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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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=Microglial+stimulation+of+glioblastoma+invasion+involves+epidermal+growth+factor+receptor+(EGFR)+and+colony+stimulating+factor+1+receptor+(CSF-1R)+signaling.">Microglial stimulation of glioblastoma invasion involves epidermal growth factor receptor (EGFR) and colony stimulating factor 1 receptor (CSF-1R) signaling.</a> Mol Med. 18, 519-527 (2012).</li><li>Miller, I. 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=Semapimod+sensitizes+glioblastoma+tumors+to+ionizing+radiation+by+targeting+microglia.">Semapimod sensitizes glioblastoma tumors to ionizing radiation by targeting microglia.</a> PLoS One. 9 (5), (2014).</li><li>Goswami, 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=Macrophages+promote+the+invasion+of+breast+carcinoma+cells+via+a+colony-stimulating+factor-1/epidermal+growth+factor+paracrine+loop.">Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop.</a> Cancer Res. 65 (12), 5278-5283 (2005).</li><li>House, R. 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The authors have nothing to disclose.

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 ...

The overall goal of this experiment is to model the interaction of glioblastoma cells with microglia and macrophages during invasion in an in vitro assay. So this method can help answer key questions in the tumor microenvironment field, such as which compounds or treatments can interfere with cancer interaction with macrophages, a cell type of the microenvironment during the progression to malignancy. One of the main advantages of this technique is that it's relatively easy to perform and can be completed at a reasonable time and seems to accurately model glioblastoma invasion in vivo.Begin by differentiating the cell line of interest using phorbol myristate acetate, as follows. First, plate two to three times 10 to the fifth THP-1 cells in 1.5 milliliters of medium in a six well culture plate. Immediately after plating, add 100 nanomolar of PMA and incubate at 37 degrees Celsius and 5%CO2 for 48 hours.After 48 hours, remove the PMA by aspirating the media, wash once with one-X PBS and add fresh media. Incubate for another 48 hours until cells are ready to use in the assay. Then equilibrate matrix pre-coated chambers by placing them in a well containing 500 microliters of media without serum and then adding 500 microliters of serum free media to the top.Incubate the chambers for two hours at 37 degrees Celsius and 5%CO2. Next, gently aspirate the media from the culture vessels containing the fluorescently-labeled glioblastoma cell lines and the microglia and macrophages. Then add 500 microliters of two millimolar EDTA and PBS and incubate for five to 10 minutes in the tissue culture incubator.Collect the cells in five milliliters of media containing 0.3%bovine serum albumin and centrifuge for five minutes at 120 times G.Following the centrifugation, aspirate the supernatant and resuspend the cell pellet in appropriate media to a concentration of one times 10 to the sixth cells per milliliter. Use the hemocytometer to count the cells. Next, add 500 microliters of the appropriate media containing 0.3%BSA to the wells of a 24 well plate.Then remove the media from the equilibrated chambers and place the chambers in the wells of the 24 well plate. If inhibitors are being used to study their effect on macrophage or microglia stimulated glioma invasion, add the appropriate concentration to both the bottom and top portions of the chamber. Depending on the cell line used, mix glioblastoma cells and mouse microglia or glioma cells, and macrophage cells according to the details in the written portion of the protocol.Add the cell mixture to the top chamber and incubate for 24 to 48 hours, depending on cell type. After the incubation, remove the cells from the top of the chamber by gentle aspiration. Then place the chambers in 3.7%formaldehyde in PBS to fix.Allow the chambers to remain in fixative for 15 minutes at room temperature. Finally, remove the fixative by gentle aspiration and replace with 500 microliters of one-X PBS. First, thaw the matrix mix at four degrees Celsius overnight.Next, prepare a 10 milligrams per milliliter concentration of matrix in the appropriate media on ice. Then, harvest the labeled cells for suspension in matrix as previously shown. Then pipette 1.5 times 10 to the fifth GL261 cells in a 150 microliter volume plus five times 10 to the fourth microglia cells in 50 microliters into a 1.5 milliliter microfuge tube.Add two times 10 to the fifth GL261 cells in a 200 microliter volume into a separate 1.5 milliliter microfuge tube. Spin the cells in a microfuge for five minutes at 120 times G.Then, resuspend the cells in 200 microliters of cold matrix on ice. For this step it is crucial to have ice tray ready in the hood, keep tubes on ice, tips in the refrigerator, make sure everything is chilled in order to prevent polymerization of the mature gel prematurely.After resuspension, plate 50, 000 cells in a 50 microliter volume onto the center of the top compartment of an eight micron pore size chamber insert. Then incubate it at 37 degrees Celsius for 30 minutes to allow polymerization to occur. After 30 minutes, add 200 microliters of serum free medium to the upper chamber and 700 microliters of serum containing cell growth medium to the lower well.After incubating for 48 hours, remove the cells from the top part of the chamber by gentle aspiration. Fix the chambers as before and replace the fixative with 500 microliters of one-X PBS before imaging the cells. This representative image shows invading mCherry expressing GL261 cells in the absence of microglia.The cells were plated on matrix coated chambers and incubated for 48 hours. The images were taken using a 10X objective and the scale bar represents 400 microns. Here the GL261 cells were combined with microglia.The number of GL261 invasive cells that cross the filter were quantified. As seen here, coculturing the mouse glioblastoma cell line GL261 with microglia enhances its invasion by up to 10 fold in the 2D assay. This image shows the effect of pharmacological inhibitors on microglia stimulated GL261 invasion compared to DMSO control.Data shown are from six independent experiments. This assay was successfully employed using human cell lines as well. U87 cells plated on matrix coated chambers and stained with CMFDA green are shown after a 24 hour incubation.Here the U87 cells were plated with differentiated THP1 macrophages. This image shows quantitation of the assay counting only invasive U87 cells. The human macrophage cell line THP1 stimulated U87 glioblastoma cell invasion.Data shown are the mean values from 10 independent experiments. This image from a 3D invasion assay of GL261 and microglia coculture is a composite from a Z-series of images and shows GL261 cells stained with CMFDA green embedded in matrix alone. Here the GL261 cells were cocultured with murine microglia labeled with CMPTX red.Here GL261 cells on the underside of the filter, which are determined to be invasive, are shown. The total number of invading GL261 cells was determined and normalized to that of GL261 cells in monoculture. Data shown represent the mean of three independent experiments performed in duplicate.So once mastered, this technique can be completed from start to finish in two hours or so. And the final results from the experiment can be obtained after 48 hours. So while performing this technique, it's very important to have everything chilled and at four degrees including the tubes and the pipettes.After watching this video, you should have a good understanding of how to establish glioblastoma in macrophage microglia coculture invasion assays.

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13.7K visualizações.  Kean University. The overall goal of this experiment is to model the interaction of glioblastoma cells with microglia and macrophages during invasion in an in vitro assay. So this method can help answer key questions in the tumor microenvironment field, such as which compounds or treatments can interfere with cancer interaction with macrophages, a cell type of the microenvironment during the progression to malignancy. One of the main advantages of this technique is that it's relatively easy to perform and can...