Name: coculture assays to study macrophage and microglia stimulation of glioblastoma invasion
Uploaded: 2016-10-20T12:30:00.000+00:00
Duration: 9 min 23 s
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 dish 24 hr before staining.</li>
	<li>Prepare fluorescent cell stain dye solution in DMSO and add 5 &#181;M dye to media, either Roswell Park Memorial Institute medium (RPMI) or Macrophage Serum Free Medium (MSFM), vortex well.</li>
	<li>Incubate cells with dye for 30 min at 37 &#176;C and 5% CO2.</li>
	<li>Remove dye containing media and add fresh media (RPMI or MSFM) to the cells.</li>
	<li>Incubate cells for 30 min. Note: Cells are now stained and ready to be used in experiment.</li>
</ol>
2. Pre-coated Matrix Chamber Coculture Invasion Assay
<ol>
	<li>Differentiate THP-1 cells using phorbol myristate acetate (PMA)15.
	<ol>
		<li>Plate 2 - 3 x 105 THP-1 cells in 1.5 ml of RPMI/10% FBS in a 6 well culture plate. Immediately after plating add 100 nM PMA and incubate at 37 &#176;C, 5% CO2 for 48 hr. 
		NOTE: After 48 hr, THP-1 cells that have successfully undergone differentiation will be adherent and spread to the bottom of the well taking on a round morphology.</li>
		<li>Remove PMA by washing once with 1x PBS and replace with fresh RPMI/10% FBS. Wait another 48 hr until cells are ready to use in assay.</li>
	</ol>
	</li>
	<li>Equilibrate matrix pre-coated chambers by placing them in a well containing 500 &#181;l of media alone (no serum) and adding 500 &#181;l of media alone to the top. Incubate chambers for 2 hr at 37 &#176;C and 5% CO2. 
	NOTE: Pre-coated chambers were purchased from the manufacturer; see Table of Materials/Equipment.</li>
	<li>To gently detach cells (fluorescently labeled glioblastoma cell lines and the microglia/macrophages), remove media from cells by aspiration. Wash cells on dish with 2 mM EDTA/PBS. Add 500 &#181;l of 2 mM EDTA/PBS and incubate for 5 - 10 min in a cell incubator at 37 &#176;C and 5% CO2.</li>
	<li>Resuspend cells in 5 ml of media containing 0.3% Bovine Serum Albumin (BSA).</li>
	<li>Centrifuge for 5 min at 120 x g.</li>
	<li>Aspirate supernatant and resuspend pellet in MSFM/0.3% BSA media (for GL261 cells and microglia) or RPMI/0.3% BSA (for U87 and THP-1 cells) such that the concentration of cells is equal to 1 x 106 cells/ml. Use a hemocytometer to count the cells.</li>
	<li>Add 500 &#181;l media/0.3% BSA per well of 24 well plate.</li>
	<li>Remove media from chambers that have been equilibrated for at least 2 hr (step 2.2). Place chambers in well containing 500 &#181;l media/0.3% BSA (step 2.7).</li>
	<li>If inhibitors are being used to study their effect on macrophage/microglia stimulated glioma invasion, add the appropriate concentration to both the bottom and top portions of the chamber. 
	NOTE: The concentrations of CSF-1R inhibitor used to fully inhibit microglia stimulated GL261 glioblastoma invasion can be found in reference 9.</li>
	<li>Depending on the cell lines used, add cells to the top chamber as described in the next two steps: mouse glioblastoma (2.10.1) and human glioblastoma (2.10.2).
	<ol>
		<li>Mix 1.5 x 105 GL261 (glioblastoma) cells (150 &#181;l) and 5 x 104 mouse microglia (50 &#181;l) (see step 2.6 for media details). Bring volume to 500 &#181;l by adding 300 &#181;l MSFM/0.3% BSA. Add cell mixture to the top chamber and incubate at 37 &#176;C , 5% CO2 for 48 hr. 
		&#8203;NOTE: Murine microglia are isolated from neonatal mice as described in 16.</li>
	</ol>
	</li>
</ol>
Mix 7.5 x 104 U87 (glioblastoma) cells (75 &#181;l) and 2.5 x 104 THP1 (macrophage) cells (25 &#181;l). Bring volume in 500 &#181;l by adding 400 &#181;l RPMI/0.3% BSA. Add the cell mixture to the top chamber. Incubate cells at 37 &#176;C, 5% CO2 for 24 hr.
<ol>
	<li>After incubation, remove cells from top of chamber by gentle aspiration and fix chambers by placing in 3.7% formaldehyde in PBS. Allow chambers to remain in fixative for 15 min at room temperature (or overnight at 4 &#176;C). Remove fixative by gentle aspiration and replace with 500 &#181;l 1x PBS. Proceed to section 4 for image analysis. 
	&#8203;NOTE: Experiment can be paused at this moment and saved for imaging in 1x PBS. Fluorescent dye signals can be detected for up to 2 weeks after fixation.</li>
</ol>
3. Invasion Assay &#34;3D&#34;-embedding Glioma and Macrophages/Microglia in Matrix
<ol>
	<li>Thaw matrix mix at 4 &#176;C overnight. Perform all further manipulations using matrix on ice in the hood.</li>
	<li>Prepare 10 mg/ml concentration of matrix in media (MSFM or RPMI) with 0.3% BSA on ice. 
	NOTE: Stock concentration of matrix is typically around 15 mg/ml.</li>
	<li>To prepare cells (labelled in step 1) for suspension in matrix, remove media from cells by aspiration. Wash cells on dish with 2 mM EDTA/PBS. Add 500 &#181;l of 2 mM EDTA/PBS to cells and incubate for 5 - 10 min in incubator at 37 &#176;C and 5% CO2.</li>
	<li>Resuspend cells in 5 ml of media containing 0.3% BSA.</li>
	<li>Centrifuge for 5 min at 120 x g.</li>
	<li>Aspirate supernatant from pellet. Resuspend pellet in media/0.3% BSA such that the concentration of cells is equal to 1 x 106 cells/ml. Use a hemocytometer to count the cells.</li>
	<li>Add 1.5 x 105 GL261 cells (in 150 &#181;l) + 5 x 104 microglia cells (in 50 &#181;l) into a 1.5 ml microfuge tube. Add 2 x 105 GL261 cells (200 &#181;l) into a separate 1.5 ml microfuge tube. 
	NOTE: GL261 cells alone without microglia serves as a control.</li>
	<li>Spin cells in microfuge for 5 min at 120 x g.</li>
	<li>Resuspend cells in 200 &#181;l cold matrix on ice.</li>
	<li>Plate 50 &#181;l (50,000 cells) onto the center of the top compartment of 8 &#181;M pore size chamber insert.</li>
	<li>Incubate at 37 &#176;C for 30 min for polymerization to occur.</li>
	<li>Add 200 &#181;l serum-free medium to the upper chamber and 700 &#181;l serum containing cell growth medium to the lower well.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 for 48 hr.</li>
	<li>After incubation, remove cells from top of chamber by gentle aspiration and fix chambers by placing in 3.7% formaldehyde in PBS. Allow chambers to remain in fixative for 15 min at room temperature (or overnight at 4 &#176;C). Remove fixative by gentle aspiration and replace with 500 &#181;l 1x PBS. Proceed to section 4 for image analysis.</li>
</ol>
4. Imaging Assays
<ol>
	<li>Remove chambers from 3.7% formaldehyde and wash in well containing fresh 500 &#181;l 1x PBS. 
	NOTE: Do not allow chambers to dry.</li>
	<li>Using a cotton tipped applicator, gently clean the non-invasive cells from the top portion of the filter (side facing the inside of the chamber) by scraping the filter surface. Start gently and increase pressure gradually. Do this at least 3 times per chamber.</li>
	<li>Place chambers in either a glass-bottomed dish or leave in a 24 well plate.</li>
	<li>Place sample on the stage of an epifluorescent or laser confocal fluorescent microscope equipped with camera and image capture software 9,10.</li>
	<li>Take several 10X or 20X images of representative fields.</li>
	<li>Using an image analysis software, such as ImageJ, count the number of glioblastoma cells that have crossed the filter to the underside 9,10.</li>
	<li>If imaging the &#34;3D&#34; invasion assay (described in section 3), generate a Z stack series using a fluorescent laser confocal microscope 5-6. To image the invasive cell population on the underside of the filter, follow the protocol steps 4.2 -&#160; 4.6 described above.</li>
</ol>

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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 faithfully replicates the ability of glioblastoma cells to physically interact with macrophages/microglia which would allow for paracrine and juxtacrine interactions (via ligand-receptor systems such as Notch-Delta and Ephrin-Ephs) that are potentially involved in bidirectional communication between the tumor cells and macrophages. Other assay formats which seek to discover the effect of macrophages on glioma cell invasion typically have the cells physically separated as the macrophages are plated on the bottom of the well and the glioblastoma cells are on the chamber 3,4. Consequently, these studies show a more modest increase in macrophage-stimulated glioma invasion (2 fold). This coculture assay improves on this effect (5 - 10 fold) likely indicating the importance of juxtacrine interactions during macrophage/microglia stimulated glioblastoma invasion.
The protocol outlined in this paper involves several steps which must be performed carefully to achieve satisfactory results. If the experiment is carried out as described here and no invading cells are seen, the fluorescent dye may have expired which will lead to very weak or non-existent staining. For the invasion assays using the pre-coated chambers, it is essential to use fresh invasion chambers which are not past the expiration date and have been kept frozen the entire time up until performing the experiment. Chambers which were left out at room temperature for over 12 hr were found to be unsatisfactory in that the matrix barrier was no longer intact and cells invaded at a much higher frequency than normal. For the 3D assay, it is absolutely crucial to keep all pipets and tubes on ice. If at room temperature for a significant length of time, the matrix will polymerize and cannot be used to resuspend the cells in it. It is recommended that a small tray filled with ice is used in the hood to hold all materials which will come into contact with the matrix. The protocol can be modified to include a higher cell density, however this may affect the length of time required to see the optimal effect on glioma invasion. It would be of interest to see if other cell types can be added in the coculture assay and what potential effects they may have on invasion. Finally, we acknowledge that the brain microenvironment is unique and difficult to replicate in vitro. One limitation of this protocol is that the invasion chambers used here lack the typical components of normal brain which glioblastoma cells will encounter during invasion (i.e., densely packed neurons and astrocytes). Although with that caveat, the stimulatory effect of macrophages/microglia in this assay is consistent with what is observed in vivo and can be used to predict which inhibitors and/or proteins may interfere with this process.
Mimicking the tumor microenvironment in culture is a daunting task given how many varied cell types and matrix proteins are present in tumors at various stages of malignancy. Yet reasonably accurate in vitro models of tumor interaction with components of the microenvironment would greatly facilitate the discovery of new therapeutic avenues. It is now appreciated that cells such as macrophages play a critical role in several aspects associated with progression to malignancy including angiogenesis, invasion, immune evasion and drug resistance. The coculture invasion assay described here uses the ratio of tumor cells to macrophages that is observed in patients with high grade glioblastomas (approximately 3:1; 5). It has been shown that the ability of macrophages/microglia to stimulate glioblastoma invasion is dependent on CSF-1R and EGFR signaling as pharmacological inhibition of these pathways using JNJ-28312141, PLX3397 (CSF-1R) and gefitinib (EGFR) strongly attenuates the increase in invasion 9. Furthermore, using the embedded matrix assay, it was demonstrated that Semapimod, a small molecule known to target macrophages and microglia, can also block microglia stimulated glioblastoma invasion 10. Results from these coculture experiments provided the impetus to validate these compounds using in vivo models. Consistent with data from the in vitro assays described in this paper, blockade of CSF-1R with the inhibitor PLX3397 (which crosses the blood brain barrier) largely blocked the ability of GL261 glioma cells to invade in the brains of C57BL/6 mice 9. Similarly, Semapimod delivered into the brain was shown to inhibit invasion and to synergize with standard radiation treatment in greatly prolonging the survival of mice harboring GL261 tumors 10. Both of these approaches may prove effective in the clinic.
It is now well appreciated that the macrophage system is quite important for progression of various cancers to malignancy 3-6, 11-13. It has been clearly established in breast and brain cancer models that tumor-associated macrophages are critical for tumor cell invasion and metastasis. In addition, macrophages are likely to be very important for mediating immune escape as macrophages (and cells of related lineage such as dendritic cells) function as professional antigen presenting cells which orchestrate adaptive immunity in response to infection 18. It is now clear that one of the normal physiological functions of the adaptive immune system is to prevent uncontrolled proliferation by destroying aberrant cells. Immunity towards the tumor cells is likely due to the fact that the highly mutagenic nature of the cancer cell generates neo-antigens that are recognized as foreign by the adaptive immune system. Indeed the process of &#34;immune evasion&#34; is postulated to be an important part of malignancy as cancer cells need to prevent the immune system (in particular cytotoxic lymphocytes, natural killer cells and inflammatory macrophages) from destroying the tumor. It is hoped the assay described in this paper will facilitates the study of glioblastoma interaction with macrophages and potentially allow the expansion of the types of questions we can address about malignant cancer using in vitro culture assays.

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><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 &amp;micro;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>0.5 M 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 invasive capacity increased by approximately 10 fold.
We observe a similar effect using human cell lines. In Figure 3, U87 cells (stained with 5-chloromethylfluorescein diacetate (CMFDA) green) shows 5-6 fold higher invasion when cocultured with differentiated THP-1 cells. THP-1 is a human monocytic cell line (derived from an acute monocytic leukemia patient) that can be differentiated into a macrophage-like state using phorbol myristate acetate (PMA) 15. Differentiated THP-1 cells display many of the characteristics of human macrophages such as cytokine secretion and the ability to carry out phagocytosis.
Alternatively, cell invasion can be measured by embedding them in matrix and plating this directly onto the filter in a chamber insert. Laser confocal microscopy can be used to produce a series of images that produces a three dimensional representation (Z-stack). Similar to what is observed in the matrix-coated chamber assay (shown in Figures 2 - 3), the coculture of microglia (stained with CMPTX red) stimulates GL261 cells (CMFDA green) to invade as measured by the number of cells that have crossed to the underside of the filter (Figure 4). This format has the added advantage of being able to visualize potential cell-cell interactions between glioblastoma cells and microglia during invasion. Indeed, microglia seem to closely associate with the glioma cells suspended in 3D matrix (Figure 5). If only endpoint analysis is required and a laser confocal is not available for use, cell chambers can be cleaned as described for the matrix coated chambers. Then the number of the remaining (invasive) cells can be determined by counting using images obtained from a standard epifluorescent microscope. Movie 1 shows the three dimensional assembly of such an assay.
<img alt="Figure 1" src="/files/ftp_upload/53990/53990fig1.jpg" /> 
Figure 1: Schematic of Two Different Protocols for Assaying Microglia/Macrophage-stimulated Glioblastoma Cell Invasion. Top panel illustrates pre-coated matrix chamber invasion assay (Section 2). Bottom panel illustrates 3D matrix invasion assay (Section 3). Figure adapted from 9. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/53990fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53990/53990fig2.jpg" /> 
Figure 2. Microglia (MG) Enhance GL261 Glioblastoma Cell Invasion. (A) Representative images of invading GL261 cells (expressing mCherry) in the absence (left panel; GL261 alone) or presence of microglia (right panel; GL261+MG) were combined and plated on matrix-coated chambers, followed by incubation for 48 hr and then evaluation of the number of cells that have crossed the filter. Images taken using 10X objective. Scale bar = 400 &#181;M. (B) Quantitation of assay counting only invasive GL261 cells (red). Data shown are Mean &#177; SEM from three independent experiments. (C) Quantitation of microglia-stimulated GL261 invasion in the presence of pharmacological inhibitors gefitinib (5 &#181;M), JNJ-28312141 (10 &#181;M), PLX3397 (1 &#181;M), PLX5562 (1 &#181;M). Data shown are Mean &#177; SEM from six independent experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/53990fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53990/53990fig3.jpg" /> 
Figure 3. THP1 Macrophages Enhance U87 Glioblastoma Cell Invasion. (A) Representative images of invading U87 cells stained with CMFDA green plated on matrix-coated chambers alone (left panel; U87) or with differentiated THP1 macrophages (right panel; U87 + THP1), followed by incubation for 24 hr and then evaluation of the number of cells that have crossed the filter. Images taken using a 10X objective. Scale bar = 400 &#181;M. (B) Quantitation of assay counting only invasive U87 cells (green). Data shown are Mean &#177; SEM from ten independent experiments.
<img alt="Figure 4" src="/files/ftp_upload/53990/53990fig4.jpg" /> 
Figure 4. 3D Invasion Assay of GL261 and Microglial Coculture. (A) Images shown are projections from a Z-series of images of GL261 cells (stained with CMFDA green) embedded in matrix alone (left panel; GL261) or with murine microglia (labeled with CMPTX red; right panel; GL261+MG). The Z-projections were of the bottom 4 slices of the image stack which encompasses the bottom of the chamber filter and therefore represents invasive cells. Scale bar = 200 &#181;M. (B) Quantitation of GL261 cells (green) determined to be on the underside of the filter as described above. The total number of invading GL261 cells was determined and normalized to that of GL261 cells in monoculture. Data shown represent the Mean &#177; SEM of 3 independent experiments, performed in duplicate (figure adapted from 10). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/53990fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53990/53990fig5.jpg" /> 
Figure 5. Image from a Slice within the 3D Stack of GL261 (green) and Microglia (red) Coculture Embedded in Matrix. GL261 and microglia cell interactions are highlighted with white arrows. Scale bar = 100 &#181;M.
<img alt="Figure 1" src="/files/ftp_upload/53990/53990movie1.jpg" /> 
Movie 1: Glioblastoma and Microglia Interaction in 3D. Rotation around the X axis of a 3D projection of entire Z series of images taken from GL261 (green) and microglia (red) coculture embedded in matrix. Slices are in 5 &#181;M increments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53990/Movie1.avi" target="_blank">Please click here to view this video.</a>

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 ...

coculture-assays-to-study-macrophage-microglia-stimulation

Nanyang Technological University

Research

JoVE Journal

Cancer Research

13.9K Views.  Kean University. 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.

Video: Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

Immunology and Infection

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 &#181;m polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 &#181;m barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Examining Macrophage-Tumor Spheroid Interactions in a 3D Collagen Matrix Setup

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Direct-Contact Co-culture of Astrocytes and Glioblastoma Cells Patterned using Polyelectrolyte Multilayer Templates

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in 2020 and 2021. GBM tumors most often arise from astrocytes and are characterized by their invasive nature, often recruiting healthy tissues into tumor tissue. Understanding communication between astrocytes and glioblastoma cells is vital for the molecular understanding of tumor progression. This protocol demonstrates a novel patterned co-culture method to investigate contact-mediated effects of astrocytes on GBM employing layer-by-layer assembly and micro-capillary-force driven patterning. Advantages include a protein-free cell culture environment and precise control of cellular interaction dictated by the pattern dimensions. This technique provides a versatile, economical, reproducible protocol for mimicking cellular interaction between glioma and astrocytes in glioma tumors. This model can further be used to tease apart changes in GBM molecular biology due to physical contact with astrocytes or with non-contact mediated soluble cofactor communication.

This protocol describes a patterned direct contact glioma-astrocyte co-culture utilizing micro-contact printing on polyelectrolyte multilayers (PEMs) to pattern U87 or A172 GBM cells and primary astrocytes.

Glioblastoma Multiforme (GBM) is the most abundant and fatal malignant brain cancer. There are more than 13,000 cases projected in the United States in ...

Bioengineering

Matrix-Based Tumor Invasion Model Using Microglia Glioblastoma Co-Culture

Source: Coniglio, S., et al.&#160;<a href="https://app-jove-com.remotexs.ntu.edu.sg/v/53990">Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion.</a> J. Vis. Exp. (2016)
In this video, glioblastoma and microglia cells are mixed with a polymer matrix solution and transferred to a porous chamber insert where a gel forms, embedding the cells. Microglia stimulate the migration of glioblastoma cells through the porous insert, mimicking tumor cell invasion into surrounding tissue, Cells are then fixed and prepared for further analysis.

Source: Coniglio, S., et al.&#160;Coculture assays to study macrophage and microglia stimulation of glioblastoma invasion. J. Vis. Exp. (2016)
In this ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Tumors

Microglial Phagocytosis Assay: An In Vitro Assay to Establish Microglial Phagocytosis Model for Neuroblastoma Cells Using iPSC Macrophages

Source: Hall-Roberts, H. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/62217/in-vitro-quantitative-imaging-assay-for-phagocytosis-dead">In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages.</a> J. Vis. Exp. (2021)
In this video, we perform the microglial phagocytosis assay for neuroblastoma using induced pluripotent stem cells. The assay helps assess the impairment of microglial function in neurodegenerative diseases.

Source: Hall-Roberts, H. et al. In vitro Quantitative Imaging Assay for Phagocytosis of Dead Neuroblastoma Cells by iPSC-Macrophages. J. Vis. Exp. (2021)
...

Cancers of the Nervous System

In vitro studies

Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Source: Guyon, J. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/62213" target="_blank">Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions.</a> J. Vis. Exp. (2021)
This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells&#39; migration on neurons in a glioma.

Source: Guyon, J. et al. Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions. J. Vis. Exp. 
...

An Assay for Analyzing Test &beta;-Glucan as an Immunotherapy Strategy against Brain Cancer Cells

Source: Folgado-Dorado, C. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64924">Isolation and Purification of Fungal &#946;-Glucan as an Immunotherapy Strategy for Glioblastoma.</a> J. Vis. Exp. (2023)
This video demonstrates an in vitro assay utilizing &#946;-glucans to activate microglial cells for the production of superoxide radicals, a reactive oxygen species. In brain cancer cells, the generated reactive oxidants actively suppress tumor cell proliferation and induce cell death.

In vitro study of &#946;-glucan-induced microglia stimulation
1. Cell culture of mouse glioblastoma and microglia cells in 8-well chamber slides
NOTE: ...

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

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