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 medulloblastoma^1-4. 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

1. Micro-dissection of Tumor-bearing Cerebellum and Dissociation of Tumor Tissue

1. Retrieval of tumor tissue
   1. Sick mice bearing medulloblastoma are often runted, and display hydrocephaly and typical neurological symptoms, including posterior paralysis and failure to regain posture when overturned. To retrieve tumor tissue, euthanize mice by carbon dioxide inhalation. It is imporant not to perform cervical dislocation, a procedure that generates pressure to the posterior skull and can compromise tumor tissue integrity.
   2. Decapitation is performed immediately after death using a pair of scissors, removing hair and muscle tissu....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 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