Tumor-selective vessel occlusions by platelets after vascular targeting chemotherapy using paclitaxel encapsulated in cationic liposomes Sebastian Strieth 1,2 * , Claudia F. Nussbaum 1 , Martin E. Eichhorn 1,3 , Martin Fuhrmann 4 , Michael Teifel 5 , Uwe Michaelis 6 , Alexander Berghaus 2 and Marc Dellian 1,2 1 Institute for Surgical Research, University of Munich (LMU), Germany 2 Department of Otorhinolaryngology, University of Munich (LMU), Germany 3 Department of Surgery, Campus Grosshadern, University of Munich (LMU), Germany 4 Center of Neuropathology and Prion Research, University of Munich (LMU), Germany 5 Zentaris GmbH, Frankfurt/Main, Germany 6 MediGene AG, Martinsried, Germany Paclitaxel encapsulated in cationic liposomes (EndoTAG-1) signifi- cantly impairs tumor growth by a significant reduction of functional tumor microcirculation and induction of endothelial cell apoptosis. The aim of the study was to analyze whether platelet activation within the tumor microcirculation contributes to the antivascular effects of vascular targeting chemotherapy using EndoTAG-1. In vitro, FACS analysis revealed a significant activation of platelets upon treatment with EndoTAG-1. In vivo, using A-Mel-3 tumors in Syrian Golden hamsters equipped with dorsal skinfold chamber preparations, the contribution of platelets to the antivascular effects of EndoTAG-1 was evaluated by fluorescence and laser-scanning microscopy. Immediately after a single treatment with EndoTAG-1 or cationic liposomes devoid of paclitaxel, an increase of platelet ad- herence in tumor microvessels was observed. This was accompanied by an acute impairment of the microcirculation within the treated tumors leading to reduced tumor perfusion. After repetitive ther- apy, an increase of platelet adherence and subsequent tumor micro- vessel occlusions occurred only after treatment with EndoTAG-1. Comparing to ‘‘tumor free’’ normal tissue controls these micro- thromboses were tumor selective. Significantly disbalancing the coagulation system within tumors by targeted induction of micro- thromboses within the tumor microcirculation appears to be an important mechanism of EndoTAG-1 therapy. ' 2007 Wiley-Liss, Inc. Key words: novel drug delivery systems; mechanisms of drug action; angiogenesis; tumor microcirculation; coagulation system; platelet; in vivo imaging; vascular targeting Currently, extensive experimental work is performed to suc- cessfully explore the potential of antivascular strategies in cancer therapy. 1 Numerous antiangiogenic substances are currently under evaluation in clinical studies. But antiangiogenic therapy is con- ceptually different from a therapeutic concept called vascular tar- geting. 2 An antiangiogenic therapy aims at the inhibition of endo- thelial cell proliferation, whereas it is the objective of a vascular disrupting strategy to destroy the existing tumor vasculature to achieve malnutrition and destruction of tumor cells. Moreover, it is currently a matter of debate whether antiangiogenic treatment ‘‘normalizes’’ tumor microcirculation. 3 In contrast, vascular targeting is considered not to stabilize or normalize tumor micro- vessels but to impair tumor microcirculation. 4 To realize such a new therapeutic concept based on the destruc- tion of tumor microvasculature drug delivery systems are of con- siderable interest: cationic liposomes have been described to selectively target angiogenic endothelial cells in tumors. 5 This property potentially enables selective drug delivery to tumor endo- thelial cells and thus vascular targeting chemotherapy. We have shown that selective uptake of cationic liposomes in tumor endo- thelium is not affected by the encapsulation of paclitaxel. 6 More- over, treatment with this liposomal paclitaxel (EndoTAG-1, for- merly MBT-0206) significantly retarded tumor growth and delayed the incidence of metastatic disease by significant micro- circulatory detraction after repetitive (3-fold) treatment. 7 Consid- ering the observation of a significantly reduced functional vessel density in tumors treated with EndoTAG-1, the mechanistic involvement and contribution of the coagulation system can be hypothesized as a mechanism of this novel treatment strategy. Blood platelets have been proposed to regulate tumor angiogen- esis by release of both potent stimulators and inhibitors of angio- genesis from their a-granules. 8,9 A prerequisite of platelet activa- tion, their aggregation and release of a-granules is fibrin-binding of platelets to activated endothelial cells or subcellular matrix components. Angiogenic tumor endothelium is characterized by endothelial gaps presenting a large variety of possible binding sites on the subcellular matrix. However, intravital microscopic studies of platelet-endothelial cell interaction in tumor microvessels surprisingly revealed only a moderate level of platelet-endothelial adherence in tumor micro- vessels during tumor angiogenesis. 10 In this context, a functional linkage between tumor microcircula- tion and intratumoral coagulation regulating tumor progression can be hypothesized which may become relevant once the ‘‘procoagu- latory state’’ within tumors 11 is significantly disbalanced towards intratumoral coagulation. Interestingly, findings of the mutual effects influencing either the coagulation system or tumor microcir- culation still remain controversial. Some investigators try to inhibit tumor growth by blocking the coagulation cascade 12 and platelet function, 13 while others follow the concept of inducing coagulation in the tumor microvasculature. 14,15,16 In line with these later studies are recent findings of other vas- cular targeting agents (VTAs) such as Combretastatin-4P, 17 or recombinant fusion proteins of procoagulatory factors and target- ing fragments directed against the tumor microvasculature. 18,19 These VTAs were shown to induce selective intravascular coagu- lation resulting in growth delay or necrosis of the treated tumors. Because of severe thromboembolic complications during the development of antiangiogenic substances 20 and in order to eluci- date the therapeutic mechanism of EndoTAG-1 with regard to the coagulation system, the aim of our study was to analyze whether the activation of the coagulation system within the tumor micro- circulation contributes to the therapeutic effects of vascular target- ing chemotherapy using EndoTAG-1. After analysis of platelet activation in vitro we therefore quantified acute effects of Endo- TAG-1 on platelet-endothelial cell interactions and microcircula- tory consequences as well as effects after repetitive treatment in vivo. In respect of tumor selectivity, measurements were performed in tumor as well as in normal ‘‘tumor free’’ tissue. S. Strieth and C.F. Nussbaum contributed equally to the study. *Correspondence to: Department of Otorhinolaryngology, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, Germany. Fax: 149-89-2180-76532. E-mail: sebastian.strieth@med.uni-muenchen.de Grant sponsor: Munich Biotech AG, Neuried, Germany; Grant sponsor: F€ oFoLe Program of the University of Munich (LMU), Germany. Grant sponsor: Novartis Foundation for Therapeutic Research, N€ urnberg, Germany. Received 5 May 2007; Accepted after revision 13 July 2007 DOI 10.1002/ijc.23088 Published online 4 October 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 452–460 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer