Mechanisms Associated With Blood Flow Modifying Effects Of Electric Pulses Used For Electrochemotherapy On Normal And Tumor Blood Vessels

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAElectropermeabilization/electroporation (EP) is a physical method that by applying external electric pulses to cells increases cell membrane permeability and thus enables the introduction of molecules into the cells. Nowadays, it is used in treatment of tumors, where its benefits are exploited in electrochemotherapy (ECT). In vivo EP also modifies blood flow, resulting in a decreased blood flow and increased permeability of blood vessel walls; whereas ECT has a vascular disrupting effect. The aim of our study was to evaluate the effects of EP on normal blood vessels and the effects of EP and ECT with bleomycin on tumor vasculature with the use of in vivo microscopy.The effects of pulse parameters used for ECT (8 pulses, 1300 V/cm, 100 μs, 1 Hz) on normal blood vessels were determined via a dorsal skinfold window chamber (DWC) with intravital two-photon microscopy after in vivo labelling of CD31 with fluorescently labeled antibodies. Furthermore, 70 kDa fluorescently labeled dextrans was used to visualize the increased permeability of blood vessel walls. Additionally, Fluorescence Lifetime Imaging (FLIM) was used to determine the stability of the used fluorochrome. In tumors the effects of ECT pulse parameters on tumor blood vessels after EP or ECT with bleomycin were determined using 70 kDa fluorescently labeled dextrans either with two-photon or widefield intravital microsocopy via DWC. Solid mouse sarcoma SA-1 tumors were induced by a subcutaneous injection of dense cell suspension into the area of DWC in mice and an intravenous injection of bleomycin (100 μg/mouse) was performed 3 min before the application of electric pulses.EP resulted in immediate constriction of the affected normal blood vessels and in a rapid increase of vascular permeability. This was accompanied by the change of the endothelial cells’ shape and a decrease of CD31 labelling in the cell-to-cell junctions. In vivo 3D FLIM showed no difference in the lifetime of the used fluorochrome before and after EP. When electric pulses or ECT were applied to tumors the increase in vascular permeability and the constriction of tumor blood vessels were not as pronounced as in normal blood vessels. However, there was a complete vascular lock that lasted for more than 30 min. Furthermore, ECT had a differential vascular disrupting effect on tumor blood vessels, leaving the larger normal blood vessels surrounding the tumors functional after the therapy.In conclusion, the results of our study provide direct in vivo evidence of the difference in vascular effects of electric pulses used for ECT in normal and tumor blood vessels, show the response of normal endothelial cells to EP with two-photon microscopy and 3D FLIM, and provide evidence of the differential vascular disrupting action of ECT.Citation Format: Bostjan Markelc, Elisabeth Bellard, Gregor Sersa, Tanja Dolinsek, Justin Teissie, Muriel Golzio, Maja Cemazar. Mechanisms associated with blood flow modifying effects of electric pulses used for electrochemotherapy on normal and tumor blood vessels. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5119. doi:10.1158/1538-7445.AM2015-5119