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

This method is dedicated to the investigation of the role of microtubules during brain cancer invasion by coupling the atopic injection of brain cancer cell in zebrafish to the subcellular intravital imaging. The main advantage of the technique lies in the high spatial temporal resolution at which the microtubule cytoskeleton is acquired. It is unique for in vivo brain cancer invasion models and it allows in depth microtubule dynamics analysis.

The subcellular imaging of in situ brain cancer cell invasion can be used for the analysis of any protein of interest potentially involved in cancer invasion. To begin, wash the glioblastoma cells cultured at 37 degrees Celsius with PBS. Detach the cells by adding one milliliter of 0.05%Trypsin EDTA and incubating for five to 10 minutes at 37 degrees Celsius in the cell incubator until all the cells are fully detached.

Resuspend the cells in five milliliters of complete glioblastoma cell medium in a 50 milliliter centrifuge tube. Add 45 milliliters of ice cold PBS and centrifuge at 134 times G for five minutes. Discard the supernatant and resuspend the cells with one milliliter of ice cold PBS by pipetting up and down thoroughly.

Add another 49 milliliters of ice cold PBS and centrifuge at 134 times G for five minutes. Discard the supernatant and resuspend the cells in 200 microliters of ice cold PBS. Store on ice for the duration of the transplantation procedure.

To micro inject the glioblastoma cells into the zebrafish larvae midbrain, fill a microinjection cast plate with six milliliters of E3 medium supplemented with 160 milligrams per liter of tricaine. Transfer a dozen DEE-KOHR-EE-UH-NAY-TED larvae into the microinjection plate. Once the larva are anesthetized and fully 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 paintbrush.

Then load five microliters of glioblastoma cells into the micro capillary using microloading tips and insert the capillary into the universal capillary holder. Place the microinjection plate containing the anesthetized larvae on the stereo microscope stage. Place the tip of the micro-capillary on the border of the microinjection plate using the micro-manipulator knobs.

Then break it with a scalpel to create a sharp entry point roughly the size of the diameter of a cell. Verify that cells are flowing out of the capillary by gently running oil in the micro-injector 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 injected and avoid filling the brain tissue with PBS.

Carefully examine the cells coming out of the micro capillary as oil is manually introduced into the injector. Define empirically the number of turns needed on the manual knob to deliver 20 to 50 cells. Typically, if cells are concentrated enough, one gentle turn is enough to eject approximately 10 cells.

Approach the tip of the capillary against the left optic tectum just above the middle cerebral vein. Gently press the capillary against the larvae until the skin membrane breaks. Once a suitable position in the optic tectum 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. Repeat the procedure for as many animals as required. Depending on how fast the experimenter is at injecting, a change of needle could be needed every 10 to 20 larvae due to rapid cell clumping in the capillary.

Once the xeno transplantation is complete, remove the larva from the microinjection plate and single them out in a 24 well plate filled with mineral source water PTU and methylene blue medium. Prepare a 1%low melting AH-KROH solution. Transfer 500 microliters of boiled 1%low melting AH-KROH solution to a 1.5 milliliter centrifuge tube, and let it cool down at 37 degrees Celsius on a heat block.

add 160 micrograms per liters of tricaine to the AH-KROHS and mix well. Transfer one to four xenografted larvae into a 3.5 centimeter Petri dish filled with mineral source water PTU and methylene medium complemented with 160 micrograms per liters of tricaine. Once the larva are anesthetized and fully unresponsive to touch, transfer them carefully into the tube containing the AH-KROHS and tricaine using a fine tip transfer pipette.

Gently mix the larva with the AH-KROHS. Using a large bulb transfer pipette, place the larvae mixed in the AH-KROHS onto the center of a glass bottomed 3.5 centimeter video imaging dish Under a stereo microscope quickly position the larvae on its back. Using a micro loading tip remove extra AH-KROHS to maintain the thinnest possible AH-KROHS layer.

Once AH-KROHS has solidified, add 2.5 milliliters of imaging medium. For the in vivo live imaging, of microtubule dynamics in invading glioblastoma cells, place the video imaging dish containing the AH-KROHS embedded xenografted larvae in the environmental chamber of an inverted confocal microscope with a temperature set at 32 degrees Celsius. Find the larvae in the video imaging dish with a 10x objective using a motorized XY stage.

Press escape to lower the objectives turret and add mineral oil onto a 60x oil objective, and press escape to go back to the initial focal position. Observe the invading glioblastoma cells in the red channel set at 561 nanometer laser source, 20%laser power, and a time exposure of 200 milliseconds. Then select a cell with a spread out microtubule network and easily distinguishable microtubule filaments.

Set the Z series settings. Using a 200 micrometer range piezo-stage, select the top and bottom positions of the microtubule network where a 10 to 30 micrometer deep Z stack is enough to visualize the microbial network in the protrusion of the migrating cell with a Z slice step of 0.3 micrometers. Set time lapse acquisition settings to allow an optimal balance between speed of acquisition, Z-stack depth and fluorescent signal to avoid rapid photo bleaching.

Acquire images of the microtubules every five to 10 seconds for several minutes and save the 5D hyper-stack. The microtubule dynamics were measured by building kymograph along growing and shrinking microtubules and manually tracking individual microtubule edges over time. Drug treatment administered in the larva medium and its reversible effect on microtubules network organization was assessed by treating with a 200 nanomolar dose of nocodazole.

This led to progressive shrinkage of the microtubule network and disappearance of glioblastoma cell protrusion after four hours. Washing out the drug restored the capacity of glioblastoma cells to form protrusions. The cells resumed migrating along the vasculature 12 hours after the washout indicating that a 200 nanomole dosage of nocodazole was sufficient to disrupt the microtubule network and rapidly block in vivo glioblastoma cell invasion.

A three day long analysis of the same treatment on global glioblastoma cell invasion revealed that the nocodazole halted long-term glioblastoma cell invasion in vivo without affecting the general health of the fish compared to a control. Remember to start with healthy three days post-fertilization larvae to concentrate the cells into the capillary, to minimize the volume injected in the brain, and to form a unique tumor mass, and try to avoid injecting in the brain ventricles.