Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery

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.

Keywords: glioblastoma; mouse models; xenograft.

Figure 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 hours 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 3 independent experiments. (B) Following treatment with 100 μM TMZ, cells were harvested at the indicated time points and processed for western blotting for MGMT and then βactin. Data reproduced with permission from (Kitange, 2009).

Figure 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 hours 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 3 independent experiments. (B) Following treatment with 100 μM TMZ, cells were harvested at the indicated time points and processed for western blotting for MGMT and then βactin. Data reproduced with permission from (Kitange, 2009).

Figure 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 are 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 3. Stereotactic injection set-up

A–B)The stereotactic injection jig with a mouse in place is shown. C) By using multiple injection jigs in an assembly line fashion, 2 or 3 technicians can implant 100 mice in 4 hours. Photographs courtesy of Cory Petell.

Figure 3. Stereotactic injection set-up

A–B)The stereotactic injection jig with a mouse in place is shown. C) By using multiple injection jigs in an assembly line fashion, 2 or 3 technicians can implant 100 mice in 4 hours. Photographs courtesy of Cory Petell.

Figure 3. Stereotactic injection set-up

A–B)The stereotactic injection jig with a mouse in place is shown. C) By using multiple injection jigs in an assembly line fashion, 2 or 3 technicians can implant 100 mice in 4 hours. Photographs courtesy of Cory Petell.

Figure 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. 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 pre-treatment 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. Data reproduced with permission from (Sarkaria, 2007).

Figure 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. 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 pre-treatment 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. Data reproduced with permission from (Sarkaria, 2007).

Figure 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. 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 pre-treatment 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. Data reproduced with permission from (Sarkaria, 2007).

Figure 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. 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 pre-treatment 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. Data reproduced with permission from (Sarkaria, 2007).

Figure 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.