Name: A 3D Spheroid Model for Glioblastoma (Video) | JoVE Journal
Uploaded: 2020-04-09T13:00:00.000+00:00
Duration: 7 min 40 s
Description: Learn a 3D glioblastoma spheroid-based invasion assay for studying brain tumor invasion into healthy tissue. This method enables quantification of migration, proliferation, and drug response evaluation.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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 articles12. These cells spontaneously form spheroids in culture.
<ol>
	<li>Wash the tumor cells with 5 mL phosphate-buffered saline (PBS) and incubate the cells with 0.5-1 mL dissociation enzyme (see Table of Materials) for 5 minutes at 37 &#176;C.</li>
	<li>Wash with 4-4.5 mL PBS and add 10 mL of the complete growth medium (complete neurobasal medium, cNBM).</li>
	<li>Count the cells using an automatic counting technique with trypan blue and a cell counting chamber slide.</li>
	<li>To generate 100 spheroids with 104 cells per spheroid (according to preferred size), mix 106 cells in 8 mL of NBM with 2 mL of 2% methylcellulose.</li>
	<li>Transfer the suspension to a sterile system container and dispense 100 &#181;L/well with a multichannel pipette into a 96 well round bottom plate.</li>
	<li>Incubate the plate at 37 &#176;C, 5% CO2 and 95% humidity. Equal sized spheroids will form and can be used after 3-4 days.</li>
</ol>
2. Three-dimensional experiments
<ol>
	<li>Proliferation
	<ol>
		<li>Preparation
		<ol>
			<li>Suspend inhibitors (e.g., rotenone as in Figure 4A) and chemicals in 100 &#181;L of medium and add to the 100 &#181;L of medium in each well (i.e., one spheroid per well).</li>
			<li>Incubate the plate at 37 &#176;C, 5% CO2, and 95% humidity.</li>
		</ol>
		</li>
		<li>Image acquisition and analysis
		<ol>
			<li>Take pictures with a video microscope in brightfield to create a series of conditions at T0 and the following times expected.</li>
			<li>Use Fiji to analyze pictures either manually or in a semiautomated manner. To do so manually, draw a circle around the spheroid core with the freehand selection tool and measure the area of each spheroid. To analyze the images in a semiautomated manner, use the macro shown in Supplementary Document 1 with //Core Area only.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Invasion
	<ol>
		<li>Preparation
		<ol>
			<li>Prepare the collagen matrix in a tube on ice with type I collagen at 1 mg/mL final concentration, 1x PBS, 0.023xVcollagen, 1 M sodium hydroxide, and sterile H2O. Incubate the solution on ice for 30 min.</li>
			<li>Collect spheroids from the round bottom well plate in 500 &#181;L tubes and wash 2x with 200 &#181;L 1x PBS.</li>
			<li>Pipette the spheroids carefully into 100 &#181;L of the collagen matrix and insert in the center of a well in normal 96 well plates.</li>
			<li>Incubate the collagen gel for 30 min at 37 &#176;C and then add cNBM on top of the gel. Inhibitors or activators (e.g., hydrogen chloride as shown in Figure 4B, 4C) can be added to the medium at this step.</li>
		</ol>
		</li>
		<li>Image acquisition and analysis
		<ol>
			<li>Take pictures sequentially with a video microscope in brightfield mode 24 h after collagen inclusion.</li>
			<li>Use Fiji to analyze pictures either manually or in a semiautomated manner. To do so manually, draw around the core and the total area of the spheroid with the freehand selection tool and measure the invasive area of each spheroid by subtract total area with core area. To analyze the images in a semiautomated manner, use the macro as indicated in Supplementary Document 1 to determine the invasive area.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Migration
	<ol>
		<li>Preparation
		<ol>
			<li>Coat a 6 well plate with Matrigel (0.2 mg/mL) in NBM for 30 min at 37 &#176;C, then remove the Matrigel and add 2 mL of cNBM.</li>
			<li>Transfer spheroids into 50 &#181;L of cNBM from the round bottom well plate to the 6 well plate.</li>
			<li>Incubate the plate at 37 &#176;C and wait 30 min for the spheroids to adhere.</li>
			<li>After 24 h of incubation, stain with 10 ng/mL of Hoechst and incubate 30 min at 37 &#176;C.</li>
		</ol>
		</li>
		<li>Image acquisition and analysis
		<ol>
			<li>Obtain images using a video microscope in brightfield. A 405 nm laser is used for visualization of Hoechst staining.</li>
			<li>Use Fiji software to analyze pictures and run the macro as indicated in Supplementary Document 1. 
			NOTE: Touching the bottom of the well or completely removing the supernatant damages the spheroids. For collagen type I gel handling, keep the gel on ice to avoid collagen polymerization, do not add an acidic component because the change in pH will affect the compactness of the gel, and pipette cells rapidly into the collagen to prevent cell death and the degradation of the gel.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Fiji Macro
NOTE: Fiji is an image analysis program developed in the public domain that allows the development of macros to speed up image analysis. Manual analysis is also possible, but this is a slow process and may introduce biases. Images can be imported by drag-and-drop in the software and quantified with the ROI Manager Tools plugin. The procedure used in this study is described below:
<ol>
	<li>Open the macro window: Plugins | Macros | Interactive Interpreter.</li>
	<li>Copy and paste the following adapted purple loop. Keep the purple sentences and add the green sentences of interest (Supplementary Document 1).</li>
	<li>To analyze the entire series, adjust the parameters in red for a specific quantification and run the macro using Macros | Run Macro or pressing Ctrl+R.</li>
	<li>Check and, if necessary, manually adapt the region of interest (ROI).</li>
</ol>
4. Electron microscopy of spheroids
NOTE: Most of the following steps must be done in a chemical hood.
<ol>
	<li>Fixation step
	<ol>
		<li>Collect the spheroid with a cut pipette tip, put it in a 1.5 mL tube, and wash 1x with 0.1 M phosphate buffer (PB).</li>
		<li>Fix the spheroid overnight at 4 &#176;C in 2% glutaraldehyde/2% paraformaldehyde (PFA) in 0.1 M PB.</li>
		<li>Replace the fixation solution with a solution of 1% PFA in 0.1 M PB followed by sample preparation.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Transfer the spheroids into a strainer and put them in a glass beaker in order to avoid spheroid damage.</li>
		<li>Carefully wash 3x with 0.1 M PB.</li>
		<li>Incubate with osmium for 2 h in the dark. Dilute osmium to 4% in 1% 0.1 M PB buffer.</li>
		<li>Carefully wash 3x with 0.1 M PB.
		<ol>
			<li>Dehydrate as follows: soak in 50% ethanol for 10 min, 70% ethanol for 10 min, 2x 90% ethanol for 15 min, 2x 100% ethanol for 20 min, and 2x acetone for 30 min.</li>
		</ol>
		</li>
		<li>Incubate the samples in a 50/50 mixture of acetone/resin for 2 h. During this step, prepare the EPON resin (Embed-812: 11.25 g; DDSA: 9 g; NMA: 4.5 g).</li>
		<li>Discard the acetone/resin mixture, replace with freshly prepared resin and incubate overnight.</li>
		<li>Replace the resin by a new one and incubate between 2-6 h.</li>
		<li>Add the spheroids in resin into a mold at 60 &#176;C for 48-72 h.</li>
	</ol>
	</li>
</ol>

<ol><li>Pelissier, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+dysfunction+in+mechanotransduction+impairs+differentiation+of+human+mammary+epithelial+progenitors%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">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/pubmed?term=((Tumor-derived+spheroids%3A+Relevance+to+cancer+stem+cells+and+clinical+applications%5BTitle%5D)+AND+%22Cancer+Science%22%5BJournal%5D)">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/pubmed?term=((Cell+and+environment+interactions+in+tumor+microregions%3A+the+multicell+spheroid+model%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Multicellular+resistance%3A+a+paradigm+for+clinical+resistance%3F%5BTitle%5D)+AND+%22Critical+Reviews+in+Oncology%2FHematology%22%5BJournal%5D)">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/pubmed?term=((Tumour+acidosis%3A+from+the+passenger+to+the+driver%27s+seat%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">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/pubmed?term=((Multicellular+tumor+spheroids%3A+an+underestimated+tool+is+catching+up+again%5BTitle%5D)+AND+%22Journal+of+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((A+Cancer+Cell+Spheroid+Assay+to+Assess+Invasion+in+a+3D+Setting%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((A+3D+Spheroid+Model+as+a+More+Physiological+System+for+Cancer-Associated+Fibroblasts+Differentiation+and+Invasion+In+Vitro+Studies%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((The+role+of+CXCR3%2FLRP1+cross-talk+in+the+invasion+of+primary+brain+tumors%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Autocrine+control+of+glioma+cells+adhesion+and+migration+through+IRE1alpha-mediated+cleavage+of+SPARC+mRNA%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">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/pubmed?term=((Increased+SPARC+expression+promotes+U87+glioblastoma+invasion+in+vitro%5BTitle%5D)+AND+%22International+Journal+of+Developmental+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Deciphering+the+complex+role+of+thrombospondin-1+in+glioblastoma+development%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Tube+travel%3A+the+role+of+proteases+in+individual+and+collective+cancer+cell+invasion%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">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/pubmed?term=((MMP+proteolytic+activity+regulates+cancer+invasiveness+by+modulating+integrins%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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/pubmed?term=((Cellular+migration+and+invasion+uncoupled%3A+increased+migration+is+not+an+inexorable+consequence+of+epithelial-to-mesenchymal+transition%5BTitle%5D)+AND+%22Molecular+and+Cellular+Biology%22%5BJournal%5D)">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/pubmed?term=((An+Experimenter%27s+Guide+to+Glioblastoma+Invasion+Pathways%5BTitle%5D)+AND+%22Trends+in+Molecular+Medicine%22%5BJournal%5D)">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/pubmed?term=((The+invasive+proteome+of+glioblastoma+revealed+by+laser-capture+microdissection%5BTitle%5D)+AND+%22Neuro-Oncology+Advances%22%5BJournal%5D)">The invasive proteome of glioblastoma revealed by laser-capture microdissection.</a> Neuro-Oncology Advances. 1 (1), vdz029(2019).</li>
</ol>

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><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 &amp;deg;C, sphere dissociation enzyme</td></tr><tr><td>B27</td><td>Gibco</td><td>12587</td><td>Stored at -20 &amp;deg;C, defrost before use</td></tr><tr><td>Basic Fibroblast Growth Factor</td><td>Peprotech</td><td>100-18B</td><td>Stored at -20 &amp;deg;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 &amp;deg;C</td></tr><tr><td>Fiji software</td><td>ImageJ</td><td></td><td>Used to analyze pictures</td></tr><tr><td>Flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Falcon</td><td>10497302</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354230</td><td>Stored at -20 &amp;deg;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 &amp;deg;C</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103049</td><td>Stored at 4 &amp;deg;C</td></tr><tr><td>Penicillin - Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Stored at 4 &amp;deg;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 &amp;deg;C</td></tr></tbody></table>

a 3d spheroid model for glioblastoma

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...

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 ...

Research

JoVE Journal

Cancer Research

This video demonstrates how gliomaspheres, derived from brain tumors, are separated into single cells and encapsulated in a hyaluronic acid-based hydrogel. This process promotes cell-cell interactions, forming a structured, three-dimensional glioblastoma model.

A Hyaluronic Acid-Based Hydrogel for 3-Dimensional Culture of Glioblastoma Cells

Source: Guyon, J., et al. A 3D Spheroid Model for Glioblastoma. J. Vis. Exp. (2020)The video demonstrates the generation of a three-dimensional spheroid model of tumor cell invasion. Spheroids are formed from human brain tumor cells and embedded in a collagen matrix. The tumor cells at the spheroid periphery adopt an invasive phenotype, secreting proteases to degrade the collagen and advancing to invade the surrounding matrix.

Generation of a Three-Dimensional Spheroid Model of Tumor Cell Invasion

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

Translational Orthotopic Models of Glioblastoma Multiforme

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Here, we establish a metabolic profiling platform integrating three-dimensional (3D) glioma spheroids and imaging-based normalization via extracellular flux analysis. This approach enables more accurate assessment of tumor metabolic responses to drug treatments and provides insights into potential mechanisms of resistance.

Advancements in the Metabolic Profiling of Three-Dimensional Brain Tumor Spheroids for Drug Screening

We present a 3D glioblastoma migration model using patient derived magnetic gliomaspheres generated from newly diagnosed and recurrent tumors. This platform allows visualization of patient specific migration dynamics and assessment of therapeutic efficacy. The assay can be multiplexed post migration to evaluate protein localization and expression patterns.

Reproducible 3D Glioblastoma Migration Assay with Magnetic Nanoparticle Mediated Spheroid Localization Under Hypoxic Conditions

Learn a 3D glioblastoma spheroid-based invasion assay for studying brain tumor invasion into healthy tissue. This method enables quantification of migration, proliferation, and drug response evaluation.

A 3D Spheroid Model for Glioblastoma

Explore More Videos