Name: Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors (Video) | JoVE Journal
Uploaded: 2020-03-01T12:00:00.000+00:00
Duration: 7 min 1 s
Description: Learn to isolate cancer stem cells from gynecological and breast tumors using polyHEMA-coated suspension cultures and sphere-forming assays. This method enables phenotypic characterization to guide personalized cancer therapy strategies.

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5 minutes

This study was funded by the Portuguese Society of Gynecology through the 2016 Research Prize and by CIMAGO. CNC.IBILI is supported through the Foundation for Science and Technology, Portugal (UID/NEU/04539/2013), and co-funded by FEDER-COMPETE (POCI-01-0145-FEDER-007440). The Coimbra Hospital and Universitary Center (CHUC) Tumor Bank, approved by CHUC's Ethics Committee for Health and by the Portuguese National Data Protection Commission, was the source of endometrial samples of patients followed at the institution's Gynecology Service. Figure 1 was produced using Servier Medical Art, available from <a href="http://www.servier.com/" target="_blank">www.servier.com</a>.

This protocol was performed complying with the ethical guidelines of the Coimbra Hospital and Universitary Center (CHUC) Tumor Bank, and was approved by CHUC's Ethics Committee for Health and by the Portuguese National Data Protection Commission.
1. Sphere-forming Protocol and Derived Adherent Populations from Continuous Cell Cultures
NOTE: Perform all procedures under strict sterile conditions.
<ol>
	<li>Preparation of non-adherent suspension culture flasks or plates by coating the growth surface with poly(2-hydroxyethyl-methacrylate (poly-HEMA)
	<ol>
		<li>Prepare a 15 mg/mL solution by stirring poly-HEMA in absolute ethanol at 65 °C. Coat cell culture flasks or plates with 50 μL/cm2.</li>
		<li>Leave to dry at 37 °C in a drying oven. If necessary, wrap the plates and store at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of the sphere culturing media (SCM)
	<ol>
		<li>Prepare a 2% solution of methylcellulose in ultrapure water and sterilize in the autoclave. Methylcellulose tends to be easier to solubilize by cooling; therefore disperse the powder in water at 80 °C and stir until cooled23.</li>
		<li>Prepare a two-times concentrated solution of SCM (stock solution). SCM working solution contains DMEM-F12, supplemented with 100 mM putrescine, 1% insulin, transferrin, selenium and 1% antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin B).</li>
		<li>To prepare the SCM, mix equal volumes of the SCM stock solution with the 2% solution of methylcellulose.</li>
		<li>Complete the medium immediately prior to use by adding 10 ng/mL epidermal growth factor (EFG) and 10 ng/mL basic fibroblast growth factor (bFGF).</li>
		<li>If more fastidious cell lines are in use, supplement the medium with 0.4% bovine serum albumin, which might be an advantage.</li>
	</ol>
	</li>
	<li>Start with a flask of MCF7 or HCC1806 breast cancer or ECC-1 or RL95-2 endometrial cancer cells (or other cancer cell line of choice) with 80% to 90% confluence.</li>
	<li>Discard the cell culture media, wash with phosphate buffered saline solution (PBS) and detach the cells with trypsin-EDTA (1 to 2 mL for a 75 cm2 cell culture flask).</li>
	<li>Add cell culture media (2 to 4 mL for a 75 cm2 cell culture flask) and centrifuge at 200 x g for 5 min to discard enzymes.</li>
	<li>Suspend the pellet in a known volume of cell culture media and pipette up and down to ensure a single cell suspension. For this purpose, a 40 μm cell strainer can be used.</li>
	<li>Count the cells in the hemocytometer and calculate the cell concentration of the cell suspension. Take advantage of this step to ensure observation of a single cell suspension. Careful cell counting is essential to accurately quantify the effects of treatments.</li>
	<li>Suspend the determined amount of cell suspension in SCM complete medium and transfer to poly-HEMA coated dishes. As a reference value for seeding density, consider 500 to 2000 cells/cm2. 
	NOTE: Optimization of seeding density and time of culture for each cell line is highly recommended24.</li>
	<li>Incubate at 37 °C and 5% CO2 for 2 days without disturbing the plates.</li>
	<li>Re-establish the concentration of growth factors by adding 10 ng/mL EFG and 10 ng/mL bFGF to the cell culture media. Repeat this step every two days.</li>
	<li>Incubate at 37 °C and 5% CO2 until 5 days after plating (this can vary from 3 to 12 days according to the cell line) to obtain spheres, which present the morphology of suspension ball-shaped cell colonies.</li>
	<li>Use or collect the spheres, by pipetting, for the experiments.</li>
	<li>To obtain derived adherent populations, place the spheres into standard culture conditions, respective of the cell line used. 1 to 2 days later, it is possible to observe a monolayer of cells growing around adherent spheres, which presents a morphology similar to the cell line of origin.</li>
</ol>
2. Sphere-forming Protocol from Human Tumor Samples
NOTE: The use of human samples for research purposes must comply with each country's legislation, and to be approved by the Ethics Committee of the Institutions involved.
<ol>
	<li>Prepare the transport media containing DMEM/F12, supplemented with 10% fetal bovine serum (FBS) and 2% antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin B).</li>
	<li>Prepare the digestion media containing DMEM/F12, supplemented with 10% FBS, 1% antibiotic antimycotic solution, 1 mg/mL type IV collagenase and 100 μg/mL DNAse I.</li>
	<li>Prepare the enzyme inactivation media containing DMEM/F12, supplemented with 10% FBS and 1% antibiotic-antimycotic solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin B).</li>
	<li>Prepare the SCM as described in section 1.2.</li>
	<li>Obtain the sample during the macroscopic study of the operative piece as soon as possible after surgical removal.</li>
	<li>Place the samples in transport media and transfer them to the laboratory for where processing. Sample processing should begin within 1 h following collection to improve the success rate of the procedure. Apply caution in sample collection. Handle the samples carefully. Avoid the use of necrotic or cauterized zones.</li>
	<li>Under the sterile flow chamber, transfer the sample to a dish and cut into smaller pieces (around 1 mm3) with a scalpel.</li>
	<li>Incubate the human tissue in a tube with digestion media in a rotating shaker up to 180 min, at 37 °C. Identify this tube as Tube A.</li>
	<li>Replace the enzyme solution every 15 min.
	<ol>
		<li>Collect the digestion media (without removing any tissue fragments) and transfer it through a 40 μm cell strainer to a new tube half-filled with enzyme inactivation media. Maintain this tube at room temperature and identify it as Tube B.</li>
		<li>Add new digestion media to Tube A and return it to the rotating shaker at 37 °C.</li>
		<li>At each collection, check cell viability using the trypan blue exclusion method.</li>
		<li>Repeat this procedure for 180 min or until cell count is significantly lower.</li>
	</ol>
	</li>
	<li>Incubate the tissue fragments in Tube A in a second digestion solution containing equal parts of accutase and trypsin-EDTA, stirring for 10 min at 37 °C.</li>
	<li>Add the enzyme inactivation media to Tube A and filter the contents through a 40 μm cell strainer into Tube B.</li>
	<li>Centrifuge the cell suspension in Tube B at 200 x g for 10 min.</li>
	<li>Suspend the pellet in SCM and check cell concentration using a hemocytometer.</li>
	<li>Suspend the determined amount of cell suspension in SCM and transfer to poly-HEMA coated dishes (see step 1.1) with a seeding density of 4000 cells/cm2.</li>
	<li>Incubate at 37 °C and 5% CO2 for 2 days without disturbing the plates.</li>
	<li>Re-establish the concentration of growth factors by adding 10 ng/mL EFG and 10 ng/mL bFGF to the cell culture media. 
	NOTE: You must do this every two days.</li>
	<li>Incubate at 37 °C and 5% CO2 until 5 days after plating (this can vary up to 12 days) to obtain spheres, which present the morphology of suspension ball-shaped cell colonies.</li>
</ol>
<img alt="Cell preparation workflow diagram; enzymatic digestion, centrifugation, counting, plating, sphere formation." class="xfigimg" src="/files/ftp_upload/60022/60022fig01.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Obtaining cancer stem cells from human endometrial tumor samples (A) and breast and gynecological cancer cell lines (B). Human tumor samples are fragmented, enzymatically digested and plated in sphere culturing medium into poly-HEMA coated dishes. Cancer cell lines are detached, cell suspensions are counted, and single cells are distributed at low density into poly-HEMA coated plates under appropriate conditions. The spheres obtained, when placed under adherent culture conditions, produce derived adherent populations. <a href="https://www.jove.com/files/ftp_upload/60022/60022fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Sphere-forming Capacity, Self-renewal, and Sphere Projection Area
NOTE: Sphere-forming capacity is the ability of a tumor cell population to produce spheres. Self-renewal is the ability of sphere cells to produce new colonies of spherical cells in suspension. The sphere projection area is representative of the area occupied by the sphere and therefore expressive of their size and the number of cell divisions undergone in a certain time period.
<ol>
	<li>Determining the sphere-forming capacity
	<ol>
		<li>After completion of the sphere-forming protocol, collect the spheres in a centrifuge tube and centrifuge at 125 x g for 5 min.</li>
		<li>Discard the SCM and gently suspend the pellet in a known volume of fresh media. With the aim of concentrating the spheres to facilitate counting, suspend the spheres in a small media volume. Be careful not to disturb the spheres.</li>
		<li>Use a hemocytometer to count the spheres with more than 40 μm in diameter. Alternatively, spheres can be counted directly on the plate by using a microscope equipped with a graticule25 or using an automated system26,27.</li>
		<li>Calculate the percentage ratio of spheres obtained vs. the number of cells initially plated.</li>
	</ol>
	</li>
	<li>Determining self-renewal
	<ol>
		<li>After completion of the sphere-forming protocol, collect the spheres in a centrifuge tube and centrifuge at 125 x g for 5 min.</li>
		<li>Discard the sphere culturing media and gently suspend the pellet in trypsin-EDTA.</li>
		<li>Incubate up to 5 min at 37 °C.</li>
		<li>Add enzyme inactivation media and pipette up and down to ensure a single cell suspension.</li>
		<li>Using a hemocytometer and the trypan blue exclusion method, count the viable cells in the suspension.</li>
		<li>Initiate the sphere-forming protocol as described in section 1.</li>
		<li>After 8 days, use a hemocytometer to count the spheres with more than 40 μm in diameter.</li>
		<li>Calculate the percentage ratio of spheres obtained vs. the number of cells initially plated.</li>
	</ol>
	</li>
	<li>Determining the sphere projection area
	<ol>
		<li>To evaluate the area occupied by the spheres, obtain images of at least 10 random fields per condition, in an inverted microscope equipped with an image acquisition module. A magnification of 100X to 400X is recommended.</li>
		<li>Analyze images using imaging software, such as ImageJ software28, by drawing areas of interest corresponding to the spheres and measuring its area in pixels.</li>
		<li>Calculate sphere projection area as the mean area of pixels measured.</li>
	</ol>
	</li>
</ol>
4. Cancer Stem Cell Marker Assessment with Flow Cytometry
NOTE: CD44+/CD24-/low phenotype was consistently associated with breast and gynecological cancer stem cells. The procedure described may be used to evaluate this and other cell surface markers.
<ol>
	<li>After completion of the sphere-forming protocol, collect the spheres in a centrifuge tube and centrifuge at 125 x g for 5 min.</li>
	<li>Discard the SCM and gently suspend the pellet in trypsin-EDTA.</li>
	<li>Incubate up to 5 min at 37 °C.</li>
	<li>Add enzyme inactivation media and pipette up and down to ensure a single cell suspension.</li>
	<li>Centrifuge at 125 x g for 5 min, discard the supernatant and gently suspend the cells in PBS.</li>
	<li>Allow the cells to rest in suspension for 30 min to ensure recovery of the membrane conformation.</li>
	<li>Using a hemocytometer and the trypan blue exclusion method, count the cells in the suspension.</li>
	<li>Adjust the cell suspension volume to 106 cells/500 µL.</li>
	<li>Incubate with the monoclonal antibodies according to the instructions of the suppliers (concentration, time, temperature, and light/dark) and considering the experiment set represented in Table 2 or the markers given in Table 1.</li>
	<li>Immediately after staining, perform the flow cytometric analysis using a flow cytometer with appropriate detection modules.</li>
	<li>Standardize cytometer setup, following protocols established by the EuroFlow Consortium29.</li>
	<li>Set up primary gates based on the forward and side scatter excluding debris and dead cells. This can be improved by concomitant labelling with annexin V and gating negative cells.</li>
	<li>Set fluorescence gates based on the unstained samples and compensation for a spectral overlap using single stained controls.</li>
</ol>
5. Cancer Stem Cell Marker Assessment with Western Blot
NOTE: In addition to ALDH1 activity, high expression of this marker was consistently associated with breast and gynecological cancer stem cells13,14. The procedure described may be used to evaluate this and other cell markers.
<ol>
	<li>After completion of the sphere-forming protocol, collect the spheres in a centrifuge tube and centrifuge at 125 x g for 5 min.</li>
	<li>Preparation of the whole cell lysates
	<ol>
		<li>Place the centrifuge tubes on ice and discard the supernatant without disrupting the pellet.</li>
		<li>Wash the pellet with 1 mL of cold PBS and discard by centrifugation.</li>
		<li>Suspend the pellet in a small volume (200-500 µL) of RIPA lysis buffer30 (NaCl 150 mM, Tris-HCl 1.50 mM pH 7.4, Triton-X100 1% vol./vol., sodium deoxycholic acid 0.5% wt./vol., sodium dodecyl sulfate 0.5% wt./vol.) supplemented with cOmplete Mini and dithiothreitol 1 mM.</li>
		<li>Maintaining the samples cold (on ice), submit them to vortex and sonication with a 30% amplitude.</li>
		<li>Centrifuge the samples for 15 min at 14,000 x g in a refrigerated centrifuge set to 4 °C.</li>
		<li>Transfer the supernatants to new, properly identified microtubes.</li>
		<li>Determine the protein concentrations using the BCA or Bradford assays31.</li>
		<li>If necessary, store the samples at -80 °C until further western blot analysis.</li>
	</ol>
	</li>
	<li>Perform sample denaturation, electrophoresis, electron transfer and protein detection according to standard western blotting protocols, as described32,33,34.</li>
</ol>

The authors have nothing to disclose.

This protocol details an approach to obtain tumorspheres from cancer cell lines and primary human samples. Tumorspheres are enriched in a sub-population with stem cell-like properties36. This enrichment in CSC is dependent on viability in an anchorage-free environment while differentiated cells are reliant on adhesion to a substrate37. As primary plating of tumor cells in a low adherence environment that imposes suspension does not ensure enrichment in CSC per se, we provid...

Cancer populations are heterogeneous, with cells presenting different morphologies, proliferation and invasion capacity, due to differential gene expression. Among these cells, a minority population exists named cancer stem cells (CSC)1, which have the capacity for self-renewal, recapitulating the heterogeneity of the primary tumor niche and producing aberrantly differentiating progenitors that do not respond adequately to homeostatic controls2. CSC properties can be directly translated in clinical practice, given the association with events, such as tumorigenicity or resistance to chemotherapy3. The identification of CSC can lead to the development of targeted therapies that may include blockage of surface markers, promotion of CSC differentiation, blocking of CSC signaling pathway components, niche destruction, and epigenetic mechanisms4.
The isolation of CSC has been performed in cells lines and in samples of primary tumors5,6,7,8. The functional profile described for CSC includes clonogenic capacity, side population and tumorosphere formation9. The CD44high/CD24low phenotype has been consistently associated with breast CSC, which has proved to be tumorigenic in vivo and has been already associated with epithelial to mesenchymal transition5,10. High ALDH activity has also been associated with stemness and epithelial to mesenchymal transition (EMT) in several types of solid tumors11. ALDH expression has been associated with resistance to chemotherapy and to CSC phenotype in vitro12,13,14,15,16. Several other markers have been linked to CSC properties in different types of tumors, such as CD133, CD49f, ITGA6, CD1663,4 and others, as described in Table 1.
The tumorspheres consist of a three-dimensional model for the study and expansion of CSC. In this model, the cell suspensions from cell lines and from blood or tumor samples are cultivated in a medium supplemented with growth factors, namely epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), without fetal bovine serum and in non-adherent conditions17. Inhibition of cell adhesion results in death by anoikis of differentiated cells18. Spheres are derived from the clonal growth of an isolated cell. For this purpose, the cells are distributed at low density to avoid cell fusion and aggregation19. Another strategy includes the use of semisolid methylcellulose20.
The sphere-forming protocol gained popularity in CSC isolation and expansion, due to time and cost and technical, profitable, and reproducible reasons21,22. Despite some reserves on the extent of which sphere formation reflects CSC, there is a propensity of stem cells to grow in non-adherent conditions with the characteristic phenotype, which resembles the native microenvironment21. None of the methods available for isolation of CSC from solid tumors has complete efficiency, highlighting the importance of developing more specific markers or combinations of methodologies and markers.
In this protocol, we detail the isolation of CSC with the sphere-forming protocol, with the principle of single-cell growth in non-adherent conditions and the capacity to produce a differentiated phenotype. A schematic representation of this procedure is represented in Figure 1. We also describe the characterization with surface markers and ALDH expression for CSC, both for breast and gynecological tumor cells lines and samples of primary tumors.

<table><tbody><tr><td>Absolute ethanol</td><td>Merck Millipore</td><td>100983</td><td></td></tr><tr><td>Accutase</td><td>Gibco</td><td>A1110501</td><td>StemPro Accutas Cell Dissociation Reagent</td></tr><tr><td>ALDH antibody</td><td>Santa Cruz Biotechnology</td><td>SC166362</td><td></td></tr><tr><td>Annexin V FITC</td><td>BD Biosciences</td><td>556547</td><td></td></tr><tr><td>Antibiotic antimycotic solution</td><td>Sigma</td><td>A5955</td><td></td></tr><tr><td>BCA assay</td><td>Thermo Scientific</td><td>23225</td><td>Pierce BCA Protein Assay Kit</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A9418</td><td></td></tr><tr><td>CD133 antibody</td><td>Miteny Biotec</td><td>293C3-APC</td><td>Allophycocyanin (APC)</td></tr><tr><td>CD24 antibody</td><td>BD Biosciences</td><td>658331</td><td>Allophycocyanin-H7 (APC-H7)</td></tr><tr><td>CD44 antibody</td><td>Biolegend</td><td>103020</td><td>Pacific Blue (PB)</td></tr><tr><td>Cell strainer</td><td>BD Falcon</td><td>352340</td><td>40 &amp;micro;M</td></tr><tr><td>Collagenase, type IV</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>cOmplete Mini</td><td>Roche</td><td>118 361 700 0</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma</td><td>43815</td><td></td></tr><tr><td>DMEM-F12</td><td>Sigma</td><td>D8900</td><td></td></tr><tr><td>DNAse I</td><td>Roche</td><td>11284932001</td><td></td></tr><tr><td>ECC-1</td><td>ATCC</td><td>CRL-2923</td><td>Human endometrium adenocarcinoma cell line</td></tr><tr><td>Epidermal growth factor</td><td>Sigma</td><td>E9644</td><td></td></tr><tr><td>Fibroblast growth factor basic</td><td>Sigma</td><td>F0291</td><td></td></tr><tr><td>Haemocytometer</td><td>VWR</td><td>HERE1080339</td><td></td></tr><tr><td>HCC1806</td><td>ATCC</td><td>CRL-2335</td><td>Human mammary squamous cell carcinoma cell line</td></tr><tr><td>Insulin, transferrin, selenium Solution</td><td>Gibco</td><td>41400045</td><td></td></tr><tr><td>MCF7</td><td>ATCC</td><td>HTB-22</td><td>Human mammary adenocarcinoma cell line</td></tr><tr><td>Methylcellulose</td><td>AlfaAesar</td><td>45490</td><td></td></tr><tr><td>NaCl</td><td>JMGS</td><td>37040005002212</td><td></td></tr><tr><td>Poly(2-hydroxyethyl-methacrylate</td><td>Sigma</td><td>P3932</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P7505</td><td></td></tr><tr><td>RL95-2</td><td>ATCC</td><td>CRL-1671</td><td>Human endometrium carcinoma cell line</td></tr><tr><td>Sodium deoxycholic acid</td><td>JMS</td><td>EINECS 206-132-7</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma</td><td>436143</td><td></td></tr><tr><td>Tris</td><td>JMGS</td><td>20360000BP152112</td><td></td></tr><tr><td>Triton-X 100</td><td>Merck</td><td>108603</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>T4049</td><td></td></tr><tr><td>&amp;#223;-actin antibody</td><td>Sigma</td><td>A5316</td><td></td></tr></tbody></table>

obtaining cancer stem cell spheres from gynecological and breast cancer tumors

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 sphere-forming protocol allows spherical colonies to be obtained in suspension from several endometrial and breast cancer cell lines (Figure 2A) or after gentle enzymatic digestion of tissue from human tumor samples (Figure 2E). In both cases, a few days after plating, monoclonal spherical colonies in suspension are obtained. Both endometrial and breast cancer spheres give rise to a cell monolayer with similar morphology to ...

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 ...

obtaining-cancer-stem-cell-spheres-from-gynecological-breast-cancer

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