Vascular disrupting action of electroporation and electrochemotherapy with bleomycin in murine sarcoma

Abstract

Electrochemotherapy has a direct cytotoxic effect on tumour cells, and presumably, a vascular disrupting effect. In this study, on the basis of the prediction of the mathematical model, histological evaluation and physiological measurements of the tumours were carried out to confirm that electroporation and electrochemotherapy of tumours have a vascular disrupting action. In the study, SA-1 solid subcutaneous sarcoma tumours in A/J mice were treated by bleomycin (BLM) given intravenously (1 mg kg(-1)), application of electric pulses (8 pulses, 1040 V, 100 micros, 1 Hz) or a combination of both - electrochemotherapy. The vascular effect was determined by laser Doppler flowmetry, power Doppler ultrasonographic imaging and Patent blue staining. The extent of tumour hypoxia was determined immunohistochemically by hypoxia marker pimonidazole and partial pressure of oxygen (pO(2)) in tumours by electron paramagnetic resonance oximetry. Electrochemotherapy with BLM induced good antitumour effect with 22 days, tumour growth delay and 38% tumour cures. The application of electric pulses to the tumours induced instant but transient tumour blood flow reduction (for 70%) that was recovered in 24 h. During this tumour blood flow reduction, we determined an increase in hypoxic tumour area for up to 30%, which was also reflected in reduced tumour oxygenation (for 70%). According to the described mathematical model, endothelial cells lining in tumour blood vessels are exposed to a approximately 40% higher electric field than the surrounding tumour cells, and therefore easily electroporated, allowing access of high BLM concentration to the cytosol. Consequently, electrochemotherapy has, besides the immediate vascular disrupting action, also a delayed one (after 24 h), as a consequence of endothelial cell swelling and apoptosis demonstrated by extensive tumour necrosis, tumour hypoxia, prolonged reduction of tumour blood flow and significant tumour growth delay, and tumour cures. Our results demonstrate that in addition to the well-established direct cytotoxic effect on tumour cells, electrochemotherapy also has an indirect vascular disrupting action resulting altogether in extensive tumour cell necrosis leading to complete regression of tumours.

Figure 1

Time course of changes in tumour necrosis in untreated control tumours, and after treatment of tumours with bleomycin, application of electric pulses or electrochemotherapy. Symbols indicate AM (arithmetic mean)±s.e. of at least three mice per point.

Figure 2

(A) Rounding up of tumour blood vessels endothelial cells 1 h after electric pulses increases vascular resistance to blood flow (arrows). (B) Apoptotic-like endothelial cells occur in tumours 8 h after electrochemotherapy (arrows).

Figure 3

The electric field strength in a capillary of 8 μm diameter (A), a vessel of 15 μm diameter (B) and in the endothelial and tumour tissue surrounding them. The plot shows the ratio between the local electric field (E) along the line a and the external field to which the tissue is exposed (E₀), for three different orientations of the vessel with respect to the field (90° – solid, 60° – dashed, 30° – dotted). The conductivities of the tissues and the blood are taken from the literature (Hirsch et al, 1950; Duck 1990; Gabriel et al, 1996). In the capillary, a somewhat lower serum content (and therefore conductivity) is assumed due to the erythrocytes filling most of the volume in the thin passages.

Figure 4

Representative power Doppler ultrasonographic images of tumours (delineated with white lines A and B) treated by bleomycin, electric pulses or electrochemotherapy at various time points post-treatment.

Figure 5

Blood flow changes after different treatments assessed by means of laser Doppler flowmetry. (A) Rapid decrease in blood flow immediately after application of electric pulses (EP). Movement artefact caused by the pulses, manipulation of the electrodes (spikes just before and after pulses) and respiration (before and after electroporation) can be observed in the raw signal along with the filtered signal. (B) Average blood flow values before and after treatment expressed as a percentage of the pretreatment blood flow (mean values with s.e. bars are shown). After time zero, the differences between the groups were statistically highly significant (P<0.001). (C) A close-up from (B): (1) pretreatment level at −5 min; (2) moment of injection at −3 min; (3) −1.5 min; (4) time zero. Relatively large scatter (nonsignificant) of values at time zero was of transient nature and was a result of movement artefacts caused by manipulation of the electrodes. Note different time scale.

Figure 6

Time course of changes in tumour perfusion in untreated tumours, and after treatment of tumours with bleomycin, application of electric pulses or electrochemotherapy. Symbols indicate AM (arithmetic mean)±s.e. of at least three mice per point.

Figure 7

Representative tumour sections 90 min after treatment with bleomycin, application of electric pulses or electrochemotherapy. Brown regions are the cells stained with pimonidazole, a marker of tumour hypoxia. Note marked differences in the extent of hypoxic regions between groups without application of electric pulses (control and bleomycin) and those with the application of electric pulses (electric pulses and electrochemotherapy).

Figure 8

Time course of changes in tumour hypoxia in untreated control tumours, and after treatment of tumours with bleomycin, application of electric pulses alone or electrochemotherapy. Excised tumours were stained with pimonidazole, and percentage of pimonidazole positive tumour areas were determined. AM (arithmetic mean)±s.e. of at least three mice per point.

Figure 9

Time course of changes in partial pressure of oxygen (pO₂) measured by EPR oximetry in tumours and subcutaneous tissue distant from the tumours, where electric pulses were not applied. The data were combined for tumour centre and tumour periphery. Animals were treated intravenously with bleomycin (1 mg kg⁻¹) and 3 min later eight electric pulses were applied to the tumours (1040 V, 100 μs, 1 Hz). Symbols indicate AM (arithmetic mean)±s.e. of six mice per point.