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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>
</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 cel...

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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

Research

JoVE Journal

Cancer Research

5.7K Views.  University Bordeaux, INSERM, LAMC, U1029, 33600, Pessac, France. 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. 

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

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Flow Cytometry-based Drug Screening System for the Identification of Small Molecules That Promote Cellular Differentiation of Glioblastoma Stem Cells

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median survival following diagnosis is less than 15 months with maximal surgical resection, radiation, and temozolomide chemotherapy. The challenges inherent in developing more effective GBM treatments have become increasingly clear, and include its unyielding invasiveness, its resistance to standard treatments, its genetic complexity and molecular adaptability, and subpopulations of GBM cells with phenotypic similarities to normal stem cells, herein referred to as glioblastoma stem cells (GSCs). Because GSCs are required for tumor growth and progression, differentiation-based therapy represents a viable treatment modality for these incurable neoplasms. 
The following protocol describes a collection of procedures to establish a high throughput screening platform aimed at the identification of small molecules that promote GSC astroglial differentiation. At the core of the system is a glial fibrillary acidic protein (GFAP) differentiation reporter-construct. The protocol contains the following general procedures: (1) establishing GSC differentiation reporter lines; (2) testing/validating the relevance of the reporter to GSC self-renewal/clonogenic capacity; and (3) high-capacity flow-cytometry based drug screening. 
The screening platform provides a straightforward and inexpensive approach to identify small molecules that promote GSCs differentiation. Furthermore, utilization of libraries of FDA-approved drugs holds the potential for the identification of agents that can be repurposed more rapidly. Also, therapies that promote cancer stem cell differentiation are expected to work synergistically with current "standard of care" therapies that have been shown to target and eliminate primarily more differentiated cancer cells.

An efficient screening protocol is presented for the identification of small molecules that promote astroglial differentiation in glioblastoma stem cells (GSCs). The assay is based on a stem cell differentiation reporter whereby the expression of the enhanced GFP (eGFP) is driven by the human GFAP promoter.

Glioblastoma (GBM) is the most common and most lethal primary brain tumor in adults, causing roughly 14,000 deaths each year in the U.S. alone. Median ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

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 ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Recent works uncover the neuronal impact on high-grade pediatric glioma (pHGG) cells and their reciprocal interactions. The present work shows the development of an in vitro model co-culturing pHGG cells and glutamatergic neurons and recorded their electrophysiological interactions to mimic those interactivities.