We would like to thank Dr. Lee Niswander and Dr. Rada Massarwa for their technical expertise and contributions to the slice culture confocal imaging protocol described here. Further thanks to Dr. Kalen Dionne who provided expertise regarding optimizing brain tumor tissue slicing and culture parameters.

Before collection of patient tissue samples is initiated, informed consent must be obtained from each patient under an approved Institutional Review Board (IRB) protocol. The authors of this protocol received consent for the work described under approved IRB protocols at the University of Colorado Hospital and Inova Fairfax Hospital. Data collected from these slice cultures were not used to direct patient care decisions.
1. Pre-slicing Preparation
<ol>
	<li>Prepare &#34;tissue processing&#34; media and &#34;slice culture maintenance&#34; media the day before planned tumor resection and tissue collection (or utilize previously generated media within 2 weeks). Add 5 mL of Penicillin-Streptomycin solution (10,000 U/mL) and 5 mL of 1 M HEPES to 500 mL of a High-Glucose DMEM to generate the tissue processing medium.</li>
	<li>Prepare 250 mL of slice culture maintenance media using a base of neuronal medium (e.g., Neurobasal) without phenol red. Supplement this medium with 10 mM HEPES, 1x B-27 supplement, 400 &#181;M L-glutamine, 600 &#181;M L-alanyl-L-glutamine dipeptide, 60 U/mL penicillin, 60 &#181;g/mL streptomycin, and 6 U/mL nystatin.</li>
	<li>Store all media at 4 &#176;C for no longer than 2 weeks.</li>
</ol>
2. Day of Surgery: Tissue Acquisition
<ol>
	<li>On the day of tissue acquisition, prepare 50 - 100 mL of 1% and 2% (wt/vol %) solutions of low melting temperature agarose in tissue processing media using autoclaved glassware and sterile technique in a laminar flow hood. Both concentrations of agarose are needed to accommodate unpredictable variation in tumor tissue consistency.</li>
	<li>Heat the agarose suspension, using a microwave, until gentle boiling is observed. Place the agarose solution in a 37 &#176;C water bath to maintain in a liquid state until use.</li>
	<li>Place 1 mL of slice culture maintenance media in each well of a 6-well plate.</li>
	<li>Use a sterile forceps to place empty PTFE culture inserts in each well. Place the plate in a humidified, water-jacketed, tissue culture incubator with a 5% CO2 atmosphere maintained at 37 &#176;C.</li>
	<li>Using a sterile Pasteur pipette in a laminar flow hood, bubble a mixture of 95% O2/5% CO2 gas into a flask containing ice cold tissue processing media for approximately 15 to 30 min, prior to tumor tissue acquisition. The use of supra-oxygenated media minimizes hypoxia in the bulk tissue during processing.</li>
	<li>Prepare labeled 50 mL conical tubes (enough for desired number of individual tumor regions to be isolated) containing 20 mL aliquots of supra-oxygenated ice-cold processing media to transport tissue between the operating room and slicing facility.</li>
	<li>In conjunction with a neurosurgeon, pre-operatively plan the tumor region for sample acquisition. Select a tumor region with contrast enhancement as demonstrated on the patient&#39;s clinical imaging (T1 post-gadolinium magnetic resonance imaging (MRI) sequences). Previous experience suggests this area yields viable tumor tissue as opposed to necrotic tissue. 
	NOTE: Generation of slices from the surrounding (peritumoral) white matter was attempted; however, background autofluorescence and decreased tumor cell density limited the experimental utility of these slices.</li>
	<li>Obtain tumor tissue towards the beginning of tumor resection. Avoid collection of tumor tissue exposed to extensive bipolar cautery, which may compromise tissue viability secondary to thermal injury. Tissue samples acquired during piecemeal tumor resection demonstrate enhanced viability when compared to tissue acquired from lengthy en bloc resections. This observation may relate to differential tumor tissue deprivation of blood flow as a result of surgical resection and extended time to acquisition.</li>
	<li>If desired, label and store tumor tissue in separate conical tubes to isolate samples from distinct tumor regions. (i.e. superficial vs. deep tumor tissue). Precise tissue locations can be recorded if/when intraoperative surgical navigation is available.</li>
</ol>
3. Slice Culture Preparation
NOTE: This protocol requires the use of fresh unfixed human tissue. All samples are presumed to be infectious, and should be handled according to universal blood borne pathogen protocols. Appropriate personal protective equipment should be donned at all times. Forceps and scalpels should be exposed to 15 min of UV light prior to use. During use, intermittently spray the tools with 70% ethanol (EtOH), allowing time for the liquid to evaporate before use. The slicing process is performed in a semi-sterile fashion utilizing a horizontal laminar flow hood with filtered air.
<ol>
	<li>Place tumor tissue pieces in a Petri dish with ice-cold processing media. To wash the tumor tissue and minimize adherent red blood cells, use a pipette to gently exchange and discard the media in the Petri dish three times.</li>
	<li>Using a scalpel, cut tumor pieces into rectangular box shapes. Trim tissue pieces to approximate 3 mm x 3 mm x 10 mm. Carefully remove any attached vessels through excision or gentle traction with fine forceps.</li>
	<li>Pipette 5 - 7 mL of 37 &#176;C agarose into a small cube-shaped (~ 2 cm3) plastic embedding mold. Confirm temperature of agarose solution is no greater than 37 &#176;C with a sterile thermometer before use.</li>
	<li>Allow agarose to sit for approximately 1 min on a bed of ice.</li>
	<li>Place 2 - 4 tumor tissue &#34;strips&#34; into agarose with long axis oriented vertically. 
	NOTE: Tissue strips will tend to &#34;sink&#34; in the agarose before solidification. To avoid this complication, temporarily hold the tissue strip in vertical orientation until agarose is further solidified.</li>
	<li>Keep the tissue containing agarose mold on a bed of ice for 2 - 5 min to facilitate solidification.</li>
	<li>Remove mold from ice and gently remove the agarose block by cutting the sides of the form with a scalpel. Avoid placing excessive force on the block, which may fracture the agarose.</li>
	<li>Use a generous drop of cyanoacrylate glue to affix the agarose block to the vibratome specimen plate. Let glue set for approximately 1 - 2 min.</li>
	<li>Fill the vibratome reservoir with ice-cold processing media to submerge the agarose block affixed to the specimen plate.</li>
	<li>During slicing, bubble 95% O2 / 5% CO2 gas mixture into the vibratome reservoir through a trimmed sterile plastic pipette.</li>
	<li>Set the vibratome slice thickness to 300 - 350 &#181;m.</li>
	<li>Adjust blade advance speed and blade amplitude according to tissue consistency. &#34;Stiffer&#34; GBM tissue requires slower blade advance speeds and higher amplitude. Exact blade speed and amplitude settings will vary according to vibratome specifications. 
	NOTE: Several issues commonly arise during slicing. If the tissue strip dislodges from the agarose block, attempt embedding the tissue in a higher concentration agarose. If tumor slices are thicker than desired (machine set), or are asymmetric, increase the amplitude and decrease speed of vibratome blade. To ensure tumor tissue is not stuck to or &#34;dragged&#34; by the blade holder, as slicing occurs, carefully position the microspatula between the blade holder and slice, as the blade moves.</li>
	<li>Transfer tissue slices to Petri dish with ice-cold processing media using a stainless steel microspatula.</li>
	<li>Obtain 6-well plates containing PTFE inserts and equilibrated slice culture media from incubator (as prepared in section 2, step 4).</li>
	<li>Plate tissue slices using a stainless steel microspatula. Minimize direct contact and manipulation of tissue by using a small fine bristle paintbrush to generate a &#34;fluid wave&#34; to gently push the slice off the spatula and onto the culture insert. 
	NOTE: Minimize the amount of processing media introduced on top of the tissue culture insert. If excessive amounts of media are transferred causing the slices to float, use a sterile Pasteur pipette to remove media.</li>
	<li>Return the 6-well plates containing tumor slices to an incubator maintained at 37 &#176;C with a 5% CO2 atmosphere. 
	NOTE: The entire embedding and slicing protocol should be completed within 90 min of tumor tissue acquisition from the operating room.</li>
</ol>
4. Slice Culture Maintenance
<ol>
	<li>After 12 - 24 h, transfer the slice cultures by grasping each insert rim with a sterile forceps, and transfer to plates containing fresh slice culture maintenance media. Ensure the slice culture maintenance media aliquoted into each well equilibrates in the incubator for at least 15 min before transferring inserts.</li>
	<li>Move inserts to new 6-well plates with fresh equilibrated slice culture media every 48 h.</li>
	<li>If desired, aliquot old slice culture media with a pipette for immediate use in biochemical assays (i.e. ELISA) or freeze at -80 &#176;C for future use.</li>
</ol>
5. Tumor Cell Labeling Via Green Fluorescence Protein Expressing Retrovirus
NOTE: Time-lapse microscopy for analysis of tumor cell migration requires stable, long-term fluorescent labeling of cells within the slice culture. Use of retrovirus is suggested because it selectively infects dividing cells, thereby enriching fluorescent labeling within the tumor cell population as opposed to microglia or other cell types present within the slice. Standardization of infection suggests that a viral titer of 104 CFUs/&#181;L results in sufficient green fluorescent protein expression for the tracking and analysis of cell migration. Increased viral titer, use of non-selective virus (i.e. adenovirus, lentivirus), or other means of labeling all cells may preclude identification of clear cell boundaries during migration, thus complicating analysis. Use of alternative fluorescent markers can be utilized and optimized as needed.
<ol>
	<li>Obtain retrovirus for infection of tumor slices either via standard protocols5,14 or from a commercially available source. Dilute the viral supernatant by adding the appropriate volume into unsupplemented neuronal medium to attain a viral titer of 104 CFUs/&#181;L.</li>
	<li>Between 7 - 10 days of culture infect the tumor slice cultures of interest with 5 - 10 &#181;L of virus (104 CFUs/&#181;L). Place the virus gently dropwise onto the surface of each tissue slice. Decrease supernatant volume added if the slice floats on the surface of the insert. Return plates containing slice cultures to incubator.</li>
	<li>Assess the slices for labeled tumor cells beginning at 24 h after viral infection (see section 6). This time delay will vary dependent on viral incorporation and fluorescence gene expression kinetics. For the viral constructs used here, 72 h was required to observe robust fluorescent signal with a standard epifluorescence microscope. 
	&#8203;NOTE: Rarely, slices display a predominantly peripheral distribution of virally labeled cells, which can complicate confocal imaging. To avoid this complication, attempt to reduce slice thickness and thickness variation across the slice. If the slice culture has a thicker region near the center, this may prevent adequate nutrient penetration, thus limiting the population of actively dividing tumor cells. A qualitative assay to screen for this complication is the addition of a tetrazolium dye reagent (i.e. MTT) to the slice culture media. Areas of the slice that do not turn blue after adding the reagent indicate a lack of metabolic activity and compromised slice health.</li>
</ol>
6. Time-Lapse Single Photon Laser Scanning Confocal Imaging of Tumor Cell Migration
NOTE: After successful transduction and health of the culture is confirmed, cells may be imaged under control conditions, followed by an equal period of imaging under treatment conditions. Using this protocol, cells were successfully imaged and tracked for 12 hours in each condition. However, shorter or longer periods of imaging and environmental manipulation may also be informative.
<ol>
	<li>Loading the Microscope
	<ol>
		<li>Before imaging, place 1 mL of fresh slice media into a glass bottom dish.</li>
		<li>Allow media in the glass bottom dish to equilibrate in an incubator for 15 min. 
		&#8203;NOTE: At this point soluble labeling agents, such as fluorescently conjugated lectins (i.e. Isolectin IB4 for microglia labeling) or ligand conjugated quantum dots, can be added to the slice culture media for identification of cellular subpopulations during imaging.</li>
		<li>Transfer the insert to be imaged to a glass bottom dish using sterile forceps in a laminar flow hood. Transport the dish to the microscope stage.</li>
		<li>Maintain slice cultures at 37 &#176;C and 5% CO2 atmosphere in a sealed microscope stage-top incubator. Utilize sterile H2O humidification of the incubation chamber if available to prevent excessive media evaporation (especially important for longer imaging experiments).</li>
		<li>Utilize a confocal microscope with a long working distance 10X air objective and laser excitation for single-photon and/or multi-photon. 
		&#8203;NOTE: Ensure that the microscope objective lens provides adequate working distance. As a result of the added height from the stage-top incubator, glass bottom dish, and tissue culture insert, long working distance objectives are critical.</li>
		<li>Secure the glass bottom plate, remove the plastic Petri dish cover, and cover with a gas-permeable membrane.</li>
	</ol>
	</li>
	<li>Image Acquisition
	<ol>
		<li>Use the microscope to visually inspect the slice, and locate a suitable field with adequate density of fluorescently labeled tumor cells between the slice edge and the center. Avoid imaging fields at the edge of the slice, due to increased susceptibility of tissue shifts during imaging. Tissue slice harps may be employed to limit this shift.</li>
		<li>Use imaging software in multidimensional analysis mode to set the first (bottom) and last (top) Z-stack boundaries, such that all positions to be imaged contain visible fluorescent cellular signal. Imaging through 150 - 200 &#181;m of the slice, with a constant Z-step of 10 &#181;m provided adequate resolution for tracking cell paths (this can be adjusted for individual imaging needs).</li>
		<li>If the microscope has a motorized stage, ensure adequate time is allowed for each Z-stack acquisition. Set the time interval between acquisitions to equal scan time per position x number of positions to image (i.e. imaged tumor regions). 
		&#8203;NOTE: For simultaneous excitation of the green and red fluorophores, utilize a dual-line laser line-scanning program, with simultaneous 488 nm and 633 nm excitation. The wavelength of laser excitation selected will vary according to individual fluorescent proteins expressed.</li>
		<li>Maintain uniform laser power and confocal pinhole settings between the imaged tumor regions. Utilize the lowest laser power setting needed to clearly demarcate the tumor cell bodies and processes to limit phototoxicity. Specific imaging parameter units (i.e. power and confocal pinhole settings) will vary according to microscope and laser source specifications.</li>
		<li>Compensate for potential media evaporation by adding 2 - 3 &#34;buffer&#34; Z-stack steps in the focal planes advancing towards the objective. This effectively prevents the tissue from leaving the range of Z-stack acquisitions in the vertical plane.</li>
	</ol>
	</li>
</ol>
7. Image Post-Processing and Tumor Cell Tracking
NOTE: Many confocal imaging systems are equipped with proprietary image-processing software. The processing steps discussed below comprise a general protocol, which can be performed across software platforms. Specific instructions will be given for the open-source platforms, NIH ImageJ and MTrackJ15.
<ol>
	<li>Open a Z-stack file. In ImageJ, click &#34;Image&#8594;Stacks&#8594;Z Project.&#34; Chose the first and last Z-stack to include, and chose &#34;Max Intensity&#34; as the projection type. The result is a rendering called a maximum intensity projection (MIP). Create an MIP from each set of Z-stack images captured at each region imaged.</li>
	<li>Concatenate the MIPs from each region to create a time series. Click &#34;Image&#8594;Stacks&#8594;Images to Stack.&#34;</li>
	<li>Manually identify the location of the cell body &#34;centroid&#34; by selecting the visually approximated center point of the tumor cell body. This is accomplished by clicking on the cell body using the &#34;add&#34; track functionality of MTrackJ (an open-source plugin-in for NIH ImageJ).</li>
	<li>Click to demarcate the cell body location in each frame of the series of images. This creates a unique &#34;track&#34; for each cell. Sequentially, mark the cell body locations of the next cell. Repeat this process until all cell migration paths are tracked.</li>
	<li>Once a population of cells is tracked in a given tumor micro region, export all cell track coordinates using the &#34;measure&#34; function of MTrackJ. Save the file as an .xls format so the raw data can be analyzed using spreadsheet or user-generated software.</li>
	<li>Use the coordinates recorded for each point along a cell&#39;s migration track, to perform quantitative analyses including derivation of migration speed, directionality, and other migration metrics, as described in Parker et al16. 
	NOTE: All calculated distances and speeds are under-estimates of actual values. This is inherent in the transformation of three-dimensional images (and migration paths) to two-dimensional data through generation of maximum intensity projections.</li>
</ol>

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The authors have nothing to disclose.

Organotypic slice cultures from human cancer tissue provide an attractive and underutilized platform for pre-clinical translational experimentation. Understanding of population-level behaviors of tumor cells with regards to migration, proliferation, and cell death in the native tumor microenvironment is lacking. Critically, studying tumor response to therapy in a dynamic, time-resolved fashion at the level of cell behavior may shed light on novel mechanisms of treatment resistance. Human tumor slice cultures provide a li...

The laboratory study of glioblastoma (GBM), is hindered by a lack of models that faithfully recapitulate the requisite pathologic characteristics of the human disease, namely tumor cell migration and invasion. Comparative studies of 2D and 3D in vitro invasion assays as well as 3D rodent slice culture models have uncovered mechanistically disparate cellular migration programs in these two contexts, potentially limiting the translatability of findings from 2D systems to the human disease1,2,3. The organotypic tumor slice culture and imaging paradigm described here allows for the study of tumor cell migration within slices of ex vivo human tumor tissue obtained from surgical resection. Thus, slice cultures of surgically resected tumor tissue in conjunction with time-lapse confocal microscopy provide a platform to study tumor cell migration in the native microenvironment without tissue dissolution or culture passaging.
There is extensive literature employing rodent brain slice culture models of GBM generated from human tumor xenografts, retroviral-induced tumors, and cellular overlays to study tumor invasion1,2,3,4,5. Recently, several groups have described the generation of organotypic slice cultures directly from human GBM tissue6,7,8,9,10. However, there is marked variation among published protocols with regards to slicing technique and culture media. Further, the use of organotypic slice cultures has focused on static experimental endpoints that have included changes in cell signaling, proliferation, and death. The protocol described herein expands upon prior slice culture paradigms by incorporating time-resolved observation of dynamic tumor cell behaviors through time-lapse laser scanning confocal microscopy. Recent discovery of inter11 and intratumoral12,13 genetic variation in human GBM underlines the importance of linking this heterogeneity with tumor cell behaviors and its implications on tumor response to therapy. Here, we report a streamlined and reproducible protocol for use of direct slice cultures from a human cancer tissue to visualize tumor cell migration in near real-time.

<table><tbody><tr><td>DMEM High Glucose&amp;nbsp;</td><td>Invitrogen (Gibco)</td><td>11960-044</td><td></td></tr><tr><td>Neurobasal-A Medium, minus phenol red</td><td>Invitrogen (Gibco)</td><td>12349-015</td><td></td></tr><tr><td>B-27 Supplement (50x), serum free</td><td>Invitrogen (Gibco)</td><td>17504-044</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Invitrogen (Gibco)</td><td>15140-122</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Invitrogen (Gibco)</td><td>35050-061</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Invitrogen (Gibco)</td><td>25030-081</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Invitrogen (Gibco)</td><td>15630-080</td><td></td></tr><tr><td>Nystatin Suspension</td><td>Sigma-Aldrich</td><td>N1638-20ML</td><td>10,000 unit/mL in DPBS, aseptically processed, BioReagent, suitable for cell culture</td></tr><tr><td>UltraPure Low Melting Point Agarose</td><td>Invitrogen (Gibco)</td><td>16520-050</td><td>Melts at 65.5 &amp;#176;C, Remains fluid at 37 &amp;#176;C, and sets rapidly below 25 &amp;#176;C.</td></tr><tr><td>Isolectin GS-IB4 from &lt;em&gt;Griffonia simplicifolia&lt;/em&gt;, Alexa Fluor 647 Conjugate</td><td>Thermo Fisher (Molecular Probes)</td><td>I32450</td><td>Used in media to label Microglia/Macrophages</td></tr><tr><td>pRetroX-IRES-ZsGreen1 Vector</td><td>Clonetech</td><td>632520</td><td></td></tr><tr><td>Retro-X Concentrator&amp;nbsp;</td><td>Clonetech</td><td>31455</td><td>Binding resin for non-ultracentrifugation concentration of viral supernatants</td></tr><tr><td>pVSG-G Vector</td><td>Clonetech</td><td>631530</td><td>part of the Retro-X Universal Retroviral Expression System</td></tr><tr><td>GP2-293 Viral packaging cells</td><td>Clonetech</td><td>631530</td><td>part of the Retro-X Universal Retroviral Expression System</td></tr><tr><td>Cyanoacrylate Glue (Super Glue)</td><td>Sigma-Aldrich</td><td>Z105899</td><td>Medium-viscosity</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Peel-A-Way Embedding Mold (Square - S22)</td><td>Polysciences, Inc.</td><td>18646A-1</td><td>Molds for tumor sample embedding</td></tr><tr><td>Stainless Steel Micro Spatulas</td><td>Fisher Scientific</td><td>S50823</td><td>Bend instrument 45 degrees at the neck of the spoon blade</td></tr><tr><td>Curved Fisherbrand Dissecting Fine-Pointed Forceps</td><td>Fisher Scientific&amp;nbsp;</td><td>08-875</td><td></td></tr><tr><td>Single Edge Razor Blade (American Safety Razors)</td><td>Fisher Scientific</td><td>17-989-001</td><td>Blade edge is 0.009&quot; thick. Crimped blunt-edge cover is removed before loading onto vibratome.</td></tr><tr><td>Leica VT1000 S Vibratome</td><td>Leica Biosystems</td><td>VT1000 S</td><td></td></tr><tr><td>Hydrophilic PTFE cell culture insert&amp;nbsp;</td><td>EMD Millipore</td><td>PICM0RG50</td><td>30 mm, hydrophilic PTFE, 0.4 &amp;micro;m pore size</td></tr><tr><td>35 mm Glass Bottom Dishes&amp;nbsp;</td><td>MatTek</td><td>P35G-1.5-20-C Sleeve</td><td>20 mm glass diameter. Coverslip glass thickness 1.5 mm</td></tr><tr><td>LSM 510 Confocal Micoscope</td><td>Zeiss</td><td>LSM 510</td><td>10x Air Objective (c-Apochromat NA 0.45)</td></tr><tr><td>PECON Stagetop Incubator</td><td>PeCON Germany</td><td>(Discontinued)</td><td>Incubator PM 2000 RBT is a comprable product designed for use with Zeiss Microscopes.</td></tr></tbody></table>

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.

Our group has successfully generated slice cultures from over 50 patients undergoing initial GBM resection. This slice generation, culture, retroviral-labeling, imaging, and migration analysis protocol has been streamlined into a reproducible workflow (Figure 1). Critically, these organotypic GBM slices demonstrate concordance with originating tumor tissue throughout culture, including maintenance of pathologic hallmarks and microglia up to 15 days in culture (Fig...

Watch this Scientific Journal Video about A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs at JoVE.com

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

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 ...

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Research

JoVE Journal

Medicine

12.8K Views.  Stanford University Hospital, Stanford University School of Medicine.  Huidige ex vivo modellen van glioblastoom (GBM) zijn niet geoptimaliseerd voor fysiologisch relevant onderzoek naar menselijke tumorinval. Hier presenteren we een protocol voor het genereren en onderhouden van organotypische plakculturen uit vers menselijk GBM-weefsel. Een beschrijving van time-lapse microscopie en kwantitatieve cel migratie analyse technieken wordt verschaft. 

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

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 ...

Cancer Research

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

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 ...

LEGO: A Human GBM Organoid Model Revealing Lipid Dysregulation and Drug Targets

Laboratory-Engineered Glioblastoma Organoid Culture and Drug Screening

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Here, we outline a comprehensive protocol for generating and utilizing laboratory-engineered glioblastoma organoids (LEGO) to investigate genotype-phenotype dependencies and screen potential drugs for glioblastoma treatment.

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Neuroscience

Using Chick Embryo Brain as a Model to Study Human Glioblastoma Cell Behavior

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

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. 
Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 &#181;m) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 &#181;m) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. 
Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

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

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

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...

Establishing a Brain Tumor in an Organotypic Slice Co-culture Model

Source: Chadwick, E. J. et al. <a href="https://app.jove.com/v/53304">A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies.</a> J. Vis. Exp. (2015).
This video demonstrates the development of a 3D co-culture system. Mouse brain slices are placed on a membrane, and fluorescently labeled human brain tumor cells are allowed to grow on it. Once the system is ready, it is stained for imaging.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plating the Slices onto the ...

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

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