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Establishment, Maintenance, and In Vitro and In Vivo Applications of Primary Human Glioblastoma Multiforme (GBM) Xenograft Models for Translational Biology Studies and Drug Discovery
Publication Name:
Current Protocols in Pharmacology
Unit Number:
Unit 14.16
DOI:
10.1002/0471141755.ph1416s52
Online Posting Date:
March, 2011
Abstract
Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short-term explant cultures or intracranial xenografts. This unit describes detailed procedures for establishment, maintenance, and utilization of a primary GBM xenograft panel for the purpose of using them as tumor models for basic or translational studies. Curr. Protoc. Pharmacol. 52:14.16.1-14.16.23. © 2011 by John Wiley & Sons, Inc.
Keywords: glioblastoma; xenograft; mouse models
Table of Contents
- Introduction
- Basic Protocol 1: Implantation of Patient Tumor Samples Into Nude Mice
- Basic Protocol 2: Serial Passage of Flank Tumor Xenografts
- Basic Protocol 3: Cryopreservation of Xenograft Tissue
- Basic Protocol 4: Establishing Short-Term Explant Cultures From Xenograft Lines
- Basic Protocol 5: Intracranial and Flank Tumor Implantation Using Short-Term Explant Cultures
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol 1: Implantation of Patient Tumor Samples Into Nude Mice Materials
NOTE: Ideally, 1-cm Basic Protocol 2: Serial Passage of Flank Tumor Xenografts Materials
Basic Protocol 3: Cryopreservation of Xenograft Tissue Materials
Basic Protocol 4: Establishing Short-Term Explant Cultures From Xenograft Lines Materials
Basic Protocol 5: Intracranial and Flank Tumor Implantation Using Short-Term Explant Cultures Materials
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Figures
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Figure 14.16.1 Analysis of TMZ resistance in short-term cell cultures. Short-term explant cell cultures from GBM12 and GBM43 were treated with TMZ. (A) The effects on cell proliferation were evaluated 96 hr after treatment with graded concentrations of TMZ using a methylene blue staining assay. Mean ± SD for relative optical density (OD) vs. TMZ concentrations are plotted from three independent experiments. (B) Following treatment with 100 µM TMZ, cells were harvested at the indicated time points and processed for immunoblotting for MGMT and then -actin.
(Source: Data reproduced with permission from Kitange et al. ( -
Figure 14.16.2 Skull anatomy of a mouse. The diagram shows the typical exposure of the skull bones following a mid-line incision. The relationship between the sagittal and coronal sutures is shown as well as the location of the bregma. The burr hole location typically used is 1-mm anterior and 2-mm lateral to the bregma. -
Figure 14.16.3 Stereotactic injection setup. (A) The stereotactic injection jig with a mouse in place is shown. (B) By using multiple injection jigs in an assembly line fashion, two or three technicians can implant 100 mice in 4 hr.
(Source: Photographs courtesy of Cory Petell..) -
Figure 14.16.4 Evaluation of erlotinib efficacy in orthotopic xenografts. The efficacy of erlotinib was evaluated in mice bearing orthotopic GBM39 tumors transduced with a luciferase-expressing lentivirus prior to implantation. (A) Kaplan-Meier survival curves for mice treated with daily oral erlotinib or placebo as shown by the horizontal arrow. (B) Moribund mice were processed for MIB-1 IHC to assess changes in tumor cell proliferation in vivo. Brown staining of the nuclei represents expression of the MIB-1 proliferation marker, which is significantly decreased in the erlotinib-treated sample. (C). Luminescence intensity overlays showing serial results for a single placebo-treated mouse and a single erlotinib-treated mouse, with images recorded at days 42, 53, and 60 days for each, and additionally at days 67, 74, and 81 for the erlotinib-treated mouse. (D) Luminescence readings were converted to normalized values by dividing each mouse's luminescence readings with its corresponding maximal pretreatment luminescence reading recorded at day 42. Mean normalized bioluminescence and corresponding standard error values for placebo and erlotinib groups have been plotted for each imaging session. The duration of treatment is indicated by the horizontal arrow.
(Source: Data reproduced with permission from Sarkaria et al. ( -
Figure 14.16.5 Changes in median survival with increasing generation. The median survival for placebo-treated mice entered into 114 intracranial survival studies involving 19 different xenograft lines is plotted relative to the generation from which any given study was derived.
Videos
Literature Cited
| Literature Cited | |
| Allen, C., Paraskevakou, G., Iankov, I., Giannini, C., Schroeder, M., Sarkaria, J., Puri, R.K., Russell, S.J., and Galanis, E. 2008. Interleukin-13 displaying retargeted oncolytic measles virus strains have significant activity against gliomas with improved specificity. Mol. Ther. 16:1556-1564. | |
| Carlson, B.L., Grogan, P.T., Mladek, A.C., Schroeder, M.A., Kitange, G.J., Decker, P.A., Giannini, C., Wu, W., Ballman, K.A., James, C.D., and Sarkaria, J.N. 2009. Radiosensitizing effects of temozolomide observed in vivo only in a subset of O6-methylguanine-DNA methyltransferase methylated glioblastoma multiforme xenografts. Int. J. Radiat. Oncol. Biol. Phys. 75:212-219. | |
| Clarke, M.J., Mulligan, E.A., Grogan, P.T., Mladek, A.C., Carlson, B.L., Schroeder, M.A., Curtin, N.J., Lou, Z., Decker, P.A., Wu, W., Plummer, E.R., and Sarkaria, J.N. 2009. Effective sensitization of temozolomide by ABT-888 is lost with development of temozolomide resistance in glioblastoma xenograft lines. Mol. Cancer Ther. 8:407-414. | |
| Danam, R.P., Howell, S.R., Remack, J.S., and Brent, T.P. 2001. Heterogeneous methylation of the O(6)-methylguanine-DNA methyltransferase promoter in immortalized IMR90 cell lines. Int. J. Oncol. 18:1187-1193. | |
| Dinca, E.B., Sarkaria, J.N., Schroeder, M.A., Carlson, B.L., Voicu, R., Gupta, N., Berger, M.S., and James, C.D. 2007. Bioluminescence monitoring of intracranial glioblastoma xenograft: response to primary and salvage temozolomide therapy. J. Neurosurg. 107:610-616. | |
| Dinca, E.B., Lu, K.V., Sarkaria, J.N., Pieper, R.O., Prados, M.D., Haas-Kogan, D.A., Vandenberg, S.R., Berger, M.S., and James, C.D. 2008. p53 Small-molecule inhibitor enhances temozolomide cytotoxic activity against intracranial glioblastoma xenografts. Cancer Res. 68:10034-10039. | |
| Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1-1.8.4. | |
| Fael Al-Mayhani, T.M., Ball, S.L.R., Zhao, J.-W., Fawcett, J., Ichimura, K., Collins, P.V., and Watts, C. 2009. An efficient method for derivation and propagation of glioblastoma cell lines that conserves the molecular profile of their original tumours. J. Neurosci. Methods 176:192-199. | |
| Frederick, L., Wang, X.Y., Eley, G., and James, C.D. 2000. Diversity and frequency of epidermal growth factor receptor mutations in human glioblastomas. Cancer Res. 60:1383-1387. | |
| Giannini, C., Sarkaria, J., Saito, A., Uhm, J., Galanis, E., Carlson, B., Schroeder, M., and James, C. 2005. Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of GBM. Neuro-Oncology 7:164-176. | |
| Huse, J.T. and Holland, E.C. 2009. Genetically engineered mouse models of brain cancer and the promise of preclinical testing. Brain Pathol. 19:132-143. | |
| Kitange, G., Carlson, B., Mladek, A., Decker, P., Schroeder, M., Wu, W., Grogan, P., Giannini, C., Ballman, K., Buckner, J., David James, C., and Sarkaria, J. 2009a. Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model. J. Neurooncol. 92:23-31. | |
| Kitange, G.J., Carlson, B.L., Schroeder, M.A., Grogan, P.T., Lamont, J.D., Decker, P.A., Wu, W., James, C.D., and Sarkaria, J.N. 2009b. Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts. Neuro-Oncology 11:281-291. | |
| Li, A., Walling, J., Kotliarov, Y., Center, A., Steed, M.E., Ahn, S.J., Rosenblum, M., Mikkelsen, T., Zenklusen, J.C., and Fine, H.A. 2008. Genomic changes and gene expression profiles reveal that established glioma cell lines are poorly representative of primary human gliomas. Mol. Cancer Res. 6:21-30. | |
| Liu, C., Sarkaria, J.N., Petell, C.A., Paraskevakou, G., Zollman, P.J., Schroeder, M., Carlson, B., Decker, P.A., Wu, W., James, C.D., Russell, S.J., and Galanis, E. 2007. Combination of measles virus virotherapy and radiation therapy has synergistic activity in the treatment of glioblastoma multiforme. Clin. Cancer Res. 13:7155-7165. | |
| Pandita, A., Aldape, K.D., Zadeh, G., Guha, A., and James, C.D. 2003. Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR. Genes Chromosomes Canc. 39:29-36. | |
| Pegg, A.E. 1990. Mammalian O6-alkylguanine-DNA alkyltransferase: Regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res. 50:6119-6129. | |
| Pollard, S.M., Yoshikawa, K., Clarke, I.D., Danovi, D., Stricker, S., Russell, R., Bayani, J., Head, R., Lee, M., Bernstein, M., Squire, J.A., Smith, A., and Dirks, P. 2009. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4:568-580. | |
| Sarkaria, J.N., Carlson, B.L., Schroeder, M.A., Grogan, P., Brown, P.D., Giannini, C., Ballman, K.V., Kitange, G.J., Guha, A., Pandita, A., and James, C.D. 2006. Use of an orthotopic xenograft model for assessing the effect of epidermal growth factor receptor amplification on glioblastoma radiation response. Clin. Cancer Res. 12:2264-2271. | |
| Sarkaria, J.N., Yang, L., Grogan, P.T., Kitange, G.J., Carlson, B.L., Schroeder, M.A., Galanis, E., Giannini, C., Wu, W., Dinca, E.B., and James, C.D. 2007. Identification of molecular characteristics correlated with glioblastoma sensitivity to EGFR kinase inhibition through use of an intracranial xenograft test panel. Mol. Cancer Ther. 6:1167-1174. | |
| Smith, J.S., Tachibana, I., Passe, S.M., Huntley, B.K., Borell, T.J., Iturria, N., O'Fallon, J.R., Schaefer, P.L., Scheithauer, B.W., James, C.D., Buckner, J.C., and Jenkins, R.B. 2001. PTEN mutation, EGFR amplification, and outcome in patients with anaplastic astrocytoma and glioblastoma multiforme. J. Natl. Cancer Inst. 93:1246-1256. | |
| TCGA. 2008. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455:1061-1068. | |
| Yamada, H., Vijayachandra, K., Penner, C., and Glick, A. 2001. Increased sensitivity of transforming growth factor (TGF) beta 1 null cells to alkylating agents reveals a novel link between TGFbeta signaling and O(6)-methylguanine methyltransferase promoter hypermethylation. J. Biol. Chem. 276:19052-19058. | |
| Yang, L., Clarke, M.J., Carlson, B.L., Mladek, A.C., Schroeder, M.A., Decker, P., Wu, W., Kitange, G.J., Grogan, P.T., Goble, J.M., Uhm, J., Galanis, E., Giannini, C., Lane, H.A., James, C.D., and Sarkaria, J.N. 2008. PTEN loss does not predict for response to RAD001 (Everolimus) in a glioblastoma orthotopic xenograft test panel. Clin. Cancer Res. 14:3993-4001. | |
