Name: coculture assays to study macrophage and microglia stimulation of glioblastoma invasion
Uploaded: 2016-10-20T12:30:00
Description: Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

We would like to thank Dr. Konstantin Dobrenis for providing murine microglia for these studies.

1. Fluorescent Labeling of Cells
NOTE: Label glioblastoma cell lines and microglia with fluorescent dyes 9. Alternatively, generate cell lines that constitutively express fluorescent proteins such as GFP/RFP as described in 14.
<ol>
	<li>Plate cells on a 6 well plate such that they will be 70 - 80% confluent on day of staining. For the murine glioblastoma cell line GL261 and human glioblastoma cell line U87, plate 1 x 106 and 1.5 x 106 cells, respectively on a 6 cm dis...

<ol>
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The highly invasive nature of high grade astrocytomas and glioblastoma make these brain cancers very deadly. It is therefore of paramount importance to understand the molecular and cellular mechanisms of glioblastoma invasion. Much has been learned about the process of glioblastoma invasion already 17. Using the assay formats detailed in this paper, our laboratory has shown in both mouse and human models that tumor associated macrophages can stimulate glioma cell invasion by 5 - 10 fold. This coculture model f...

Glioblastoma multiforme is an aggressive human brain cancer with a median survival of approximately 12 months from the time of diagnosis 1,2. Glioblastoma is one of the most deadly and clinically challenging cancers as it is refractory to standard chemotherapy and surgical resection. The diffuse nature of glioblastoma enables tumor cells to spread throughout the normal brain making the advanced tumor practically impossible to surgically resect completely. This highly invasive aspect is a hallmark feature of glioblastoma and other advanced astrocytomas. Therefore, the focus of much research has been on the molecular mechanism of glioblastoma cell invasion. The glioblastoma tumor microenvironment plays vital roles in establishing malignancy 3-6. Tumor associated macrophages/microglia were shown to be responsible for promoting glioblastoma invasion 7,8. Most of these studies however measured the effect of macrophages/microglia using assays which physically separate them from the glioblastoma cells. Our laboratory has set out to generate improved assays which allow us to study how glioblastoma invasion is dependent on macrophages/microglia in cocultures and enable us to image the physical interaction between them during invasion.
Classic assays to measure cell invasion include the &#34;standard&#34; Boyden chamber chemotaxis and chemoinvasion formats. Here the cells to be studied are plated in a plastic chamber which contains a polycarbonate filter on the bottom that has pores of a specified size (generally between 0.4 and 8 &#181;M in diameter). The process of cell invasion involves a physical barrier, usually composed of extracellular matrix protein. In the chemoinvasion assay, the preferred matrix used is Matrigel (hereafter referred to as &#34;matrix&#34;), an extracellular matrix protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and consists largely of collagen type IV and laminin. The chambers are then placed in a tissue culture well which contains cell culture media with or without growth factors that are suspected to stimulate invasion. Cells which have a higher invasive capacity will invade through the extracellular matrix coated filter at a higher frequency and adhere to the underside of the filter. We have modified this assay in order to assess the role of microglia and tumor associated macrophages on glioblastoma cell invasion.
We have been able to determine using coculture assays described within this paper that microglia can stimulate the invasion of two glioblastoma cell lines by 5 - 10 fold 9,10. This reflects what is observed in animal models of glioblastoma. Furthermore, we developed a three dimensional invasion assay where the interactions between glioblastoma cells and macrophages/microglia can be examined more directly. The extent of glioblastoma cell invasion stimulated by macrophages/microglia in the 3D assay is comparable to what is seen using the matrix coated chamber approach. Similar assays were previously developed to study breast carcinoma interactions with macrophages during invasion 11-13. Both methods described in this paper should aid in the ability to dissect the molecular mechanism(s) of macrophage/microglia-stimulated invasion of glioblastoma cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Corning BioCoat Matrigel Invasion Chamber: With BD Matrigel Matrix</td>
			<td>Corning/Fisher Scientific</td>
			<td>Cat: 354481</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage Serum Free Media (MSFM) (500 ml)</td>
			<td>Life Technologies</td>
			<td>12065-074</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red CMTPX Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C2925</td>
			<td></td>
		</tr>
		<tr>
			<td>GL261 cell line</td>
			<td>National Cancer Institute (NCI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U87 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium (500 ml)</td>
			<td>Life Technologies/Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polycarbonate membrane cell culture inserts (8 &#181;M pore) 48 per pack.</td>
			<td>Corning</td>
			<td>CLS3422</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, PathClear</td>
			<td>Trevigen</td>
			<td>3445-005-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>Cat: 10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, Fraction V, Heat Shock Treated</td>
			<td>Fisherscientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma Aldrich</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture assays to study macrophage and microglia stimulation of glioblastoma invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Using the methods outlined here, we have shown that microglia and macrophages can substantially stimulate glioblastoma cell invasion. Two different invasion assays are employed and are depicted in Figure 1. In Figure 2, GL261 cells that constitutively express the fluorescent protein mCherry were plated on pre-coated chambers with and without microglia for 48 hr. GL261 cells were minimally invasive on their own however when cultured with microglia the inva...

Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

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