve in DMSO</td></tr><tr><td>7-AAD (or propidium iodide - PI)</td><td>BioLegend</td><td>420403</td><td>Ready to use</td></tr><tr><td>Standard cell culture reagents (&lt;em&gt;e.g.&lt;/em&gt;, medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, &lt;em&gt;etc.&lt;/em&gt;)</td><td>Different suppliers</td><td></td><td></td></tr><tr><td>Sample/cells of interest (&lt;em&gt;e.g.&lt;/em&gt;, human or murine cancer cell line, human or murine tumor tissue)</td><td>Different suppliers</td><td></td><td></td></tr><tr><td>Flow cytometric instrument (FACS; analyzer or cell sorter)</td><td>Different suppliers</td><td></td><td>Violet laser source required</td></tr><tr><td>FACS tubes (round-bottom)</td><td>Different suppliers</td><td></td><td>Tubes with cap recommended</td></tr><tr><td>70 &amp;micro;m cut-off strainers</td><td>Different suppliers</td><td></td><td>Optional but recommended</td></tr><tr><td>Digestion enzyme mix (&lt;em&gt;e.g.&lt;/em&gt;, collagenase/dispase/Dnase)</td><td>Different suppliers</td><td></td><td>Only relevant for tissue dissociation</td></tr><tr><td>Flurochrome-conjugated antibodies against surface markers of interest</td><td>Different suppliers</td><td></td><td>Optional</td></tr><tr><td>ALDEFLUOR Kit</td><td>STEMCELL Technologies</td><td>01700</td><td>Optional</td></tr></tbody></table>

harnessing the dna dye-triggered side population phenotype to detect and purify cancer stem cells from biological samples

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP&#160;cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP&#160;assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.

Provided is a representative SP analysis of A2780 cells, a human ovarian cancer cell line previously shown to harbor a SP accounting for less than 1% of cells9. Cells were cultured at 37 °C in RPMI 1640 supplemented with 10% (v/v) FBS, 2 mM L-glutamine and 1x penicillin/streptomycin and harvested at 85% confluency using treatment with 0.05% trypsin-EDTA. Cells were washed in PBS and filtered through 70 µm cut-off strainers. The cell count was determined us...

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

The overall goal of this procedure is to detect putative cancer stem cell populations in biological samples based on dye extrusion capacity of the side population cells. This method addresses a key question in cancer stem cell research regarding reliable means for detection and purification of cancer stem cells from biological samples. The main advantage of this technique is that it deploys functional markers and therefore providing better resolution.In addition, expression of these ABC drug transporters is highly conserved among cancer stem cells and therefore we can use this technique for many different cancer entities. The implications of this technique extends toward the discovery of anti-cancer stem cell therapeutics because it facilitates downstream analysis such as gene expression profiling and target validation in highly purified cancer cells. So this method can provide insight into the characteristics of cancer stem cells.We can also use this method to characterize as a systems comprising ABC drug transporter expressing populations such as the bone marrow. Demonstrating the procedure will be Elisabeth Hoflehner, a technician from my laboratory. To begin, culture the desire

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Research

JoVE Journal

Cancer Research

Cancer stem cells (CSCs) have been identified in a number of malignancies.  In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts.  These viable cells can then be used in functional and analytical studies.

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PDAC Stem Cell Isolation: A Method to Isolate Cancer Stem Cells from Pancreatic Tumors

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Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

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Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

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Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Understanding the role of cancer stem-like cells in tumor recurrence and resistance to therapies has become a topic of great interest in the last decade. This article describes the isolation and characterization of the sub-population of cancer stem-like cells from head and neck squamous carcinoma cell lines (HNSCC).

Isolation and Characterization of a Head and Neck Squamous Cell Carcinoma Subpopulation Having Stem Cell Characteristics

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

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Analysis of Side Population in Solid Tumor Cell Lines

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Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Here, we show that high-power 375 nm and 405 nm lasers can effectively excite Hoechst 33342 and serve as a viable alternative to the 355 nm laser for side population (SP) cell detection, thereby expanding the range of available lasers in flow cytometry applications.

Effective Detection of Hoechst Side Population Cells by Flow Cytometry

Learn to detect and isolate cancer stem cells using a violet dye-based side population assay with flow cytometry. This functional method enables downstream analyses and therapeutic target discovery in cancer research.

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

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