This protocol enables the user to study the impact of invasive surgical procedures used in the clinic on tumor cell behaviors, such as migration, invasion, and proliferation. This method uniquely allows visualization of the same tumor before and after an invasive procedure, providing an insight that could be missed in a non-longitudinal approach. After confirming a lack of response to toe pinch in an anesthetized adult mouse, mount the mouse on a stereotactic frame and secure the head with a nose clamp and two ear bars.
Use a heating lamp to preserve the body temperature, and apply ointment to the animal's eyes. Using sharp scissors, shave the fur on the skull from the eyes to the base of the skull, and use 70%ethanol to sterilize the exposed skin. Cut the skin in a circular manner, and use a cotton swab to scrape away the exposed periosteum.
Treat the surgical area with a drop of 1%lidocaine, and a 1:100, 000 concentration of epinephrine for five minutes before removing the excess solution with a cotton swab. Use cyanoacrylate glue to adhere the edges of the skin to the skull, and place the stereotactic frame under a dissection stereo microscope with a 4x magnification. Next, carefully drill a superficial, five millimeter diameter, circular groove over the right parietal bone.
And apply a drop of cortex buffer to the groove. Use thin forceps to lift the bone flap to visualize the brain surface, and use curved, tapered, very fine point forceps to remove the dura mater. If bleeding occurs, use an absorbable gelatin sponge to achieve hemostasis.
For tumor cell injection, load about three microliters of the cells into a five microliter gas-tight syringe equipped with a point style two needle. Fix the needle on the stereotaxic manipulator arm, and remove the cortex buffer from the exposed tissue. Place the tip of the syringe in the middle of the craniotomy and insert the needle at a 0.5 millimeter depth from the surface of the skull.
Then, add a drop of cortex buffer to the craniotomy, and use a microsyringe pump injector to deliver the cells at a 250 to 400 nanoliter per minute rate. To prepare the cranial imaging window, replace the cortex buffer with a drop of silicone oil to the craniotomy site to avoid air bubbles under the window. Seal the exposed brain with a six millimeter coverslip, and apply cyanoacrylate glue between the coverslip and the skull.
Then use fine tweezers to gently press the coverslip against the skull. At the appropriate experimental timepoint, place the mouse face-up in an imaging box, and set the 25x water objective to the lowest z-position. Add a large drop of water to the objective, and transfer the imaging box into the 37 degree Celsius dark climate chamber of the microscope.
Bring the objective to the cranial imaging window coverslip until the water drop touches the coverslip. And using the epifluorescence mode, observe the tumor through the eyepiece to bring the cells into focus. Tune the laser to the correct wavelength and select live mode.
After selecting several positions of interest for imaging, record their coordinates in the software. Define a z-stack for each position to acquire the maximal volume of tumor cells without compromising the tumor cell resolution, with the step size between images set to three micrometers. Then, acquire images of the tumor volume at different positions every 20 minutes for two hours, adding water to the objective before each image acquisition.
After the last image is acquired, transfer the mouse to a heating pad with monitoring until full recovery. One day after the first imaging session, secure the anesthetized mouse in the stereotaxic frame as demonstrated, and use an acetone-soaked cotton swab to wipe the edges of the coverslip until the glue is softened. Slide a 25 gauge needle under the coverslip and use thin point forceps to lift it, and hydrate the craniotomy with fresh cortex buffer.
Puncture the tumor to a one millimeter depth with a 25 gauge needle, stopping any bleeding with a sterile gelatin sponge, and seal the brain's surface with fresh silicone oil. Then glue a new six millimeter coverslip over the wound. For image analysis, open the time-lapse LIF file in the microscope software program, and select the tab Process, Process Tools, and Merge.
Select the first image of the time sequence and click first. Select the second image of the time sequence and click second. In Merge Dimensions, select t for time, then click apply.
A new file with two timepoints will be generated. After tracking each time-lapse series for three consecutive z-stacks separately, drag the folder containing the time-lapse images to ImageJ. Select the tab Plugins, Tracking, and MtrackJ.
To track each individual cell, select Add and click on each cell at each timepoint. Then click measure, and save the file to extract the measure of the tracks. The migration of individual tumor cells can be determined by tracking the migration path over time in different xy planes of the z-stack and plotted as a percentage of the migratory cells pre and post biopsy.
The tumor cell proliferation rate can be quantified based on H2B-tagged Dendra2 condensation upon mitosis and plotted as a percentage of the dividing cells pre and post biopsy. Comparing the distribution of the migration velocity before and after biopsy in the same tumor, reveals that the number of migratory cells increases after the intervention, with an associated decrease in the number of slow to nonmigratory cells. On average per tumor, a 1.75-fold increase in the percentage of migratory cells is observed when a biopsy-like injury is performed, compared to control, nonbiopsied mice.
Although the percentage of migratory tumor cells eventually decreases in both control and biopsied mice, biopsied mice still exhibit a higher migratory capacity than control mice overall. Tumor cell proliferative behavior also demonstrates a 1.52-fold increase in the number of mitotic events upon biopsy over time, relative to nonbiopsied control mice. Of note, no induction of tumor cell migration or proliferation is observed after cranial imaging window replacement without biopsy, indicating that the boost in tumor cell proliferation and migration rates is specifically triggered by the biopsy-like injury.
It is important to work with high precision and surgical skill during the cranial imaging, implantation, and replacement steps. Extensive practice may be necessary.
