Name: a 3d spheroid model for glioblastoma
Uploaded: 2020-04-09T13:00:00
Description: Watch this Scientific Journal Video about A 3D Spheroid Model for Glioblastoma at JoVE.com

This work was supported by Transcan 2017, ARC 2017, Ligue Contre le Cancer (Comit&#233; de la Gironde et de la Charente-Maritime). Joris Guyon is a recipient of fellowship from the Toulouse University Hospital (CHU Toulouse).

Informed written consent was obtained from all patients (from the Haukeland Hospital, Bergen, Norway according to local ethics committee regulations). Our protocol follows the guidelines of our institution&#8217;s human research ethics committee.&#160;
1. Generation of uniform size tumor spheroids
NOTE: Stem-like cells are cultured in neurobasal medium complemented with B27 supplement, heparin, FGF-2, penicillin, and streptomycin, as described in previous articles<sup...

<ol>
	<li>Pelissier, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+dysfunction+in+mechanotransduction+impairs+differentiation+of+human+mammary+epithelial+progenitors.">Age-related dysfunction in mechanotransduction impairs differentiation of human mammary epithelial progenitors.</a> Cell Reports. 7, 1926-1939 (2014).</li><li>Ishiguro, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+spheroids:+Relevance+to+cancer+stem+cells+and+clinical+applications.">Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications.</a> Cancer Science. 108, 283-289 (2017).</li><li>Sutherland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+environment+interactions+in+tumor+microregions:+the+multicell+spheroid+model.">Cell and environment interactions in tumor microregions: the multicell spheroid model.</a> Science. 240, 177-184 (1988).</li><li>Desoize, B., Jardillier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+resistance:+a+paradigm+for+clinical+resistance?.">Multicellular resistance: a paradigm for clinical resistance?.</a> Critical Reviews in Oncology/Hematology. 36, 193-207 (2000).</li><li>Corbet, C., Feron, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+acidosis:+from+the+passenger+to+the+driver's+seat.">Tumour acidosis: from the passenger to the driver's seat.</a> Nature Reviews Cancer. 17, 577-593 (2017).</li><li>Hirschhaeuser, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> Journal of Biotechnology. 148, 3-15 (2010).</li><li>Berens, E. B., Holy, J. M., Riegel, A. T., Wellstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cancer+Cell+Spheroid+Assay+to+Assess+Invasion+in+a+3D+Setting.">A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting.</a> Journal of Visualized Experiments. (105), e53409 (2015).</li><li>Cavaco, A. C. M., Eble, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Spheroid+Model+as+a+More+Physiological+System+for+Cancer-Associated+Fibroblasts+Differentiation+and+Invasion+In+Vitro+Studies.">A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies.</a> Journal of Visualized Experiments. (150), e60122 (2019).</li><li>Boye, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+CXCR3/LRP1+cross-talk+in+the+invasion+of+primary+brain+tumors.">The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors.</a> Nature Communications. 8, 1571 (2017).</li><li>Dejeans, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+control+of+glioma+cells+adhesion+and+migration+through+IRE1alpha-mediated+cleavage+of+SPARC+mRNA.">Autocrine control of glioma cells adhesion and migration through IRE1alpha-mediated cleavage of SPARC mRNA.</a> Journal of Cell Science. 125, 4278-4287 (2012).</li><li>Golembieski, W. A., Ge, S., Nelson, K., Mikkelsen, T., Rempel, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+SPARC+expression+promotes+U87+glioblastoma+invasion+in+vitro.">Increased SPARC expression promotes U87 glioblastoma invasion in vitro.</a> International Journal of Developmental Neuroscience. 17, 463-472 (1999).</li><li>Daubon, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+complex+role+of+thrombospondin-1+in+glioblastoma+development.">Deciphering the complex role of thrombospondin-1 in glioblastoma development.</a> Nature Communications. 10, 1146 (2019).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tube+travel:+the+role+of+proteases+in+individual+and+collective+cancer+cell+invasion.">Tube travel: the role of proteases in individual and collective cancer cell invasion.</a> Cancer Research. 68, 7247-7249 (2008).</li><li>Das, A., Monteiro, M., Barai, A., Kumar, S., Sen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP+proteolytic+activity+regulates+cancer+invasiveness+by+modulating+integrins.">MMP proteolytic activity regulates cancer invasiveness by modulating integrins.</a> Scientific Reports. 7, 14219 (2017).</li><li>Schaeffer, D., Somarelli, J. A., Hanna, G., Palmer, G. M., Garcia-Blanco, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+invasion+uncoupled:+increased+migration+is+not+an+inexorable+consequence+of+epithelial-to-mesenchymal+transition.">Cellular migration and invasion uncoupled: increased migration is not an inexorable consequence of epithelial-to-mesenchymal transition.</a> Molecular and Cellular Biology. 34, 3486-3499 (2014).</li><li>de Gooijer, M. C., Guillen Navarro, M., Bernards, R., Wurdinger, T., van Tellingen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Experimenter's+Guide+to+Glioblastoma+Invasion+Pathways.">An Experimenter's Guide to Glioblastoma Invasion Pathways.</a> Trends in Molecular Medicine. 24, 763-780 (2018).</li><li>Daubon, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+invasive+proteome+of+glioblastoma+revealed+by+laser-capture+microdissection.">The invasive proteome of glioblastoma revealed by laser-capture microdissection.</a> Neuro-Oncology Advances. 1 (1), vdz029 (2019).</li></ol>

Tumor spheroid assays are well adapted to study tumor characteristics including proliferation, invasion, and migration, as well as cell death and drug response. Cancer cells invade the 3D matrix forming an invasive microtumor, as seen in Figure 4B,C. During the invasive process, matrix metalloproteinases (MMP) digest matrices surrounding tumor cells13, and MMP inhibitors (e.g., GM6001 or Rebimastat) may impair cell invasion but not migration<sup class...

In most studies using primary or commercially available cell lines, assays are performed on cells grown on plastic surfaces as monolayer cultures. Managing cell culture in 2D represents disadvantages, as it does not mimic an in vivo 3D cell environment. In 2D cultures, the entire cell surface is directly in contact with the medium, altering cell growth and modifying drug availability. Furthermore, the nonphysiological plastic surface triggers cell differentiation1. Three-dimensional culture models have been developed to overcome these difficulties. They have the advantage of mimicking the multicellular architecture and heterogeneity of tumors2, and thus could be considered to be a more relevant model for solid tumors3. The complex morphology of spheroids contributes to better evaluate drug penetrance and resistance4. The tumor heterogeneity in the spheroid impacts the diffusion of oxygen and nutrients, and the response to pharmacological agents (Figure 1A). Diffusion of oxygen is altered when the spheroid size reaches 300 &#181;m, inducing a hypoxic environment in the center of the spheroid (Figure 1A,C). Metabolites are also less penetrating through the cell layers and compensating metabolic reactions take place5. When the diameter of the spheroid increases, necrotic cores can be observed, further mimicking characteristics found in many solid cancers, including the aggressive brain cancer glioblastoma (GBM)6.
Several 2D or 3D invasion assays for glioblastoma have been reported in the literature7,8. Two-dimensional assays are mainly for studying invasion in a horizontal plane on a thin matrix layer or in a Boyden chamber assay9. Three-dimensional assays have been described with 3D spheroid cultures using classical glioblastoma cell lines10. More complex variants are represented by invasion of brain organoids by tumor spheroids in confrontation cultures11. However, it is still important to develop an easy-to-use and reproducible assay available to any laboratory. We have developed a protocol to generate glioblastoma stem-like cells from patient samples. The quantification of these assays is easily manageable and only requires open-access online software. Briefly, tumor pieces are cut into small pieces and enzymatically digested. Single cells derived from the digestion are cultivated in neurobasal medium. After 4-7 days, spheroid structures form spontaneously. Upon intracranial implantation in mice models, they form tumors exhibiting a necrotic core surrounded by pseudo-palisading cells12. This closely resembles the characteristics found in GBM patients.
In this article, we describe our protocol to produce spheroids from a determined number of cells to ensure reproducibility. Two complementary matrices can be used for this purpose: Matrigel and collagen type I. Matrigel is enriched in growth factors and mimics the mammalian basal membrane required for cell attachment and migration. On the other hand, collagen type I, a structural element of stroma, is the most common fibrillary extracellular matrix and is used in cell invasion assays. Herein, we illustrate our GBM spheroid model by performing migration and proliferation assays. Analysis was done not only at fixed time points but also by monitoring spheroid expansion and cell movement by live imaging. Furthermore, electron microscopy was done to visualize morphological details.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 well round-bottom plate</td>
			<td>Falcon</td>
			<td>08-772-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Gibco</td>
			<td>A11105-01</td>
			<td>Stored at 4 &#176;C, sphere dissociation enzyme</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>12587</td>
			<td>Stored at -20 &#176;C, defrost before use</td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Stored at -20 &#176;C, defrost before use</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 10X</td>
			<td>Pan Biotech</td>
			<td>P04-53-500</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>ImageJ</td>
			<td></td>
			<td>Used to analyze pictures</td>
		</tr>
		<tr>
			<td>Flask 75 cm2</td>
			<td>Falcon</td>
			<td>10497302</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Stored at -20 &#176;C, diluted to a final concentration of 0.2 mg/mL in cold NBM</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma</td>
			<td>M0512</td>
			<td>Diluted in NBM for a 2% final concentration</td>
		</tr>
		<tr>
			<td>NBM</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>ThermoFisher</td>
			<td>T10282</td>
			<td>Used to cell counting</td>
		</tr>
		<tr>
			<td>Type I Collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

a 3d spheroid model for glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Spheroids were prepared as described in the protocols section and observations were made regarding migration, invasion, proliferation, and microscopy. To measure hypoxia in distinct areas of the spherical structure, carboxic anhydrase IX staining was used for determining hypoxic activity (Figure 1A-C). More CAIX-positive cells were observed in the spheroid center (Figure 1A-C). Hypoxic cells loca...

Watch this Scientific Journal Video about A 3D Spheroid Model for Glioblastoma at JoVE.com

A 3D Spheroid Model for Glioblastoma

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

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