Abstract 2866: Volumetric optoacoustic imaging of tumor cell death using a targeted imaging agent
Authors: Xie Bangwen , Tomaszewski Michal , Neves André A. , Mullins Stefanie R. , Tice David , Sainson Richard , Bohndiek Sarah , Wilkinson Robert W. , Brindle Kevin M. 
Published: July 2017

Abstract
Multispectral optoacoustic tomography (MSOT) generates high-resolution cross-sectional images in less than a second. MEDI3039, is a newly described agonist of the TNF-related apoptosis-inducing ligand receptor2 (TRAILR2), which has been shown in preclinical studies to preferentially induce cell death in cancer versus normal cells1. We show here that MSOT, when used with a near infra-red (NIR) fluorophore-labelled protein domain of Synaptotagmin-I (C2Am-750, ~15kDa) that binds to phosphatidylserine (PS) exposed by apoptotic and necrotic cells2, can be used to image MEDI3039-induced cell death within the entire tumor region. Non-specific probe retention was assessed using a site-directed mutant (iC2Am-680 or iC2Am-750).
PS-specific binding of C2Am-750 to MEDI3039-treated TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 human colon adenocarcinoma cells in vitro was evaluated by flow cytometry and confocal microscopy. The capability of C2Am-750 to detect cell death in vivo was assessed in mice bearing Colo205 or HT-29 xenografts, following a single dose (0.4 mg/kg, i.v.) of MEDI3039. All mice then received an i.v. injection of a 1:1 mixture of 0.1 µmole/kg C2Am-750 and iC2Am-680 16 h post treatment, followed by fluorescence imaging (FLI) measurements using an IVIS 200 camera at 0 and 3 h post probe injection. FLI measurements in treated Colo205 xenografts showed significantly increased retention of C2Am-750 uptake versus size-matched untreated tumors (6.0-fold, P value &lt;0.0001), but there was no significant difference between treated and untreated HT-29 xenografts. iC2Am-680 retention was negligible in all groups. Next, volumetric MSOT measurements of C2Am-750 and iC2Am-750 (0.2 µmole/kg) retention in MEDI3039-treated Colo205 tumors were made using an iTheraMedical inVision 256 system at different wavelengths (660-900nm) and time points (3-24 h). C2Am-750 signal was markedly increased in tumors at 3 h post probe injection. Maximal C2Am-750 signal appeared in the center of treated tumors, whereas there was negligible iC2Am-750 retention. Subsequent histological analyses showed that C2Am-750 signal was strongly correlated with both cleaved caspase-3 and TUNEL staining of dead cells.
We have shown that real-time volumetric MSOT measurements with a NIR fluorophore-labelled C2Am, can be used to detect early tumor cell death within the entire tumor region following TRAILR2 agonist treatment. This study shows that this imaging technique can be used to characterize tumor heterogeneity and to determine the most appropriate therapy at an early stage post drug administration.
1.
Swers, J.S. et al. Multivalent scaffold proteins as superagonists of TRAIL receptor 2-induced apoptosis. Molecular cancer therapeutics 12, 1235-1244 (2013).
2.
Alam, I.S. et al. Comparison of the C2A domain of synaptotagmin-I and annexin-V as probes for detecting cell death. Bioconjugate chemistry 21, 884-891 (2010).
Citation Format: Bangwen Xie, Michal Tomaszewski, André A. Neves, Stefanie R. Mullins, David Tice, Richard Sainson, Sarah Bohndiek, Robert W. Wilkinson, Kevin M. Brindle. Volumetric optoacoustic imaging of tumor cell death using a targeted imaging agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2866. doi:10.1158/1538-7445.AM2017-2866