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>
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The authors declare that they have no competing financial interests.

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 ...

Two-dimensional cell cultures do not adequately mimic in vivo tumor growth. To improve, 3D procedures have been developed using spheroids. Our 3D glioblastoma spheroid-based assay is particularly well-suited to investigate brain tumor invasion into the healthy brain tumor environment.Three-dimensional culture models can be used to recapitulate a multicellular architecture, heterogeneity and metabolic state of tumors allowing more rigorous evaluation of drug effects. Be sure not to touch the bottom of the well or to completely remove the supernatant, to keep the gel on ice, and to add the cells to the gel quickly. To generate uniformly sized tumor spheroids, first wash the tumor cell culture of interest with five milliliters of PBS and treat the cells with 0.5 to one milliliter of dissociation enzyme.After five minutes at 37 degrees Celsius, stop the reaction with four to 4.5 milliliters of PBS and transfer the detached cells into a 15 milliliter conical tube containing 10 milliliters of complete growth medium. After counting, resuspend the cells at a one times 10 to the sixth cells per eight milliliters of complete growth medium supplemented with two milliliters of 2%methylcellulose concentration and transfer the suspension to a sterile system container. Then add 100 microliters of cells to each well of a 96-well round bottom plate and place the plate into a 37 degree Celsius 5%carbon dioxide incubator for three to four days.To perform an invasion assay, when the tumor cells have formed uniformly sized spheroids, transfer each cell structure into a 500 microliter tube and wash the spheroids once with 200 microliters of PBS. After the second wash, transfer the spheroids carefully into 100 microliters of freshly prepared collagen matrix and add each spheroid suspension into the center of each well of a flat bottom 96-well plate. After a 30-minute incubation at 37 degrees Celsius, add 100 microliters of complete growth medium on top of the gel in each well.Be sure to place all of the spheroids into the center of each well for the best imaging of the tumor cell invasion. To perform a migration assay, when the tumor cells have formed uniformly sized spheroids, coat a six-well plate with 0.2 milligrams per milliliter of Matrigel in growth medium for 30 minutes at 37 degrees Celsius. At the end of the incubation, remove the Matrigel and add two milliliters of complete growth medium to each well.Transfer the spheroids from the round bottom plate in 50 microliter volumes into individual wells of the six-well plate and place the plate into the cell culture incubator for 24 hours. The next day, stain the spheroids with 10 nanograms per milliliter of Hoechst for 30 minutes at 37 degrees Celsius. For image acquisition after an invasion or migration assay, place the plate onto the stage of a Brightfield confocal microscope equipped with a video camera and obtain images over the appropriate experimental period.To analyze the images in a semi-automated manner, import the images into Fiji and click Plugins, Macros, Interactive Interpreter. Copy and paste the adapted purple loop. Keep the purple sentences and add the green sentences of interest as necessary.Then to measure the area of each spheroid, click Analyze and Measure. To measure hypoxia in distinct areas of the spherical structure, carbonic anhydrase IX staining can be used to determine the hypoxic activity. In this representative analysis, more carbonic anhydrase IX positive cells were observed in the spheroid center.Hypoxic cells located in the spheroid core tend to be more glycolytic than the cells surrounding the core. Mitochondria can be imaged by electron microscopy for further analysis. The spheroid diameter is directly correlated to the density of the cells that make up the 3D cell structure and Fiji macros can be used to quantify the proliferation, invasion or migration of the cells within each spheroid or migration of the cells within each spheroid.Increases in the spheroid core reflect the stimulation of cell proliferation. Upon the inhibition of complex I of the mitochondrial respiratory chain, the vast majority of ATP production in the mitochondria is compromised reducing proliferation by 20%after 72 hours. Hydrogen chloride treatment enhances collagen type I invasion over a period of 24 hours but reduces the migratory area of the spheroids 1.5-fold compared to control untreated spheroids.As assessed by live imaging, the spheroids demonstrate high internal dynamics and move quickly. The size of the spheroids should be relatively uniform and care should be taken to manipulate the spheroids to the center of the wells for the best imaging results. This method can be also used to investigate tumor cell proliferation, migration and death as well as the expression of extracellular matrix, cell receptors or intracellular proteins of interest.The spheroids may be also encapsulated and the effect of increased cell surface tension can be investigated upon removal of the capsules.

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Cancer Research

14.2K vistas.  University Bordeaux, LAMC. Los cultivos celulares bidimensionales no imitan adecuadamente el crecimiento tumoral in vivo. Para mejorar, se han desarrollado procedimientos 3D utilizando esferoides. Nuestro ensayo basado en el esferoide de glioblastoma 3D es particularmente adecuado para investigar la invasión de tumores cerebrales en el entorno saludable del tumor cerebral.Los modelos de cultivo tridimensionales se pueden utilizar para recapitular una arquitectura multicelular, heterogeneidad y estado metabólic...