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/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>

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 ...

Research

JoVE Journal

Cancer Research

14.5K Views.  University Bordeaux, LAMC. 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.

Video: A 3D Spheroid Model for Glioblastoma

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Glioblastoma Relapse Post-Resection Model for Therapeutic Hydrogel Investigations

Tumor recurrence is an important factor indicative of a poor prognosis in glioblastoma (GBM). Many studies are attempting to identify effective therapeutic strategies to prevent the recurrence of GBM after surgery. Bioresponsive therapeutic hydrogels capable of sustaining locally released drugs are frequently used for the local treatment of GBM after surgery. However, research is limited due to the lack of a suitable GBM relapse post-resection model. Here, a GBM relapse post-resection model was developed and applied in therapeutic hydrogel investigations. This model was constructed based on the orthotopic intracranial GBM model, which is widely used in studies on GBM. Subtotal resection was performed on the orthotopic intracranial GBM model mouse to mimic the clinical treatment. The residual tumor was used to indicate the size of the tumor growth. This model is easy to build, can better mimic the situation of GBM surgical resection, and can be applied in various studies on the local treatment of GBM relapse post-resection. As a result, the GBM relapse post-resection model provides a unique GBM recurrence model for effective local treatment studies of relapse post-resection.

The present protocol establishes a glioblastoma (GBM) relapse post-resection model using microscopy to investigate the therapeutic effect of an injectable, bioresponsive hydrogel in vivo.