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.
