Targeting VPAC1 receptors for imaging glioblastoma

Objectives: Scintigraphic imaging of human glioblastoma continues to be challenging. Objective was to develop a genomic tracer to efficiently image glioblastoma at an early stage.

Methods: Expression of VPAC1 receptors on the mouse GL-261 and human U-87 glioma cell lines was determined by western blot. Cell binding assay was performed by incubating ⁶⁴Cu-TP3805 with 1.2 x 10⁶ GL-261 and U-87 cells. GL-261 cell line which mimics human glioblastoma was chosen and was grown in tissue culture. 10⁵ GL-261 cells were then implanted in the right brain lobe of T-bet knockout C57BL/6 mice (N=5). Tumors were allowed to grow for 2-3 weeks. PET/CT imaging and tissue distribution studies were performed using F-18-FDG and 24 hrs later using Cu-64-TP3805, which has been shown in our laboratory to efficiently image breast and prostate cancers in humans. On day 10 to 14 after GL-261 implantation mice were injected i.v., first with 150 µCi of F-18-FDG and 24 hrs later with 150 µCi of Cu-64-TP3805. Mice were imaged using Micro-PET/CT 2 hr later, sacrificed and % ID/g of tumor and normal brain were calculated. To prepare Cu-64-TP3805, 30 µL of ⁶⁴CuCl₂ in 0.1M HC1, was added to peptide-conjugate (20 µg) in 200 µL of 0.2M glycine buffer (pH=9.27), and incubated at 70º C for 90 min. The radiochemical purity was determined by radio-HPLC.

Results: The labeling efficiency of 64-TP3805 as determined by radio-HPLC was 96.3±0.5% with retention time for ⁶⁴Cu-peptide at 5.3 min and for free ⁶⁴CuCl₂ at 3.4 min. We found strong signal for VPAC1 receptor on western blot for GL-261 and U-87 cells (Fig-1). The cell binding for ⁶⁴Cu-peptide was 86±1.5% for both the cell lines. Micro-PET/CT image analyses and tissue distribution showed that the Cu-64-TP3805 tumor uptake at 2 hrs post injection was 7±2% ID/g as compared to 0.96±0.3% ID/g for normal brain. The normal tissue distribution showed a similar pattern as observed in PET imaging. F-18-FDG brain images were unclear for tumor imaging. Conclusion: Western blot studies confirmed high VPAC1 expression on the glioblastoma cell lines examined. ⁶⁴Cu-TP3805 showed excellent labeling efficiency, and high uptake in glioblastoma. Targeting VPAC1 receptors using ⁶⁴Cu-TP3805 for PET imaging of glioblastoma is a promising approach and calls for further investigation. Acknowledgements: The research, in part, was supported by NIH/NCI R01CA157372 (MLT), NIH/NCI 1S10OD012406 (MLT) and NIH/NCI S10RR23709 (MLT).

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