Abstract 4557: In vivo near-infrared fluorescent quantification of therapy-induced apoptosis using Annexin-Vivo 750

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Drug-induced ex vivo apoptotic tests are widely used pre-clinically to monitor chemotherapeutic efficacy. Real-time non-invasive in vivo imaging and quantification of therapy-induced tumor apoptosis would impact drug discovery significantly. As a key apoptotic marker, phosphatidylserine (PS) translocated to the outer leaflet of the plasma membrane can be detected by fluorescent-labeled Annexin A5 specifically with high affinity. We synthesized an in vivo imaging contrast agent Annexin-Vivo 750 by conjugating a human recombinant Annexin A5 to a near-infrared fluorophore with em 770 nm. Fluorescence microscopy showed 4h anti-FAS Ab treatment significantly increased the number of Annexin-Vivo 750 positive Jurkat cells, confirming that the agent binds to PS on apoptotic cells. Similarly, flow cytometry showed anti-FAS Ab increased Annexin-Vivo 750 positive cells from 15% to 55%. TUNEL positive cells were increased from 0% to 65.14 ± 3.93% (p=0.0004) by anti-FAS antibody. The TUNEL data correlate very well with flow cytometry data and are consistent with the fluorescence microscopy data. In vivo imaging of Annexin-Vivo 750 was performed in nude mice implanted with human colorectal carcinoma HT-29 cells. Once tumors reached a volume of 142.36 + 8.3 mm3 mice were randomized and treated intraperitoneally with cyclophosphamide (170 mg/kg) or vehicle at time 0h. The agent was injected at 24h with a dose of 2.4 nmol/animal and the tumors were imaged at 26h with an FMT2500 quantitative tomography imager for in vivo real-time 3D Annexin-Vivo 750 quantification. Tumor fluorescence signal was 2.55 fold higher with than without cyclophosphamide (p=0.0002), while tumor volumes were the same (p=0.35). Fluorescent signal decreased significantly by 24h and returned to baseline at 48h post agent injection. Likewise, intratumoral injection of TNFα (2.5 μg) plus IFNγ subcutaneous injection (2500 units) also significantly increased HT29 tumor Annexin-Vivo 750 fluorescence (3 fold compared to control, p=0.0099, 24h after treatment, 2h post-agent injection). TUNEL positive HT-29 cells were increased from 11.16 ± 7.05 % to 41.77 ± 8.78 % (p=0.0004) by cyclophosphomide, indicating apoptosis. These results demonstrate that Annexin-Vivo 750 in vivo imaging can be used for detecting, quantifying and monitoring apoptosis following cancer treatments, underscoring its value in drug discovery and development in cancer and potentially in other clinically important areas, such as cardiovascular-, autoimmune-, inflammatory- and neurodegenerative diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4557.