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1Department of Cell and Developmental Biology, Vanderbilt University, 2Department of Neurology, Vanderbilt University
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This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.
Huang, X., Sarangi, A., Ketova, T., Litingtung, Y., Cooper, M. K., Chiang, C. Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice. J. Vis. Exp. (44), e2153, doi:10.3791/2153 (2010).
Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron precursors (CGNPs) as the cell-of-origin for medulloblastoma1-4. However, the diverse clinical presentations of medulloblastoma subtypes in human patients (nodular, desmoplastic, classical and large cell/anaplastic), and the fact that medulloblastoma is found in a subset of human patients with no ectopic expression of CGNP marker5, suggest that the cellular and molecular origins of medulloblastoma are more complex and far from being completely deciphered. Therefore, it is essential to determine whether there is an alternative medulloblastoma tumor cell-of-origin based on which cell-type specific therapeutic modality can be developed. To this end, intracranial orthotopic allografting of genetically marked tumor cell types followed by subsequent analyses of secondary tumor development in recipients will allow determination of the cellular origin of tumor-initiating cells. Here we describe the experimental protocol for intracranial orthotopic allografting of medulloblastoma cells derived from primary tumor tissue, and this procedure can also be used for transplanting cells from established cell lines.
1. Micro-dissection of Tumor-bearing Cerebellum and Dissociation of Tumor Tissue
Note: all instruments are disinfected in 95% ethanol and steam autoclaved before use.
Resuspend the cell pellet in freshly prepared neural stem cell medium consisting of Neurobasal medium with glutamine, Penicillin-Streptomycin, N2, B27, human EGF (25 ng/mL) and basic FGF (25 ng/mL). Calculate the cell density of the solution and make appropriate further dilutions with additional neural stem cell medium such that one can load 4 μL of solution with an appropriate number of dissociated tumor cells, e.g. 5x105, into a sterile bevel-tipped 10μL syringe. The exact number of cells to be inoculated should be determined by the researcher according to specific experimental requirements. Keep the cell solution in a mini 200μL microcentrifuge tube (PCR tube) on ice.
2. Anesthetic Procedures for the Recipient Mice
For the surgical procedure, a disinfected area dedicated to rodent surgery is needed for the duration of the procedure. Ideally, set up a long bench with separate but adjoining areas for animal preparation, operating field and animal recovery. Surgical gloves, not exam gloves, are required for the surgery. Aseptic technique is maintained by holding gloved hands above the waist and used only to handle sterile objects from a dry sterile surface.
3. Intracranial Grafting of the Tumor Cells
4. Representative Results
Secondary medulloblastomas developed in recipient mice highly resembled those of the primary tumors. The cerebella bearing secondary tumors were often enlarged, amorphous with ectopic blood vessels apparent when viewed as whole-mounts. Immunohistochemical analyses of the secondary tumor tissue revealed that the tumor cells are highly proliferative, express strong SmoM2-YFP and markers of cerebellar granule neuron precursors, such as Math1 and Pax6. When dissociated and cultured using reported methods ("Isolation, enrichment and maintenance of medulloblastoma stem cells", JoVE in press), the secondary medulloblastoma tumor cells can be expanded rapidly, express multiple stem cell markers, are clonogenic and can further initiate tumor formation when transplanted into additional immunocompromised recipients.
Figure 1. Typical whole-mount and sectional views of a secondary medulloblastoma tissue
A typical example of secondary medulloblastoma tissue developed 26 days after injection of 5x105 primary tumor cells. Hematoxylin staining reveals the typical cellular morphology of medulloblastoma, including high nuclear/cytoplasmic ratio and nuclear polymorphism. These secondary tumor cells are highly proliferative as indicated by Ki67 expression and they also robustly express membranous SmoM2-YFP.
Figure 2. Secondary medulloblastoma cells can be cultured and expanded in vitro
Using the protocol described for primary mouse medulloblastoma cells ("Isolation, enrichment and maintenance of medulloblastoma stem cells", JoVE in press), these secondary medulloblastoma cells can be cultured and expanded for multiple passages. The picture shows the same representative tumor colony cultured at 4 days and 14 days. The cultured tumor cells are bipolar, elongated with scant cytoplasm and often radiate from a dense core of cellular aggregate. They do not exhibit growth contact inhibition. The small round cells are red blood cells.
Several critical factors to ensure successful medulloblastoma cell allografting that yields secondary tumor formation are as follows: First, use only 50% Accutase for tissue dissociation and do not over-digest the tissue as prolonged enzyme treatment leads to significant reduction in cell viability Also use only the 1 mL size Pipetman as smaller tips can also generate physical damage to the dissociated cells. It is fine to have a mixture of single cells and small aggregates for allografting. Second, mix and equilibrate the tumor cell solution each time before loading the Hamilton syringe. Do not use more than 4 μL for each injection as a larger volume would generate too much intracranial pressure and resistance. Last, it is important to wait an additional 2 minutes to let the injected solution dissipate into the cerebellar tissue after each injection.
The procedures described here allow for the determination of genetically marked cell type(s) that can initiate and propagate tumors. This protocol also applies to situations where the cells to be injected are not derived directly from primary tumor tissue, but from established cultured cell lines One can prepare the appropriate number of cells by either enzyme digestion and/or repetitive pipetting, and start from Step 2 of this protocol. Therefore, we can also test the functions of candidate genes and the inhibitory effect of chemical compounds with an in vivo tumor-growth readout.
No conflicts of interest declared.
This study was supported by grants from the Vanderbilt-Ingram Cancer Center Support Grant (P30 CA068485), the Childhood Brain Tumor Foundation and the National Institutes of Health (NS042205).
|Stereotaxic instrument||Stoelting Co.||51730|
|Foredom flexible shaft drill, hang-up style||Stoelting Co.||58650|
|Large probe holder||Stoelting Co.||51633|
|10 μL syringe, 26 ga., bevel tip||Stoelting Co.||51105|
|Drill bit .75mm||Stoelting Co.||51455-3|
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