Lauren D. Stegman

Molecular Imaging of Gene Expression and Efficacy following Adenoviral-Mediated Brain Tumor Gene Therapy

Cancer gene therapy is an active area of research relying upon the transfer and subsequent expression of a therapeutic transgene into tumor cells in order to provide for therapeutic selectivity. Noninvasive assessment of therapeutic response and correlation of the location, magnitude, and duration of transgene expression in vivo would be particularly useful in the development of cancer gene therapy protocols by facilitating optimization of gene transfer protocols, vector development, and prodrug dosing schedules. In this study, we developed an adenoviral vector containing both the therapeutic transgene yeast cytosine deaminase (yCD) along with an optical reporter gene (luciferase). Following intratumoral injection of the vector into orthotopic 9 L gliomas, anatomical and diffusion-weighted MR images were obtained over time in order to provide for quantitative assessment of overall therapeutic efficacy and spatial heterogeneity of cell kill, respectively. In addition, bioluminescence images were acquired to assess the duration and magnitude of gene expression. MR images revealed significant reduction in tumor growth rates associated with yCD/5-fluorocytosine (5FC) gene therapy. Significant increases in mean tumor diffusion values were also observed during treatment with 5FC. Moreover, spatial heterogeneity in tumor diffusion changes were also observed revealing that diffusion magnetic resonance imaging could detect regional therapeutic effects due to the nonuniform delivery and/or expression of the therapeutic yCD transgene within the tumor mass. In addition, in vivo bioluminescence imaging detected luciferase gene expression, which was found to decrease over time during administration of the prodrug providing a noninvasive surrogate marker for monitoring gene expression. These results demonstrate the efficacy of the yCD/5FC strategy for the treatment of brain tumors and reveal the feasibility of using multimodality molecular and functional imaging for assessment of gene expression and therapeutic efficacy.

Experimental Gliosarcoma Induces Chemokine Receptor Expression in Rat Brain

Macrophage/microglial infiltration is a characteristic feature of brain tumors. The functional role(s) of these cells is complex and could include both trophic and suppressive effects on tumors. Information has recently emerged about the molecular signals that regulate the accumulation and function of monocytes in pathological disorders. Recent data indicate that the chemokine, monocyte chemoattractant protein-1 (MCP-1), a potent monocyte activating and chemotactic factor, is a primary regulator of the macrophage response in brain tumors. We hypothesized that if MCP-1 regulates macrophage/microglial infiltration, then expression of the specific MCP-1 receptor, CCR2, will be induced in peritumoral tissue and/or within brain tumors. Identification of a specific receptor that is preferentially expressed in brain tumors could be important both in terms of tumor biology and as a potential therapeutic target. We used an established experimental gliosarcoma model, induced by intracranial transplantation of cultured 9L cells into adult rat brain, to test this hypothesis. RT-PCR analysis showed high levels of both MCP-1 and CCR2 mRNA and Western blot analysis demonstrated increased CCR2 protein in tumor extracts. Immunocytochemistry showed CCR2 immunoreactive microglia in peritumoral tissue and, unexpectedly, that intrinsic tumor cells, rather than monocytes, were the predominant source of CCR2. These results demonstrate that CCR2 expression is markedly upregulated in this brain tumor model.

In Vivo 31P MRS Evaluation of Ganciclovir Toxicity in C6 Gliomas Stably Expressing the Herpes Simplex Thymidine Kinase Gene

Phosphorus MRS was evaluated as a monitor of tumour therapeutic response to the herpes simplex virus thymidine kinase suicide gene therapy paradigm. In vivo 31P spectra were obtained from subcutaneous rat C6 gliomas constitutively expressing the HSVtk gene post treatment with ganciclovir (GCV, 15 mg/kg i.p., twice‐daily). Significant regression (p<0.1) of tumour volume was observed 10 days after beginning GCV administration. However, no changes in tumour pH or energy metabolites from pre‐treatment values were observed. High‐resolution 31P spectra of tumour extracts revealed a statistically significant reduction in the phosphocholine to phosphoethanolamine ratio six days post‐GCV administration. These results indicate that the HSVtk/GCV‐induced killing of tumours is not associated with corresponding changes in 31P MRS‐observable energy metabolites and pH. The observed reduction in the PE/PC ratio may provide a non‐invasive in vivo indicator of therapeutic efficacy.