Combinatorial effects of doxorubicin and retargeted tissue factor by intratumoral entrapment of doxorubicin and proapoptotic increase of tumor vascular infarction

Janine Stucke-Ring,Eva Wardelmann,Wolfgang E Berdel,Saliha Harrach,Georg Lenz,Verena Mantke,Rolf M Mesters,Lars Henning Schmidt,Christian Schwöppe,Carsten Müller-Tidow,Torsten Kessler,Caroline Brand,Bernhard Wünsch,Christoph Schliemann,Wolfgang Hartmann,Julian Ronnacker,Heike Hintelmann,Carsten Höltke

doi:10.18632/oncotarget.12559

Abstract

Truncated tissue factor (tTF), retargeted to tumor vasculature by GNGRAHA peptide (tTF-NGR), and doxorubicin have therapeutic activity against a variety of tumors. We report on combination experiments of both drugs using different schedules. We have tested fluorescence- and HPLC-based intratumoral pharmacokinetics of doxorubicin, flow cytometry for cellular phosphatidylserine (PS) expression, and tumor xenograft studies for showing in vivo apoptosis, proliferation decrease, and tumor shrinkage upon combination therapy with doxorubicin and induced tumor vascular infarction. tTF-NGR given before doxorubicin inhibits the uptake of the drug into human fibrosarcoma xenografts in vivo. Reverse sequence does not influence the uptake of doxorubicin into tumor, but significantly inhibits the late wash-out phase, thus entrapping doxorubicin in tumor tissue by vascular occlusion. Incubation of endothelial and tumor cells with doxorubicin in vitro increases PS concentrations in the outer layer of the cell membrane as a sign of early apoptosis. Cells expressing increased PS concentrations show comparatively higher procoagulatory efficacy on the basis of equimolar tTF-NGR present in the Factor X assay. Experiments using human M21 melanoma and HT1080 fibrosarcoma xenografts in athymic nude mice indeed show a combinatorial tumor growth inhibition applying doxorubicin and tTF-NGR in sequence over single drug treatment. Combination of cytotoxic drugs such as doxorubicin with tTF-NGR-induced tumor vessel infarction can improve pharmacodynamics of the drugs by new mechanisms, entrapping a cytotoxic molecule inside tumor tissue and reciprocally improving procoagulatory activity of tTF-NGR in the tumor vasculature via apoptosis induction in tumor endothelial and tumor cells.

Highlights

Denekamp et al first proposed tumor vessels and endothelial cells as a target for antitumor therapy [1]
Our results show that doxorubicin is effectively entrapped by Truncated tissue factor (tTF)-peptide GNGRAHA (NGR)-induced vascular infarction inside tumor tissues leading to a longer exposure time of the tumor to the drug with increased and prolonged tumor cell apoptosis
We hypothesized that tumor vessel occlusion with retargeted tissue factor tTF-NGR will entrap this organic cytotoxic molecule inside tumor tissue and prolong and amplify its antitumor effect

Summary

Introduction

Denekamp et al first proposed tumor vessels and endothelial cells as a target for antitumor therapy [1]. We have constructed a series of fusion proteins consisting of short NGR-peptide sequences coupled to the C-terminal end of tTF [4,5,6,7,8]. Among others from this series, tTF-NGR (HIStag-tTF1-218-GNGRAHA) as a model fusion protein retains its procoagulatory activity in vitro, binds to the respective targets on endothelial and tumor cells, and upon intravenous infusion induces vascular infarction in blood vessels of human tumors of various histologies growing in athymic mice with subsequent tumor growth retardation or regression. Intravenous infusion of tTF-NGR in late-stage cancer patients at dose levels without side effects was shown to reduce tumor blood flow in situ [5]

Methods

Results

Conclusion