Name: obtaining cancer stem cell spheres from gynecological and breast cancer tumors
Uploaded: 2020-03-01T12:00:00
Description: Watch this Scientific Journal Video about Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors at JoVE.com

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 o...

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 &#181;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>&#65533;&#65533;-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 ...

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