This work was supported by Fondation ARC 2020, Ligue Contre le Cancer (Comite de la Gironde), ARTC, Plan Cancer 2021, INCA PLBIO. Alveole is supported by Agence Nationale de la Recherche (Grant Labex BRAIN ANR-10-LABX-43). 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). This protocol follows the guidelines of Bordeaux University human and animal research ethics committees. Pregnant rats were housed and treated in the animal facility of Bordeaux University. Euthanasia of an E18-timed pregnant rat was performed by using CO2. All animal procedures have been done according to the institutional guidelines and approved by the local ethics committee. All commercial products are referenced in the Table of Materials.
1. Preparation of the patterned slides
<ol>
	<li>Substrate preparation for micropatterning
	<ol>
		<li>Treat 18 mm circular glass coverslips by air/plasma activation for 5 min. Place the coverslips in a closed chamber with 100 &#181;L of (3-aminopropyl) triethoxysilane in a desiccator for 1 h.</li>
		<li>Incubate with 100 mg/mL of poly (ethylene glycol)-succinimidyl valerate (molecular weight 5,000 (Peg-SVA)) in 10 mM carbonate buffer, pH &#62; 8, for 1 h. Rinse extensively with ultrapure water, and dry under a chemical hood. 
		&#8203;NOTE: At this stage, the sample can be stored at 4 &#176;C in the dark for further use.</li>
		<li>Add the photoinitiator, 4-benzoylbenzyl-trimethylammonium chloride (PLPP), at 14.7 mg/mL in phosphate-buffered saline (PBS). 
		NOTE: A concentrated form of PLPP, a PLPP gel, can also be used. It results in a shorter ultraviolet (UV) illumination time required to degrade the PEG brush (100 mJ/mm2).</li>
	</ol>
	</li>
	<li>Photoinitiator gel deposition
	<ol>
		<li>Prepare a mixture of 3 &#181;L of PLPP gel and 50 &#181;L of absolute ethanol to deposit in the center of the slide. Place the sample under a chemical hood until complete evaporation of the absolute ethanol. 
		NOTE: At this stage, the sample can be stored at 4 &#176;C in the dark for further use.</li>
	</ol>
	</li>
	<li>Glass slide micropatterning
	<ol>
		<li>Mount the coverslip in a Ludin chamber, and place it on the motorized stage of a microscope equipped with an auto-focus system.</li>
		<li>Load images corresponding to the envisioned micropatterns into the software. Apply these parameters: replication 4 x 4 times, spacing of 200 &#181;m, UV dose of 1,000 mJ/mm2. After the automatic UV-illumination sequence, rinse the PLPP away with multiple PBS washes. 
		NOTE: If PLPP gel was used, remove it by extensive washes with deionized water, dry in a stream of N2, and store at 4 &#176;C.</li>
		<li>Incubate with laminin (50 &#181;g/mL in PBS) for 30 min. Wash extensively with PBS. 
		&#8203;NOTE: A fluorescent solution of purified green fluorescent protein (GFP, 10 &#181;g/mL in PBS) can be mixed with laminin to visualize the micropatterns by fluorescence microscopy.</li>
	</ol>
	</li>
</ol>
2. Preparation of neurons and GBM cells for co-culture
<ol>
	<li>Culture of embryonic rat hippocampal neurons
	<ol>
		<li>Dissect the hippocampus of embryonic (E18) rats, and transfer the tissue into a Hank&#39;s balanced salt solution (HBSS)/1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/penicillin-streptomycin solution in a 15 mL tube. Remove excess solution without drying the hippocampus.</li>
		<li>Add 5 mL of trypsin-ethylenediamine tetraacetic acid (EDTA) supplemented with penicillin (10,000 units/mL)/streptomycin (10,000 &#181;g/mL) and 1 mM HEPES, and incubate for 15 min at 37 &#176;C. Wash 2x with the HBSS/HEPES/penicillin-streptomycin solution, and let the tissue remain in this solution for 2-3 min.</li>
		<li>Dissociate the tissue using two flame-polished Pasteur pipettes, by pipetting up and down 10x with each tissue, taking care to minimize foaming. Count the cells, and evaluate the viability of the cell suspension. Plate the neurons on micropatterned coverslips as indicated below. 
		NOTE: Cell viability rate is 85-90% after extraction.</li>
	</ol>
	</li>
	<li>Cell culture for neurons on micropatterned coverslips
	<ol>
		<li>Rehydrate micropatterned glass slides with PBS, and incubate complete neuronal cell culture medium.</li>
		<li>Seed the hippocampal neurons obtained from E18 Sprague-Dawley rats directly over the micropatterned glass coverslip at a density of 50,000 cells per cm2 in neurobasal medium (NBM) enriched with 3% horse serum. Place the micropatterned neurons in the incubator (37 &#176;C, 5% CO2) for 48 h. 
		NOTE: After ~6 h, primary hippocampal neurons can be seen adhering to the laminin micropatterns.</li>
	</ol>
	</li>
	<li>Co-culture of human GBM stem-like cells on neurons 
	&#8203;NOTE: For this study, membrane GFP-positive and nuclear-tomato&#160;patient-derived GBM cells were grown according to previous published protocols10.
	<ol>
		<li>As the spheroid-shaped cells grow in suspension, centrifuge the suspension for 5 min at 200 &#215; g. Wash the spheroids with 5 mL of PBS, and incubate the cells with 0.5 mL of the cell dissociation reagent (see the Table of Materials) for 5 min at 37 &#176;C.</li>
		<li>Add 4.5 mL of complete NBM (complemented with B27 supplement, heparin, fibroblast growth factor 2, penicillin, and streptomycin, as described previously8), and count the cells using an automatic counting technique.</li>
		<li>Seed 1,000 GBM cells over the micropatterned neuronal culture in NBM enriched with 3% horse serum. Incubate the plate at 37 &#176;C, 5% CO2, and 95% humidity.</li>
	</ol>
	</li>
</ol>
3. Live cell imaging
<ol>
	<li>Immediately after GBM cell seeding, place the sample on the stage of an inverted microscope equipped with a thermostat chamber. Perform live-cell imaging on the microscope equipped with a motorized stage for recording multiple positions by using a multidimensional acquisitions toolbox in the software. Acquire brightfield and epifluorescence GFP/Tomato images every 5 minutes over 12 h with a 20x objective in a temperature (37 &#176;C) and gas-controlled (5% CO2) environment.</li>
</ol>
4. Image analysis
NOTE: Using Fiji, two-dimensional (2D) image&#160;stack were semi-automatically preprocessed or processed by using a homemade and user-friendly tool (available at this address: https://github.com/Guyon-J/Coculture_Gliomas-Neurons/blob/main/README.md), which is written in IJ1 macro language (Figure 2A). The automated workflow and procedures are summarized in Figure 2B.
<ol>
	<li>Neuronal network analysis (Figure 2Bi)
	<ol>
		<li>Select one image of the stack. Right-click on the Network tool to open the corresponding Options dialog box and adjust the settings (e.g., Threshold = Triangle, Li, Huang&#8230;, Gaussian Blur, and Median filters = 1, 2, 3&#8230;) to produce a precise segmentation of images. Then, click on OK.</li>
		<li>Left-click on the Network tool to automatically activate the following procedure.
		<ol>
			<li>Duplicate the selected image, and split it into three color channels (Red-Grey-Green).</li>
			<li>Select the grey channel (brightfield), and perform contrast stretch enhancement (CSE) to enhance the separation between different areas. Use the Sobel edge detector (SED) to perform the 2D signal processing convolution operation already grouped under the Find Edge command.</li>
			<li>For double-filtering (F), apply a Gaussian blur and median filter to reduce noise and smooth the object signal. Convert to Mask (CM) by executing adapted threshold algorithms to obtain a binary picture (BIN-grey) with black pixels (cell area) and white pixels (background). Skeletonize (Sk) the cell area into a simple network (NET), and filter particles (EP) in a NET image by removing small, non-networked particles.</li>
			<li>For red and green channels (nucleus and membrane), perform double-filtering, convert to Mask using the adapted thresholding method, and allow BIN-green to determine cell morphology with the Analyze Particles&#160;command.</li>
			<li>Merge all channels using their region of interest (ROI) with the OR (combine) operator, and readjust their initial color into a simple RGB image.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Single-cell motility analysis (Figure 2Bii)
	<ol>
		<li>Right-click on the Single Cell Tracking tool to open the corresponding Options dialog box, and adjust the settings (e.g., Trail type = Nucleus or Membrane, Threshold = Triangle, Li, Huang&#8230;, Z projection = Max intensity, Sum Slices&#8230;, Gaussian Blur and Median filters = 1, 2, 3&#8230;) to produce a precise segmentation of images. Then, click on OK.</li>
		<li>Left-click on the Single Cell Tracking tool to automatically activate the following procedure.
		<ol>
			<li>Remove the grey channel. Apply a Z projection on the stack, which will generate an image corresponding to an image stack according to the time (T-stack). Double-filter and convert to Mask the trails left by cells. Remove small particles to the BIN-red/green image.</li>
			<li>By using ROI, select each contour of the cell trace, and check the box Skip edge detection in the Options dialog box to skip this preprocessing step for subsequent steps.</li>
			<li>Isolate the red channel (Trail type = Nucleus) on the original stack. Select one ROI and remove the outer area. Double-filter all images and convert to Mask (BIN-red). Determine the centroid X/Y position of each binarized nucleus.</li>
		</ol>
		</li>
		<li>Using an already published macro for spreadsheet software13, calculate the mean square displacement, directionality ratio, and average speed for this cell.</li>
	</ol>
	</li>
	<li>Multiple-cell tracking analysis (Figure 2Biii)
	<ol>
		<li>Right-click on the Tracking tool to open the corresponding Options dialog box and adjust the settings (e.g., Threshold = Triangle, Li, Huang&#8230;, Gaussian Blur and Median filters = 1, 2, 3&#8230;) to produce a precise segmentation of images. Then, click on OK.</li>
		<li>Left-click on the Tracking tool to automatically activate the following procedure.
		<ol>
			<li>Remove the grey channel.</li>
			<li>Split the red and green channels, double-filter, and convert to Mask.</li>
			<li>Merge the channels using Image Calculator&#8230; command with the AND operator, leaving only the nucleus signal found in the membranes. 
			NOTE: Several plugins in Fiji can be used to determine the X/Y position of several cells in this binary preprocessed image at the same time (see the Table of Materials).</li>
		</ol>
		</li>
		<li>Using a previously described macro13, calculate the trajectory plot, mean square displacement, directionality ratio, and average speed for these cells.</li>
	</ol>
	</li>
	<li>Spheroid migration on the neural mat (Figure 2Biv)
	<ol>
		<li>Right-click on the Migration tool to open the corresponding Options dialog box and adjust the settings (e.g., Threshold = Triangle, Li, Huang&#8230;, Gaussian Blur and Median filters = 1, 2, 3&#8230;) to produce a precise segmentation of images. Then, click on OK.</li>
		<li>Left-click on the Migration tool to automatically activate the following procedure.
		<ol>
			<li>Remove the red channel.</li>
			<li>Split the green and grey channels.</li>
			<li>For the grey channel, draw manually the contour of the neuronal mat, and measure its area.</li>
			<li>For the green channel, double-filter the stack and convert to Mask. Remove the area outside the pattern (BIN), and determine the binarized cell area for each image. 
			&#8203;NOTE: Parameters described above are calibrated by changing their values by left-clicking on the icon of interest. This processing can be done manually. However, for a large number of images (approximately a hundred per acquisitions), channels (generally 3 channels), and processing steps, an automated or semi-automated tool would be preferable.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no conflicts of interest.

Glioblastomas extensively invade the parenchyma by using different modes: co-option of surrounding blood vessels, interstitial invasion, or invasion on WMTs18. This latter mode is not well characterized in the literature because of the difficulty in finding suitable in vitro or in vivo models related to WMT invasion. Here, a simplified model has been proposed in which cultured rodent neurons were patterned on laminin-coated surfaces, and fluorescent GBM stem-like cells were seeded on top of the neurons. A grid-shape pattern was used in this study to improve the analysis of tumor cell attachment, invasion, and proliferation. GBM cells migrated more efficiently on top of the neurons than directly on the matrix, which was laminin in these experiments. The cell shape changed throughout the recording process, and the cell surface area increased at the same time. GBM stem-like cells are likely to be attracted by neuron tracts via the activation of specific signaling pathways (i.e., NOTCH2/SOX2)4 or secreted factors and signals from the neurons themselves. This system is well-suited to analyze the molecular exchanges between GBM cells and neurons, which may include metabolites, neurotransmitters, or cytokines.
Recently, Venkatesh and collaborators described the formation of a neuroglioma synapse in which glutamate activates its receptor, AMPAR6. Are similar processes involved during WMT invasion of GBM cells? This can be investigated with this experimental system using pharmacological inhibition or genetic approaches.
The following critical points should be noted. First, during the PEGylation steps, the substrate should not be allowed to dry out to avoid affecting the integrity of the anti-adhesive coating. Of note, it is possible to extensively wash the PEG-SVA solution with ultrapure water and to dry it out under a stream of nitrogen before storing it at 4 &#176;C in the dark. Second, the macro developed for analyzing GBM cell migration on neurons is in an open-access mode and is compatible with FiJi software. Although this macro and its updates are available on GitHub, it requires an appropriate calibration for detecting cells. Hence, it may be useful to check the samples manually as a quality control while starting the analysis. The flexibility of the system used here allows different shapes of the pattern, with parallel lines separating the neurons to different extents. With this approach, the shape of several cerebral structures can be mimicked, as observed in the corpus callosum-the largest white matter structure in human brain-where WMT invasion is mainly observed. Alternatively, the same UV-light projection apparatus has been shown to structure UV-sensitive non-adhesive hydrogels in a z-controlled manner15, allowing the standardization of spheroid formation.
In this context, 3D neurospheres could be generated to test GBM invasion. This technique can be also applied for patterning other cerebral cells, such as brain endothelial cells, to reproduce vessel-like shape or mimic microglial or other immune cells. Thus, synergistic or inhibitory effects of cerebral cells can be observed when co-cultured with GBM cells. One limitation of this study is the use of embryonic rat neurons in co-culture with human GBM cells, which may not mimic true physiological conditions. One way to overcome this drawback would be to use human induced pluripotent stem cell-derived neurons to avoid species cross-reactivity16. However, GBM cells quickly adhere and efficiently migrate on rat neurons, as shown in these experiments. It has also been demonstrated than rat cerebral cells (Schwann cells) could be efficiently co-cultured with human neurons17. Other methods include the use of 3D nanofibers, which offer a good model to study glioma cell migration12, but limit cell-cell contact as nanofibers are considered non-living structures.
Furthermore, 2D cultures are reductionistic, simplify the observation of cellular processes, and may limit their validity for the in vivo context10. Thus, 3D co-cultures of GBM cells and neurons are better representatives of the in vivo situation. Complex brain organoids, such as mini-brains18, have been used in confrontation-culture invasion assays19. The main advantage of the strategy described herein is the reproducibility of the co-culture approach, i.e., the primary neurons are geometrically constrained on size-controlled micropatterns, and the interaction with the injected GBM cells cannot occur elsewhere. Furthermore, the spatial organization of neurons can be tuned because of the versatility of the UV-projection system, allowing for further optimization. Ultimately, the development and validation of such biomimetic approaches could also help in reducing the number of animal models used in biomedical research.

Primary malignant gliomas, including GBMs, are devastating tumors, with a medium survival rate of 12 to 15 months reported for GBM patients. Current therapy relies on large tumor mass resection and chemotherapy coupled with radiotherapy, which only extends the survival rate by few months. Therapeutic failures are intimately related to poor drug delivery across the blood-brain barrier (BBB) and to invasive growth in perivascular spaces, meninges, and along WMTs1. Perivascular invasion, also called vascular co-option, is a well-studied process, and the molecular mechanisms are beginning to be elucidated; however, the process of GBM cell invasion along WMTs is not well understood. Tumor cells migrate into the healthy brain along Scherer&#39;s secondary structures2. Indeed, almost one century ago, Hans-Joachim Scherer described the invasive routes of GBM, which are now referred to as perineuronal satellitosis, perivascular satellitosis, subpial spread, and invasion along the WMT (Figure 1A).
Some chemokines and their receptors, such as stromal cell-derived factor-1&#945; (SDF1&#945;) and C-X-C motif chemokine receptor 4 (CXCR4), but not vascular endothelial growth factor (VEGF), seem to be implicated in WMT invasion3. More recently, a transcellular NOTCH1-SOX2 axis has been shown to be an important pathway in WMT invasion of GBM cells4. The authors described how GBM stem-like cells invade the brain parenchyma on partially unmyelinated neurons, suggesting the destruction of myelin sheaths by GBM cells. A milestone was reached in 2019 when three articles wereconsecutively published in Nature journal, underlining the role of electrical activity in glioma development5,6. Seminal work by Monje and collaborators shed light on the central role of electric activity in the secretion of neuroligin-3, which promotes glioma development.
Winkler and collaborators described connections between GBM cells (microtubes) being crucial in invasive steps, and lately, interactions between GBM cells and neurons via newly described neuroglioma synapses. Those structures favor glutamatergic stimulation of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors located at the GBM cell membrane, which promotes tumor development and invasion. Tumor cell invasion is a central process in the dissemination of metastases or distant secondary foci, as observed in GBM patients. Several factors have been identified to be important in GBM invasion such as thrombospondin-1, a transforming growth factor beta (TGF&#946;-regulated matricellular protein, or the chemokine receptor CXCR3)7,8.
Here, a simplified biomimetic model has been described for studying GBM invasion, in which neurons are patterned on tracks of laminin, and GBM cells are seeded onto it, as single-cells or as spheroids (Figure 1B). The two experimental settings are aimed at recapitulating invasion on neurons, which is observed in GBM9,10,11. Such models have been developed in the past as aligned nanofiber biomaterials (core-shell electrospinning) that allow studying cell migration by modulating mechanical or chemical properties12. The co-culture model described in this article allows a better understanding of how GBM cells escape on neurons by defining new molecular pathways involved in this process.

<table><tbody><tr><td>(3-aminopropyl) triethoxysilane</td><td>Sigma</td><td>440140-100ML</td><td>The amino group is useful for the bioconjugation of mPEG-SVA</td></tr><tr><td>96-well round-bottom plate</td><td>Sarstedt</td><td>2582624</td><td>Used to prepare spheroids</td></tr><tr><td>Accutase</td><td>Gibco</td><td>A11105-01</td><td>Stored at -20 &amp;deg;C (long-term) or 4 &amp;deg;C (short-term), 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 ChamberSlides</td><td>Invitrogen</td><td>C10283</td><td>Used to cell counting</td></tr><tr><td>Coverslips</td><td>Marienfeld</td><td>111580</td><td>Cell culture substrate</td></tr><tr><td>Dessicator cartridges</td><td>Sigma</td><td>Z363456-6EA</td><td>Used to reduce mosture during (3-aminopropyl) triethoxysilane treatment</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, MTrack2 macro</td><td>ImageJ</td><td></td><td>Used to analyze pictures</td></tr><tr><td>Flask 75 cm&amp;sup2;</td><td>Falcon</td><td>10497302</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H8264-500ML</td><td></td></tr><tr><td>Heparin sodium</td><td>Sigma</td><td>H3149-100KU</td><td>Stored at 4 &amp;deg;C</td></tr><tr><td>Laminin</td><td></td><td>114956-81-9</td><td>Promotes neuronal adhesion</td></tr><tr><td>Leonardo software</td><td></td><td></td><td>loading of envisioned micropatterns</td></tr><tr><td>MetaMorph Software&amp;nbsp;</td><td>Molecular Devices LLC</td><td>NA</td><td>Microscopy automation software</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>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td>Stored at 4 &amp;deg;C</td></tr><tr><td>Nikon TiE (S Fluor, 20x/0.75 NA)</td><td></td><td></td><td>inverted microscope equipped with a motorized stage&amp;nbsp;</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>PLPP</td><td>Alveole</td><td>PLPPclassic_1ml</td><td>Photoinitiator used to degrade the PEG brush</td></tr><tr><td>Poly(ethylene glycol)-Succinimidyl Valerate (mPEG-SVA)</td><td>Laysan Bio</td><td>VA-PEG-VA-5000-5g</td><td>Used as an anti-fouling coating</td></tr><tr><td>PRIMO</td><td>Alveole</td><td>PRIMO1</td><td>Digital micromirror device (DMD)-based UV projection apparatus</td></tr><tr><td>Trypan blue 0.4%</td><td>ThermoFisher</td><td>T10282</td><td>Used for cell counting</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma</td><td>T4049-100ML</td><td>Used to detach adherent cells</td></tr></tbody></table>

co-culture of glioblastoma stem-like cells on patterned neurons to study migration and cellular interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Patterned neurons co-cultured with fluorescent GBM cells were prepared as described in the protocol section, and tracking experiments were performed. GBM cells quickly modified their shape while migrating on the neurons (Figure 1B: panel 6 and Video 1). Cells migrated along the neuronal extensions, in a random motion (Video 1). Fluorescent GBM cells and non-fluorescent neurons can be easily distinguished, and this allowed the tracking of cell movements by using the Fiji macro, as described in the protocol section (Figure 2). Fiji is an open license software that facilitates image processing and analysis. Manual procedures that are relatively time-consuming for the analysis of numerous images can be automatized in a macro. Images are imported by drag-and-drop procedure, processed, and quantified, generating data which are available using an ROI manager.
When deposited in the Fiji.app | macros | toolsets folder, the Gliomas-Neurons tool was available in the More Tools menu and displayed several icons (Figure 2A). A workflow of the image processing, obtained by clicking on the icons, is illustrated in Figure 2B (i-iv). Cell shape can be analyzed on the neural network (Figure 2B, i). Several parameters from cell tracking can be obtained for one (Figure 2B,&#160;ii) or several cells (Figure 2B,iii) by using two distinct image processes. The speed of recovery by spheroid GBM cells on the neurons can be also analyzed (Figure 2B, iv). Cells seeded onto neurons displayed an elongated shape with multiple protrusions following neuron tracts (Figure 3A, i), but had a round shape when cultured directly on laminin (Figure 3A, ii). Cells cultured on neurons efficiently modified their shape, although they were not elongated when cultured on laminin (Figure 3A, iii-v). Thin protrusions, sometimes linking two cells, were seen in cells co-cultured with neurons at later stages (Video 1).
The migratory capacity of GBM cells seeded on neurons was compared to cells directly seeded on laminin. Cells seeded on neurons had greater migratory capacities than on laminin alone (Figure 3B, i,ii). Random movement of P3 cells was detected in both conditions, with greater distance for P3 cells on the neurons, as shown in the trajectory plot (Figure 3B, i,ii). Cell motility was quantitatively estimated by the mean square displacement (MSD), and its log representation was fitted with a linear function14&#160;(Figure 3B, iii). Directionality and average speed were also calculated for both conditions (Figure 3B, iv,v). Cell migration of P3 spheroids was also followed by detecting the fluorescent area over time on the neurons and compared with migration on laminin alone (Figure 3C, i,ii and Video 2). Half of the pattern with neurons was covered with GBM cells after 500 min in a linear profile; however, spheroids did not adhere to the laminin pattern (Figure 3D, iii and Video 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62213/62213fig1v2.jpg" /> 
Figure 1: Experimental setup of glioblastoma cells migrating on patterned neurons.&#160;(A) Representation of glioblastoma cells invading the contralateral hemisphere through the corpus callosum. (B) Experimental setup. In step 1, the plate surface is coated with an antifouling PEG layer. The photoinitiator is added in step 2, covering the entire coating. In step 3, a UV widefield image is projected through the objective of the microscope, which locally activates the photoinitiator molecules. The activated photoinitiator locally cleaves PEG molecules and allows the subsequent adsorption of laminin. In step 5, neurons are seeded and adhere on the laminin arrays. P3 (GFP/Tomatonuclear) glioblastoma cells are then deposited on the neuronal pattern, and images are acquired (step 6). Abbreviations: PEG = polyethylene glycol; UV = ultraviolet; GFP = green fluorescent protein.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62213/62213fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62213/62213fig2v2.jpg" /> 
Figure 2: Fiji tool presentation and analysis workflow.&#160;(A) The tool called Gliomas-Neurons is available in the More Tools menu when the macro is added to Fiji.app folder | macros | toolsets (left panel). It is composed of several action tools described below (right panel). (B) i. Network tool: image processing, which is used to draw the neural network and glioma cells in a simplified representation. ii. Single-cell tracking tool: image processing, which is used to draw and select the cell movement area for analyzing the displacement of single cells. iii. Tracking tool: image preprocessing steps for the use of pre-installed Fiji tracking plugins. iv. Relative migration tool: image processing, which is used to determine the relative cell migration on a manually selected pattern. Some parameters can be calibrated with a left click on the tool button. Abbreviations: CSE = Contrast Stretch Enhancement; SED = Sobel Edge Detector; F = double Filtering; CM = Convert to Mask; Sk = Skeletonize; EP = Elimination of particles; OR (combine) = Union operator on selected&#160;ROIs; AND = Conjunction operator on selected ROIs.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62213/62213fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62213/62213fig3v2.jpg" /> 
Figure 3: Comparison of P3 cells or spheroids on patterned neurons vs. laminin coating.&#160;(A) Shape descriptors of dissociated P3 GFP/Tomatonuclear cells on neurons or on laminin. Example of processed networks of P3 cells on i. patterned neurons or on ii. laminin coating. Scale bar = 50 &#181;m. iii. Average cell area on patterned neurons or on laminin. iv. Formfactor is the ratio of the circumference to the area normalized to a circle, providing parameters on cell elongation and cell branching. v. Aspect ratio is the ratio of the major axis to the minor axis of the cell. In iii, iv, and v, 15 cells were analyzed per field; 4 independent patterns; data are represented as mean &#177; S.E.M. (B) Tracking analysis of dissociated P3 cells. One representative chart plot of cells on (i) patterned neurons and (ii) on laminin coating. iii. MSD of P3 cells migrating on neurons or on laminin coating. X/Y values are in logarithmic scales. iv. Directionality ratio. v. Average cell speed migration. In iii, iv, and v, 15 cells were analyzed per field; 4 independent patterns; data are represented as mean &#177; S.E.M. (C) Migration analysis of P3 GFP spheroids. Representative images at different time points of P3 spheroids on (i) patterned neurons or on (ii) laminin coating. Scale bar = 100 &#181;m. iii. Spheroid migration represented by the pattern confluency; 4 independent patterns; data are represented as mean &#177; S.E.M. Abbreviations: GFP = green fluorescent protein; S.E.M. = standard error of the mean; MSD = Mean Square Displacement.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62213/62213fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: P3 cells on neurons or laminin recorded over 8 h (imaged every 5 min).&#160;The video shows P3 single-cell migration on neurons (left) and on laminin coating (right). Cells expressed green fluorescent protein (GFP, green color) and nuclear Tomato (red color). Bar = 50 &#181;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62213/Dissoc_cells.mp4" target="_blank">Please click here to download this video.</a>
Video 2: P3 spheroids on neurons or laminin recorded over 8 h (imaged every 5 min).&#160;The video shows P3 spheroid migration on neurons (left) and on laminin coating (right). Cells expressed green fluorescent protein (GFP, green color). Bar = 100 &#181;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62213/Spheroid.mp4" target="_blank">Please click here to download this video.</a>

Watch this Scientific Journal Video about Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions at JoVE.com

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

co-culture-glioblastoma-stem-like-cells-on-patterned-neurons-to-study

Nanyang Technological University

Research

JoVE Journal

Cancer Research

5.8K Views.  University Bordeaux, INSERM, LAMC, U1029, 33600, Pessac, France. Glioblastomen zijn verwoestende hersentumoren met een hoog invasief profiel. Dit co-kweeksysteem bootst de migratie van glioblastoomcellen op neuronen na om een van de invasieve routes die bij patiënten worden waargenomen, samen te vatten. De geometrie en samenstelling van ons co-cultuurmodel wordt nauwkeurig gecontroleerd.Het vergemakkelijkt een betere reproduceerbaarheid en ook een eenvoudige kwantificering van complexe biologische processen. Deze methode kan worden aangepast aan de...

Video: Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

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

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

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.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in 2020 and 2021. GBM tumors most often arise from astrocytes and are characterized by their invasive nature, often recruiting healthy tissues into tumor tissue. Understanding communication between astrocytes and glioblastoma cells is vital for the molecular understanding of tumor progression. This protocol demonstrates a novel patterned co-culture method to investigate contact-mediated effects of astrocytes on GBM employing layer-by-layer assembly and micro-capillary-force driven patterning. Advantages include a protein-free cell culture environment and precise control of cellular interaction dictated by the pattern dimensions. This technique provides a versatile, economical, reproducible protocol for mimicking cellular interaction between glioma and astrocytes in glioma tumors. This model can further be used to tease apart changes in GBM molecular biology due to physical contact with astrocytes or with non-contact mediated soluble cofactor communication.

This protocol describes a patterned direct contact glioma-astrocyte co-culture utilizing micro-contact printing on polyelectrolyte multilayers (PEMs) to pattern U87 or A172 GBM cells and primary astrocytes.

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in ...

Bioengineering

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Neuroscience

Matrix-Based Tumor Invasion Model Using Microglia Glioblastoma Co-Culture

Source: Coniglio, S., et al.&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53990">Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion.</a> J. Vis. Exp. (2016)
In this video, glioblastoma and microglia cells are mixed with a polymer matrix solution and transferred to a porous chamber insert where a gel forms, embedding the cells. Microglia stimulate the migration of glioblastoma cells through the porous insert, mimicking tumor cell invasion into surrounding tissue, Cells are then fixed and prepared for further analysis.

Source: Coniglio, S., et al.&#160;Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion. J. Vis. Exp. (2016)
In this ...

JoVE Encyclopedia of Experiments

Neuropathology

Tumors

Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Source: Guyon, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/62213" target="_blank">Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions.</a> J. Vis. Exp. (2021)
This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells&#39; migration on neurons in a glioma.

Source: Guyon, J. et al. Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions. J. Vis. Exp. 
...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

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