Abstract LB-223: Intratumoral accumulation/entrapment of doxorubicin by tumor vascular infarction with retargeted tissue factor tTF-NGR

Abstract

Abstract Truncated tissue factor (tTF) retargeted to tumor vasculature by C-terminal GNGRAHA peptide (tTF-NGR) is able to induce tumor vessel infarction. Here, we describe the identification of the optimum therapeutic regimen for the use of tTF-NGR (or a random-PEGylated derivative of tTF-NGR) in combination with the chemotherapeutic agent doxorubicin in vitro and in vivo. First, we treated human HT1080 xenografts subcutaneously transplanted in athymic CD-1 mice with doxorubicin (5 mg/kg body weight (bw), i.v.) and excised the tumors at different times after injection. Fluorescence spectrometric analyses of the autofluorescence of doxorubicin revealed that intratumoral maximum of doxorubicin accumulation was reached approximately 6 hours after injection. While tTF-NGR given before doxorubicin inhibits the uptake of the latter into HT1080 human fibrosarcoma xenografts in vivo, the reverse sequence does not influence the uptake of doxorubicin in tumor tissue, but significantly inhibits the late wash-out phase entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with various concentrations of doxorubicin in vitro increases concentrations of phosphatidylserine (PS) in the outer layer of the lipid bilayer induced by pro-apoptotic stress. In Factor X activation kinetics, both HUVEC and HT1080 cells showed a significantly higher procoagulatory efficacy when used with equimolar concentrations of tTF-NGR upon doxorubicin incubation and higher PS externalization than identical numbers of control cells with low PS externalization. For proof of a causal relation between doxorubicin-induced PS externalization and procoagulant behaviour of the cells, we could show that preincubation with an phosphatidylserine-inhibiting antibody completely abolished this elevated procoagulatory state of the cells. Thus, we hypothesized that both drugs by the reciprocal mechanisms described mutually increase their therapeutic anti-tumor efficacy in vivo. Therapeutic experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in CD1 nude mice revealed a significantly increased tumor growth inhibition applying suboptimal doses of doxorubicin and tTF-NGR in sequence over single drug treatment. In conclusion, combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by a new mechanism, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in endothelial and tumor cells. Citation Format: Janine Stucke-Ring, Julian Timo Ronnacker, Caroline Brand, Verena Mantke, Carsten Höltke, Christoph Schliemann, Torsten Kessler, Rolf M. Mesters, Christian Schwöppe, Wolfgang E. Berdel. Intratumoral accumulation/entrapment of doxorubicin by tumor vascular infarction with retargeted tissue factor tTF-NGR. [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 LB-223.