Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice
Summary
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.
Abstract
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.
Protocol
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Neurobasal medium | Invitrogen | 21103049 | ||
| hEGF | Invitrogen | PHG0311 | 25ng/ml | |
| bFGF | Invitrogen | PHG0023 | 25ng/ml | |
| N2 | Invitrogen | 17502048 | 1X | |
| B27(-RA) | Invitrogen | 12587010 | 1X | |
| Accutase | Invitrogen | A1110501 | 50% | |
| Glutamine | Invitrogen | 25030081 | 2mM | |
| Stereotaxic instrument | Stoelting | 51730 | ||
| Foredom flexible shaft drill, hang-up style | Stoelting | 58650 | ||
| Large probe holder | Stoelting | 51633 | ||
| 10 μL syringe, 26 ga., bevel tip | Stoelting | 51105 | ||
| Drill bit .75mm | Stoelting | 51455-3 |
References
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