Antivascular effects of electrochemotherapy: implications in treatment of bleeding metastases

Figures & data

Figure 1. Electrochemotherapy (ECT) of tumors.

(A) The main mechanism of ECT and (B1–5) an example of a clinical ECT procedure are presented. In this example a subcutaneous melanoma metastasis on the lower leg was treated on an outpatient basis by ECT with cisplatin: (B1) tumor before treatment; (B2) application of local anesthesia; (B3) intratumoral injection of cisplatin; (B4) application of electric pulses using parallel plate electrodes; (B5) treated area immediately after treatment. Note the bluish-pale color of the treated area in the last photograph (indication of locally arrested tumor blood flow and the beginning of edema formation). For an example of the long-term effect of ECT on a bleeding melanoma tumor see Figure 7.

EP: Electroporation.

Figure 2. Changes in tumor blood flow after different treatments.

(A) An example of rapid decrease in blood flow immediately after application of EP pulses. Movement artifacts caused by electric pulses and respiration can be seen in the unfiltered raw signal. (B–D) The effects of different treatments on blood flow over three different time frames. All data were measured in subcutaneous Sa-1 tumors in anesthetized A/J mice. OxyFlo laser Doppler flowmeter with bare fiber probes was used in A–C (Oxford Optronix, Oxford, UK) and PBV staining technique in D. For electrochemotherapy (ECT) bleomycin (1 mg/kg) was injected intravenously 3 min prior to application of EP. EP (eight pulses, amplitude 1040 V, duration 100 µs, 1 Hz, plate electrodes, 8 mm distance) was delivered at time 0. In B–D the data points represent the mean average blood flow and standard error of the mean (N ≥13 in figures B & C and N ≥3 in figure D). In figures B & C the values are expressed relatively as percentage of the pretreatment value.

EP: Electroporation.

Redrawn from [66] in accordance with the ‘License to publish’ of British Journal of Cancer.

Figure 3. The effect of EP on human umbilical vein endothelial cells.

The endothelial cell monolayer was exposed to three electric pulses (duration 100 µs, repetition frequency 1 Hz, amplitude 40 V, interelectrode distance 4 mm) in situ. Microtubules and actin filaments were stained with an anti-β-tubulin antibody and phalloidin respectively and analyzed by fluorescence microscopy. Before EP, cells displayed intact actin filament and microtubule networks. After application of EP, staining of actin fibers with phalloidin appeared diffused demonstrating dissociation of actin fibers, while microtubules became fragmented. Microtubule and microfilament networks recovered their structural composition within 1 h after exposure to electric pulses at amplitude of 40 V.

EP: Electroporation.

Reproduced with permission from [86].

Figure 4. A model of two-component decrease in blood flow after EP.

The total perfusion change (thicker line) observed in solid tumors after EP is modeled here as a sum of two simple biexponential functions with different time constants (thin lines). For comparison with experimental data, the data points for EP from Figure 2C and 2D are superimposed on the model. A more elaborate mathematical representation of the two components in the model would have to be used to fully describe the actual blood flow data, but the general adequacy of a two-phase model to describe the experimental data is obvious.

EP: Electroporation.

Figure 5. Electrochemotherapy with cisplatin and bleomycin: comparison of the effects on blood flow in tumors.

The results of the PBV-staining technique are shown. Symbols represent mean values of at least six mice with standard error of the mean. Time 0 corresponds to the pretreatment value. Bleomycin and cisplatin were injected 3 min prior to EP at doses 5 mg/kg and 4 mg/kg, respectively. EP protocol: eight pulses, amplitude 1040 V, duration 100 µs, 1 Hz, plate electrodes, interelectrode distance 8 mm.

ECT: Electrochemotherapy; EP: Electroporation.

Based on combined data from [67,81,82].

Figure 6. Model of blood flow changes in tumors induced by EP and ECT.

The effects of EP alone and of ECT are presented on the level of a microcirculatory blood vessel. Application of the drug and electric pulses are indicated by the block arrows. The general sequence of physiological changes and their consequences runs from the left to the right.

ECT: Electrochemotherapy; EP: Electroporation.

Figure 7. The effect of electrochemotherapy (ECT) with bleomycin on bleeding melanoma metastasis.

It can be observed that bleeding of this tumor was stopped immediately after the application of ECT. Gradually a crust was formed in place of the tumor (within 1 week), which fell off 3 weeks after the treatment. This particular tumor reduced in size dramatically and remained in partial response during the 21 weeks observation time.

ECT: Electrochemotherapy.

Reproduced with permission from [100].

Table 1. Short description of the experimental methods for assessment of blood flow and related parameters in tumors referred to in the text.

Method	Description	Ref.
Contrast-enhanced MRI	Systemically administered macromolecular contrast-enhancing agents, such as albumin-(Gd-DTPA)30 or gadomer-17, provide stable enhancement of the tissue blood pool in conventional MRI images and can be used for measurement of blood flow-related parameters, such as fractional tumor blood volume and microvascular permeability surface area product at different intervals after injection of the agent	[70,81,83,122,123]
Rubidium (^86Rb) extraction technique	Extraction of the radioactive rubidium isotope ^86Rb by tissue at a predefined interval after systemic injection of the tracer is used to quantify relative perfusion of excised tissue samples as a proportion of cardiac output	[72,124]
Patent blue-violet staining technique	A simple in vivo staining technique using patent blue-violet biological dye (otherwise commonly used in lymphography) as a contrast agent for in vivo visualization of perfused versus nonperfused tissue areas and for qualitative estimation of blood flow in excised tumors	[66,67]
Laser Doppler flowmetry	A highly sensitive optical method for continuous monitoring of relative microcirculation based on the Doppler-shift effect occurring when photons of a single wavelength (laser light) scatter on moving red blood cells	[91,125]
Power Doppler ultrasonographic imaging	A method for noninvasive assessment of gross tumor blood flow changes and for visualization of the distribution of areas with different perfusion rates. Contrast agents are used to achieve contrast enhancement of perfused areas	[66,126]
Electron paramagnetic resonance oximetry	A method related to NMR for absolute pO2 measurement. When tissue is exposed to appropriate external magnetic excitation, the resulting electron paramagnetic resonance spectra of the paramagnetic material implanted in tissue depends on local oxygenation and can be measured by a surface coil resonator	[81,89,127-129]
Luminescence-based fiber-optic oximetry	A novel method for absolute pO2 monitoring. A ruthenium-based luminophore is encapsulated at the tip of a fiber-optic probe and brought into tissue. Quenching of the externally stimulated fluorescence of the luminophore is directly related to pO2 in tissue	[84,130]
Polarographic oximetry	A ‘gold standard’ method for absolute tissue oxygenation measurement. In the steady-state, the electric current flowing between the negatively polarized active electrode and the reference electrode is proportional to tissue pO2 at the site of insertion of the active electrode. Prognostic value of oxygenation profiles in tumors measured by this method has been demonstrated in clinical settings	[131,132]
Hypoxia marker pimonidazole	Pimonidazole is an exogeneous hypoxia marker which binds preferentially to hypoxic tumor cells. Administered in vivo it can later be used after immunohistochemical staining to detect hypoxic regions or cells in histological preparations of excised tumors	[92,133]
Hypoxia marker glucose transporter-1	Glucose transporter 1 is an endogenous hypoxia marker. It is upregulated in hypoxic tumor conditions and can be immunohistochemically detected also in archived material	[92,134,135]

References cited in the last column contain a more detailed description of the method and/or the use of the method for assessment of the effects of electroporation and electrochemotherapy on tumor blood flow.

Table 2. Antitumor effectiveness of electroporation and electrochemotherapy with bleomycin (at doses of 1 and 5 mg/kg) and cisplatin (at a dose of 4 mg/kg) on Sa-1 tumors in A/J mice.

Treatment	Mice (n)	Tumor doubling time (days)	Tumor growth delay (days)	Cure rate (%)
Control	20	1.8 ± 0.1		0
EP	17	3.1 ± 0.2^*	1.3 ± 0.2	0
Bleomycin (1 mg/kg)	16	1.2 ± 0.1	-0.6 ± 0.1	0
Bleomycin (5 mg/kg)	20	1.9 ± 0.1	0.1 ± 0.1	0
Cisplatin (4 mg/kg)	10	3.7 ± 0.4^*	1.9 ± 0.4	0
ECT with bleomycin (1 mg/kg)	16	23.7 ± 4.6^*	21.9 ± 4.6	38
ECT with bleomycin (5 mg/kg)	17	34.5 ± 2.9^*	32.7 ± 2.9	70
ECT with cisplatin (4 mg/kg)	10	12.1 ± 1.6^*	10.3 ± 1.6	0

Tumor growth delay was calculated with respect to the control group. In the case of ECT with bleomycin, cured tumors were excluded from growth delay calculation. Mean ± standard error of the mean values are given.

^*p < 0.05.

ECT: Electrochemotherapy; EP: Electroporation.

Reproduced with permission from [82].

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