Abstract 4203: Non-invasive imaging of tumor cell death induced by a TRAILR2 agonist

Abstract

Abstract Imaging tumour cell death can give an early indication of treatment efficacy. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand receptor2 (TRAILR2) is a member of the TNF receptor superfamily, which interacts with death receptors (DRs) and induces apoptosis in a broad range of cancer cells but not normal cells[1]. MEDI3039, a newly described agonist of TRAILR2, was used to induce tumour cell death. A NIRF fluorophore-labelled phosphatidylserine (PS)-binding protein (∼15kDa), the C2A domain of Synaptotagmin-I (C2Am-750), which binds to the PS exposed by apoptotic and necrotic cells, was used to image MEDI3039-induced cell death[2]. Non-specific binding was assessed using a site-directed mutant that is inactive in PS binding, which was conjugated to a different fluorophore (iC2Am-680). Binding of C2Am-750 to MEDI3039-treated human colon and breast adenocarcinoma cells in vitro (Colo205 and MDA-Dual respectively) was assessed by flow cytometry and confocal microscopy. Both methods showed that C2Am-750 labelled MEDI3039-treated Colo205 cells, while the inactive iC2Am-680 showed only low non-specific binding. C2Am-750 and iC2Am-680 were also used to monitor the effect of treatment in a Colo205 xenograft model. One cohort of mice (n = 5) received a single dose of MEDI3039 (0.4 mg/kg), while another untreated cohort (n = 5) served as a control group. All mice received a single i.v. injection of a 1:1 mixture of 0.1 μmole/kg C2Am-750 and iC2Am-680 at 16 h after drug treatment, followed by whole body fluorescence imaging (FLI) measurements using an IVIS200 camera at 0 and 3 h post probe injection. FLI measurements in MEDI3039-treated Colo205 tumours showed significant increases in the uptake of C2Am-750 relative to iC2Am-680, both in vivo (4-9 fold, P value <0.0001) and ex vivo (7-11 fold, P value <0.0001). Uptake of iC2Am-680 was low in both treated and untreated groups. Subsequent histological analyses showed that the C2Am-750 signal was strongly correlated with both cleaved caspase-3 and TUNEL staining of dead cells. NIRF fluorophore-labelled C2Am showed a favourable biodistribution profile, with good tumour penetration and quick clearance of unbound material in vivo. The probe can be used to investigate the efficacy of targeted therapy, or other anti-tumour therapies, at an early stage following treatment. 1. Prasad, S., Kim, J.H., Gupta, S.C. & Aggarwal, B.B. Targeting death receptors for TRAIL by agents designed by Mother Nature. Trends in pharmacological sciences 35, 520-536 (2014). 2. Alam, I.S., Neves, A.A., Witney, T.H., Boren, J. & Brindle, K.M. 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, Andre Neves, Stefanie R. Mullins, David Tice, Danielle Carroll, Robert W. Wilkinson, Kevin M. Brindle. Non-invasive imaging of tumor cell death induced by a TRAILR2 agonist. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4203.