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.

a-human-glioblastoma-organotypic-slice-culture-model-for-study-tumor

Research

JoVE Journal

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 management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

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

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 treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

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

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

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 malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer 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 by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

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

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

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

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

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 rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

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

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 cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

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 lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on 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 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 ...

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 element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

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