Cationic lipid complexed paclitaxel (EndoTAG TM -1) is a novel vascular targeting agent for the treatment of cancer. Here, the aim was to investigate intratumoral drug distribution after EndoTAG TM -1 therapy and analyze the impact of EndoTAG TM -1 scheduling on antitumoral efficacy. The therapeutic effect of EndoTAG TM -1 in combination with conventional gemcitabine or cisplatin therapy was evaluated in L3.6pl orthotopic pancreatic cancer and a subcutaneous Lewis lung (LLC-1) carcinoma model. Oregon Green paclitaxel encapsulated in cationic liposomes in combination with intravital fluorescence microscopy clearly exhibited delivery of the drug by EndoTAG TM -1 to the tumor endothelium, whereas Oregon Green paclitaxel dissolved in cremophor displayed an interstitial distribution pattern. The therapeutic efficacy of EndoTAG TM -1 was critically dependent on the application schedule with best therapeutic results using a metronomic rather than a maximum tolerated dose application sequence. The combination of EndoTAG TM -1 therapy and cytotoxic chemotherapy significantly enhanced antitumoral efficacy in both tumor models. Interestingly, only EndoTAG TM -1 in combination with gemcitabine was able to inhibit the incidence of metastasis in pancreatic cancer. In conclusion, vascular targeting tumor therapy by EndoTAG TM -1 combined with standard small molecular chemotherapy results in markedly enhanced antitumoral efficacy. Therefore, this combination represents a promising novel strategy for clinical cancer therapy.Angiogenesis, the formation of new blood vessels from the endothelium of the existing vasculature, is fundamental in tumor growth, progression and metastasis. 1 The complex network of tumor blood microvessels guarantees an adequate supply of tumor cells with nutrients and oxygen and provides efficient drainage of metabolites.Therapeutic strategies that target and disrupt already formed vessel networks of growing tumors are therefore actively pursued. In contrast with the antiangiogenesis approach, the aim of vascular targeting is to destroy the established tumor vasculature thus causing a rapid and extensive decrease in tumor blood flow, followed by secondary tumor cell death. 2,3 Ligand-directed vascular targeting agents and small molecular tubulin-binding agents have been successfully developed to induce a vascular shutdown of tumor microvessels. 4 In addition to these compounds, drug delivery systems are of considerable interest in realizing such a new therapeutic concept: cationic lipid complexes have been described to target angiogenic endothelial cells in tumor preferentially. [5][6][7] This property potentially enables selective delivery of cytotoxic drugs to tumor endothelial cells and thus vascular targeting chemotherapy. We have previously shown that vascular targeting therapy can be realized by paclitaxel or camptothecin encapsulated in cationic lipid complexes. 8,9 Treatment with these liposomal compounds significantly retarded primary tumor growth and delayed metastatic disease by an antivascular...

The aim of the study was to quantitatively assess tumor microcirculation upon vascular targeting tumor therapy by non-destructive contrast enhanced ultrasonography (CEUS) and to validate this technology by correlation with high-resolution intravital fluorescence microscopy (IVM). Subcutaneous Lewis Lung carcinomas (LLC-1) carcinomas were established in mice. A-MEL-3 melanomas were grown in dorsal skinfold chambers of hamsters to permit bimodal imaging of tumor microcirculation by CEUS and IVM. Animals were treated by i.p. injection of ZD6126 and CEUS imaging after bolus injection of microbubbles was performed. Red blood cell velocity (VRBC), segmental blood flow (Q) and microcirculatory perfusion (PI) of tumors was quantified by IVM. Change in signal intensity (SI) from baseline (ΔSI), rate of SI increase (RSI) and area below intensity time curves (AUC) were calculated in tumors by analysis of CEUS data. Microvessel density was measured by quantitative analysis of CD31 immunohistochemistry. The Mann-Whitney test was used to evaluate differences between groups. Spearman correlation test was used to investigate the relation between CEUS and IVM parameters or histologic CD31 count. ΔSI, RSI and AUC values in ZD6126 treated tumors were lower compared to untreated controls. Comparing central and peripheral tumor regions a vascularized viable rim in the tumor periphery could be detected by CEUS imaging. For the entire cohort ΔSI, RSI and AUC values positively correlated with VRBC, Q and PI quantified by IVM. In LLC-1 carcinomas a positive correlation between ΔSI, RSI and AUC and histological assessment of tumor vascularity was found. In conclusion tumor vascular response to vascular targeting therapy can be quantified non-invasively by CEUS. Bimodal tumor imaging by intravital microscopy and CEUS represents an experimental tool to further develop molecular imaging of tumor microcirculation by CEUS.

Neo-vascular targeting by cationic colloidal carriers enables to realize an innovative approach for tumor therapy. EndoTag-2 is a novel vascular targeting agent, comprising the mammalian topoisomerase I inhibitor camptothecin in its carboxylate form complexed to cationic lipid (cationic lipid complexed camptothecin). Here we studied tumor vascular targeting properties, antitumoral effects and mode of action of EndoTag-2. Tumor vascular targeting properties of fluorescently labelled EndoTag-2 were investigated by in vivo microscopy using A-MEL-3 tumors grown in the dorsal skinfold chamber preparation and by fluorescence histology of s.c. LLC-1 carcinomas. Therapeutic effects have been investigated in the s.c. LLC-1 carcinoma model and the L3.6pl human pancreatic cancer model implanted orthotopically in athymic nude mice. Antivascular effects have been studied by histological investigation of tumor microvessel density and non invasive investigation of tumor blood flow by dynamic contrast enhanced MRI imaging (DCE-MRI). EndoTag-2 selectively targeted tumor microvessels as confirmed by quantitative fluorescence microscopy. Compared to controls EndoTag-2 revealed remarkable antitumoral efficiency in s.c. LLC-1 carcinomas implanted in C57/Bl6 mice. Growth and metastasis of orthotopic L3.6pl human pancreatic tumors was significantly inhibited by EndoTag-2 treatment. Quantitative analysis of tumor microvessel density revealed significant reduction of microvessel density in lewis lung carcinomas up to 50%. DCE-MRI confirmed significant reduction of intratumoral vascular volume as well as tumor perfusion upon EndoTag-2 treatment. In conclusion this study shows that cationic lipid complexed camptothecin (EndoTag-2) is a markedly active antitumor agent based on an innovative vascular targeting approach.

Exposure to static magnetic fields (SMFs) results in a reduced blood flow in tumor vessels as well as in activation and adherence of platelets. Whether this phenomenon may have a significant functional impact on tumors has not been investigated as yet. The aim of our study was to evaluate the effects of prolonged exposure to SMFs on tumor angiogenesis and growth. Experiments were performed in dorsal skinfold chamber preparations of Syrian Golden hamsters bearing syngenic A-Mel-3 melanomas. On 3 d following tumor cell implantation one group of animals was immobilized and exposed to a SMF of 586 mT for three h. Control animals were immobilized for the same duration without SMF exposure. Using in vivo-fluorescence microscopy the field effects on tumor angiogenesis and microcirculation were analyzed for seven days. Tumor growth was assessed by repeated planimetry of the tumor area during the observation period. Exposure to SMFs resulted in a significant retardation of tumor growth ( approximately 30%). Furthermore, histological analysis showed an increased peri- and intratumoral edema in tumors exposed to SMFs. Analysis of microcirculatory parameters revealed a significant reduction of functional vessel density, vessel diameters and red blood cell velocity in tumors after exposure to SMFs compared to control tumors. These changes reflect retarded vessel maturation by antiangiogenesis. The increased edema after SMF exposure indicates an increased tumor microvessel leakiness possibly enhancing drug-uptake. Hence, SMF therapy appears as a promising new anticancer strategy-as an inhibitor of tumor growth and angiogenesis and as a potential sensitizer to chemotherapy.

Anti-angiogenic therapy by blocking VEGF signalling combined with standard chemotherapy is a novel strategy for clinical cancer treatment. The mechanisms for enhanced antitumoral effects are still a matter of controversial debate. Tumor vessel "normalization" upon anti-angiogenic therapy leading to improved drug delivery has been proposed as possible mechanism. Therefore, aim of the study was to investigate tumor microvascular function upon anti-VEGFR treatment in highly vascularized melanomas. A detailed intravital-microscopic analysis of tumor microcirculation including the distribution pattern of vessel diameters and blood flow velocities was performed in melanomas grown in dorsal skinfold chambers of hamsters. Animals with highly vascularized established tumors were treated by a VEGFR tyrosin kinase inhibitor (SU5416) on 3 repetitive days. Tumor tissue oxygenation was measured by phosphorescence quenching technique. Overall tumor microcirculation of subcutaneous tumors was investigated by contrast enhanced MRI (CE-MRI). Vessel density was significantly decreased in treated animals. A significant shift in the distribution patterns towards increased vessel diameters and faster red blood cell velocities in remaining tumor vessels was observed upon anti-VEGF treatment, compensating reduced vascular density. Moreover, a trend towards elevated pO(2) levels in treated tumors was observed. Compared to controls, inflow kinetics of tumors quantified by CE-MRI as well as overall uptake of contrast agent in tumor tissue were significantly increased following short-term SU5416 treatment. In conclusion the results confirm temporarily improved tumor microvascular function in highly vascularized melanomas upon short term anti-VEGFR treatment leading to enhanced tumor blood supply and oxygenation potentially improving the efficacy of simultaneous chemo- or radiotherapy.

Red blood cell flow in striated muscle capillaries is reduced during exposure to strong static magnetic fields (SMFs). Intratumoral microcirculation is characterized by tortuous microvessels with chaotic architecture and by irregular, sluggish blood flow with unstable rheology.It was the aim of this study to analyze SMF exposure effects on tumor microcirculation with regard to interactions of corpuscular blood components with tumor microvessel walls. In vivo fluorescence microscopy was performed in A-Mel-3 tumors growing in dorsal skinfold chamber preparations of Syrian Golden hamsters. SMFs with varying field strength (<600 mT) were generated by changing the distance between a strong NdFeB rod magnet and the tissue region of interest.Short-time exposure above a magnetic flux density of about 150 mT resulted in a significant reduction of red blood cell velocity (v RBC ) and segmental blood flow in tumor microvessels. At the maximum strength of 587 mT, a reversible reduction of v RBC (~40%) and of functional vessel densitiy (~15%) was observed.Prolongation of the exposure time from one minute to up to 3 h resulted in comparable reductions. Microvessel diameters and leukocyte-endothelial cell interactions remained unaffected by SMF exposure. However, in contrast to tumor-free striated muscle controls, exposure at the maximum flux density induced a significant increase in platelet-endothelial cell adherence in a time-dependent manner that was reversible after reducing SMF strength.These reversible changes may have implications for functional measurements of tumor microcirculation by MRI and new therapeutic strategies using strong SMFs.