We would like to thank Dr. Silvia Soddu and Dr. Giulia Federici (Unit of Cellular Networks and Molecular Therapeutic Targets, IRCCS-Regina Elena National Cancer Institute, Rome, Italy) for access to the IncuCyte S3 Live Cell Imaging System in the preliminary set up of the imaging protocol. Moreover, we thank Bernadett Kolozsvari for the technical advice. The study was supported by the Children with Cancer UK grant (16-234) and The Italian Ministry of Health Ricerca Corrente. M Vinci is a Children with Cancer UK Fellow. R Ferretti is a recipient of Fondazione Veronesi Fellowship (2018 and 2019). The authors acknowledge Fondazione Heal for their support and the Children&#39;s Hospital Foundation for funding the Queensland Children&#39;s Tumor Bank.

This protocol follows the guidelines of the institutions&#39; human research ethics committees.
NOTE: This study was performed using Incucyte S3 and/or SX5 Live-Cell Analysis Instrument (referenced as live cell analysis instrument).
1. Generation of reproducibly sized tumor spheroids
NOTE: The protocol (section 1) described by Vinci et al. 20157,12, was used as repo...

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The live-3D-cell immunocytochemistry we have adopted here for pediatric DMG invasion and migration could be easily adapted also for other highly invasive tumor cell types, including breast and colon cancer cell lines. 
Different from previously performed live-2D-cell immunocytochemistry assays10, when working in 3D, it is suggested to pay attention to some critical steps. In particular, for the invasion assay we describe, it is advised to add the ALR/antibody mix direct...

The ability of tumor cells to evade and disseminate through the surrounding tissue is a hallmark of cancer1. In particular, tumor cell motility is a characteristic feature of malignant tumors, whether it is a metastatic tumor type such as breast2 or colorectal cancer3 or a locally invasive type such as diffuse glioma4,5.
Imaging has a central role in the investigation of many aspects of tumor cell phenotypes; however, live-cell imaging is definitely to be preferred when studying dynamic cellular processes such as migration and invasion, when changes in morphology and cell-cell interaction6,7 occur and can be more easily examined over time. For live-cell imaging, different optical microscopy systems can be used, from phase contrast to confocal fluorescent microscopes, and image acquisition performed over a short or long period of time on an inverted microscope equipped with a chamber for temperature and CO2 control, or in high-content image analysis systems which have built-in chambers, or alternatively in image systems that can sit in the incubator without the need to disturb the cells during the whole duration of the experiment. The choice of the system used is often dictated by a number of factors such as resolution needed, length of the overall acquisition time and time intervals, vessel type used and throughput of the assay (single chamber or multi-well plate), the sensitivity of the cells used (precious and/or rare cells) and phototoxicity of the cells if fluorophores are present.
With regard to fluorescent imaging in live mode, this can be achieved by transducing cells for the expression of fluorescent proteins either for stable expression or as an inducible system8, by transient cell transfection, or by using cell dyes which are now available for live-cell labeling7, for live-cell tracking as well as for labeling subcellular organelles9.
A useful approach has been recently developed for live-cell immunocytochemistry, where an antibody recognizing a surface marker of choice can be bound to a labeling reagent, and upon addition to the culture media, cells expressing the specific marker can be readily imaged in real-time by live-cell imaging. The visualization and quantification of marker expression using such a system can be easily achieved when cells are grown in two-dimensional (2D) culture conditions10.
In this study, we optimized protocols for live-3D-cell immunocytochemistry invasion and migration of pediatric diffuse midline glioma (DMG) patient-derived cells11,12. DMG are highly aggressive brain tumors affecting children, for the vast majority associated with the driver mutation K27M in histone H3 variants. DMG arise in the brain stem and the midline regions of the central nervous system (CNS) and are characterized by a highly infiltrative nature. This invasive capacity has been shown to be at least in part mediated by the intratumor heterogeneity and the cancer-stem-like features of DMG cells7.
To exemplify our assays, an antibody labeling reagent (ALR) was used in combination with an antibody for CD44. CD44 is a transmembrane glycoprotein and adhesion molecule expressed on stem-cell and other cell types, associated with cancer stem cell phenotype and tumor cell migration and invasion13. The protocols include the sample preparation, the image acquisition in brightfield and fluorescent mode, and the analysis on a live-cell analysis instrument that allowed to quantitatively measure in real-time the overall CD44 expression on the DMG cell membrane during 3D invasion and migration. The assays also allowed the possibility to visualize the intermittent fluorescent signal of CD44 on individual cells while migrating and invading. Interestingly an effect of the anti-CD44 antibody was also observed, which potentially acting as a blocking antibody, also seemed to reduce cell migration and invasion as well as to induce a switch of the invasion pattern from a collective mesenchymal-like to a more single-cell amoeboid-like phenotype.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96 Well TC-Treated Microplates</td>
			<td>Corning</td>
			<td>3595</td>
			<td>size 96 wells, polystyrene plate, flat bottom</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Euroclone</td>
			<td>ECB3056D</td>
			<td>solution for neurosphere dissociation</td>
		</tr>
		<tr>
			<td>Burker chamber</td>
			<td>Mv medical</td>
			<td>FFL16034</td>
			<td>cell counting chamber</td>
		</tr>
		<tr>
			<td>CD-44 (156-3C11)</td>
			<td>Cell Signaling Technology</td>
			<td>3570</td>
			<td>Mouse mAb IgG2a</td>
		</tr>
		<tr>
			<td>Corning Matrigel Matrix</td>
			<td>Corning</td>
			<td>356237</td>
			<td>Basement Membrane Matrix (BMM), Phenol Red-free, LDEV-free</td>
		</tr>
		<tr>
			<td>Fabfluor-488 Antibody Labeling Dye</td>
			<td>Incucyte</td>
			<td>4743</td>
			<td>Antibody labelling reagent (ALR): Mouse IgG2a 488 antibody for Live-Cell Immunocytochemistry</td>
		</tr>
		<tr>
			<td>Incucyte S3 and/or SX5 Live-Cell Analysis Instrument</td>
			<td>Sartorius</td>
			<td>-</td>
			<td>The Incucyte S3 and/or SX5 Instrument is used for real-time cell monitoring and surveillance, cell health and viability, migration and invasion, plus a wide range of phenotypic cell-based assays.</td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td></td>
			<td>-</td>
			<td>any inverted microscope</td>
		</tr>
		<tr>
			<td>Opti-Green Background Suppressor Reagent</td>
			<td>Incucyte</td>
			<td>6500-0045</td>
			<td>Backgroung suppressor reagent (BSR)</td>
		</tr>
		<tr>
			<td>Tumor stem cell (TSM) medium</td>
			<td>-</td>
			<td>-</td>
			<td>growth cell medium (see reference in the text for details)</td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Multiple Well Plate</td>
			<td>Corning Costar</td>
			<td>7007</td>
			<td>size 96 well, round bottom clear</td>
		</tr>
	</tbody>
</table>

live-3d-cell immunocytochemistry assays of pediatric diffuse midline glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

Live-3D-Cell Immunocytochemistry protocol for invasion and migration is summarized in a straightforward and reproducible workflow in Figure 1. By seeding the DMG cells in ULA 96-well round-bottom plates, reproducible sized NS are obtained and used in the steps displayed. When the NS have reached the ideal size of ~300 &#181;m (approximately 4 days post-seeding) the invasion12 and migration14 assays are initiated. The addition of the ALR/antibod...

Watch this Scientific Journal Video about Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma at JoVE.com

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

Diffuse midline glioma is a highly invasive brain tumor. This method helps to study in real time how DMG cells migrate and invade in a 3D context with specific insights on the potential role of a key adhesion molecule. By using a labeling reagent and a specific antibody anti-CD44, we can study by live cell imaging the expression of CD44 on plasma membrane on the DMG cells also in 3D migration and invasion.The application of this technique as highlighted the potential role of CD44 in the motility of these cells, suggesting it could represent a valuable and invasive molecular target. To begin, rehydrate the ALR by adding 100 microliters of sterile water and mix the solution by pipetting. Mix the antibody with the ALR in the TSM medium in a round bottom multi-well plate or in an amber tube and protect it from light.Prepare enough quantity of the medium to dispense 25 microliters per well at three times final assay concentration and then incubate at room temperature for 15 minutes. Dilute BSR in the TSM medium at 1.5 millimolar concentration to obtain a final concentration of 0.5 millimolar at the end of the assay. Then gently and slowly remove 75 microliters of the medium from each well, avoiding touching the bottom of the well where the NS sits and checking the presence of the NS visually.Gently add 25 microliters of the BSR and ALR antibody complex to each well and let the ALR antibody complex mix with the medium for two to three minutes. Using an inverted microscope, ensure that each NS is centrally located at the bottom of the well. Avoid the formation of bubbles by removing them using a needle.Place the plate on ice for five minutes to let the bottom of the plate become cold. Dispense 75 microliters of BMM per well by placing a pre-cooled P200 tip on the internal wall of the well, avoiding bubble formation and touching the bottom of the well. Leave the plate on ice for five minutes to let the BMM mix with the medium.Using an inverted microscope, check the location of NS.And if not present centrally, centrifuge the plate at four degrees Celsius at 180 times G for five minutes. Then transfer the plate in the live cell analysis instrument placed within the incubator set at 37 degrees Celsius, 5%carbon dioxide and 95%humidity. Once the wells are coated with BMM, cut a P200 tip.Take 50 microliters of the cell medium and NS from each selected well and transfer it to a coated flat bottom well. Check the presence and the position of the NS in each well visually. Gently add 50 microliters of the diluted BSR and ALR antibody to each well, waiting for two to three minutes to let the reagents mix.And using an inverted microscope, ensure that most of the replicate NS are centrally located in the well. After ensuring the absence of any bubble, gently transfer the plate in the live cell analysis instrument as demonstrated previously. On the live cell analysis instrument software, select the option schedule to acquire, then click on the plus tab and select scan on schedule to scan the plates with intervals as mentioned in the text manuscript.On the software window, create or restore vessel and then click on the option New. Select spheroid scan type, phase plus brightfield, green image channels for the invasion assay, and 4X objective. Select dilution cloning scan, type 4X objective, and phase and green for the migration assay.Select the plate type and define the wells to be scanned by highlighting them on the plate map, then set up the scanning frequency. Click on Add to Schedule and start the scan. Select the tab Create New Analysis Definition.Then select spheroid invasion or basic analyzer application for invasion and migration respectively on the tab. Select the invasion and migration appropriate channels in the image channel. Select a few representative images from three to four wells for previewing and refining the analysis setting.For the invasion assay, in the analysis definition tab, adjust the application settings in the brightfield and green channels with the settings mentioned in the text manuscript to generate a precise segmentation between the spheroid and invading cells. For migration assay, adjust the application settings as mentioned in the text manuscript in the phase and green channels to generate a precise segmentation between the confluence and green cells. Check that the analysis settings are correct for the NS by clicking randomly on several wells.Select the wells and time points to analyze. Save the analysis definition and click on Finish. The representative immunofluorescent confocal images of CD44 expression in primary DMG patient-derived cells demonstrated the role of CD44 in cell migration and invasion.The live 3D cell immunochemistry allows visualizing CD44 expression. The representative frames of the time lapse for both migration and invasion show the presence and absence of the green fluorescent signal on the same cell observed over time, suggesting the expression of CD44 is on and off while cells are migrating and invading. The migration and invasion processes are followed over 96 hours, showing QCTB-R059 cells with a high level of CD44.The quantification of CD44 expression and its increase over time was measured by the overall green fluorescence signal associated with the ALR for both migration and invasion. When the anti-CD44 antibody is used on live cells, it affects cell morphology, inducing a transition from mesenchymal-like to amoeboid-like invasion and a reduction of the invasive and migratory capacity of these cells. The most critical step of the method are the mixing of labeling reagent with an antibody, the addition of the mix to the Matrigel and of the medium, and access to the live cell imaging instrument.This method can be further implemented by the use of two or more distinctive labeling reagents and antibodies to investigate the DMG cell interaction mediated by direct cells and contact. Our method offers new tools to investigate in 3D the cellular and molecular mechanisms of DMG migration and invasion, providing new directions for inhibiting their infiltrative phenotype.

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Cancer Research

2.4K Views.  IRCCS. Il glioma diffuso della linea mediana è un tumore cerebrale altamente invasivo. Questo metodo aiuta a studiare in tempo reale come le cellule DMG migrano e invadono in un contesto 3D con approfondimenti specifici sul ruolo potenziale di una molecola di adesione chiave. Utilizzando un reagente di marcatura e un anticorpo specifico anti-CD44, possiamo studiare mediante imaging cellulare vivo l'espressione di CD44 sulla membrana plasmatica sulle cellule DMG anche nella migrazione e invasione 3D...