Name: a human glioblastoma organotypic slice culture model for study of tumor cell migration and patient-specific effects of anti-invasive drugs
Uploaded: 2017-07-20T12:30:00
Description: 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

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...

<ol>
	<li>Beadle, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+myosin+II+in+glioma+invasion+of+the+brain.">The role of myosin II in glioma invasion of the brain.</a> Mol Biol Cell. 19, 3357-3368 (2008).</li><li>Farin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+glioma+cells+migrate+and+proliferate+on+host+brain+vasculature:+a+dynamic+analysis.">Transplanted glioma cells migrate and proliferate on host brain vasculature: a dynamic analysis.</a> Glia. 53, 799-808 (2006).</li><li>Panopoulos, A., Howell, M., Fotedar, R., Margolis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+motility+occurs+in+the+absence+of+actin+polymer.">Glioblastoma motility occurs in the absence of actin polymer.</a> Mol Biol Cell. 22, 2212-2220 (2011).</li><li>Ivkovic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+inhibition+of+myosin+II+effectively+blocks+glioma+invasion+in+the+presence+of+multiple+motogens.">Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens.</a> Mol Biol Cell. 23, 533-542 (2012).</li><li>Assanah, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+progenitors+in+adult+white+matter+are+driven+to+form+malignant+gliomas+by+platelet-derived+growth+factor-expressing+retroviruses.">Glial progenitors in adult white matter are driven to form malignant gliomas by platelet-derived growth factor-expressing retroviruses.</a> J Neurosci. 26, 6781-6790 (2006).</li><li>Chaichana, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+glial+cytoarchitecture+from+ex+vivo+human+tumor+and+non-tumor+cerebral+cortical+explants:+A+human+model+to+study+neurological+diseases.">Preservation of glial cytoarchitecture from ex vivo human tumor and non-tumor cerebral cortical explants: A human model to study neurological diseases.</a> J Neurosci Methods. 164, 261-270 (2007).</li><li>Grube, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+fatty+acid+synthase+in+human+gliomas+correlates+with+the+WHO+tumor+grade+and+inhibition+with+Orlistat+reduces+cell+viability+and+triggers+apoptosis.">Overexpression of fatty acid synthase in human gliomas correlates with the WHO tumor grade and inhibition with Orlistat reduces cell viability and triggers apoptosis.</a> J Neurooncol. 118, 277-287 (2014).</li><li>Hovinga, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+notch+signaling+in+glioblastoma+targets+cancer+stem+cells+via+an+endothelial+cell+intermediate.">Inhibition of notch signaling in glioblastoma targets cancer stem cells via an endothelial cell intermediate.</a> Stem Cells. 28, 1019-1029 (2010).</li><li>Merz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+cultures+of+human+glioblastoma+reveal+different+susceptibilities+to+treatments.">Organotypic slice cultures of human glioblastoma reveal different susceptibilities to treatments.</a> Neurooncol. 15, 670-681 (2013).</li><li>Xu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vorinostat+modulates+cell+cycle+regulatory+proteins+in+glioma+cells+and+human+glioma+slice+cultures.">Vorinostat modulates cell cycle regulatory proteins in glioma cells and human glioma slice cultures.</a> J Neurooncol. 105, 241-251 (2011).</li><li>Verhaak, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+genomic+analysis+identifies+clinically+relevant+subtypes+of+glioblastoma+characterized+by+abnormalities.">Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities.</a> in PDGFRA, IDH1, EGFR, and NF1. Cancer cell. 17, 98-110 (2010).</li><li>Gill, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI-localized+biopsies+reveal+subtype-specific+differences+in+molecular+and+cellular+composition+at+the+margins+of+glioblastoma.">MRI-localized biopsies reveal subtype-specific differences in molecular and cellular composition at the margins of glioblastoma.</a> Proc Natl Acad Sci USA. 111, 12550-12555 (2014).</li><li>Snuderl, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+amplification+of+multiple+receptor+tyrosine+kinase+genes+in+glioblastoma.">Mosaic amplification of multiple receptor tyrosine kinase genes in glioblastoma.</a> Cancer cell. 20, 810-817 (2011).</li><li>Kakita, A., Goldman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+and+dynamics+of+SVZ+cell+migration+in+the+postnatal+forebrain:+monitoring+living+progenitors+in+slice+preparations.">Patterns and dynamics of SVZ cell migration in the postnatal forebrain: monitoring living progenitors in slice preparations.</a> Neuron. 23, 461-472 (1999).</li><li>Meijering, E., Dzyubachyk, O., Smal, I., van Cappellen, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+in+cell+and+developmental+biology.">Tracking in cell and developmental biology.</a> Sem Cell Dev Biol. 20, 894-902 (2009).</li><li>Parker, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gefitinib+selectively+inhibits+tumor+cell+migration+in+EGFR-amplified+human+glioblastoma.">Gefitinib selectively inhibits tumor cell migration in EGFR-amplified human glioblastoma.</a> Neurooncol. 15, 1048-1057 (2013).</li><li>Brat, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudopalisades+in+glioblastoma+are+hypoxic,+express+extracellular+matrix+proteases,+and+are+formed+by+an+actively+migrating+cell+population.">Pseudopalisades in glioblastoma are hypoxic, express extracellular matrix proteases, and are formed by an actively migrating cell population.</a> Cancer Res. 64, 920-927 (2004).</li><li>Shweiki, D., Itin, A., Soffer, D., Keshet, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+induced+by+hypoxia+may+mediate+hypoxia-initiated+angiogenesis.">Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.</a> Nature. 359, 843-845 (1992).</li><li>Shamir, E. R., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+culture:+experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: experimental models of mammalian biology and disease.</a> Nat Rev Mol Cell Biol. 15, 647-664 (2014).</li><li>Charles, N. A., Holland, E. C., Gilbertson, R., Glass, R., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+tumor+microenvironment.">The brain tumor microenvironment.</a> Glia. 60, 502-514 (2012).</li><li>Di Cristofori, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vacuolar+H++ATPase+is+a+novel+therapeutic+target+for+glioblastoma.">The vacuolar H+ ATPase is a novel therapeutic target for glioblastoma.</a> Oncotarget. 6, 17514-17513 (2015).</li><li>Vaira, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> Proc Natl Acad Sci USA. , 8352-8356 (2010).</li><li>Gerlach, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+cultures+from+head+and+neck+squamous+cell+carcinoma:+a+novel+test+system+for+drug+susceptibility+and+mechanisms+of+resistance.">Slice cultures from head and neck squamous cell carcinoma: a novel test system for drug susceptibility and mechanisms of resistance.</a> Br J Cancer. 110, 479-488 (2014).</li><li>Holliday, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+practicalities+of+using+tissue+slices+as+preclinical+organotypic+breast+cancer+models.">The practicalities of using tissue slices as preclinical organotypic breast cancer models.</a> J Clin Pathol. 66, 253-255 (2013).</li><li>Maund, S. L., Nolley, R., Peehl, D. 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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 fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM High Glucose&#160;</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 C, Remains fluid at 37 C, and sets rapidly below 25 C.</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 from Griffonia simplicifolia, 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&#160;</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</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&#160;</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&#34; 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&#160;</td>
			<td>EMD Millipore</td>
			<td>PICM0RG50</td>
			<td>30 mm, hydrophilic PTFE, 0.4 &#181;m pore size</td>
		</tr>
		<tr>
			<td>35 mm Glass Bottom Dishes&#160;</td>
			<td>MatTek</td>
			<td>P35G-1.5-20-C Sleeve</td>
			<td>20mm glass diameter. Coverslip glass thickness 1.5</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 ...

The overall goal of this procedure is to observe and analyze invasive migratory behaviors in primary human glioblastoma cells. This method can help answer key questions in the field of neurooncology, such as what are the phenotypic and molecular properties underlying patient-specific differences in disease progression and treatment responses? The main advantage of this technique is that it allows the direct manipulation of primary cells while preserving the in vivo tissue architecture and cellular heterogeneity of glioblastoma.Begin by using sterile forceps to place empty PTFE culture inserts into each well of a six-well plate containing one milliliter of slice culture maintenance medium per well. When all of the inserts have been added, place the plate in a humidified water-jacketed tissue culture incubator at 37 degrees Celsius and 5%CO2. Next, place the tumor tissue pieces in a Petri dish containing ice-cold processing medium, and use a pipette to gently wash the tissue three times with fresh medium to remove any adherent red blood cells.After the third wash, use the scalpel to cut the tumor pieces into approximately three by three by 10 millimeter strips, carefully removing any attached vessels and avoiding necrotic or cauterized tissue. When all of the tissue has been trimmed, add five to seven millimeters of 37 degree Celsius liquid agarose into a two-cubed centimeter plastic embedding mold, and chill the agarose on a bed of ice for one minute. Place two to four strips of tissue into the agarose with the long axis oriented vertically, leaving the tissues on ice for another two to five minutes after their placement.With the agarose has solidified, cut the sides of the form with a scalpel to gently remove the agarose from the mold. And use a generous drop of cyanoacrylate glue to fix the agarose block to a vibratome specimen plate. When the glue has set, submerge the agarose block in ice-cold processing medium and use a trimmed sterile plastic pipette to bubble a 5%CO2, 95%O2 mixture into the medium within the vibratome reservoir.Set the vibratome slice thickness to 300 to 350 microns, and adjust the blade advance speed and blade amplitude according to the tissue consistency and vibratome specifications. Obtain the appropriate number of slices according to the planned experimental analysis, using a stainless steel micro spatula to transfer the slices into a Petri dish containing ice-cold processing medium as they are acquired. When all of the slices have been acquired, use the micro spatula to transfer each slice onto a single PTFE insert.After a 12 to 24 hour incubation, equilibrate a new six-well plate containing fresh slice culture maintenance medium in the cell culture incubator for 15 minutes. Then use sterile forceps to grasp each insert by the rim and transfer the inserts into individual wells of the new six-well plate. Between seven to 10 days of culture, gently add five to 10 microliters of GFP-expressing retrovirus dropwise onto the surface of each tissue slice and return the plate to the incubator.When the signal is apparent, equilibrate one milliliter of fresh slice medium in a glass-bottom dish and use sterile forceps to transfer the inserts into the dish. Place the dish into a 37 degree, 5%CO2 sealed microscope stage top incubator of a confocal microscope. Use a long working distance, 10x air objective in conjunction with single or multi-photon imaging to capture three-dimensional time-resolved images of cell migration.Visualize the slice with the microscope, locating a suitable field with an adequate density of fluorescently-labeled tumor cells between the slice edge and the center of the tissue. Then in the multidimensional analysis mode of the imaging software, set the center of the Z-stack such that all of the positions to be imaged contain a visible fluorescent cellular signal, and set an adequate time for each Z-stack acquisition. To begin, open a Z-stack file in ImageJ and select Image, Stacks, and Z Project.Select the first and last time frame of the Z-stack for analysis, and choose Max Intensity as the projection type to generate a maximum intensity projection or MIP rendering. To generate MIPs for each time point in the series at once, check the box All time frames. Create an MIP from each set of Z-stack images captured at each region imaged.Open MTrackJ from the Plugins menu, and click the Add button. To manually identify the cell body centroid in the tumor cell of interest, click on the visually approximated center point of the cell. Demarcate the cell body location in each timeframe of the series of images to create a unique track for each cell, sequentially marking the cell body locations of the next cell.Once a population of cells has been tracked in a given tumor microregion, use the measure function to export all of the cell track coordinates and save the files in the xls format for later analysis. Finally, use the coordinates recorded for each point along a cell's migration track to perform quantitative analyses of the tumor invasion. Organotypic glioblastoma slices demonstrate a concordance with the originally tumor tissue throughout the culture, including the maintenance of pseudopalisading necrosis and microglia for up to 15 days.Under hypoxic conditions, the slices mount a rapid physiologic response, inducing the release of vascular endothelial growth factor into the medium, a process that occurs abundantly within the glioblastoma microenvironment in vivo. The quantitative measurement of time-lapsed image GFP-expressing tumor cells allows the calculation of the migration speed and the directionality of the glioblastoma cells across tumor regions in samples. Tracking the migration of intermingled but morphologically distinct motile tumor cells in microglia within a slice culture reveal cell type-specific patterns of movements.Imaging the slice regions approximately every 10 minutes also provides an adequate temporal resolution for recording time lapse images of the tumor cells undergoing cell division. For example, here are a pair of dividing cells that were captured as they paused for migration, completed mitosis, and reinitiated migration in their daughter cells without delay, all within a three-hour time frame. Once mastered, this technique can be completed in 90 minutes if it's performed properly.

Research

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Medicine

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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 ...

Assessing Specificity of Anticancer Drugs In Vitro

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...