Name: co-culture of glioblastoma stem-like cells on patterned neurons to study migration and cellular interactions
Uploaded: 2021-02-24T14:30:00
Description: 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

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 ...

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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 seede...

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.

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	<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 &#176;C (long-term) or 4 &#176;C (short-term), 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 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 &#176;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&#178;</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 &#176;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&#160;</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 &#176;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&#160;</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Stored at 4 &#176;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>
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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 cel...

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 ...

Glioblastomas are devastating brain tumors with a high invasive profile. This co-culture system mimics glioblastoma cells migration on neurons to recapitulate one of the invasive routes observed in patients. The geometry and composition of our co-culture model is precisely controlled.It does facilitate a better reproducibility and also a straightforward quantification of complex biological processes. This method can be adapted to clinical diagnosis by co-culturing freshly dissociated glioblastoma cells and neurons. It defines a clinical index, which is very important as clinical outcome.Our method can also be used to quantify other migrating cells, such as fibroblasts or immune cells. Demonstrating the procedure will be Joris Guyon, a PhD student from my team. To make a substrate for the micropatterning, treat 18 millimeter circular glass coverslips by air or plasma activation for five minutes before placing the coverslips in a desiccator with 100 microliters of triethoxysilane for one hour, then incubate a solution of PEG-SVA at 100 milligrams per milliliter for one hour.For gel deposition, at the end of the incubation, add three microliters of PLPP and 50 microliters of absolute ethanol onto the center of the slide and wait until it dries completely. For glass slide micropatterning, mount the coverslip in a Ludin chamber and place the chamber onto the stage of a microscope equipped with an auto-focus system. After imaging, load the micropattern images into the software.After automatic UV illumination sequencing, use a pipette to wash the PLPP from the coverslip extensively with PBS. Then incubate the coverslip with 50 micrograms per milliliter of laminin for 30 minutes, followed by another wash with PBS as demonstrated. To set up an embryonic rat hippocampal neuron culture on the micropattern coverslips, after the last wash, rehydrate the glass slides with neuronal cell culture medium.Seed five times 10 to the fourth rat hippocampal neurons suspended in Neurobasal medium enriched with 3%horse serum per square centimeter onto each micropatterned coverslip for a 24-hour incubation in a 5%carbon dioxide incubator at 37 degrees Celsius. Centrifuge dissociated glioblastoma cells for five minutes at 1, 000 RPM and resuspend the pellet in glioblastoma cell culture medium, then deposit one times 10 to the third GBM cells over the micropatterned neurons. For live cell imaging of the cells, place the co-culture onto the stage of an inverted microscope equipped with a 37 degree Celsius thermostat chamber and select the 20X objective.Then use the multi-dimensional acquisitions toolbox in the microscope software to acquire live Brightfield and epifluorescence GFP tomato images every two minutes for 12 hours in 16 different positions based on the number of patterns with neurons. For neuronal network analysis, after imaging, select one image from the stack. Right-click on the network tool to open the corresponding options dialog box and adjust the settings to produce a precise segmentation of the images.Click OK.Left-click on the network tool to duplicate the selected image and split the image into the red, gray, and green color channels. Select the gray channel and perform contrast stretch enhancement to improve the separation between the different areas. Use the Sobel edge detector to perform the 2D signal processing convolution as grouped under the find edge command.For double filtering, apply Gaussian blur and a median filter to reduce the noise and to smooth the object signal. To convert to mask, execute adapted threshold algorithms to obtain a binary picture with black and white pixels. Next, skeletonize the cell area into a simple network and use filter particles to remove small non-networked particles in the results.Network filter particles in a network image. To obtain red and green channels, perform double filtering and convert to mask as demonstrated using the adapted thresholding method. Use analyze particles to determine the cell morphology in the binary green image.Use the or operator to merge all of the channels using their regions of interest and readjust their initial color into a simple RGB image. To perform a single-cell motility analysis, right-click on the single cell tracking tool to open the corresponding options dialog box and adjust the settings to produce a precise segmentation of images. Click OK and left-click on single-cell tracking to remove the gray channel.To generate an image corresponding to an image stack according to the time, apply Z projection and double filter and convert to mask the trails left by the cells. Remove the small particles from the binary red and green image as demonstrated. Use the region of interest tool to select each contour of the cell trace and check the skip edge detection box in the options dialog window.Isolate the red channel on the original stack and select one region of interest. Double filter all of the images and convert to mask to allow the centroid XY position of each binarized nucleus to be determined. The XY positions can be used to calculate the mean square displacement, directionality ratio, and average speed for the cell.For multiple cells tracking analysis, right-click on the tracking tool to open the corresponding options dialog box and adjust the settings to produce a precise segmentation of images. Left-click on the tracking tool to remove the gray channel. Split the red and green channels, double filter, and convert to mask.Then use the image calculator command with the and operator to merge the channels leaving only the nucleus signal located within the membranes and calculate the trajectory plot, mean square displacement, directionality ratio, and average speed for the cells as demonstrated. Fluorescent GBM co-cultured with patterned neurons quickly modify their shape and show migration along neuronal extensions in a random motion. GBM cells seeded onto neurons display an elongated shape with multiple protrusions that follow the neuron tracks while the cells retain their rounded shape when cultured on laminin.At later stages of culture, thin protrusions linking to cells can be observed in GBM neuronal co-cultures. GBM cells seeded onto neurons demonstrate a greater migratory capacity than GBM cells seeded onto laminin as observed in these trajectory plots. Analysis of the fluorescent confluence of the cells demonstrates that over a 500-minute observation period, more cell migration is observed when the spheroids are co-cultured with neurons than when the cells are cultured on laminin alone.Indeed by the end of the analysis, nearly half of the pattern is covered with GBM cells while the spheroids cultured on laminin remain unadhered to the coverslip. Depending on the biological questions you want to answer, one should take care that the pattern design and also the cell density are representative of the in vivo conditions. The cells can be fixed and imaged by confocal microscopy.Live imaging is also possible since our method does not impair the imaging capabilities. This technique is well-suited for studying molecular interaction, such as metabolic exchanges between glioblastoma cells and neurons. It allows the exploration of function biological processes.High-throughput experiments can be also done for clinical purposes.

Research

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