R. C. Bynum1, T. Husarova1,2, E. J. Sanderson1, M. M. Bonds1, A. Jain1, B. Edil1, L. R. McNally1 1University Of Oklahoma College Of Medicine, Department Of Surgery, Oklahoma City, OK, USA 2Military University Hospital 16902 Prague, Department Of Surgery, Prague, Czech Republic
Introduction:
Intraoperative imaging in hepatobiliary surgery is an area of increasing research. Its utilization here may have a significant clinical benefit by improving negative margin resection rates. However, intraoperative ultrasound and current near-infrared fluorescent imaging (NIR), such as indocyanine green or methylene blue, are limited by steep operator learning curves and limited depth of imaging respectively. Optoacoustic imaging is a novel intraoperative imaging method which offers deeper visualization compared to NIR imaging, and opportunity for a more specific imaging paradigm. In our study, we synthesized V3 and V7 pH low insertion peptides (pHLIPs) as probes to image pancreatic cancer using multispectral optoacoustic tomography (MSOT) in orthotopic murine models and ex vivo in human pathology specimens.
Methods:
V3 and V7 pHLIP peptides were synthesized using microwave chemistry and conjugated to HiLyte 750 dye. Uptake of V3-750 and V7-750 probes was measured in S2VP10 and S2013 pancreatic cancer cell lines grown in pH specific media (6.6, 6.8, 7.4). The specificity of V3-750 and V7-750 were also tested in ex vivo surgical specimens from pancreatic cancer resection using fluorescence imaging. Female athymic mice bearing orthotopically implanted with S2VP10 or S2013 cells (3 mm tumor size) were intravenously injected with V3-750 or V7-750 and were imaged using MSOT. Accumulation of probe in tumor was confirmed by ex vivo NIR fluorescence. Statistical analysis was performed using ANOVA and the Wilcoxon test.
Results:
In vitro, V3-750 demonstrated significantly higher fluorescence centered at pH 6.8 (p=0.011) and V7-750 significantly higher fluorescence at pH 6.6 (p=0.015). V3-750 and V7-750 probes were tested on human tumor tissue and non-involved pancreas from ex vivo surgical specimens showing tumor specific uptake of probe V3-750 at pH 6.8 (p=0.01) and V7-750 at pH 6.6 (p=0.01) as shown in Figure 1A. We further imaged orthotopic pancreatic tumors in mice using MSOT. V3-750 and V7-750 both demonstrated tumor specificity in vivo in S2VP10 and S2013 orthotopic murine models of pancreatic cancer (p=0.011, p=0.021), shown in Figure 1B and 1C.
Conclusion:
This study demonstrates the specificity of fluorescent dye bound V3 and V7 pHLIPs in imaging of pancreatic tumors using MSOT. Additionally, high accumulation of probe was observed in pancreatic tumor allowing for effective imaging with MSOT in a murine model and ex vivo human tissue. This imaging modality is currently undergoing translation into large animal models with the V7 pHLIP, a promising next step in the translation of this imaging modality from preclinical to clinical use.
