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Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

Published: July 29, 2022 doi: 10.3791/64093


With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.


Cell motility is a stereotyped process requiring polarity axis establishment and force-generating cytoskeletal rearrangements. Actin polymerization and its association with myosin are recognized as the main contributors to protrusive and contractile forces required for cell movement1. Microtubules are considered to be the main actors in cell polarization and directional persistence during migration2. In recent years, MTs have also been shown to create and stabilize protrusions to support mechanocompressive forces during cell invasion in 3D3. More recently, MTs have been directly involved in mechanotransduction at focal adhesions and mechanosensitive migration4. The dynamic instability that characterizes MT-plus end dynamics is made of repeated phases of polymerization (growth) and depolymerization (shrinkage), which are controlled by a plethora of microtubule-binding proteins and intracellular signaling cascades, such as those governed by RHO-GTPases5,6,7. The role of the MT network in cell migration and invasion has made the investigation of MT dynamics a key element to better understand the mechanisms of immune cell homing, wound healing, and cancer invasion.

The ability of cancer cells to escape the primary tumor core, spread in the tissues, and generate secondary tumors is a critical step in preventing global success in the war against cancer declared 50 years ago8,9. One of the biggest hurdles has been understanding how cancer cells actively invade the tissue. Key invasion mechanisms rely on the same principles as those governing non-tumorous cell migration10. However, cancer cell migration specificities have emerged11, triggering the need for better characterization of this type of migration. Specifically, because the tumor microenvironment appears as a key player in cancer progression12, observing and analyzing cancer cell invasion in a relevant physiological context is essential to unravel the mechanisms of cancer cell dissemination.

MTs are central to cancer progression, to sustain both proliferation and invasion. Precise analysis of MT dynamics in situ can help identify MT-altering agents (MTA) in both processes. MT dynamics vary drastically upon a change in environment. In vitro, treatment with MT-destabilizing agents such as nocodazole prevents cell protrusion formation when cells are embedded in gels in 3D, whereas it has little effect on 2D cell migration13,14. Although technically challenging, advances in intravital imaging permit in vivo analysis of MT dynamics during cancer cell invasion. For instance, the observation of MTs in subcutaneously xenografted fibrosarcoma cells in mice revealed that tumor-associated macrophages affect MT dynamics in tumor cells15. However, these mouse models involve extensive surgical procedures and remain unsatisfying for less accessible cancers, such as the highly invasive brain tumor, GBM.

Despite a dismal 15 month average survival time16, little is known about GBM's mode of dissemination within the brain parenchyma or the key molecular elements sustaining GBM cell invasion in the brain tissue. Improvement in the mouse orthotopic xenograft (PDX) model and the establishment of cranial windows offered new prospects for GBM cell invasion studies17,18. However, due to suboptimal imaging quality, this model has mostly permitted longitudinal imaging of superficial xenografts and has not been successfully used to study subcellular imaging of cytoskeleton proteins so far. Furthermore, in the wake of the "3Rs" injunction to reduce the use of rodents and replace them with lower vertebrates, alternative models have been established.

Taking advantage of the primitive immunity observed in zebrafish (Danio rerio) larvae, orthotopic injection of GBM cells in the fish brain was developed19,20,21. Injection in the vicinity of the ventricles in the developing midbrain recapitulates most of human GBM pathophysiology21, and the same preferred pattern of GBM invasion as in humans-vessel co-option-is observed22. Thanks to the transparency of the fish larvae, this model allows the visualization of GBM cells invading the brain from the peri-ventricular areas where most GBMs are thought to arise23.

Because MTs are essential for GBM cell invasion in vitro24,25, a better characterization of MT dynamics and the identification of key regulators during cell invasion is needed. However, to date, the data generated with the zebrafish orthotopic model has not included subcellular analysis of MT dynamics during the invasion process. This paper provides a protocol to study MT dynamics in vivo and determine its role during brain cancer invasion. Following stable microtubule labeling, GBM cells are microinjected at 3 dpf in zebrafish larvae's brains and imaged in real time at high spatio-temporal resolution during their progression in the brain tissue. Live imaging of fluorescent MTs allows the qualitative and quantitative analysis of MT plus-end dynamics. Furthermore, this model makes it possible to assess the effect of MTAs on MT dynamics and on the invasive properties of GBM cells in real time. This relatively non-invasive protocol combined with a large number of larvae handled at a time and the ease of drug application (in the fish water) makes the model an asset for preclinical testing.

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Animal experiments were conducted according to European Union guidelines for the handling of laboratory animals. All protocols were approved by the Ethical Committee for Animal Experimentation of Institut Pasteur - CEEA 89 and the French Ministry of Research and Education (permit #01265.03). During injections or live imaging sessions, animals were anaesthetized with Tricaine.At the end of the experimental procedures, they were euthanized by anesthetic overdose. See the Table of Materials for details related to the materials, equipment, and software used in this protocol. The general workflow of the protocol is described in Figure 1.

1. Generation of glioblastoma cells stably expressing α-tubulin-mKate2

NOTE: The following steps are performed in a biosafety cabinet BSL2+.

  1. Produce lentiviral particles expressing mKate2-tubulin using the calcium phosphate method for the transfection of 7 × 106 HEK-293T cells.
    1. In a 1.5 mL microcentrifuge tube, add 10 µg of 2nd generation packaging plasmid psPAX2, 5 µg of viral envelope plasmid pMD2.G, and 10 µg of the plasmid of interest, pGK-mKate2-human tubulin α1a with 50 µL of CaCl2 (2.5 M stock). Adjust to 500 µL with sterile DNAse-free H2O.
      NOTE: It is important to add CaCl2 last.
    2. Mix by inverting the tube a few times and incubate for 20 min at room temperature (RT).
    3. During the incubation, prepare another 1.5 mL microcentrifuge tube with 500 µL of HEPES-buffered saline (HBS), pH 7.0 (2x).
    4. After 20 min, add the CaCl2/DNA mix drop by drop into the HBS (2x) solution. Mix by inverting the tube a few times. Incubate for 12 min at RT.
    5. After 12 min, gently add the CaCl2/DNA mixed with HBS directly onto a 70% confluent 10 cm dish of HEK-293T cells, drop by drop.
    6. Leave the cells in the humidified incubator at 37 °C with 5% CO2 for 36-48 h.
    7. Collect the supernatant and spin it down at 3,000 × g for 5 min to remove cellular debris.
    8. To concentrate the viral particles, load the supernatant into an ultracentrifuge tube and spin it down at 47,508 × g for 90 min at 4 °C.
    9. Discard the supernatant and place the tube upside down on paper to dry the inside of the tube.
    10. Add 30 µL of PBS onto the viral pellet. Do not resuspend the pellet. Leave at 4 °C for 2 h.
    11. Gently resuspend the viral particles by pipetting up and down. Avoid making bubbles. Store at -80 °C.
      NOTE: Larger amounts of viral particles can be produced by transfecting multiple plates of HEK-2-93T cells.
  2. Infect glioblastoma cells with lentiviral particles.
    NOTE: This protocol is written for commercial glioblastoma cell lines such as U-87 MG, U-373 MG, or T98. To use primary patient-derived glioblastoma cells, use specific coating of the plates and non-serum-based medium26.
    1. Prepare a 70% confluent 10 cm dish of U-87 MG glioblastoma cells grown in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum, penicillin-streptomycin (100 units/mL final concentration for penicillin and 100 µg/mL for streptomycin), and non-essential amino-acids (1x).
    2. Add the viral particles at a 1 in 5,000 dilution directly onto the cells. Mix with a gentle swirl. Allow the viruses to infect the cells for no more than 20 h.
    3. Remove the virus-containing medium and replace it with fresh culture medium. Allow the expression of the tagged tubulin for 48-72 h.
      NOTE: The following steps are to be performed in a biosafety cabinet BSL2.
    4. FACS-sort the cells to select the brightest 15% of mKate2+ cells. After removing debris and doublet cells using the forward and side scatter (FCS vs SSC) gating, apply another gating on the brightest 30% mKate2+ cells to keep the top 15%.
    5. Amplify the MT-labeled cells.
      ​NOTE: Alternatively, clonal selection based on high levels of tubulin-mKate2 can be performed under an epifluorescence microscope.

2. Prepare zebrafish larvae for microinjection

  1. Generate zebrafish eggs.
    1. Place three males and four females of the desired zebrafish strain in a mating tank supplemented with marbles 4 days before the xenotransplantation, late in the afternoon.
      NOTE: The Tg(fli1a:gfp), Tg(gfap:gfp), or Tg(Huc:gfp) lines are used to mark endogenous vessels, neural stem cells/astrocytes, and neurons, respectively.
    2. Collect the eggs produced by marble-induced spawning the morning after.
    3. Clean the eggs by transferring them to a 50 mL centrifuge tube filled with mineral source water27 supplemented with bleach (0.004% final). Gently invert the tube for 5 min. Wash twice with mineral water only.
    4. Transfer the eggs into a Petri dish containing the mineral water supplemented with 0.28 mg/mL methylene blue.
    5. Remove the unfertilized and development-arrested eggs. Incubate the embryos at 28 °C.
  2. Create transparent larvae.
    1. Introduce N-phenylthiourea (PTU) (0.003% final) to the medium 8 h later, to prevent melanin pigmentation and ensure optical transparency. Keep PTU in the medium for the rest of the protocol.
      NOTE: Because PTU treatment can cause developmental defects, select only the normally developed larvae for xenotransplantation. As an alternative to chemical treatment, one can use the mutant zebrafish strain casper (nacre and roy orbison double mutant), in which pigmentation is absent28.
    2. Raise the incubating temperature to 29 °C.
    3. Increase the temperature every day by 1 °C so that the 3 dpf larvae reach 32 °C on the day of injection.
  3. Prepare the microinjection plate.
    1. Prepare 20 mL of 1% agarose + 0.28 mg/mL methylene blue with mineral water.
    2. Pour the agarose into a 10 cm Petri dish and quickly apply the microinjection plastic mold upside down to create 2.5 mm wide V-shaped trenches (Figure 2B).
      NOTE: The plastic mold is available commercially or can be built in-house following the design described in the Zebrafish Book29.
    3. Carefully remove the plastic mold when the agarose has solidified.
      NOTE: Microinjection cast plates can be stored at 4 °C for up to 2 months.
  4. Prepare the larvae for xenotransplantation.
    1. On the day of injection, screen for selected fluorescent transgene expression in the 3 dpf larvae. Remove the non-fluorescent, abnormal-looking animals.
    2. Dechorionate the larvae manually with fine-tipped watchmaker forceps. At 3 dpf, gently poke or tear open the chorions with two pairs of fine-tipped forceps to free the larvae from the chorion.
      NOTE: Alternatively, enzymatic treatment with pronase A can be used to dechorionate larvae, usually at 24 hpf.
    3. Maintain the dechorionated larvae in mineral water medium with methylene blue (0.28 mg/mL) and PTU (0.003% final).

3. Xenotransplantation procedure

  1. Prepare microinjection needles.
    1. Take a borosilicate glass capillary without a central filament and place it in a vertical needle puller.
    2. Using the following settings-increase 8.5 and heater 3-stretch the capillary to turn it into a microinjection needle.
  2. Set up the microinjector.
    1. Load mineral oil in a manual microinjector. Remove air bubbles.
    2. Plug a universal capillary holder to the microinjector and firmly attach it to a mechanical micromanipulator (Figure 2A).
  3. Harvest the glioblastoma cells.
    NOTE: The following steps are conducted in a biosafety cabinet BSL2.
    1. Prepare a 10 cm plate of glioblastoma cells so that they reach 80% confluence on the day of transplantation.
    2. (Optional)Transiently label the cell nuclei by adding Hoechst 35480 (200 ng/mL) to the cells. Incubate for 20 min at 37 °C in the humidified cell incubator and wash 2x with PBS.
    3. Take the plate of cells out of the incubator and wash once with PBS. Detach the cells by adding 1 mL of 0.05% trypsin-EDTA and incubating for 5-10 min at 37 °C in the cell incubator until all the cells are fully detached.
      NOTE: This step is critical as poor trypsinization will result in cell aggregates remaining stuck in the needle.
    4. Resuspend the cells in 5 mL of complete glioblastoma cell medium in a 50 mL centrifuge tube. Add 45 mL of ice-cold PBS and centrifuge at 134 × g for 5 min.
    5. Discard the supernatant and resuspend the cells with 1 mL of ice-cold PBS by pipetting up and down thoroughly.
      NOTE: This step of mechanical dissociation greatly helps to prevent the risk of clogging in the capillary during microinjection.
    6. Add 49 mL of ice-cold PBS and centrifuge at 134 × g for 5 min. Discard the supernatant and resuspend the cells in 200 µL of ice-cold PBS. Store on ice for the duration of the transplantation procedure.
  4. Microinject the glioblastoma cells into the zebrafish larvae midbrain.
    1. Fill a microinjection cast plate with 6 mL of E3-medium30 supplemented with 160 mg/L tricaine.
    2. Transfer a dozen dechorionated larvae into the microinjection plate. Once unresponsive to touch, align them in the trenches on their side, head up, and the yolk sac pushed against the wall of the trench, with a size 00 paint brush (Figure 2B,C).
    3. Resuspend the glioblastoma cells. Load 5 µL of cells into the microcapillary using microloading tips and insert the capillary into the universal capillary holder.
    4. Place the microinjection plate containing the unresponsive larvae under the stereomicroscope. Place the tip of the microcapillary on the border of the microinjection plate using the micromanipulator knobs. Break it with a scalpel to create a sharp entry point, roughly the size of the diameter of a cell.
    5. Verify that cells are flowing out of the capillary by gently running oil in the microinjector and plunging the tip of the needle into the medium. Concentrate the cells at the tip of the capillary to maximize the number of cells injected per volume ejected and avoid filling the brain tissue with PBS (Figure 2C).
    6. Carefully examine the cells coming out of the microcapillary as oil is manually introduced into the injector. Define empirically how much turn is needed on the manual knob to deliver 20 to 50 cells. Typically, if cells are concentrated enough, one gentle turn is enough to eject ~10 cells.
      NOTE: If liquid does not properly flow out of the microcapillary, try to loosen the attachment of the microcapillary in the capillary holder. By doing so, oil might leak and drip down along the capillary.
    7. Approach the tip of the capillary against the left Optic Tectum (OT), just above the Middle Cerebral Vein (MCeV, Figure 2F).
    8. Gently press the capillary against the larvae until the skin membrane breaks (Figure 2D,E).
      NOTE: Do not push too hard as this will result in injecting the cells too deep in the brain where the lower optical clarity will prevent observing MTs in detail. The imaging technique is possible for a depth reaching 250-300 µm. However, it is recommended not to inject deeper than 100 µm from the fish surface.
    9. Once a suitable position in the OT is reached, eject the cells. Carefully observe the tip of the capillary to visualize the stream of cells going inside the animal, thereby ensuring successful injection.
      NOTE: Be careful not to inject into the ventricles (Figure 2E). Once in the ventricles, the cells tend to accumulate and get stuck instead of infiltrating the tissue (Figure 2I). Ventricle injection is characterized by intense swelling of the brain and observable dissemination of injected cells to the forebrain and the hindbrain (Figure 2J).
    10. Repeat the procedure from steps 3.4.9-3.4.11 for as many animals as required. Proceed quickly to prevent clumping of cells in the capillary.
      NOTE: Depending on how fast the experimenter is at injecting, a change of needle could be needed every 10 to 20 larvae.
    11. Once the xenotransplantation is complete, remove the larvae from the microinjection plate and single them out in a 24-well plate filled with mineral source water + PTU + methylene blue medium.
    12. Validate successful injection by observing the larvae under a fluorescent stereomicroscope (Figure 2G). Select only xenografts containing a single tumor mass formed by 20-50 cells located in the top 200 µm (in z) of the OT (Figure 2H vs unsuccessful injection in Figure 2I-K).
      NOTE: The yield of successfully localized xenografts varies from 10% at the beginning to almost 100% with practice.
    13. Let the larvae recover for at least 4 h at 32 °C before imaging.
      ​NOTE: Adding antibiotics in the medium does not increase the survival rate of the larvae. At this stage, if microinjection has been correctly performed, almost 100% of the fish survive.

4. Intravital imaging of the glioblastoma xenografts

  1. Mount the larvae for live imaging.
    NOTE: Larvae can be imaged from 4 h post injection (hpi). Live imaging of MT is usually performed from 20 hpi, when invasive GBM cells have started to extend protrusions and migrate away from the tumor mass.
    1. Prepare a 1% low-melting agarose solution. Transfer 500 µL of boiled 1% low-melting agarose solution to a 1.5 mL centrifuge tube and let it cool down at 37 °C on a heat block. Add tricaine (112 µg/mL) to the agarose and mix well.
    2. Transfer one to four xenografted larvae in a 3.5 cm Petri dish filled with mineral source water + PTU + methylene medium complemented with tricaine (112 µg/mL). Once the larvae are unresponsive to touch, transfer them carefully into the tube containing the agarose and the tricaine using a fine-tip transfer pipette.
      NOTE: Keep the volume of medium to a minimum to limit diluting the agarose.
    3. Gently mix the larvae with the agarose. Using a normal (large bulb) transfer pipette, place the larvae mixed in the agarose onto the center of a glass-bottomed 3.5 cm video-imaging dish. Under a stereomicroscope, quickly position the larvae on its back using a microloading tip to manipulate the fish.
      NOTE: Because an inverted spinning-disk confocal microscope is used for intravital brain imaging in this protocol, the larvae are mounted dorsally. Adjust the positioning of the larvae accordingly if using an upright confocal microscope.
    4. Remove extra agarose to maintain the thinnest possible agarose layer. Once the agarose has solidified, add 2.5 mL of mineral source water + PTU + 0.2x tricaine (imaging medium) and proceed to the next step.
  2. In vivo live imaging of MT dynamics in invading glioblastoma cells
    NOTE: The optical quality of the images greatly depends on the performance of the microscope being used. The protocol is written for an inverted spinning-disk confocal microscope equipped with an sCMOS camera (pixel size 6.5 µm, 2048 x 2044 pixels), long working distance objective, and temperature-controlled environmental chamber.
    1. Place the video-imaging dish containing the agarose-embedded xenografted larvae in the environmental chamber of an inverted confocal microscope, with the temperature set at 32 °C. Find the larvae in the video-imaging dish with a 10x objective, using a motorized XY stage.
    2. Press ESC to lower the objectives turret, add mineral oil onto a 60x oil objective (1.4 NA, working distance: 0.13 mm), and press ESC to go back to the initial focal position.
    3. Observe the invading glioblastoma cells in the red channel (561 nm laser source, 20% laser power, time exposure: 200 ms) and select a cell with a spread-out MT network and easily distinguishable MT filaments (Figure 3B).
    4. Set the z-series settings. Using a 200 µm range piezo stage, select the top and bottom positions of the MT network: a 10-30 µm deep z-stack is enough to visualize the microtubule network in the protrusion of the migrating cell, with a z-slice step of 0.3 µm.
    5. Set time-lapse acquisition settings to allow an optimal balance between speed of acquisition, z-stack depth, and fluorescent signal to avoid rapid photobleaching. Acquire images of MTs every 5-10 s for several minutes. Acquire and save the 5D (x,y,z,t,c) hyperstack.
      NOTE: To avoid drifts in z during acquisition, use a microscope equipped with a perfect focus system as hardware focus stabilization.
  3. (Optional) Determine the effects of the microtubule-altering agents (MTAs) on MTs.
    NOTE: The following steps permit testing the effects of the MTAs on an MT network in migrating glioblastoma cells in real time.
    1. Gently remove the imaging medium from the video-imaging dish.Do not touch the bottom of the dish, as the xyz position will be lost.
    2. Prepare new imaging medium containing the MTA at different concentrations. Gently add the MTA-containing medium into the video-imaging dish drop by drop.
    3. Acquire long-term movies (2-16 h, one image every 10-20 min) to observe the effects of each concentration of the MTA on the MT network and cell migration (Figure 4A).
    4. (Optional) Wash out the MTA by gently removing the medium and adding 2.5 mL of fresh imaging medium without the MTA. Repeat the washout procedure 3x to clear any trace of MTA in the medium.
    5. (Optional) Acquire a similar long-term movie as in 4.3.3 (Figure 4B).
      NOTE: The above steps determine the minimal concentration altering the MT network without affecting the larvae survival. The washout steps define whether the effect of the drug is reversible.
  4. Assess MTA impact on glioblastoma invasion by sequential imaging.
    1. Follow steps 4.1 to 4.2.1 4 h after microinjection.
      NOTE: This part of the protocol can also be achieved with other fluorescently tagged glioblastoma cell lines. Ideally, co-label the GBM with a cytosolic tag and a nucleus tag to ensure detection of the global morphology of the cell.
    2. Switch to a long working distance 40x water objective (NA:1.15, WD: 0.6 mm).
    3. Set the z-series range to acquire the OT region. Acquire the z-stack using a high sensitivity sCMOS camera (pixel size 11 µm,1,200 x 1,200 pixels, quantum efficiency 95%).
      NOTE: The z-stack usually starts at the most dorsal part of the OT (close to the surface) and ends deep enough in the brain to include the whole tumor cell mass.
    4. Remove the video-imaging dish from the microscope. Free the larvae from the agarose by using a microloading tip and gently poking the agarose around the animal.
      NOTE: As 3 dpf larvae are still very fragile, be careful when removing them from the agarose.
    5. Once the animal is freed from the agarose, gently transfer it into a single well of a 24-well plate filled with mineral source water + methylene blue + PTU medium. Mark the well to identify the larvae for subsequent imaging.
    6. Add the MTA of interest in the medium at the concentration determined previously (step 4.3.3). Refresh the medium supplemented with the drug every day. Repeat the imaging procedure from steps 4.4.1 to 4.4.5 every day for 3-4 days.

5. Image analysis

  1. Analyze MT dynamics with freely available FIJI plugins.
    NOTE: Many reviews and protocols describe the methods of MT dynamics analysis in cells31,32,33,34 and can be applied at this stage. This protocol will briefly refer to two methods to measure basic MT dynamic properties.
    1. Open the 5D hyperstack and generate a 4D stack where the z-slices have been projected in a single plane to create a maximum intensity projection (MIP) in z (Image | Stacks | Z project | Max Intensity) (Figure 3B).
    2. Open the 4D MIP stack and manually track the end of a protrusion-based MT using the manual tracking function in FIJI (Plugins | Tracking | Manual Tracking). (Figure 3D). Extract the parameters of MT dynamics such as growth speed, shrinkage speed, rescue frequency, and catastrophe frequency.
    3. Alternatively, draw a segmented line of 10 pixels wide along the MT to analyze (Figure 3B). Use the Multi Kymograph function (Analyze | Multi Kymograph) (Figure 3C) in FIJI. Observe and measure the MT growing phases (G), the length of the pauses (P), and the frequency of MT catastrophes.
  2. Analysis of long-term glioblastoma invasion
    NOTE: This analysis requires the use of 4D visualization and analysis with bioimaging software.
    1. Convert the raw 4D z-stack from step 4.4.3 (4 hpi) into the appropriate software format, using the File Converter software.
    2. Open the software and import the converted z-stack file in the Surpass view (Figure 5A).
    3. To segment the tumor cells and discard irrelevant autofluorescence in the red channel, click on Add new Surfaces and follow the 5-step process.
      1. Validate the default settings by clicking on the next arrow once the window pops up. Proceed to step 2/5.
        NOTE: If the fluorescent signal is acquired in the 561 channel, autofluorescence from residual pigmentation in the eye can be strong and alter the automatic segmentation process. This is problematic if the xenograft is located close to the eye. In that case, segmentation has to be finished manually by cutting and deleting non-glioblastoma cell signals.
      2. Navigate to Source channel | 561 Channel. Smoothen the image (Gaussian Filter) by adding 1.50 µm in Surfaces Detail and by adding 2.5 µm for the diameter of the spheres in Substract background. Click on the next arrow and proceed to step 3/5.
      3. Depending on the intensity of the signal, manually adjust the Threshold (background substraction) to include every cell process. Proceed to step 4/5.
      4. Filter the segmented signal by removing the events that do not represent part of a cell (e.g., debris, autofluorescence). Click on Filter type | volume and adjust the threshold manually to exclude all the events below the smallest volume representing part of a cell. Proceed to step 5/5.
        NOTE: If the autofluorescence signals are bigger in volume than the smallest cell part, proceed anyway and manually remove the undesired signals by left-clicking and deleting them.
      5. Discard the classification step and finish the segmentation wizard by clicking on the double green arrow to finish creating a Surface view of the tumor cells (Figure 5B).
    4. If the segmented cells do not touch each other and do not form a unique object, merge them artificially to create a tumor cell mass object. In brief, click on Statistics | detailed | specific values | volume. Select all events by left-clicking on the top one and then holding Shift and left clicking on the last one. Navigate to edit | selection | unify.
    5. Define the centroid of the tumor cell mass. Click on Add new Spots | Skip automatic creation | Add (cursor intersects with) | Center of Object. Hold shift and left click to create the spot, which is the centroid of the segmented tumor cell mass.
    6. Measure the 3D distance between each GBM cell and the centroid of the tumor mass. To do so, create a distance to centroid channel by remaining on the Spot view and selecting the tool icon | Distance Transformation. Wait for a new distance to centroid channel to be created, alongside the 488 and the 561 channel (in blue, Figure 5C).
      NOTE: The intensity of every voxel in this channel corresponds to the 3D distance between the voxel to the centroid of the tumor cell mass.
    7. Measure the 3D distance to the centroid of every segmented cell by clicking on Add new Spots | Skip automatic creation | Add (cursor intersects with) | specific channel 561. Hold shift and left click on the cell nucleus area. Perform this task for every cell (Figure 5D).
      NOTE: Remove the Surface view to better appreciate the fluorescence signal of the cells. Alternatively, if present, use the nucleus signal (405 nm or 647 nm signal) for better accuracy.
    8. Click on Statistics | Detailed | Specific values | intensity mean Ch=distance to centroid.
      NOTE: The value of the intensity is the 3D distance in µm between the cell nucleus area and the centroid.
    9. Average these intensities to calculate the radius of the tumor mass at 4 hpi. Repeat the procedure for every time point of the analysis (24 hpi, 48 hpi, and 72 hpi).
      NOTE: Only measure the 10 or 20 most disseminated cells to avoid underestimating cell invasion. Indeed, the forced compaction of the cells during injection may prevent cells in the center of the tumor mass to migrate.
    10. Calculate the invasion index (II) as the ratio between the mean 3D distances at t = 24 hpi/48 hpi/72 hpi of the most disseminated cells over the mean radius of the tumor mass at t = 4 hpi (Figure 5E).

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Representative Results

To analyze the role played by MTs during in vivo GBM invasion, we describe here the major steps to perform stable MT labeling in GBM cells by lentiviral infection, orthotopic xenotransplantation of GBM cells in 3 dpf zebrafish larvae, high-resolution intravital imaging of MT dynamics, MTA treatment and its effects on GBM invasion, and image analysis of MT dynamics and in vivo invasion (Figure 1). MT dynamics are measured either by building kymographs along growing and shrinking MTs (Figure 3C) or by manually tracking individual MT edges over time (Figure 3D). An example of drug treatment administered in the larvae medium and its reversible effect on MT network organization is given in Figure 4. Treatment with a low dose of nocodazole (200 nM) leads to progressive shrinkage of the MT network and disappearance of glioblastoma cell protrusion 4 h later (Figure 4A). Washing out the drug restored the capacity of glioblastoma cells to form protrusions. The cells resumed migrating along the vasculature 12 h after the washout (Figure 4B). These data suggest treatment with 200 nM nocodazole is sufficient to disrupt the MT network and rapidly blocks in vivo glioblastoma cell invasion. A 3 day-long analysis of the same treatment on global glioblastoma cell invasion reveals that nocodazole 200 nM halts long-term glioblastoma cell invasion in vivo, without affecting the general health of the fish, compared to a control (Figure 4C).

Figure 1
Figure 1: Protocol workflow diagram. Abbreviations: dpf = days post fertilization; hpi = hours post injection; MT = microtubule; MTA = microtubule-altering agent; GBM = glioblastoma multiforme. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Microinjecting glioblastoma cells into 3 dpf zebrafish larvae brains. (A) Photograph of the equipment used for xenotransplantation: 1, oil microinjector; 2, mechanical micromanipulator; 3, universal capillary holder; 4, glass capillary. The microinjection plate is under a stereomicroscope. (B) Photograph showing anesthetized 3 dfp zebrafish larvae aligned in a trench and ready to be microinjected. The tip of a microcapillary loaded with a red dye is visible on the right of the photograph. Details of the patterned trenches built in the agarose plate are seen in the inset on the bottom right corner. Scale bar = 3 mm. (C) Scheme of a representative microinjection plate. Ready-to-be-injected larvae are placed laterally (center of the plate). Note that cells are concentrated at the tip of the microcapillary (black arrow) before proceeding to the injection. Injected larvae are shown on the right of the plate, placed ventrally. (D) Scheme of a transversal slice in the microinjection plate (along the black dotted line in C) showing the trenches where the larvae are placed during injection. (E) Photograph showing the tip of the capillary ready to penetrate the optic tectum (dotted line). The ventricles are delineated by the white lines. Scale bar = 150 µm. (F) Scheme of a 3 dpf fli1a:gfp larva expressing gfp in the endothelial cells, indicating the OT region where the cells are injected, just above the middle cerebral vein. (G) Fluorescence image of a 3 dpf gfap:gfp larva (gfp expressed in neural stem cells) placed laterally after microinjection of U87-mkate2 cells (white circle and white arrow). High autofluorescence in red is caused by the iridophores in the eyes. Scale bar = 100 µm. (H) Confocal fluorescence image of a successfully injected fli1a:gfp larva at 16 hpi. Scale bar: 50 µm. (I-K) Confocal fluorescence images of unsuccessfully injected fli1a:gfp larvae at 96 hpi (I) and 4 hpi (J,K). GBM cells have been injected in ventricles (I,J) or in multiple foci in the brain (K). Scale bars = 50 µm. Abbreviations: dpf = days post fertilization; OT = optic tectum; MCeV = middle cerebral vein; gfp = green fluorescent protein; gfap = glial fibrillary acidic protein; hpi = hours post injection. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Visualizing in vivo microtubule dynamics in glioblastoma cells. (A) Representative fluorescence image of xenografted U87 cells expressing tubulin-α1-mkate2 in the OT of a fli1a:GFP zebrafish larva at 20 hpi. (B) Maximum intensity projected fluorescence image of the MT network in a single xenografted U87 cell. (C) Kymograph along the red dotted line in B, showing the growing, the pause, and the catastrophe phases of MT dynamic instability. (D) Time-lapse sequence of the boxed region in B highlighting the tracking of three MT + ends. Scale bars = 10 µm. Abbreviations: OT = optic tectum; GFP = green fluorescent protein; hpi = hours post injection; MT = microtubule; G = growing phase; P = pause phase; C = catastrophe phase. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Visualizing the effects of the microtubule altering agent on glioblastoma invasion in vivo. (A) Time-lapse sequence of U87 cells expressing tubulin-α1a-mkate2 in zebrafish larvae treated with nocadazole (200 nM). Arrows point to the extremity of the protrusion in two different cells invading the brain along a blood vessel. Note the retraction of the protrusion upon treatment with nocodazole. Scale bar = 10 µm. (B) Time-lapse sequence representing the effect of nocodazole washout on U87 cell invasion. At 500 min after the washout, the cell marked with the white asterisk elongates an MT-based protrusion (white arrow), which permits its resumption of the invasion along a blood vessel. Scale bar = 20 µm. (C) 3D representations of a xenografted larvae brain treated with DMSO or nocodazole (200 nM) for 72 h. U87 cells signal has been segmented (in red) and integrated to the fli1a-GFP fluorescence signal (in white). Note the decreased dissemination of U87 cells treated with nocodazole. Scale bar = 30 µm. Abbreviations: GFP = green fluorescent protein; hpi = hours post injection; MT = microtubule. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Image analysis of in vivo glioblastoma invasion. (A) Fluorescent images of xenografted U87 cells expressing cytosolic mKate2 in fli1a-GFP zebrafish larvae, 4 hpi. White asterisks underline typical autofluorescence from eye iridophores. Scale bar = 40 µm. (B) Fluorescent image of fli1a-GFP larvae coupled with the segmented surface corresponding to the U87 cells signal in A. The centroid of the tumor mass appears green. Scale bar = 40 µm. (C) Fluorescent image representing the red channel signal in A and the newly defined "distance to centroid" channel (in blue). Scale bar = 30 µm. (D) Red channel fluorescence image superimposed with colored spots, whose color represents the distance of the cell to the centroid (in green), violet being the closest to the centroid and white being the furthest apart.Scale bar = 20 µm. (E) An example of sequential analysis of global GBM invasion. 3D distances are determined at 4 hpi and 72 hpi, and the invasion index (II) is calculated according to the formula in the inbox. Scale bar = 20 µm. Abbreviations: GFP = green fluorescent protein; hpi = hours post injection. Please click here to view a larger version of this figure.

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Imaging tumor xenografts at single-cell resolution is likely to become an indispensable tool to improve our understanding of GBM biology. Live imaging in mouse PDX models has led to valuable discoveries on how GBM collectively invades the brain tissue18. However, to date, the spatiotemporal resolution is not high enough to reveal the dynamics of proteins controlling GBM invasion. We reasoned that by coupling the orthotopic engrafting of GBM cells in transparent zebrafish larvae with high-resolution intravital imaging, cytoskeletal proteins such as MTs could be analyzed in sufficient detail to analyze their dynamics during in situ GBM invasion.

The critical steps of the methodology lie in the preparation of the GBM cells and the microinjection procedure. Unhealthy and inadequately dissociated cells will stick together or to the capillary borders and block the flow of injection. In addition, cells need to be sufficiently concentrated in the capillary to minimize the injected volume and implant them in bulk. Injecting a higher volume of more diluted cells will result in multiple, sometimes intermixed tumor foci, whose invasive indexes become difficult to measure. In our hands, manual handling of the oil-based microinjector allows better control of the injection flow than a pressurized air-based electronic microinjector that has been used previously in a similar model19. This is critical to prevent excess flow pressure inside the brain, thereby avoiding subsequent tissue damage and ventricular aggregation of the injected cells.

Some limitations of this model include the necessity to perform the experiment at suboptimal temperatures for both species. Zebrafish are usually raised at 28 °C, whereas human cells are cultured at 37 °C. Above 32 °C, zebrafish embryo development is altered and these changes can be lethal35. However, similar to what is done in adult zebrafish xenograft models36, sequentially acclimating the zebrafish larvae to a temperature of 32 °C increases the survival of transplanted animals compared to the rapid change in temperature post transplantation from 28 °C to 32 °C. However, increasing the temperature further leads to increased animal deaths in accordance with the sensitivity of zebrafish embryos to temperatures above 32 °C35.

Interpreting the in vivo MT dynamics data has to be done carefully as MT dynamics change when the temperature drops below 37 °C37. Parallel in vitro measurement of MT dynamics at 37 °C and 32 °C in the same GBM cells with the same MTA treatment will help validate the differences seen between various GBM cells or between in vivo treatments. It should help confirm that the differences are not caused by variation in temperature sensitivity but by different regulation pathways (for GBM comparison analysis) or by MTA treatment (for MTA effect analysis). This will be of interest should the MT dynamics heterogeneities be linked to different invasion abilities.

Another limitation is the short time window during which invasion can be monitored (72 to 96 h), preventing the measurement of invasion plasticity driven by potential changes in MT dynamics38. After 96 h, we noticed a sharp decrease in GBM cell invasion. At 6 days post injection, the number of GBM cells declined rapidly, presumably due to a host immune response caused by the accumulation of neutrophils and macrophages in the tumor microenvironment39. Delivering MTAs to the whole brain is likely to affect nearby neuronal host cells, which depend on MTs for their activity and whose alteration might subsequently affect GBM invasion40. This approach needs to be complemented with shRNA or optogenetics assays restricting MT alteration to the GBM cells, but remains a good platform to screen for new anti-invasive compounds.

The orthotopic injection of MT-labeled GBM cells in the zebrafish brain is of particular interest to decipher the role of MTs during cancer cell invasion, as very few animal models permit in situ subcellular imaging of cancer cell migration in their tissue of origin15. To date, studies of MT functions during GBM migration rely mostly on in vitro and ex vivo assays and lack in vivo validation24,41,42,43. Coupled with gene-of-interest knock down or an unbiased gene-based screening approach, the assay presented here will help reveal new MT regulators that are important for GBM invasion in vivo.

GBMs are highly heterogeneous tumors whose invasive properties differ greatly between specimens44. Understanding the molecular mechanisms underlying their different mode of invasion will help define ad hoc therapeutic treatments to block GBM dissemination. Systematic measurement of the invasive index, mode of invasion, and cytoskeletal properties such as MT dynamics in various GBM samples will reveal new correlations between frequent genomic mutational profiles and cell invasion patterns relying on specific cytoskeletal properties. Revealing how these mutations affect the change in MT dynamics not only would add to our knowledge on MT regulation during cell migration45,46 but could also lead to long-awaited patient-specific, anti-invasive therapeutics.

The relative ease of microinjecting in zebrafish combined with the high number of larvae available and the ease of injection of drugs make this procedure suitable for personalized medicine47,48. Moreover, in contrast to intravital imaging of GBM xenografts in mice, that only occurs in the upper 500 µm part of the cortex49,50, using zebrafish allows for the visualization of GBM infiltration in the whole CNS. The model presented here meets the criteria to become an invaluable tool for rapid analysis of glioblastoma's invasive capacities and its response to treatments.

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The authors have no conflicts of interest to disclose.


We are extremely grateful to Dr. P. Herbomel (Institut Pasteur, France) and his laboratory, especially Valérie Briolat, and Emma Colucci-Guyon for providing us with the zebrafish lines and the plastic mold for microinjection plates, and for their valuable expertise on zebrafish experimental procedures. We gratefully acknowledge the UtechS Photonic BioImaging (C2RT, Institut Pasteur, supported by the French National Research Agency France BioImaging, and ANR-10-INBS-04; Investments for the Future). This work was supported by the Ligue contre le cancer (EL2017.LNCC), the Centre National de la Recherche Scientifique, and Institut Pasteur and by the generous donations of Mrs. Marguerite MICHEL and Mr. Porquet.


Name Company Catalog Number Comments
Glioblastoma cell culture
Foetal calf serum Eurobio CVFSVF00-01 Reagent
MEM NEAA Gibco 11140-050 Reagent
Modified Eagle's medium Eurobio CM1MEM18-01 Reagent
Penicillin–streptomycin Gibco 15140-122 Reagent
U-87 MG ECACC 89081402-1VL Cells
Lenitivirus production
BD FACSAria III BD bioscience Instrument
BD FACSDiva software v8.0 BD bioscience Software
HEK-293T Merck 12022001 Cells
pMD2.G Addgene Plasmid #12259 Reagent
psPAX2 Addgene Plasmid #12260 Reagent
Ultracentrifuge Optima XPN-80 Beckman Coulter Instrument
Cell passaging and staining
dPBS Gibco 14190-094 Chemical
Hoechst 34580 Sigma-Aldrich 63493 Chemical
Trypsin-EDTA (0,05%) Gibco 25300-054 Reagent
Zebrafish husbandry
Fluorescence stereomicroscope LEICA M165FC LEICA https://www.leica-microsystems.com/fr/produits/stereomicroscopes-et-macroscopes/informations-detaillees/leica-m165-fc/ Instrument
Methylene Blue hydrate Sigma-Aldrich M4159 Chemical
N-Phenylthiourea (PTU) Sigma-Aldrich P7629-25G Chemical
Transfer Pipettes fine tips Samco Scientific 232 Equipment
Transfer Pipettes Large Bulb3mL Samco Scientific 225 Equipment
Tricaine (Ethyl 3-aminobenzoate methanesulfonate) Sigma-Aldrich Cat#: A5040 Chemical
Volvic Source Water DUTSCHER DOMINIQUE SAS 999556 Reagent
24-well plate TPP 92024 Equipment
Borosilicate glass capillaries (1.0 ODx0.58IDx150L mm) Harvard Apparatus (#30-0017 GC100-15 Equipment
CellTram oil vario microinjector Eppendorf 5176000.025 Instrument
Microloading pipet tips (Microloader) 20µL Eppendorf  5242956003 Equipment
Micromanipulator NARISHIGE https://products.narishige-group.com/group1/injection/english.html Equipment
Mineral Oil Sigma M8410-100ml Equipment
Stereomicroscope Olympus KL 2500 LCD Instrument
Universal capillary holder Eppendorf 5176190002 Equipment
Vertical Pipette puller KOPF (Roucaire) Model 720 Instrument
Intravital Imaging
3.5cm glass-bottom videoimaging dish MatTek Life Sciences, MA, USA P35G-1,5-14-C Equipment
Acquisition software: NIS-Elements-AR version 5.21 Nikon Software
Heat-Block Techne DRI-BLOCK DB-2D Equipment
Microscope head Nikon Ti2E Nikon Instrument
sCMOS camera Prime 95B Photometrics Instrument
sCMOS camera Orca Flash 4 Hammatsu Instrument
Ultrapure Low melting point agarose Invitrogen 16520-050 Chemical
Yokagawa CSU-W1 spinning disk unit Hammatsu Instrument
Drug Treatment
DMSO Sigma-Aldrich D2650-100ML Chemical
Nocodazole Sigma-Aldrich M1404-2MG Chemical
Image Analysis
Imaris 9.5.1 software Oxford Instruments Software
ImarisFileConverter 9.5.1 Oxford Instruments Software



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Peglion, F., Coumailleau, F., Etienne-Manneville, S. Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain. J. Vis. Exp. (185), e64093, doi:10.3791/64093 (2022).More

Peglion, F., Coumailleau, F., Etienne-Manneville, S. Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain. J. Vis. Exp. (185), e64093, doi:10.3791/64093 (2022).

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