An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

This protocol allows for the formation of brain metastatic tumor cells and the generation of ex vivo brain slices that allows for rapid testing of drug efficacy and radiation on a preformed tumor. The main advantage of this technique is the ability to rapidly test multiple drugs or treatments in a physiologically relevant setting that maintains brain-tumor-host interactions. This method may help understand how brain metastatic cells grow, survive, and invade the brain parenchyma and understand the role of the brain tumor microenvironment in cancer growth and spread.

Demonstrating the procedure will be Lorela Ciraku and Emily Esquea, both PhD candidates from my laboratory. To begin, with place the anesthetized mice into a stereotaxic frame and immobilize the head using standard ear bars. Sterilize the surface of the head thrice by repeated alternating application of iodine swabs and 70%ethanol prior to incision.

Using a surgical scalpel, make a one-centimeter midline incision at a diagonal angle to the imaginary axis dividing the mice's brain into two symmetrical halves to expose the skull. After wiping any blood with a cotton swab, deflect the skin laterally to expose the injection site. Use a 0.73 millimeter burr bit to penetrate the skull and drill a hole using slight pressure and twisting motion.

To inject five microliters of cells stably expressing GFP luciferase, insert a high-precision syringe to 3.5 millimeters depth in the brain. Allow it to settle for two minutes and then pull the syringe up about one millimeter. After two minutes, slowly inject the first half-volume and wait for another two minutes before injecting the rest of the volume.

After three minutes, slowly pull the syringe. Apply bone wax to the injection site on the skull and suture the tissue with polypropylene sutures. Monitor the behavior of the mouse right after the surgery periodically to determine if special treatment, including saline injection and soft food, are needed to help quick recovery.

To monitor the tumor growth via bioluminescence imaging, first turn on the oxygen and isoflurane on the imaging system and allow both to be distributed to the separate chamber outside of the imaging box. Then, after turning on the software and initializing the instrument, choose the appropriate option for visualizing and capturing the whole mouse body. Next, transfer the mice to the chamber outside the imaging box supplied with oxygen and isoflurane.

Once the mice are under the anesthetic effect, inject the mice intraperitoneally with 200 microliters of 30 milligrams per milliliter of luciferin. Place the mice in the imaging chamber with stomach facing down to the imaging chambers nose lip. Lock the chamber, choose two minutes time exposure to start imaging, and then complete the imaging.

After placing the tissue slicer in a sterile laminar flow hood, clean all the tools and instruments with 70%ethanol. After euthanizing the mouse, remove and place the brain rapidly into ice-cold sucrose-ACSF solution. Using a spatula, transfer the brain on an ACSF-wetted filter paper on a 60-millimeter dish lid.

Using a sharp, sterile razor blade, cut the excess parts of the brain that do not contain any tumor, including the bottom part of the brain, to bring the shape of the brain to a non-perfect cube. After placing several sheets of ACSF-wetted filter paper onto the cutting platform, place the blocked tissue upon the filter paper. To slice the tissue into 200-to 250-micrometers-thick sections, set the cut size to 2 or 2.5 units on the provider ruler and start cutting.

Use a paint brush to transfer brain slices into a dish containing sucrose-ACSF and separate the slices under a light microscope using 27-gauge needles. Identify the GFP-positive slices under the fluorescent microscope. Then, using a sterile one-milliliter broken-off pipette with a wide opening, transfer identified slices to a new 60-millimeter dish containing two to three milliliters of adult slice media.

Then, transfer three to five slices onto each tissue culture insert in each well of a six-well plate at a safe distance. After removing the excess medium from the surface of the insert, add one milliliter of equilibrated adult mouse slice medium underneath each insert. Incubate the tissues at 37 degrees Celsius, 5%carbon dioxide, and 95%humidity for 24 hours before imaging.

Pipette five microliters of 30 milligrams per milliliter luciferin solution into the medium underneath the inserts inside a sterile hood. Place the six-well plate with the lid inside the imaging chamber of the instrument below the stationary camera and lock the chamber door. Open the software and initialize the instrument.

After choosing camera settings allowing visualization and capturing of only one well per image, place the well to be imaged directly under the camera. Use the ROI tool, fit it around the tumor shape and size, measure the ROI, and report the total readings to quantify the bioluminescent signal of each tumor on the slice. After bringing the plate back to the sterile laminar flow hood, use forceps to slightly lift the insert from the well.

After aspirating the medium, replace the medium with one milliliter of fresh medium and place the inset back in the well. For the experiments where the slices are treated with various compounds, such as inhibitors and metabolites, prepare the appropriate reagent concentration in 1.2 milliliters of brain slice media in a 1.5-milliliter tube, and then transfer one milliliter of reagent to the well containing the slice. The MDA-MB-231 BR-GFP luciferase cells were injected into nude mice, tumors were allowed to grow, brains with tumors were dissected out, sliced, grown ex vivo, and tumor growth was monitored by bioluminescence imaging.

The H&E staining and GFP-positive fluorescence confirmed the presence of a tumor in the brain slice. The increased Ki-67 staining confirmed the presence of highly proliferative cancer cells. The tumors in brain slices that were not exposed to irradiation continued to grow ex vivo, while tumors exposed to irradiation showed a stalled growth.

Brain slices containing tumors were also monitored via live imaging to visualize the tumor growth following the irradiation treatment. Consistent with the bioluminescent imaging, control cancer cells rapidly grew as GFP intensity increased and the cells started invading the brain parenchyma. In contrast, the cancer cells in brain slices treated with irradiation were cytostatic and GFP intensity was maintained.

Large multi-nucleated cancer cells and an increase in staining of DNA damage marker in IR-treated cancer cells were detected. Also, an increase in reactive astrocytes was detected. The IR treatment-induced apoptosis was not detected in the tumor-free mouse brain slices.

The brain slices containing tumors were treated with different concentrations of paclitaxel, a chemotherapeutic drug. Treatment decreased the tumor's size significantly following day 10 of treatment. An increase in apoptosis was detected in paclitaxel-treated tumor cells.

The inhibitor treatment did not alter brain tissue viability. No increase in apoptosis was measured by cleaved caspase-3 staining in paclitaxel-treated tumor-free mouse brain slice. When the treatment with the drugs of interest has terminated, slices can be analyzed for protein expression through immunocytochemistry, omic studies, or achieve single-cell suspension suitable for flow cytometry.