2018 Synaptic vesicle 2 receptors as a novel targets for neuroendocrine cancer therapy

OBJECTIVES/SPECIFIC AIMS: (1) To delineate the function of the heavy-chain receptor binding domain (HCR), a portion of botulinum neurotoxin type A (BoNT/A) and synaptic vesicle 2 (SV2) signaling pathway, which provide a novel multipurpose biologic with potential clinical applications in tumor detection/imaging, inhibition of tumor progression, and reduction of bioactive hormone secretion in metastatic neuroendocrine (NE) cancers. (2) To evaluate the expression pattern of SV2 receptors in NE cancer patient-derived tissues for prediction of patient response for recombinant HCR (rHCR) treatment. (3) To assess the in vivo efficacy and toxicity of rHCR in a NE cancer liver metastasis mouse models and in the NE patient-derived 3D MicroTumor system. (4) To collect preclinical data to design and conduct a clinical trial with NE cancer patients, a major goal toward translating our discoveries into much needed therapies. METHODS/STUDY POPULATION: Recombinant botulinum heavy chain (rHCR) was produced using an IPTG-inducible expression vector in E. coli BL21. The rHCR was His-Tag purified and stored in PBS buffer before usage. Cytotoxicity: H727, TT, and MZ cells were plated at a density of 5000 cells/well in 96-well plates and incubated under standard conditions overnight. The next day, cells were treated with 10, 100, or 500 nmol/L of rHCR and incubated for 72 hours. Following incubation, cell viability was assessed by ATP quantification using the CellTiter-Glo (Promega) assay. Fresh NE tumors were dissociated and injected into polydimethylsiloxane bioreactors in a matrigel and collagen suspension for 3D culture experiments. The viability of 3D cultures incubated with various doses of rHCR was assessed by measuring the uptake of the near-infrared dye IR-783 using an IVIS imaging system. Western blot: H727, TT, and MZ cells were seeded in 6-well plates at a density of 3×105 cells/well for 24 hours followed by treatment with 100 nmol/L for 72 hours. Total cellular proteins were isolated and analyzed to assess the level of SV2A expression and the effect of rHCR on the expression levels of NET marker proteins. Immunohistochemistry: Deparaffinized tissue culture slides were incubated with SV2A primary antibody in 1% BSA and incubated overnight at 4°C. Slides were rinsed twice with TBS containing 0.025% Triton, followed by 0.3% H 2 O 2 for 15 minutes. Slides were then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Detection of protein-pro