J. Whitt1, J. Ou2, X. Liu2, H. Chen1, R. Jaskula-Sztul1 1University Of Alabama at Birmingham,Department Of Surgery,Birmingham, Alabama, USA 2University Of Alabama at Birmingham,Biomedical Engineering,Birmingham, Alabama, USA
Introduction: Neuroendocrine (NE) malignancies may arise from hormone producing cells located throughout the body. Although advances in diagnostics have led to an increased ability to detect localized and well-differentiated tumors, many neuroendocrine tumors (NETs) don’t produce symptoms until after metastasis. Moreover, patients with liver metastases have a 5-year overall survival rate of less than 30 percent. The identification of a new theranostic agent is necessary to improve the clinical outcome for patients. We propose the use of synaptic vesicle membrane protein 2(SV2A) as an alternative therapeutic target for advanced NETs.
Methods:
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 hr. 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 h followed by treatment with 100nmol/L for 72 h. 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% H2O2 for 15 min. Slides were then incubated with HRP-conjugated secondary antibody for 1 h at room temperature.
Detection of protein-protein interaction: Precleared cell lysate was incubated with glutathione-agarose beads in the presence of 10 ug of GST-tagged rHCR for 2 h at 4?C with end-over end mixing. Bound proteins were eluted with 20 mM reduced glutathione and analyzed by SDS-PAGE.
Results:All NET cell lines and tumor samples showed SV2A expression. In vitro and 3D bioreactor studies showed no significant change in cell viability at 100 nmol/L. SV2A was detected by Western blot after incubation with GST-tagged rHCR and glutathione-agarose beads.
Conclusion:The detection of SV2A in neuroendocrine cell lines and patient derived samples indicates that SV2A is a potential new therapeutic target for neuroendocrine tumors. The lack of cytotoxicity of rHCR supports its use for targeted delivery of drugs to NETs.