This work is supported by grants from the NIH (P01CA142536 to RAS, T32CA009361 to DPY) and the Pediatric Low Grade Astrocytoma foundation.

Ethics Statement: The following procedure involving animal subjects were done in accordance with the National Institutes of Health guidelines and were approved by the Dana-Farber Cancer Institutional Animal Care and Use Committee. All human subjects work was reviewed by the Institutional Review Board Committees of the Brigham and Women&#39;s Hospital and Dana-Farber Cancer Institute, and by Stanford University for appropriate use, that informed consent was obtained from all subjects when required, and appropriate waiver ...

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	<li>Heddleston, J. 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=Glioma+stem+cell+maintenance:+the+role+of+the+microenvironment.">Glioma stem cell maintenance: the role of the microenvironment.</a> Curr Pharm Des. 17 (23), 2386-2401 (2013).</li><li>Sasai, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shh+pathway+activity+is+down-regulated+in+cultured+medulloblastoma+cells:+implications+for+preclinical+studies.">Shh pathway activity is down-regulated in cultured medulloblastoma cells: implications for preclinical studies.</a> Cancer Res. 66 (8), 4215-4222 (2006).</li><li>Louis, D. 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=The+2007+WHO+classification+of+tumours+of+the+central+nervous+system.">The 2007 WHO classification of tumours of the central nervous system.</a> Acta Neuropathol. 114 (2), 97-109 (2007).</li><li>Duffau, H., Capelle, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+brain+locations+of+low-grade+gliomas.">Preferential brain locations of low-grade gliomas.</a> Cancer. 100 (12), 2622-2626 (2004).</li><li>Chourmouzi, D., 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=Manifestations+of+pilocytic+astrocytoma:+a+pictorial+review.">Manifestations of pilocytic astrocytoma: a pictorial review.</a> Insights Imaging. 5 (3), 387-402 (2014).</li><li>Buonamici, 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=Interfering+with+resistance+to+smoothened+antagonists+by+inhibition+of+the+PI3K+pathway+in+medulloblastoma.">Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma.</a> Sci Transl Med. 2 (51), 51ra70 (2010).</li><li>Choi, Y., Borghesani, P. R., Chan, J. A., Segal, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+from+a+mitogenic+niche+promotes+cell-cycle+exit.">Migration from a mitogenic niche promotes cell-cycle exit.</a> J Neurosci. 25 (45), 10437-10445 (2005).</li><li>Chan, J. 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=Proteoglycan+interactions+with+Sonic+Hedgehog+specify+mitogenic+responses.">Proteoglycan interactions with Sonic Hedgehog specify mitogenic responses.</a> Nat Neurosci. 12 (4), 409-417 (2009).</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> J Neurosci Methods. 37 (2), 173-182 (1991).</li><li>Kenny, H. 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=Quantitative+high+throughput+screening+using+a+primary+human+three-+dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three- dimensional organotypic culture predicts in vivo efficacy.</a> Nat Commun. 6, 6220 (2015).</li><li>Liu, X., 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=ROCK+inhibitor+and+feeder+cells+induce+the+conditional+reprogramming+of+epithelial+cells.">ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.</a> Am J Pathol. 180 (2), 599-607 (2012).</li></ol>

This protocol describes how brain tumor cells can be fluorescently labeled and plated on a sagittal brain section of a P6 mouse and then monitored for one week in culture. This brain tumor/organotypic slice co-culture assay can be used to determine the effect of regional microenvironment on tumor cell number and may also be used as a system for measuring the efficacy of new drug treatments on human tumor growth. Previous studies have used a similar strategy to assess the role of brain micro-environment on neural precurso...

Recent cancer research has made significant advancements in identifying genetic mutations, molecular changes and possible treatments for a variety of brain tumors. Despite this progress, brain tumors remain one of the top causes of cancer-related mortality for adults and children. Limiting factors in brain tumor research include the restricted availability of primary patient samples and cell lines and the difficulty in replicating the unique and heterogeneous brain microenvironment in accessible experimental systems. For many brain tumors the conditions required to maintain tumor cells over time are not yet known. Even for brain tumors that can be grown in cell suspension as neurospheres, culture conditions may affect the tumor cells1,2. Indeed, the addition of basic fibroblast growth factor or epidermal growth factor to encourage proliferation and inhibit differentiation may alter gene expression1. Other methods for tumor cell growth such as tumor propagation in mice via orthotopic or subcutaneous xenograft of tumor cells are valuable assays, but are limited by factors such as time of tumor development (especially for low grade tumors), cost, and the number of tumor cells that can be injected and studied. Thus current methods for growing human brain tumor cells are inadequate for maintaining certain tumor types, and often provide artificial environments that do not closely mimic in vivo tumor environments.
Distinct types of pediatric brain tumors grow in highly specialized locations within the brain[3, 4] and this is likely to reflect distinct microenvironmental requirements for tumor growth[5]. This protocol describes a novel system where cells that are difficult to propagate in normal culture conditions can be grown in an organotypic brain microenvironment which mimics in vivo tumor growth conditions. In this quantitative assay, fluorescently labeled brain tumor cells are plated on juvenile mouse brain organotypic slices and monitored over time. This assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies in a clinically relevant brain microenvironment.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen&#160;</td>
			<td>17504044</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Invitrogen</td>
			<td>17502048</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Pennicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14185-052</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Life Technologies</td>
			<td>17502-048</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td rowspan="2">Neurobasal-A- Medium minus phenol red</td>
			<td rowspan="2">Invitrogen&#160;</td>
			<td rowspan="2">12349015</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Low Melting Point Agarose</td>
			<td>Promega</td>
			<td>V2111</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Slice Culture Inserts&#160;</td>
			<td>Milipore</td>
			<td>PICM0RG50</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>laminin</td>
			<td>Invitrogen</td>
			<td>23017015</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Cm-DiI&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>V22888</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>EDU (Labeling and Detection)&#160;</td>
			<td>Life Technologies</td>
			<td>c10337</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Microspheres&#160;</td>
			<td>Life Technologies</td>
			<td>F-21010</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Vibratome&#160;</td>
			<td>Leica</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Confocal Microscope&#160;</td>
			<td>Nikon Eclipse Ni C2si</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>Image J software&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td colspan="3"></td>
		</tr>
		<tr>
			<td>5mm Cover Glasses&#160;</td>
			<td>Fisher Scientific</td>
			<td>64-0700 (CS-5R)</td>
			<td colspan="3"></td>
		</tr>
	</tbody>
</table>

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.

This section exemplifies the type of results to be expected from utilizing the brain tumor/organotypic slice co-culture to investigate regional microenvironment preference as well as to test new therapies. We show that the assay is designed to replicate the microenvironment for brain tumors, as the tissue organization and proliferative state of the slice is maintained (Figure 3). We also demonstrate that an increase in the number of tumor cells on the slice over time may partially be due to the migration...

Watch this Scientific Journal Video about A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies at JoVE.com

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

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 ...

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10.9K vistas.  Dana-Farber Cancer Institute. 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.  