This work was funded in part by a grant to D.S.G. from the National Cancer Institute (R03CA227312) and by a generous grant from the Lisa Dean Moseley Foundation. Live GBM specimens were obtained with patient consent through the Tissue Procurement Center of the Helen F. Graham Cancer Center and Research Institute. Funding to A.R. was provided by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health (UL1TR003107). Summer undergraduate research fellowships to N.P., A.L., Z.W., and K.S. were provided by the University of Delaware Undergraduate Research Program.

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

<ol>
	<li>Cretu, A., Fotos, J. S., Little, B. W., Galileo, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+rat+glioma+growth,+invasion,+and+vascularization+in+a+novel+chick+embryo+brain+tumor+model.">Human and rat glioma growth, invasion, and vascularization in a novel chick embryo brain tumor model.</a> Clinical &amp; Experimental Metastasis. 22 (3), 225-236 (2005).</li><li>Yang, 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=L1+stimulation+of+human+glioma+cell+motility+correlates+with+FAK+activation.">L1 stimulation of human glioma cell motility correlates with FAK activation.</a> Journal of Neuro-Oncology. 105 (1), 27-44 (2011).</li><li>Mohanan, V., Temburni, M. K., Kappes, J. C., Galileo, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1CAM+stimulates+glioma+cell+motility+and+proliferation+through+the+fibroblast+growth+factor+receptor.">L1CAM stimulates glioma cell motility and proliferation through the fibroblast growth factor receptor.</a> Clinical &amp; Experimental Metastasis. 30 (4), 507-520 (2013).</li><li>Anderson, H. J., Galileo, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+FGFR,+integrins+and+FAK+selectively+decrease+L1CAM-stimulated+glioblastoma+cell+motility+and+proliferation.">Small-molecule inhibitors of FGFR, integrins and FAK selectively decrease L1CAM-stimulated glioblastoma cell motility and proliferation.</a> Cellular Oncology. 39 (3), 229-242 (2016).</li><li>Pace, K. R., Dutt, R., Galileo, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+L1CAM+stimulates+glioblastoma+cell+motility,+proliferation,+and+invasiveness.">Exosomal L1CAM stimulates glioblastoma cell motility, proliferation, and invasiveness.</a> International Journal of Molecular Sciences. 20 (16), 3982 (2019).</li><li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Ohnishi, T., Matsumura, H., Izumoto, S., Hiraga, S., Hayakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+glioma+cell+invasion+using+organotypic+brain+slice+culture.">A novel model of glioma cell invasion using organotypic brain slice culture.</a> Cancer Research. 58 (14), 2935-2940 (1998).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Aaberg-Jessen, 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=Invasion+of+primary+glioma-+and+cell+line-derived+spheroids+implanted+into+corticostriatal+slice+cultures.">Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures.</a> International Journal of Clinical and Experimental Pathology. 6 (4), 546-560 (2013).</li><li>Matsumura, H., Ohnishi, T., Kanemura, Y., Maruno, M., Yoshimine, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+glioma+cell+invasion+by+confocal+laser+scanning+microscopy+in+a+novel+brain+slice+model.">Quantitative analysis of glioma cell invasion by confocal laser scanning microscopy in a novel brain slice model.</a> Biochemical and Biophysical Research Communications. 269 (2), 513-520 (2000).</li><li>Ren, B., 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=Invasion+and+anti-invasion+research+of+glioma+cells+in+an+improved+model+of+organotypic+brain+slice+culture.">Invasion and anti-invasion research of glioma cells in an improved model of organotypic brain slice culture.</a> Tumori. 101 (4), 390-397 (2015).</li><li>Fayzullin, 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=Time-lapse+phenotyping+of+invasive+glioma+cells+ex+vivo+reveals+subtype-specific+movement+patterns+guided+by+tumor+core+signaling.">Time-lapse phenotyping of invasive glioma cells ex vivo reveals subtype-specific movement patterns guided by tumor core signaling.</a> Experimental Cell Research. 349 (2), 199-213 (2016).</li><li>Jensen, S. 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=Establishment+and+characterization+of+a+tumor+stem+cell-based+glioblastoma+invasion+model.">Establishment and characterization of a tumor stem cell-based glioblastoma invasion model.</a> PloS One. 11 (7), e0158746 (2016).</li><li>Marques-Torrejon, M. A., Gangoso, E., Pollard, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+glioblastoma+tumour-host+cell+interactions+using+adult+brain+organotypic+slice+co-culture.">Modelling glioblastoma tumour-host cell interactions using adult brain organotypic slice co-culture.</a> Disease Models &amp; Mechanisms. 11 (2), 031435 (2018).</li><li>Tamura, R., 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=Visualization+of+spatiotemporal+dynamics+of+human+glioma+stem+cell+invasion.">Visualization of spatiotemporal dynamics of human glioma stem cell invasion.</a> Molecular Brain. 12 (1), 45 (2019).</li><li>Yang, 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=Organotypic+slice+culture+based+on+in+ovo+electroporation+for+chicken+embryonic+central+nervous+system.">Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system.</a> Journal of Cellular and Molecular Medicine. 23 (3), 1813-1826 (2019).</li><li>Murrell, W., 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=Expansion+of+multipotent+stem+cells+from+the+adult+human+brain.">Expansion of multipotent stem cells from the adult human brain.</a> PloS One. 8 (8), e71334 (2013).</li><li>Fotos, J. 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=Automated+time-lapse+microscopy+and+high-resolution+tracking+of+cell+migration.">Automated time-lapse microscopy and high-resolution tracking of cell migration.</a> Cytotechnology. 51 (1), 7-19 (2006).</li><li>Tufan, A. C., Akdogan, I., Adiguzel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+culture+of+the+chick+embryo+as+a+model+system+in+the+study+of+developmental+neurobiology.">Shell-less culture of the chick embryo as a model system in the study of developmental neurobiology.</a> Neuroanatomy. 3 (1), 8-11 (2004).</li><li>Shoin, K., 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=Chick+embryo+assay+as+chemosensitivity+test+for+malignant+glioma.">Chick embryo assay as chemosensitivity test for malignant glioma.</a> Japanese Journal of Cancer Research. 82 (10), 1165-1170 (1991).</li><li>Hagedorn, 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=Accessing+key+steps+of+human+tumor+progression+in+vivo+by+using+an+avian+embryo+model.">Accessing key steps of human tumor progression in vivo by using an avian embryo model.</a> Proceedings of the National Academy of Sciences. 102 (5), 1643-1648 (2005).</li><li>Balci&#363;niene, N., 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=Histology+of+human+glioblastoma+transplanted+on+chicken+chorioallantoic+membrane.">Histology of human glioblastoma transplanted on chicken chorioallantoic membrane.</a> Medicina. 45 (2), 123-131 (2009).</li><li>De Magalh&#227;es, N., 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=Applications+of+a+new+In+vivo+tumor+spheroid+based+shell-less+chorioallantoic+membrane+3-D+model+in+bioengineering+research.">Applications of a new In vivo tumor spheroid based shell-less chorioallantoic membrane 3-D model in bioengineering research.</a> Journal of Biomedical Science and Engineering. 3 (1), 20-26 (2010).</li><li>Szmidt, 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=Morphology+of+human+glioblastoma+model+cultured+in+ovo.">Morphology of human glioblastoma model cultured in ovo.</a> Journal of Veterinary Research. 56 (2), 261-266 (2012).</li><li>Jaworski, 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=Comparison+of+tumor+morphology+and+structure+from+U87+and+U118+glioma+cells+cultured+on+chicken+embryo+chorioallantoic+membrane.">Comparison of tumor morphology and structure from U87 and U118 glioma cells cultured on chicken embryo chorioallantoic membrane.</a> Journal of Veterinary Research. 57 (4), 593-598 (2013).</li><li>Yuan, Y. J., Xu, K., Wu, W., Luo, Q., Yu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+chick+embryo+chorioallantoic+membrane+in+neurosurgery+disease.">Application of the chick embryo chorioallantoic membrane in neurosurgery disease.</a> International Journal of Medical Sciences. 11 (12), 1275-1281 (2014).</li><li>Urba&#324;ska, K., 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+effect+of+silver+nanoparticles+(AgNPs)+on+proliferation+and+apoptosis+of+in+ovo+cultured+glioblastoma+multiforme+(GBM)+cells.">The effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells.</a> Nanoscale Research Letters. 10, 98 (2015).</li><li>DeBord, L. 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+chick+chorioallantoic+membrane+(CAM)+as+a+versatile+patient-derived+xenograft+(PDX)+platform+for+precision+medicine+and+preclinical+research.">The chick chorioallantoic membrane (CAM) as a versatile patient-derived xenograft (PDX) platform for precision medicine and preclinical research.</a> American Journal of Cancer Research. 8 (8), 1642-1660 (2018).</li><li>Han, J. M., Jung, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+anticancer+effect+of+a+combination+of+berbamine+and+arcyriaflavin+A+against+glioblastoma+stem-like+cells.">Synergistic anticancer effect of a combination of berbamine and arcyriaflavin A against glioblastoma stem-like cells.</a> Molecules. 27 (22), 7968 (2022).</li><li>Ruiz-Onta&#241;on, P., 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=Cellular+plasticity+confers+migratory+and+invasive+advantages+to+a+population+of+glioblastoma-initiating+cells+that+infiltrate+peritumoral+tissue.">Cellular plasticity confers migratory and invasive advantages to a population of glioblastoma-initiating cells that infiltrate peritumoral tissue.</a> Stem Cells. 31 (6), 1075-1085 (2013).</li><li>Cage, T. 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=Distinct+patterns+of+human+medulloblastoma+dissemination+in+the+developing+chick+embryo+nervous+system.">Distinct patterns of human medulloblastoma dissemination in the developing chick embryo nervous system.</a> Clinical &amp; Experimental Metastasis. 29 (4), 371-380 (2012).</li><li>Darrigues, 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=Biobanked+glioblastoma+patient-derived+organoids+as+a+precision+medicine+model+to+study+inhibition+of+invasion.">Biobanked glioblastoma patient-derived organoids as a precision medicine model to study inhibition of invasion.</a> International Journal of Molecular Sciences. 22 (19), 10720 (2021).</li></ol>

None of the authors have any conflicts of interest.

Critical steps in the protocol for the injection of cells into the midbrain (optic tectum) ventricle include not damaging the blood vessels in the chorioallantoic membrane in the egg or surrounding the embryo before and during injection, although the amnion membrane immediately surrounding the embryo can be gently pulled and held to position the head when injecting the cells into the midbrain. The amnion is relatively tough and can be pulled with fine forceps to position the head and hold it steady with one hand, for the injection of cells with the other hand into the optic tectum, which is the large, round structure in the middle of the brain. Generally, the viability of injected embryos ranges from 25% to 75%, depending upon unknown factors, and practically every embryo that survives contains at least a small tumor in the optic tectum. Critical steps in generating viable brain slices include blotting the tissue of excess liquid so that the agarose adheres to the brain during slicing and to keep tissue and slices cold until placed on the membrane insert. As different cell types form spheroids differently (in speed and size), the plated cell density on poly-HEMA plates and the length of time before harvesting spheroids should be optimized for each cell type.
The work here has not been subject to a formal longitudinal study of brain slice viability. Yang et al. used chick embryo brain slice cultures similar to the ones used here and showed good viability of the slices for at least 7 days16. Previous work showed that when OT tissue was kept in suboptimal media, many pyknotic nuclei appeared in the tissue, which did not occur in the slices in the work here. Additionally, when slices degenerate in suboptimal conditions, the blood vessels fragment and appear as rows of laminin-positive spheres (not shown). Thus, although the viability here has not been checked by methods such as electrophysiology or active caspase-3 expression, none of the indicators of cell death that were seen under suboptimal culture conditions appeared here.
The OT has been focused on for in vivo brain tumor experiments because it is the most easily injected region with the largest ventricle. At E5, which is the latest day that the embryo is small enough to remain accessible on top of the yolk, injections must be made into a ventricle, as all brain regions are nothing more than a thin ventricular zone. Nevertheless, these injections result successfully in embedded tumors with cells that invade the brain parenchyma. Sometimes, resulting tumors are found in the forebrain or cerebellum, but this is not common. Ex vivo slices of E15 optic tectum have been primarily used for experiments here, so that the ex vivo co-culture results can be correlated with the in vivo injection experiments. However, forebrain slices are also suitable and have a larger surface area and a very thin ventricle compared to the optic tectum, which might make the forebrain more suitable for ex vivo co-cultures that are not being correlated with in vivo injections.
It has been demonstrated here that in vivo injections, followed by tissue fixation, vibrating tissue slicer sectioning, and immunostaining for laminin and other markers, resulted in high-resolution images of GBM cells and GSCs in brain tissue in close proximity to blood vessels. The ability to determine the interrelationships between tumor cells and blood vessels was greatly facilitated by creating 3D volume renders from z-stacks of confocal optical sections using the confocal software and manufacturer&#39;s instructions. Time-lapse imaging using widefield fluorescence microscopy of GFP, mCherry, and DiD labeled cells was possible; however, migrating cells that were in close proximity to the highly fluorescent spheroids were sometimes obscured by the &#34;glow&#34; from the spheroid. This undesired effect can be somewhat minimized by carefully adjusting the exposure times for collecting widefield images. Time-lapse imaging using confocal z-stacks over time (4D) eliminated the out-of-focus glow from the spheroids and resulted in sharply defined migrating cells with a dark background. This was not described in the protocol, but was carried out similarly to widefield time-lapse imaging, which was performed while brain slices were on the transparent membrane inserts in a 6-well plastic plate. Although confocal time-lapse imaging results in markedly clearer images of individual cells and their behavior, a multi-point time-lapse experiment collecting z-stacks of 10 z-planes/point, at 10 min intervals over a 20 h period, is an extensive use of the scan head galvanometers. As this could significantly decrease the lifespan of the galvanometers, this method is used judiciously.
Although the chick embryo system is very suitable for both in vivo injection and ex vivo co-culture experiments that investigate GBM cell behavior, there are several limitations to this model system. As with any xenograft system, the environment in which human cells are implanted is not the human brain, but GBM cell behavior appears to mimic that in rodent models and in human patients. After performing in vivo injection experiments on E5, tumors are normally allowed to form for 10 days, until E15. This clearly is not enough time to study all aspects of tumorigenesis and cell invasion. However, it has been demonstrated here that solid tumors form in the brain parenchyma, cells interact and rearrange themselves within the tumor, and significant brain invasion occurs both along blood vessels and diffusely within this relatively short time period. Another limitation to the in vivo chick embryo system is that it is not suitable for drug or other treatments because of the large yolk and extraembryonic circulatory system that operates during chick embryo development. Topical liquid drug treatments would result in a highly variable and unknown concentration in the brain due to diffusion away from the embryo into the much larger yolk mass. Similarly, intravenous injection of drugs into the very delicate extraembryonic circulatory system would leak or diffuse out of the blood vessels and also result in unknown concentrations in the brain. This is one of the main reasons that the ex vivo slice culture method was adopted-so that not only could cell behavior be observed and tracked via time-lapse microscopy, but also so that treatments that have been successful in altering GBM cell behavior in a dish4 could be tested in a more relevant brain tissue environment.
The development of the chick embryo orthotopic brain tumor model system is seen as a significant addition to the systems and tools available for the study of GBM tumor formation and invasive cell behavior. Fertilized chicken eggs are likely to be readily available in most areas, they are inexpensive compared to rodents, there are no animal care costs, the embryos are very resilient and resistant to infection (i.e., most work is done on a bench top), the embryos are highly manipulable and can be grown in shell-less culture19, and chick embryos are not considered vertebrate animals and so do not require IACUC approval by NIH guidelines (institutional requirements may vary). Thus, these multiple advantages make the chick embryo system very attractive if one confines their questions and experiments to those that fall within its limitations. Multiple GBM cell studies have been performed by others using the chick embryo, but these almost exclusively have utilized the chorioallantoic membrane (CAM) of the embryo20,21,22,23,24,25,26,27,28,29 and limb bud30, and not the brain. There has also been a report implanting medulloblastoma into the chick brain on E231. Undoubtedly, the use of the chick embryo as an orthotopic xenograft model system, as described here, should yield results that are much more meaningful to human GBM tumor biology than studies using the CAM.
Although these studies have only begun to fully utilize the chick embryo brain tumor model system for studies of human GBM cell and GSC behavior, it is hoped that others will extend the uses and find further potential applications. One could imagine that not only will this system uncover mechanisms that regulate GBM tumor formation and cell behavior, but will also allow preclinical testing of specific drugs and substances on specific patients&#39; cells. For instance, if brain slice cultures were set up in advance, then tumor cells, pieces from surgical tumor resections, or patient-derived GBM organoids32 could be placed directly in ex vivo co-culture, and various treatments could be assessed in a matter of days. Similarly, dissociated patient cells could be injected directly into E5 midbrains in ovo to assess their ability to form tumors and invade brain parenchyma. Thus, it is hoped that the descriptions of the methods and representative results here will facilitate and encourage increased use of this highly underutilized system for brain cancer research.

In vitro studies of cancer cell behaviors are often used to dissect potential mechanisms that operate during the more complex behavior that is observed during tumor formation and cell invasion in in vivo xenograft models. For example, with glioblastoma (GBM), in vitro studies have uncovered mechanisms of how L1CAM potentially operates during tumor formation and brain invasion in a novel chick embryo xenograft brain tumor model1,2,3,4,5. Although in vitro and in vivo experiments complement each other in useful ways, they leave a substantial gap in how the results can be correlated. For instance, mechanistic analyses of GBM cell motility on a dish is a highly artificial situation, and in vivo xenograft models can only reveal static time point or endpoint analyses of tumor formation and cell behavior. In vivo studies using either rodents or chick embryos do not easily lend themselves to monitoring cell behavior while the cells invade brain tissue in these xenograft models. Nevertheless, the chick embryo xenograft model has demonstrated that the adhesion protein L1CAM plays a stimulatory role in the invasive ability of human T98G GBM cells2,5.
A suitable solution to this problem can be reached by bridging both in vivo and in vitro methods using an organotypic brain slice culture model, referred to as an ex vivo model. In this ex vivo model, live brain tissue can be maintained at a thickness of several hundred microns for up to a few weeks, making it possible to implant cancer cells, observe their behavior in actual tissue over time, and then perform a more detailed marker analysis at the endpoint of the experiment.
A popular organotypic slice culture method has been to culture a several hundred micron-thick brain slice on top of a translucent or transparent porous membrane, leaving the tissue exposed to air, yet allowing nutrient media to sustain the tissue from below the membrane (refer to Stoppini et al.6). Different variations of this method have been used for different studies, including using different media or different membrane inserts. Different membrane inserts include a 30 mm diameter, porous (0.4 &#181;m) membrane insert in a 35 mm culture dish6, and cell culture inserts (0.4 &#181;m) for 6-well plates7. Different media include 50% MEM/HEPES + 25% heat-inactivated horse serum + 25% Hanks balanced salt solution (HBSS)8, 50% reduced serum media + 25% horse serum + 25% HBSS9, as well as others. If a translucent or transparent membrane is used along with fluorescently labeled GBM cells, then such cultures can be imaged from below using an inverted widefield or confocal fluorescence microscope10,11,12,13,14,15.
While many in vivo orthotopic brain tumor xenograft and ex vivo organotypic brain slice culture models have been established using rodents, as cited above, the chick embryo (Gallus gallus) has been underutilized for these purposes. However, the chick embryo has been demonstrated to be capable of being used as an in vivo orthotopic xenograft model for the study of both human and rat glioma invasion1,2,5. Xenografted cells into chick embryo brains have exhibited invasion patterns similar to those observed in rodent models, further supporting the use of chick embryos as an in vivo model for GBM tumor cell analysis. Chick embryos also are inexpensive, can be more easily maintained than rodents (i.e., in their egg shells in a lab incubator), and are much easier to work with, making them an attractive option for short-term in vivo GBM studies. A recent article has described the use of chick embryo brain slice cultures for the formation and growth of axons during normal brain development where the slices were viable for at least 7 days16. However, the use of such chick embryo brain slice cultures for ex vivo analysis of GBM cell behavior in a tissue environment is lacking. In this article, both the transplantation of human GBM cells and GBM stem cells (GSCs) into the early chick embryo brain in vivo, as well as the introduction of GBM cells onto live chick embryo brain slice cultures ex vivo, are described. Some representative examples of the resulting tumors and cell invasion patterns obtained from these preparations are also provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 cm x 1 cm square hole paper punch</td>
			<td>Birabira</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm biopsy punch pen</td>
			<td>Robbins Instruments</td>
			<td>20335</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well insert plate (Corning Transwell)</td>
			<td>Millipore Sigma</td>
			<td>CLS3450</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; Disposable Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Corning Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher BioReagents</td>
			<td>BP1423-500</td>
			<td>for embedding fixed brains</td>
		</tr>
		<tr>
			<td>Alexafluor 488-conjugated GAM IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-605-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexafluor 647-conjugated GAM IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-545-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>ReynoldsWrap</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>A-9518</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-integrin alpha-6 monoclonal antibody GOH3</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-19622</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-L1CAM monoclonal antibody UJ127</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53386</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-laminin monoclonal antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3H11</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-nestin monoclonal antibody 10c2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23927</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sox2 monoclonal antibody E-4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365823</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement without vitamin A</td>
			<td>GIBCO</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>bisbenzimide (Hoechst 33258)</td>
			<td>Sigma-Aldrich</td>
			<td>B2883</td>
			<td>nuclear stain</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Forma</td>
			<td></td>
			<td>standard humidified CO2 incubator</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon Instruments</td>
			<td>C2si+</td>
			<td>With custom-made cell incubator chamber</td>
		</tr>
		<tr>
			<td>Confocal microscope objective lenses</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td>Plan Apo lenses, except S Plan Fluor ELWD 20x 0.45 NA objective lens for confocal time-lapse imaging</td>
		</tr>
		<tr>
			<td>Confocal microscope software</td>
			<td>Nikon Instruments</td>
			<td>NIS Elements</td>
			<td>Version 5.2</td>
		</tr>
		<tr>
			<td>Curved foreceps</td>
			<td>World Precision Intruments</td>
			<td>504478</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved spatula</td>
			<td>Fisher Scientific</td>
			<td>14-375-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Krazy Glue</td>
			<td>KG-585 12R</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>DiD far red fluorescent dye</td>
			<td>Invitrogen</td>
			<td>V22887</td>
			<td>Vybrant DiD</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Sigma Aldrich</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>egg incubator</td>
			<td>Humidaire</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electrical tape (10 mil thick/254 &#181;m)</td>
			<td>Scotch</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast green FCF dye</td>
			<td>Avocado Research Chemicals</td>
			<td>16520</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini Bio-products</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>filter paper</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Dynarex</td>
			<td>3353</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries for microinjection</td>
			<td>World Precision Instruments</td>
			<td>TW100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP228-1</td>
			<td>for mounting media</td>
		</tr>
		<tr>
			<td>GSCs (human glioblastoma stem cells)</td>
			<td>Not applicable</td>
			<td></td>
			<td>Isolated from patient GBM specimens in Galileo laboratory in GSC media and then transduced with a GFP encoding lentiviral vector.&#160; Cells used were between passage 10 and 30.</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher Scientific</td>
			<td>BP2524-100</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum (HI)</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-2</td>
			<td>BioVision</td>
			<td>4037-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF-&#945;</td>
			<td>BioVision</td>
			<td>4339-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope</td>
			<td>Nikon Instruments</td>
			<td>TMS</td>
			<td>for routine viewing of cultured cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P284</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory film</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labquake Shaker</td>
			<td>LabIndustries</td>
			<td>T400-110</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glut:Pen:Strep</td>
			<td>Gemini Bio-products</td>
			<td>400-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melt agarose</td>
			<td>Fisher Scientific</td>
			<td>BP1360</td>
			<td>for embedding live brains</td>
		</tr>
		<tr>
			<td>Matrix</td>
			<td>Corning Matrigel</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Corning</td>
			<td>10-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal vibratome block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips (20, 200, 1,000 &#181;L)</td>
			<td>Fisherbrand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettors (20, 200, 1,000 &#181;L)</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverglass (no. 1.5 thickness)</td>
			<td>Fisherbrand</td>
			<td>12544A</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 + H2O</td>
			<td>Fisher Scientific</td>
			<td>S369</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>BP328</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Millipore Sigma</td>
			<td>526-M</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>Sigma Aldrich</td>
			<td>P3130</td>
			<td>for mounting media</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plain Microscope slides</td>
			<td>Fisherbrand</td>
			<td>12-550-A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic 35 mm Petri dish</td>
			<td>Becton Dickinson</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>pneumatic picopump</td>
			<td>World Precision Intruments</td>
			<td>PV830</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(2-hydroxyethyl methacrylate)&#160; (poly-HEMA)</td>
			<td>Sigma Aldrich</td>
			<td>P-3932</td>
			<td></td>
		</tr>
		<tr>
			<td>razor blade- double edge</td>
			<td>PACE</td>
			<td></td>
			<td>for cutting fixed brain slices</td>
		</tr>
		<tr>
			<td>sapphire knife</td>
			<td>Delaware Diamond Knives</td>
			<td></td>
			<td>for cutting live brain slices</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>TruMed</td>
			<td>1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cacodylate buffer 0.2 M pH 7.4</td>
			<td>Electron Microscopy Sciences</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen chamber for vibratome</td>
			<td>custom-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Dissecting Microscope</td>
			<td>Nikon Instruments</td>
			<td>SMZ1500</td>
			<td>Equipped with epifluorescence</td>
		</tr>
		<tr>
			<td>straight foreceps</td>
			<td>World Precision Intruments</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>straight scissors</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Mallinckrodt</td>
			<td>7723</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-lapse fluorescence microscope (widefield fluorescence)</td>
			<td>Nikon Instruments</td>
			<td>TE2000-E</td>
			<td>With custom-made cell incubator chamber (see Fotos et al., 2006)</td>
		</tr>
		<tr>
			<td>Tissue culture dish polystyrene 100 mm</td>
			<td>Thermo Fisher Scientific</td>
			<td>130182</td>
			<td>for cell culturing</td>
		</tr>
		<tr>
			<td>Tissue culture dish polystyrene 60 mm</td>
			<td>Becton Dickinson</td>
			<td>353004</td>
			<td>for cell culturing</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>American Central Scientific Co.</td>
			<td>FFP011</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent tape</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (0.25%) + 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>U-118 MG human GBM cell line</td>
			<td>ATCC</td>
			<td>HTB-15</td>
			<td>Cells were transduced with a lentiviral vector encoding the entire ectodomain sequence of the L1CAM adhesion protein and then with lentiviral vector pUltra-hot encoding mCherry.&#160; Passage numbers are unknown.</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Cole-Parmer</td>
			<td>EW-07532-40</td>
			<td>&#34;Air Cadet&#34;</td>
		</tr>
		<tr>
			<td>Vibrating tissue slicer</td>
			<td>Vibratome</td>
			<td>3000</td>
			<td>for cutting live and fixed brain slices</td>
		</tr>
	</tbody>
</table>

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  

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

The scope of our research is to try to uncover mechanisms that promote glioblastoma cell spread in brain tissue. Specific questions revolve around differences in glioblastoma cell types or states and molecular mechanisms that promote these cells to be highly invasive. For example, how does L1CAM expression promote invasiveness?Both in vitro and in vivo models are insufficient for accurately analyzing glioblastoma cell behavior. In vitro models almost certainly modify and oversimplify in vivo cell behaviors. While in vivo models do not allow for easy observation of behaviors as they occur, instead allowing for analyses after the behaviors have occurred.We have shown that the chick embryo brain is a good model for studying human brain cancer cell behavior both in vivo and in ex-vivo slice cultures. We also have established that L1CAM expression by glioblastoma cells can have profound effects on cell proliferation, invasion, and arrangement within the tumor. Besides several advantages of using chick embryos, our ex-vivo spheroid protocol introduces cells onto live slices with no additional damage, whereas other methods of cell introduction involve piercing the tissue and implanting cells.Additionally, the protocol allows for live time-lapse imaging that in vivo techniques do not. Our findings suggests that the chick embryo is a good model for cancer research, particularly brain cancer, and is extremely beneficial for the scientific community, as it is much more readily available to those who may not have the funds, facilities, or expertise for rodent models. We will continue to focus on molecular mechanisms that control glioblastoma cell invasiveness in brain tissue, particularly along blood vessels.Another crucial question is whether glioblastoma stem cells are a distinct and stable phenotype or a functional state that can be adopted when needed. That has huge implications for treatments.

Watch this Scientific Journal Video about Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior at JoVE.com

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 ...

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