Tumor Vascular Permeabilization by Vascular-Targeting Photosensitization: Effects, Mechanism, and Therapeutic Implications

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Abstract
Purpose: Loss of vascular barrier function has been observed shortly following vascular-targeting photodynamic therapy. However, the mechanism involved in this event is still not clear, and the therapeutic implications associated with this pathophysiologic change have not been fully explored. Experimental Design: The effect of vascular-targeting photodynamic therapy on vascular barrier function was examined in both s.c. and orthotopic MatLyLu rat prostate tumor models and endothelial cells in vitro, using photosensitizer verteporfin. Vascular permeability to macromolecules (Evans blue-albumin and high molecular weight dextran) was assessed with dye extraction (ex vivo) and intravital microscopy (in vivo) methods. Intravital microscopy was also used to monitor tumor vascular functional changes after vascular-targeting photodynamic therapy. The effects of photosensitization on monolayer endothelial cell morphology and cytoskeleton structures were studied with immunofluorescence staining. Results: Vascular-targeting photodynamic therapy induced vascular barrier dysfunction in the MatLyLu tumors. Thus, tumor uptake of macromolecules was significantly increased following photodynamic therapy treatments. In addition to vascular permeability increase, blood cell adherence to vessel wall was observed shortly after treatment, further suggesting the loss of endothelial integrity. Blood cell adhesion led to the formation of thrombi that can occlude blood vessels, causing vascular shutdown. However, viable tumor cells were often detected at tumor periphery after vascular-targeting photodynamic therapy. Endothelial cell barrier dysfunction following photodynamic therapy treatment was also observed in vitro by culturing monolayer endothelial cells on Transwell inserts. Immunofluorescence study revealed microtubule depolymerization shortly after photosensitization treatment and stress actin fiber formation thereafter. Consequently, endothelial cells were found to retract, and this endothelial morphologic change led to the formation of intercellular gaps. Conclusions: Vascular-targeting photodynamic therapy permeabilizes blood vessels through the formation of endothelial intercellular gaps, which are likely induced via endothelial cell microtubule depolymerization following vascular photosensitization. Loss of endothelial barrier function can ultimately lead to tumor vascular shutdown and has significant implications in drug transport and tumor cell metastasis.