Establishment of Orthotopic Patient-derived Xenograft Models for Brain Tumors using a Stereotaxic Device

In my research, we focus on developing new treatments for aggressive childhood brain cancers like diffuse midline gliomas, high-grade gliomas, ependymomas, and ameloblastomas. Our aim is to find new drug targets and develop therapies that can make a difference in patients, while also studying how these tumors may affect the surrounding brain environment. In pediatric brain tumor research, we developed patient-derived xenograft models using intracranial injections, advanced imaging, and high-throughput drug screening.

Techniques like single-cell and special transcriptomics help us understand how the tumor impacts the brain environment and treatment effects, allowing us to explore new therapies. Our brain tumor group at Children's Cancer Institute has developed many pediatric brain tumor pediatrics models through the Zero Childhood Cancer Personalized Medicine Program. We found that diffuse midline gliomas are resistant to drug treatments due to an intact blood-brain barrier.

Promising therapies targeting the epigenetic and oncogenic pathways improved pediatric survival and are now in clinical development. Using orthotopic brain tumor models, we would like to understand how tumors affect the brain vasculature. By applying the techniques like special transcriptomics, we would like to identify pathways impacted by brain tumors.

This knowledge will help us identify blood-brain barrier modulators that can loosen the barrier, allowing drug treatments to penetrate more effectively. To begin, thaw cultured neurosphere-forming brain tumor cells. In a biosafety cabinet, carefully pipette out the cultured cells and culture medium from the tissue culture flask into a 50 milliliter conical tube with a serological pipette.

Centrifuge the conical tube at 300 G for up to five minutes at room temperature. With a 25 milliliter pipette, aspirate the medium above the pellet, leaving approximately 0.5 milliliters in the tube. Gently pipette the cells up and down 10 times with a one milliliter pipette to dissociate the cell pellet.

Add trypan blue to the cell suspension and count with a hemocytometer. Aliquot cell suspension containing two million cells into a fresh tube. Add 0.5 of a milliliter of PBS to the cell suspension.

Then centrifuge at 300 G for five minutes at four degrees Celsius. Discard the supernatant and wash again. After the final wash, aspirate as much PBS as possible.

Then place the cell pellet and 50 microliters of extracellular matrix hydrogel on ice. Mix the cell pellet with the extracellular matrix hydrogel promptly to prepare for intracranial injection. To begin, mount an anesthetized mouse onto a stereotaxic device.

Ensure the front teeth are fixed in the incisor bar and the nose cone secures the animal in place. Disinfect the mouse's head with an iodine-soaked cotton tip, followed by an isopropanol wipe. Then apply corneal eye ointment to both eyes to prevent drying.

Make an incision at the base of the cerebellum and extend it across the cranium to the midpoint to create a one centimeter long cut along the superior aspect of the skull. Tighten the skin between the ears to expose the skull and then tighten the ear bards to secure the head. Clean the skull surface and dry it thoroughly with a cotton bud.

Secure the drill onto the stereotaxic frame and locate the bregma or lambdoid structure using the drill. Once the coordinates are established, carefully drill a small burr hole in the bone at the designated site. Resuspend the prepared brain tumor injection cell suspension multiple times using a pipette, ensuring air bubbles are avoided.

Draw two microliters of the cell mixture into a pre-washed cold glass microsyringe and attach the syringe to the stereotaxic frame. Adjust the needle to the skull tip to prepare for injection. Within 30 seconds, inject the cells into the drilled region.

Wipe away any refluxed cell suspension with an isopropanol wipe during the injection. Leave the syringe in place for one minute to prevent backflow and allow the extracellular matrix hydrogel to settle. Then remove the syringe and clean the wound with an isopropanol wipe.

Seal the incision with skin glue or wound clips if necessary. Place the mouse in a recumbent position in a clean recovery cage with a heat lamp or heating pad to maintain warmth. Monitor the animal continuously until it begins moving independently and normally.

Following the intracranial injection, monitor the mice five days per week to assess their general wellbeing. Record parameters such as weight loss, activity level, posture, signs of dehydration, and fur condition on a designated monitoring sheet. Animals exhibited distinct symptoms based on tumor type and injection site, such as head tilting in brainstem tumors and forebrain enlargement for cortical gliomas or ependymomas.

Intracranial injections of medulloblastoma D425 cells at different densities revealed a direct correlation between cell density and tumor engraftment speed with 100, 000 cells leading to the shortest median survival of 15 days. High-grade glioma cells injected into the cortex resulted in a median survival of approximately 25.5 days with immunohistochemical analysis revealing a large highly nucleated tumor mass and increased vascularization, as well as abundant Ki-67 positive proliferative cells. Brainstem injections of high-grade glioma cells resulted in a median survival of approximately 26 days with immunohistochemical analysis indicating a tumor mass in the upper pons and leptomeningeal infiltration, as well as proliferative Ki-67 positive cells in infiltrated areas.