Our group is focused on neurofibromatosis type one and understanding the pathology of the cancers associated with the disease, plexiform neurofibromas and malignant peripheral nerve sheath tumors. To do so, we employ several techniques, including the use of a genetically-engineered mouse model, P0-GGF-beta-3. Our lab and others studying plexiform neurofibromas and malignant peripheral nerve sheet tumors face similar challenges.
Limited access to patient-derived tumor samples hinders our work. As a result, creating and characterizing genetically-engineered mouse models is crucial for ongoing research into disease mechanisms and the potential development of treatments. Identifying peripheral nerve tumors can be tricky and are often misdiagnosed as other cancers.
To avoid this, we have developed a detailed protocol using histology, immunohistochemistry, and grading to accurately identify and grade these tumors. This improves the reliability of future research studies. Our work provides a framework to investigate and validate genetically-engineered mouse model-derived tumors to ensure they properly mimic the human disease.
We will use current genetically-engineered mouse models to understand tumor microenvironment and the factors essential to tumor transformation, what causes plexiform neurofibromas to progress to malignant peripheral tumors. Begin by identifying large visible tumors in P0-GGF-beta-3 mice. After euthanizing the mouse, dissect the tumor under sterile conditions.
Check if the tumor is associated with a peripheral nerve and excise the tumor. Then divide the visibly noticeable tumors into three pieces. Allocate one piece for histology and one for early passage cultures.
Fix the tissue sample for histology in a 4%solution of paraformaldehyde in PBS. Freeze the third piece for potential future experiments. Place the tissue sample and remaining parts of the animal's body in a paraformaldehyde PBS mixture overnight at four degrees Celsius.
P0-GGF-beta-3 mouse with a large, grossly evident tumor on the right flank and the MRI scan of this mouse showing the tumor connected to the sciatic nerve is shown. The tumor was identified as a neurofibroma upon histologic examination. A fleshy, MPNST originating from the brachial plexus of the animal was identified.
To begin, take the paraffin-embedded tumor sections of P0-GGF-beta-3 mice and deparaffinize them in a d-limonene-based solvent for 10 minutes at room temperature. Then incubate the sections in a fresh volume of d-limonene-based solvent for another 10 minutes. After staining the sections with Eosin, use a xylene-based mounting medium to mount the coverslips while keeping the slide damp with the d-limonene-based solvent.
To examine the spinal cord and nerve roots for nerve root tumors, incubate the vertebrae column, associated ribs, and soft tissues in 0.3 molar EDTA and 4%paraformaldehyde solution. Then rinse the samples using PBS. Try to pierce the vertebrae column with a needle to confirm decalcification.
The needle should penetrate without resistance in completely decalcified bone. Cut the decalcified vertebral column and associated structures into blocks to fit in a tissue cassette and perform the desired staining. Appropriate labeling for MPNST markers was observed in independently arising MPNSTs.
In properly decalcified vertebral columns, the spinal cord, nerve roots, vertebral body, and skeletal muscles were clearly visible. A dorsal nerve root neurofibroma was also observed in a P0-GGF-beta-3 mouse. Examine the Hematoxylin and Eosin stained tumor slides of P0-GGF-beta-3 mice under brightfield microscopy to identify potential peripheral nerve sheath tumors.
Ensure microscopic neurofibromas and MPNSTs are associated with a peripheral nerve. Identify blocks containing potential peripheral nerve sheath tumors to special stains for diagnosis confirmation or refutation. Differentiate potential PNSTs from MPNSTs based on the histological features.
Human and P0-GGF-beta-3 mouse PNs have elongated wavy nuclei with loosely packed cells separated by mixoid extracellular material. The presence of mitosis and hypercellular tumor indicates MPNST or high-grade neoplasm. Then examine the immunostained sections using brightfield microscopy.
Search for uniform or patchy immunoreactivity for markers S100 beta, Nestin, and SOX10, indicating schwannian differentiation in MPNSTs. Examine the sections for prominent hypercellularity, brisk mitotic activity, and cytologic atypia for grade four MPNSTs. Neurofibromas are composed of neoplastic Schwann cells and other non-neoplastic elements as indicated by S100 beta staining of a cell subpopulation.
Neoplastic Schwann cells are also immunoreactive for the intermediate filament Nestin and the transcription factor SOX10. Luna stain performed on a P0-GGF-beta-3 plexiform neurofibroma highlighted the presence of mast cells usually absent in MPNSTs. In contrast, Ki67 immunoreactivity is virtually non-existent in plexiform neurofibromas Nuclear Ki67 labeling is typically present in a very high fraction of tumor cells as seen in the microscopic MPNST arising in the trigeminal ganglion of a P0-GGF-beta-3 mouse.
To begin, place sterile coverslips in the wells of six-well tissue culture dishes and pour an adequate volume of poly-l-lysine and laminin solution into the wells to cover the coverslip. Next, seal the plate with plastic wrap to curtail evaporation and incubate at four degrees Celsius overnight. The next morning, wash the coverslips three times with sterile PBS.
Add two milliliters of cell suspension and growth media to plate 10, 000 cells to each well containing the coverslip. After washing the coverslips with PBS, fix the cells with a 4%paraformaldehyde solution and PBS for 18 minutes. Rinse the coverslips three times with PBS for five minutes before immunostaining.
Incubate the immunostained and washed coverslips in one to five micrograms of Hoechst dye for 10 minutes. Then wash the coverslips with PBS for an additional five minutes. Mount the slides using an equal ratio of PBS and glycerol mixture and capture the images using a fluorescent microscope.
Treat the cells with a non-enzymatic cell dissociation solution for 30 seconds to one minute. Then add five milliliters of DMEM for every one milliliter of the non-enzymatic cell dissociation reagent. Count cells using a hemocytometer.
Centrifuge the cells at five G for five minutes. Resuspend the cells at a concentration of one to two times 10 of the sixth cells per 100 microliters of DMEM. Place the animal on its stomach and sterilize the injection site with 70%ethanol.
Inject one to two times 10 to the sixth cells subcutaneously in the right flank. Place the mouse alone in a bedding-free cage and allow it to recover. To prevent hypothermia, place a portion of the cage on the heating pad.
Low and high-power images of early-passage P0-GGF-beta-3 MPNST cells are shown. The cells were found to be tumorigenic, forming colonies in soft agar and grafts in immunodeficient mice.
