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 reported below, with some modifications:
<ol>
	<li>Collect the DMG H3K27M-mutant neurospheres (NS) and centrifuge at 170 x g for 10 minutes (min) at room temperature (RT).</li>
	<li>Incubate the NS with 500 &#181;L of the accutase solution for 3 min at 37 &#176;C to break them up.</li>
	<li>Neutralize the accutase solution with tumor stem cell (TSM) medium7 and centrifuge the cell suspension at 355 x g for 5 min at RT.</li>
	<li>Resuspend the cell pellet in 1 mL of TSM medium and then count the cells using a cell counting chamber.</li>
	<li>Dilute the cell suspension to obtain 2.5-5 x 103cells/mL and seed 100 &#181;L/well into ultra-low attachment (ULA) 96-well round-bottom plates (see Table of Materials). Use a proper cell density to obtain individual NS of ~300 &#181;m diameter, 4 days after cell seeding (250-500 cells/well for highly aggressive glioma cells).</li>
	<li>Visually confirm the NS formation by using an inverted microscope 4 days after cell seeding.</li>
</ol>
2. Preparation of the ALR/antibody complex and setup for the invasion assay
NOTE: For the antibody labeling procedure, the antibody labeling dyes protocol10 for live-cell Immunocytochemistry is used with some modifications, as reported below. For the invasion assay, the protocol previously described by Vinci et al. 201512 is followed.
<ol>
	<li>Consider the number of wells (e.g., 60 wells) to analyze and calculate the volume needed for each reagent. Also include the wells for the negative control (samples with ALR but without antibody).</li>
	<li>Add 100 &#181;L of sterile water to the ALR to rehydrate the reagent (final concentration = 0.5 mg/mL). Pipette to mix the solution. 
	NOTE: The reagent is light-sensitive; therefore, keep in the dark. Aliquot the leftover reagent and store at -80 &#176;C (avoid freezing and thawing).</li>
	<li>Mix the antibody with the ALR in the TSM medium (or appropriate cell growth media for the cell line of choice) in a round bottom multi-well plate or in an amber tube and protect from light.</li>
	<li>Prepare enough quantity of the medium to dispense 25 &#181;L/well at 3x final assay concentration. Incubate at RT for 15 min. 
	NOTE: A 1:3 molar ratio of antibody to ALR is recommended, with a final (1x) concentration of the test antibody &#60;1.5 &#181;g/mL. For the experiments in this protocol an anti-human CD44 mouse antibody is used (starting concentration 86 &#181;g/mL) at a final concentration of 0.1 &#181;g/mL (3x concentration = 0.3 &#181;g/mL).Add the reagents in the following order: i) antibody; ii) ALR; iii) TSM medium. Mix by pipetting.</li>
	<li>Dilute the background suppressor reagent (BSR) in TSM medium (or appropriate cell growth media for cell line of choice) at 1.5 mM (3x) to obtain at the end of the assay a final concentration of 0.5 mM.</li>
	<li>Perform the invasion assay directly in the ULA 96-well round-bottom plate where cells were initially seeded. Check the NS visually using an inverted microscope before starting.</li>
	<li>Gently and slowly remove 75 &#181;L/well of the medium, avoiding touching the bottom of the well where the NS sits. Check the presence of the NS visually.</li>
	<li>Gently add 25 &#181;L of the BSR to each well.</li>
	<li>Gently add 25 &#181;L of the ALR/antibody complex to each well. Wait 2 or 3 min to let the ALR/antibody complex mix with the medium.</li>
	<li>Check visually using an inverted microscope to ensure that each NS is centrally located at the bottom of the well. Avoid the formation of bubbles. If any bubble is present, remove it by using a needle.</li>
	<li>Place the plate on ice and wait 5 min to let the bottom of the plate become cold.</li>
	<li>With a pre-cooled p200 tip, dispense 75 &#181;L/well of basal membrane matrix (BMM), placing the pipette tip on the internal wall of the well and avoiding touching the bottom of the well. Avoid the formation of bubbles and remove with a sterile needle the existing ones. 
	NOTE: Make sure to have thawed the BMM at 4 &#176;C from the night before.</li>
	<li>Leave the plate on ice for 5 min to let the BMM mix with the medium. Check visually using an inverted microscope the presence of the NS and that they are centrally located in the well. If not, centrifuge the plate at 4 &#176;C at 180 x g for 5 min.</li>
	<li>Transfer the plate in the live-cell analysis instrument (Table of Materials) placed within the incubator at 37 &#176;C, 5% CO2, 95% humidity.</li>
</ol>
3. Preparation of the ALR/antibody complex and setup for the migration assay
NOTE: For the antibody labeling procedure, the Labeling Dyes protocol10 for Live-Cell Immunocytochemistry is used.
<ol>
	<li>Consider the number of wells to analyze and calculate the volume needed for each reagent. Include also the wells needed for the negative controls. Check the NS visually using an inverted microscope before starting.</li>
	<li>Use flat-bottom 96-well plates. Perform the coating procedure as described by Vinci et al., 201314. For this study, the BMM is used as a thin coating.</li>
	<li>Once the coating is ready, remove the excess of BMM coating with a p200 tip placing the tip in the edge of the well and avoiding touching the bottom. If working with multiple wells, use a multichannel pipette.</li>
	<li>Cut a p200 tip, take 50 &#181;L of the cell medium + 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. 
	NOTE: Each NS must be centrally located in the well. Avoid leaning on the tip on the edge of the well during the transfer but drop the medium centrally in the well without touching the bottom. For highly migratory cells, consider a higher number of replicates than the standard three replicates. This is because when NS sits too close to the edge of the well, the migrating cells may cover a smaller area of the well.</li>
	<li>Rehydrate the ALR as described above (steps 2.2-2.3). 
	NOTE: Reagent is light sensitive. See above for good handling procedures.</li>
	<li>Mix the antibody with ALR in the appropriate complete cell growth media in a round bottom multi-well plate or in an amber tube and protect from light. Prepare enough quantity to dispense 50 &#181;L/well at 3x final assay concentration. Incubate at RT for 15 min. 
	NOTE: Add the reagents in the order as indicated above (step 2.4).</li>
	<li>Follow the same procedure as reported in step 2.5.</li>
	<li>Gently add 50 &#181;L of the BSR to each well.</li>
	<li>Gently add 50 &#181;L of the ALR/antibody to each well. Wait 2 or 3 min to let the reagents mix and check visually using an inverted microscope to ensure that most of the replicate NS are centrally located in the well.</li>
	<li>Avoid the formation of bubbles and remove any existing ones by using a needle. Gently transfer the plate in the live-cell analysis instrument placed within the incubator at 37 &#176;C, 5% CO2, 95% humidity.</li>
</ol>
4. Live-cell analysis instrument setting for image acquisition
<ol>
	<li>Scan the plates using the live-cell analysis instrument (for specifications, see Table of Materials) with scanning intervals starting from time point zero (t0) of the invasion and migration assays set up, respectively, after step 2.14. and 3.10. up to 96 h. 
	NOTE: Ensure to be able to dispose of the live-cell analysis instrument immediately after the starting of the invasion and migration assay. Depending on the tumor type, cells can start to invade or migrate from the NS already within 1 h from the assay setup.</li>
	<li>On the live-cell analysis instrument software, select the option Schedule to Acquire. Click on the + tab and select the option Scan on Schedule.</li>
	<li>On the software window Create or Restore Vessel, click on the option New.</li>
	<li>Select the specific application in the live-cell analysis instrument for the invasion and migration acquisition. Select Spheroid scan type, 4x objective, Phase+Brightfield and Green image channels for the invasion assay. Select Dilution Cloning scan type, 4x objective and Phase and Green for the migration assay.</li>
	<li>Select the plate type and define the wells to be scanned by highlighting them on the plate map.</li>
	<li>Set up the scanning frequency (for the experiments in this protocol scanning frequency was 15 min for invasion and 30 min for migration).</li>
	<li>Click on Add to Schedule and start the scan.</li>
</ol>
5. Live-cell analysis instrument setting for image analysis
<ol>
	<li>Select the tab Create New Analysis Definition.</li>
	<li>Select Spheroid Invasion or Basic Analyzer application for invasion and migration, respectively, on the tab.</li>
	<li>Select the invasion and migration appropriate channels (for Invasion: Phase+Brightfield-Green; for Migration: Phase-Green) in the image channel.</li>
	<li>Select few representative images from 3-4 wells for previewing and refining the analysis setting.</li>
	<li>For the invasion assay, in the Analysis Definition tab, adjust the application settings in the Brightfield and Green channels with the following setting to generate a precise segmentation between the Whole Spheroid and Invading Cells (see Figure 5; blue mask): 
	Brightfield Segmentation: Whole Spheroid sensitivity = 50; Invading Cell sensitivity = 100; Clean Up = default. 
	Whole Spheroid Filters: set all parameters as default. 
	Invading Cells Filters: set all parameters as default. 
	Green Segmentation: Radius = 900.</li>
	<li>For migration assay, adjust the application settings in the Phase and Green channels to generate a precise segmentation between the Confluence and Green Cells (see Figure 5, yellow and pink masks) with the following setting: 
	Phase: set all parameters as default. 
	Green Segmentation: Radius = 300; Threshold = 1000 
	Cleanup: Hole Fill = 400; Filters = default. 
	Whole Well: set all parameters as default.</li>
	<li>Check that the analysis settings are correct for the NS by clicking randomly on several wells. The segmentation must outline the spheroid. If not, adjust the setting accordingly.</li>
	<li>Select the wells and time points to analyze.</li>
	<li>Save the Analysis Definition and click on Finish.</li>
</ol>

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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

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 directly to the medium with NS in each well, prior to the addition of the BMM, and not in the BMM or on top of the BMM once gelled. This is to allow a good mix of the reagents with the medium and ensure more direct access of the reagents to the cell surface. Moreover, to ensure a better quality of the imaging, although the protocol includes the use of the BSR, we advise using phenol red-free medium and BMM. 
Another point to consider for the live-cell immunocytochemistry is that any antibody binding an extracellular membrane protein on live cells may affect the protein function by altering its conformation or by occupying the binding site of a ligand or of a protein on an adjacent cell, therefore acting as a "blocking agent"18,19. While this approach may be useful as a therapeutic strategy19, it may not be the primary goal of any experimental setup. Therefore, prior to performing a large set of experiments, different antibodies binding distinct epitopes of the same protein should be tested to also verify any potential "blocking" effect. In this study, we used a specific antibody to follow in real-time the expression of CD44 on the cell membrane of a highly aggressive pediatric DMG cell line in 3D invasion and migration. The protocol used allowed us to quantitatively measure the expression of CD44 over time on cells invading and migrating. Interestingly, in the presence of the anti-CD44 antibody, we also noted a reduction in cell motility in comparison to the cells with the ALR but in the absence of the antibody. We cannot exclude though also an inhibitory effect on cell proliferation. The acquisition of a different invasion pattern with a switch from mesenchymal-like to amoeboid-like cell morphology20 was also observed in the presence of anti-CD44 antibodies. These unexpected results suggest that blocking CD44 may contribute to mesenchymal to amoeboid transition in pediatric DMG. 
With regard to the limitations of this protocol, taking into consideration the resolution of the CCD camera of the Incucyte Live-Cell Analysis Instrument and its limited z-stack capability, the setup we present for the live-3D-cell immunocytochemistry assays may be used as a preliminary approach, on a large scale multi-well format, before moving on to a more in-depth analysis using either more powerful fluorescent imaging systems (e.g., confocal microscopes and high-content imaging system with a confocal modality such as the Operetta CLS or the Opera Phoneix) or a more refined approach for studying in real-time the expression of a surface protein via a reporter assay21.
A broader application of the live-3d-cell immunocytochemistry presented here as a monoculture, could be a 3D co-culture assay established to image and analyze in real-time direct cell-cell interactions. In this case, two different ALR could be used with different fluorophores to bind proteins specifically expressed on the cell membrane on different cell types (e.g., tumor cell and immune cells). In this case, direct cell-cell contact may be analyzed with live imaging by the co-localization of the two different ALR/antibody complexes.

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><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/antibody complex together with the background suppressor in the medium of the individual NS, allows following the specific marker expression on the cell membrane, in live imaging and over time. The surface marker expression during the cell invasion and migration is easily monitored at intervals starting from t = 0 up to 96 h using the live-cell analysis instrument. This imaging system allows a fully automated image analysis.
A primary patient-derived cell line, QCTB-R059, was used to exemplify the invasion and migration proprieties of pediatric DMG tumor dissemination. QCTB-R059 was originally indicated as a pediatric thalamic glioblastoma (GBM) cell line15. Later on, it has been indicated as H3-K27M thalamic glioma cell line16 or diffuse midline glioma (DMG) H3-K27M cell line11, following the 2016 World Health Organization classification of brain tumors with the introduction of DMG H3F3A K27M-mutant as a new entity17.
CD44, an adhesion molecule known to be involved in cell migration and invasion, was investigated. CD44 is expressed by QCTB-R059 cells as demonstrated by confocal images of immunofluorescent (IF) staining on 3D cell migration onto (Figure 2), and invasion into (Figure 3) BMM.
Taking into consideration that 3D invasion and migration are both non-static processes, we thought to investigate the expression of CD44 over time when cells are in movement. To do this we employed the live-cell immunocytochemistry assay and adapted the protocol for 3D assays. By using the ALR in complex with an anti-CD44 antibody, we are able to follow in real-time the expression of CD44 when the protein is expressed on the cell membrane while the cells evade the neurospheres and spread onto and into the BMM.
The live-3D-cell immunocytochemistry allows visualizing CD44 expression (Supplementary Video 1 and Supplementary Video 2). The representative frames of the time-lapse, for both migration and invasion (Figure 4A,B), show more in detail the intermittent expression of CD44 on the cell membrane. In particular, it is possible to see the green fluorescent signal to be on (green circle) and then off (black circle) on the same cell observed over time, suggesting that the expression of CD44 is on and off while cells are migrating and invading.
The migration and invasion processes are followed over 96 h, and as shown in Figure 5, QCTB-R059 cells show a high level of CD44, demonstrating that overall the expression observed with the live-cell immunocytochemistry is in line with the expression of CD44 obtained by IF shown on confocal images in Figure 2 and Figure 3. Interestingly though, we also noticed that when the anti-CD44 antibody is used on live cells, it affects cell morphology, inducing a transition from mesenchymal-like to amoeboid-like invasion. It induces a reduction of the invasive and migratory capacity of these cells (Supplementary Figure&#160;1). We cannot exclude, though, that the reduction in cell migration and invasion observed is also in part due to an inhibition of cell proliferation.
The automated image analysis performed on the live-cell analysis instrument shows the quantification of CD44 expression and its increase over time, measured by the overall green fluorescent signal associated with the ALR (Figure 5B,C) for both migration and invasion. The quantifications are achieved as exemplified in Figure 5B and Figure 5C, with the automated image analysis set to segment all the area covered by the CD44 green-migrated cells (Figure 4B) and all the spread area covered by the CD44 green-invaded cells but excluding the neurosphere core (Figure 5C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63091/63091fig01.jpg" /> 
Figure 1: Schematic workflow of Live-3D-Cell Immunocytochemistry Assays. The workflow shows the steps involved in the 3D invasion and migration live imaging methods, including representative images of pediatric primary DMG patient-derived cells (QCTB-R059) after invasion into (top panel; t = 96 h) and migration onto (bottom panel; t = 96 h) BMM. Bars = 800 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/63091fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63091/63091fig02.jpg" /> 
Figure 2: CD44 expression in 3D tumor cell migration. Representative immunofluorescent confocal images of CD44 expression in primary DMG patient-derived cells (QCTB-R059) upon migration onto BMM. Timepoint = 96 h (red:CD44; blue: nuclei). Scale bars: 500 &#181;m (upper panel) and 200 &#181;m (lower panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/63091fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63091/63091fig03.jpg" /> 
Figure 3: CD44 expression in 3D tumor cell invasion. Representative immunofluorescent confocal images of CD44 expression in primary DMG patient-derived cells (QCTB-R059) upon invasion into BMM. Time point = 96 h (red:CD44; blue: nuclei). Scale bars: 250 &#181;m (upper panel) and 100 &#181;m (lower panel). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/63091fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63091/63091fig04.jpg" /> 
Figure 4: CD44 expression over time. Selected frames of QCTB-R059 migration (A) and invasion (B) time-lapse. Images were obtained on the live-cell imaging instrument. Green circle indicates the expression of CD44, black circle indicates no CD44 expression on the cell membrane of the same cell observed over time. Scale bars: 200 &#181;m (A) and 100 &#181;m (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/63091fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63091/63091fig05.jpg" /> 
Figure 5: Live-3D-Cell Immunocytochemistry Assays for CD44: migration and invasion. (A) Representative brightfield, fluorescent (ALR with anti-CD44 antibody), and merge images of QCTB-R059 cell immunocytochemistry migration, and invasion (96 h) are shown. Scale bars: 400 &#181;m. (B) Quantification of CD44 overall expression relatively to migration (B) and invasion (C), determined by ALR-anti-CD44 image analysis on the live-cell imaging instrument. The curves show the Green Mean Intensity of CD44 expression over time. Values are mean &#177; SD. The two small figures in the plots display the segmentation applied for the analysis of the migration (B) where all area was considered and the invasion (C) for which the NS core part was excluded. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/63091fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Effect of anti-CD44 antibody on cell morphology and degree of cell motility. Representative images of QCTB-R059 invasion and migration assay show the effect of anti-CD44 antibody used for the live-3D-cell immunocytochemistry. Cells display a reduced invasion and migration capacity as well as the transition from a more mesenchymal-like to amoeboid-like invasion pattern between the negative control (without anti-CD44 antibody) and CD44 (plus anti-CD44 antibody). Lower panel shows higher power magnification displaying a more clear view on the morphological appearance of the cells in the absence and the presence of the anti-CD44 antibody (white arrows). Scale bars: 800 &#181;m upper panel and 100 &#181;m lower panel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/Supplementary Figure 1.png" target="_blank">Please click here to download this File.</a>
Supplementary Video 1: Time-lapse video of QCTB-R059 3D cell migration on BMM in the presence of anti-CD44 antibody. Fluorescent green signal, representing the expression of CD44 on the cell membrane, is visualized over time by the conjugation of the anti-CD44 antibody with ALR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/Supplementary video 1_QCTB-R059_migration_video.avi" target="_blank">Please click here to download this Video.</a>
Supplementary Video 2: Time-lapse video of QCTB-R059 3D cell invasion in BMM in the presence of anti-CD44 antibody. Fluorescent green signal, representing the expression of CD44 on the cell membrane, is visualized over time by the conjugation of the anti-CD44 antibody with ALR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63091/Supplementary video 2_QCTB-R059_invasion video.avi" target="_blank">Please click here to download this Video.</a>

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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 ...

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.5K Views.  IRCCS. 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. 

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

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.

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 ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Medicine

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically complex tumors, with a high degree of proteomic variability across both the tumor itself and its heterogenous microenvironment. The malignant potential of these tumors is enhanced by the dysregulation of proteins involved in several key pathways, including processes that maintain cellular stability and preserve the structural integrity of the microenvironment. Although there have been numerous bulk and single-cell glioma analyses, there is a relative paucity of spatial stratification of these proteomic data. Understanding differences in spatial distribution of tumorigenic factors and immune cell populations between the intrinsic tumor, invasive edge, and microenvironment offers valuable insight into the mechanisms underlying tumor proliferation and propagation. Digital spatial profiling (DSP) represents a powerful technology that can form the foundation for these important multilayer analyses.
DSP is a method that efficiently quantifies protein expression within user-specified spatial regions in a tissue specimen. DSP is ideal for studying the differential expression of multiple proteins within and across regions of distinction, enabling multiple levels of quantitative and qualitative analysis. The DSP protocol is systematic and user-friendly, allowing for customized spatial analysis of proteomic data. In this experiment, tissue microarrays are constructed from archived glioblastoma core biopsies. Next, a panel of antibodies is selected, targeting proteins of interest within the sample. The antibodies, which are preconjugated to UV-photocleavable DNA oligonucleotides, are then incubated with the tissue sample overnight. Under fluorescence microscopy visualization of the antibodies, regions of interest (ROIs) within which to quantify protein expression are defined with the samples. UV light is then directed at each ROI, cleaving the DNA oligonucleotides. The oligonucleotides are microaspirated and counted within each ROI, quantifying the corresponding protein on a spatial basis.

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Diffusely infiltrating gliomas are associated with high morbidity and mortality due to the infiltrative nature of tumor spread. They are morphologically ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Biology

Immunocytochemistry-Based Imaging for Visualizing Pediatric Glioma Cell Migration

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/63091/live-3d-cell-immunocytochemistry-assays-pediatric-diffuse-midline'>Source: Pericoli, G. et. al., Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma. J. Vis. Exp. (2021)</a>This video demonstrates live cell imaging to observe pediatric diffuse midline glioma cell migration using immunohistochemical labeling with anti-CD44 antibodies.

Source: Pericoli, G. et. al., Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma. J. Vis. Exp. (2021)This video demonstrates live ...

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

Summary

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Chapters in this video

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

A 3D Spheroid Model for Glioblastoma

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in Clinically Relevant Animal Models and 3D Immunochemistry

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

Immunocytochemistry-Based Imaging for Visualizing Pediatric Glioma Cell Migration