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&#39;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 &#176;C. Coat cell culture flasks or plates with 50 &#956;L/cm2.</li>
		<li>Leave to dry at 37 &#176;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 &#176;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 &#956;g/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 &#956;m 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 &#176;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 &#176;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&#39;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 &#956;g/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 &#956;g/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 &#956;g/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 &#176;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 &#956;m 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 &#176;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 &#176;C.</li>
	<li>Add the enzyme inactivation media to Tube A and filter the contents through a 40 &#956;m 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 &#176;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 &#176;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="Figure 1" class="xfigimg" src="/files/ftp_upload/60022/60022fig01.jpg" /> 
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 &#956;m 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 &#176;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 &#956;m 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 &#176;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 &#181;L.</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 &#181;L) 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 &#176;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 &#176;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

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 ...

Watch this Scientific Journal Video about Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors at JoVE.com

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

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

Research

JoVE Journal

Cancer Research

10.1K Views.  University of Coimbra. Dit protocol maakt de identificatie en isolatie van kankerstamcellen op een robuuste manier mogelijk met behulp van functionele testen en fenotypische karakterisering. Kanker stamcelisolatie van een tumor monster kan een platform voor het begeleiden van de klinische toepassing van specifieke therapieën op individuele tumoren, het voorspellen van resistentie en de daaruit voortvloeiende herhaling. Om niet-aanhangende suspensieculturen voor te bereiden, u eerst de celkweekcontainers met 50 mic...

Video: Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Genetics

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Neuroscience

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

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 ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Medicine

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Primary Mammospheres: A Technique to Determine Cell Potency

Source: Lombardo et. al., <a href="https://www.jove.com/v/52671/mammosphere-formation-assay-from-human-breast-cancer-tissues-cell">Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines.</a> J. Vis. Exp. (2015).
This video describes isolating cancer stem cells from breast cancer cell lines. We culture these cancer stem cells to generate primary mammospheres for assessing self-renewal and calculating sphere-forming efficiency, allowing comparison across different seeding densities.&#160;

Source: Lombardo et. al., Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines. J. Vis. Exp. (2015).
This video describes isolating ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Samenvatting

Meer video's verkennen