Bioluminescence monitoring of intracranial glioblastoma xenograft: response to primary and salvage temozolomide therapy

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Object

Bioluminescence imaging (BLI) offers a rapid and accurate means for longitudinal study of tumor cell growth and response to therapy in rodent models. Because this technology has only recently come into use in the field of small animal imaging, applications in this area have been limited. In the current study we have applied BLI to the analysis of clinically relevant issues involving use of the DNA methylating agent temozolomide (TMZ) in a mouse model.

Methods

An invasive glioblastoma multiforme xenograft was modified for BLI via transduction with a luciferase-encoding lentivirus. Supratentorial tumors were established in athymic nude mice that were subsequently assigned randomly to control and TMZ treatment groups, and the extent of intracranial tumor was monitored using BLI.

Results

In an experiment designed to compare the extent of antitumor effect between a single high-dose TMZ treatment and a protracted low-dose TMZ regimen, BLI revealed the protracted regimen as having superior antitumor effect, and this interpretation was consistent with results from a survival comparison between the two TMZ treatment groups. In a second experiment designed to assess the utility of BLI for testing therapies against recurrent glioblastoma multiforme, mice with intracranial tumors were retreated with TMZ at a time when BLI monitoring revealed tumor regrowth following initial TMZ treatment, and retreatment was successful in providing additional survival benefit.

Conclusion

The results of these experiments indicate that BLI monitoring can be used as a surrogate for predicting survival benefit from TMZ treatment, permits early determination of relative survival benefit associated with distinct TMZ therapeutic regimens, and offers a means of investigating secondary/salvage therapy efficacy following tumor regrowth from initial therapy.

Abbreviations used in this paper:BLI = bioluminescence imaging; GBM = glioblastoma multiforme; MGMT = O6-methylguanine DNA methyltransferase; PBS = phosphate-buffered saline; TMZ = temozolomide.

Article Information

Address reprint requests to: C. David James, Ph.D., Department of Neurological Surgery, Brain Tumor Research Center, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, California 94143. email: david.james@ucsf.edu.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Accuracy of BLI as an indicator of intracranial tumor volume. Sixteen mice received intracranial injection of 7.5 × 105 luciferase-modified U87 cells. Groups of four mice each were killed at 1-week intervals immediately following BLI. Paraffin-embedded brains were sectioned, and serially-registered sections were evaluated for surface area and tumor volume, with tumor volumes plotted against corresponding BLI measurements obtained when the animals were killed. A: Photomicrographs of examples of single sections containing the largest areas of tumors from mice killed at Weeks 1, 2, 3, and 4. H & E, original magnification × 5. B: Luminescence images obtained in mice immediately before they were killed. C: Graph illustrating the relationship between tumor volumes and corresponding luminescence values. The correlation coefficient shows a significant association between BLI and tumor volume. Curved lines represent the 95% confidence interval for this comparison.

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    Variability in BLI measurements between mice and consistency of intracranial tumor luminescence growth-response patterns. A: Graph illustrating the individual BLI values from 30 mice with signal acquired at initial imaging, 7 days subsequent to injection of an estimated 3 × 105 GBM 14 cells in each mouse. Minimum BLI values recorded for animals receiving a single 120 mg/kg administration or five 50 mg/kg administrations of TMZ, as well as the last BLI values obtained for each mouse prior to its becoming moribund, are also shown. Arrows indicate the low and high luminescence values obtained during the course of this experiment. B: Graph showing the consistent pattern of luminescence increase in untreated mice (black lines) and the consistent pattern of luminescence decrease followed by increase in mice receiving a single administration of 120 mg/kg TMZ (gray lines).

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    Use of BLI for comparing the efficacy of different TMZ treatment regimens and as a surrogate for survival analysis. Thirty mice receiving intracranial injections of GBM 14 cells were randomly assigned into groups that received no treatment, a single treatment of 120 mg/kg TMZ at Day 18, or treatment with 50 mg/kg TMZ for five consecutive days (Days 18–22). A: Graph showing the mean normalized BLI values associated with longitudinal monitoring of intracranial tumor growth for each treatment group. Regrowth of intracranial tumors in mice receiving a single administration of 120 mg/kg TMZ administration was initially seen at Day 42 (asterisk), whereas the first indication of intracranial tumor regrowth in mice receiving five doses of 50 mg/kg TMZ was seen at day Day 56 (downward-pointing arrow). B: Representative luminescence intensity images obtained in individual control and treatment group mice on the indicated days along with stained sections from control and treatment group mice killed on Day 23 (second column). Inset: Enlargement of the boxed area in the photomicrograph of the control group mouse demonstrates the infiltrative intracranial growth pattern of this xenograft. H & E, original magnification × 5. C: Graph showing the corresponding survival curves and revealing the consistency between relative survival benefit associated with the two TMZ treatment regimens and the corresponding patterns of bioluminescence increase for each group of mice (shown in A). Note that the survival analysis does not reveal the more efficacious TMZ regimen until Day 64 (upward-pointing arrow).

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    Application of BLI to monitoring tumor recurrence and assessing the effect of treating recurrent tumor with secondary or salvage therapy. Mice receiving intracranial injections of GBM 14 were randomly assigned into groups that received no treatment, a single 120 mg/kg TMZ treatment, or two 120 mg/kg TMZ treatments, with the second treatment delivered when tumor recurrence was revealed by BLI monitoring (Day 57). A: Graph illustrating the contrasting longitudinal patterns of BLI for the three treatment groups and highlighting the effect of retreatment (Day 57) on suppressing tumor regrowth, relative to the effect of a single dose of TMZ. B: Representative luminescence intensity images obtained in individual control and treatment-group mice on the days indicated. C: Graph showing the corresponding survival curves for the three treatment groups and demonstrating an easily discernible association between luminescence patterns (A) and survival benefit from a second TMZ treatment against recurrent tumor. Note, however, that the survival benefit from retreatment is substantially less than the benefit associated with initial treatment (mean survival difference between control group and single-dose treatment group 56.2 days; mean survival difference between single- and two-dose treatment groups 21.8 days).

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