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>

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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)</td>
			<td>BioLegend</td>
			<td>420403</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Standard cell culture reagents (e.g., medium, FBS, L-glutamine, antibiotics, PBS, trypsin-EDTA, etc.)</td>
			<td>Different suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sample/cells of interest (e.g., 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 (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 &#181;m cut-off strainers</td>
			<td>Different suppliers</td>
			<td></td>
			<td>Optional but recommended</td>
		</tr>
		<tr>
			<td>Digestion enzyme mix (e.g., 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

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 &#176;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 &#181;m cut-off strainers. The cell count was det...

Watch this Scientific Journal Video about Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples at JoVE.com

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

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 ...

harnessing-dna-dye-triggered-side-population-phenotype-to-detect

Research

JoVE Journal

Cancer Research

7.6K Views.  Kantonsspital St. Gallen. 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.

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

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Modeling Osteosarcoma Using Li-Fraumeni Syndrome Patient-derived Induced Pluripotent Stem Cells

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including osteosarcoma--one of the most frequent primary non-hematologic malignancies in the childhood and adolescence. Therefore, LFS provides an ideal model to study this malignancy. Taking advantage of iPSC methodologies, LFS-associated osteosarcoma can be successfully modeled by differentiating LFS patient iPSCs to mesenchymal stem cells (MSCs), and then to osteoblasts--the cells of origin for osteosarcomas. These LFS osteoblasts recapitulate oncogenic properties of osteosarcoma, providing an attractive model system for delineating the pathogenesis of osteosarcoma. This manuscript demonstrates a protocol for the generation of iPSCs from LFS patient fibroblasts, differentiation of iPSCs to MSCs, differentiation of MSCs to osteoblasts, and in vivo tumorigenesis using LFS osteoblasts. This iPSC disease model can be extended to identify potential biomarkers or therapeutic targets for LFS-associated osteosarcoma.

Here, we present a protocol for the generation of induced pluripotent Stem Cells (iPSCs) from Li-Fraumeni Syndrome (LFS) patient derived fibroblasts, differentiation of iPSCs via mesenchymal stem cells (MSCs) to osteoblasts, and modeling in vivo tumorigenesis using LFS patient-derived osteoblasts.

Li-Fraumeni syndrome (LFS) is an autosomal dominant hereditary cancer disorder. Patients with LFS are predisposed to a various type of tumors, including ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

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.

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Analysis of Side Population in Solid Tumor Cell Lines

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.

A convenient, fast, and cost-effective method to measure the proportion of side population cells in solid tumor cell lines is presented.

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

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

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, protein amounts, or other relevant molecules. These methods have shown to be substantial alternatives to PCR or ELISA for clinical and research applications. Moreover, the detection of protein amount (by Western blot or immunohistochemistry) is often insufficient to provide information for cancer diagnosis, whereas the measurement of enzyme activity represents a valuable biomarker. Measurement of enzyme activity also allows for the diagnosis and potential treatment of pathogen-borne diseases. In all eukaryotes, topoisomerases are the key DNA-binding enzymes involved in the control of the DNA topological state during important cellular processes and are among the important biomarkers for cancer prognosis and treatment.
Over the years, topoisomerases have been substantially investigated as a potential target of antiparasitic and anticancer drugs with libraries of natural and synthetic small-molecule compounds that are investigated every year. Here, the rolling circle amplification method, termed rolling circle enhanced enzyme activity detection (REEAD) assay that allows for the quantitative measurement of topoisomerase 1 (TOP1) activity in a simple, fast, and gel-free manner is presented.By cleaving and ligating a specially designed DNA substrate, TOP1 converts a DNA oligonucleotide into a closed circle, which becomes the template for rolling circle amplification, yielding ~103 tandem repeat rolling circle products. Depending on the nucleotide incorporation during the amplification, there is the possibility of different readout methods, from fluorescence to chemiluminescence to colorimetric. As each TOP1-mediated cleavage-ligation generates one closed DNA circle, the assay is highly sensitive and directly quantitative.

A protocol for the sensitive and quantitative detection of topoisomerase 1 activity using the rolling circle enhanced enzyme activity detection assay is described. The method allows detection of topoisomerase 1 activity from purified components or cell/tissue extracts. This protocol has wide-ranging applications in any field involving detection of enzymatic activity.

Isothermal amplification-based techniques such as the rolling circle amplification have been successfully employed for the detection of nucleic acids, ...