Julie Gehl

Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation

Over the last decade a new cancer treatment modality, electrochemotherapy, has emerged. By using short, intense electric pulses that surpass the capacitance of the cell membrane, permeabilization can occur (electroporation). Thus, molecules that are otherwise non-permeant can gain direct access to the cytosol of cells in the treated area.A highly toxic molecule that does not usually pass the membrane barrier is the hydrophilic drug bleomycin. Once inside the cell, bleomycin acts as an enzyme creating single- and double-strand DMA-breaks. The cytotoxicity of bleomycin can be augmented several 100-fold by electroporation. Drug delivery by electroporation has been in experimental use for cancer treatment since 1991. This article reviews 11 studies of electrochemotherapy of malignant cutaneous or subcutaneous lesions, e.g., metastases from melanoma, breast or head- and neck cancer. These studies encompass 96 patients with altogether 411 malignant tumours. Electroporation was performed using plate or needle electrodes under local or general anaesthesia. Bleomycin was administered intratumourally or intravenously prior to delivery of electric pulses. The rates of complete response (CR) after once-only treatments were between 9 and 100% depending on the technique used. The treatment was well tolerated and could be performed on an out-patient basis.

In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution

In vivo electroporation is increasingly being used to deliver small molecules as well as DNA to tissues. The aim of this study was to quantitatively investigate in vivo electroporation of skeletal muscle, and to determine the threshold for permeabilization. We designed a quantitative method to study in vivo electroporation, by measuring uptake of (51)Cr-EDTA. As electrode configuration influences electric field (E-field) distribution, we developed a method to calculate this. Electroporation of mouse muscle tissue was investigated using either external plate electrodes or internal needle electrodes placed 4 mm apart, and eight pulses of 99 micros duration at a frequency of 1 Hz. The applied voltage to electrode distance ratio was varied from 0 to 2.0 kV/cm. We found that: (1) the threshold for permeabilization of skeletal muscle tissue using short duration pulses was at an applied voltage to electrode distance ratio of 0.53 kV/cm (+/-0.03 kV/cm), corresponding to an E-field of 0.45 kV/cm; (2) there were two phases in the uptake of (51)Cr-EDTA, the first indicating increasing permeabilization and the second indicating beginning irreversible membrane damage; and (3) the calculated E-field distribution was more homogeneous for plate than for needle electrodes, which was reflected in the experimental results.

Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer.

Gene transfer using electrical pulses is a rapidly expanding field. Many studies have been performed in vitro to elucidate the mechanism of DNA electrotransfer. In vivo, the use of efficient procedures for DNA electrotransfer in tissues is recent, and the question of the implied mechanisms is largely open. We have evaluated the effects of various combinations of square wave electric pulses of variable field strength and duration, on cell permeabilization and on DNA transfection in the skeletal muscle in vivo. One high voltage pulse of 800 V/cm, 0.1 ms duration (short high pulse) or a series of four low voltage pulses of 80 V/cm, 83 ms duration (long low pulses) slightly amplified transfection efficacy, while no significant permeabilization was detected using the (51)Cr-EDTA uptake test. By contrast, the combination of one short high pulse followed by four long low pulses led to optimal gene transfer efficiency, while inducing muscle fibers permeabilization. These results are consistent with additive effects of electropermeabilization and DNA electrophoresis on electrotransfer efficiency. Finally, the described new combination, as compared to the previously reported use of repeated identical pulses of intermediate voltage, leads to similar gene transfer efficiency, while causing less permeabilization and thus being likely less deleterious. Thus, combination of pulses of various strengths and durations is a new procedure for skeletal muscle gene transfer that may represents a clear improvement in view of further clinical development.

Elevated neutrophil and monocyte counts in peripheral blood are associated with poor survival in patients with metastatic melanoma: a prognostic model

We aimed to create a prognostic model in metastatic melanoma based on independent prognostic factors in 321 patients receiving interleukin-2 (IL-2)-based immunotherapy with a median follow-up time for patients currently alive of 52 months (range 15–189 months). The patients were treated as part of several phase II protocols and the majority received treatment with intermediate dose subcutaneous IL-2 and interferon-α. Neutrophil and monocyte counts, lactate dehydrogenase (LDH), number of metastatic sites, location of metastases and performance status were all statistically significant prognostic factors in univariate analyses. Subsequently, a multivariate Coxs regression analysis identified elevated LDH (P<0.001, hazard ratio 2.8), elevated neutrophil counts (P=0.02, hazard ratio 1.4) and a performance status of 2 (P=0.008, hazard ratio 1.6) as independent prognostic factors for poor survival. An elevated monocyte count could replace an elevated neutrophil count. Patients were assigned to one of three risk groups according to the cumulative risk defined as the sum of simplified risk scores of the three independent prognostic factors. Low-, intermediate- and high-risk patients achieved a median survival of 12.6 months (95% confidence interval (CI), 11.4–13.8), 6.0 months (95% CI, 4.8–7.2) and 3.4 months (95% CI, 1.2–5.6), respectively. The low-risk group encompassed the majority of long-term survivors, whereas the patients in the high-risk group with a very poor prognosis should probably not be offered IL-2-based immunotherapy.

Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery.

In vivo electroporation (EP) is gaining momentum for drug and gene delivery. In particular, DNA transfer by EP to muscle tissue can lead to highly efficient long-term gene expression. We characterized a vascular effect of in vivo EP and its consequences for drug and gene delivery. Pulses of 10-20,000 micros and 0.1-1.6 kV/cm were applied over hind- and forelimb of mice and perfusion was examined by dye injection. The role of a sympathetically mediated vasoconstrictory reflex was investigated by pretreatment with reserpine. Expression of a transferred gene (luciferase), permeabilization (determined using (51)Cr-EDTA), membrane resealing and effects on perfusion were compared to assess the significance of the vascular effects. Above the permeabilization threshold, a sympathetically mediated Raynaud-like phenomenon with perfusion delays of 1-2 min was observed. Resolution of this phase followed kinetics of membrane resealing. Above a second threshold, irreversible permeabilization led to long perfusion delays. These vascular reactions (1) affect kinetics of drug delivery, (2) predict efficient DNA transfer, which is optimal during short perfusion delays, and (3) might explain electrocardiographic ST segment depressions after defibrillation as being caused by vascular effects of EP of cardiac muscle.

Electric pulse-mediated gene delivery to various animal tissues.

Electroporation designates the use of electric pulses to transiently permeabilize the cell membrane. It has been shown that DNA can be transferred to cells through a combined effect of electric pulses causing (1) permeabilization of the cell membrane and (2) an electrophoretic effect on DNA, leading the polyanionic molecule to move toward or across the destabilized membrane. This process is now referred to as DNA electrotransfer or electro gene transfer (EGT). Several studies have shown that EGT can be highly efficient, with low variability both in vitro and in vivo. Furthermore, the area transfected is restricted by the placement of the electrodes, and is thus highly controllable. This has led to an increasing use of the technology to transfer reporter or therapeutic genes to various tissues, as evidenced from the large amount of data accumulated on this new approach for non-viral gene therapy, termed electrogenetherapy (EGT as well). By transfecting cells with a long lifetime, such as muscle fibers, a very long-term expression of genes can be obtained. A great variety of tissues have been transfected successfully, from muscle as the most extensively used, to both soft (e.g., spleen) and hard tissue (e.g., cartilage). It has been shown that therapeutic levels of systemically circulating proteins can be obtained, opening possibilities for using EGT therapeutically. This chapter describes the various aspects of in vivo gene delivery by means of electric pulses, from important issues in methodology to updated results concerning the electrotransfer of reporter and therapeutic genes to different tissues.

Enhancement of cytotoxicity by electropermeabilization: an improved method for screening drugs.

Electropermeabilization (EPN), also termed electroporation, is a physical method to overcome the barrier of the cell membrane by applying short and intense electric pulses. It is the basis for a new cancer treatment modality, electrochemotherapy, where uptake of chemotherapeutics is enhanced by EPN. Preclinical and clinical trials have shown that application of electric pulses in vivo is feasible and that electrochemotherapy is highly efficient. The aim of this study was to develop an improved method of screening drugs on electropermeabilized versus non-electropermeabilized cells. In this study we describe an easy protocol which gives high cell viability, good reproducibility and a high rate of cell permeabilization. Cell cytotoxicity is simply determined by the MTT assay. Cell death due to the EPN procedure was less than 4% and more than 90% of cells were permeabilized. For daunorubicin, doxorubicin, etoposide and paclitaxel, no effect of EPN was found. For carboplatin and cisplatin the effect of EPN was a factor 3 and 2.3, respectively, on the IC50 (inhibitory concentration 50%). For bleomycin we found a dramatic effect of EPN of the magnitude of a factor 300 on the IC50. In conclusion, we have established a new, easy and reliable protocol to test new drugs for cytotoxicity with or without the limitations of the cell membrane. Our data support the role of bleomycin as the drug of choice for electrochemotherapy.

Efficient palliation of haemorrhaging malignant melanoma skin metastases by electrochemotherapy.

Electric pulses can cause transient permeabilization of cell membranes (electroporation) and this can be utilized to increase the uptake of chemotherapy (electrochemotherapy). Preclinical studies have shown that in vivo electroporation causes transient shut down of blood flow both in normal and, in particular, malignant tissues. We report the successful palliation of a malignant melanoma patient with bleeding skin metastases using electrochemotherapy. In an on-going study of combined electrochemotherapy and low dose interleukin-2, one patient with bleeding skin metastases was included. Nine skin metastases, of which seven were ulcerated, were treated. After intratumoral bleomycin injection, needle electrodes with two arrays 4 mm apart were inserted into the tumours. Eight square wave electric pulses each 99 μs in duration and with an applied voltage to electrode distance ratio of 1.2 kV/cm were administered. In all the treated lesions, bleeding immediately stopped on administration of the electric pulses and did not recur. The treated metastases developed crusts and the lesions healed in a matter of weeks. Treatments were given under local anaesthesia, lasted a few minutes, and patient discomfort was brief and modest. In conclusion, we propose that electrochemotherapy should be considered for the palliation of haemorrhaging metastases as it is an efficient, tolerable, brief, outpatient, once-only treatment.

Efficiency of High- and Low-Voltage Pulse Combinations for Gene Electrotransfer in Muscle, Liver, Tumor, and Skin

Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and electrophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investigate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommendations for gene transfer to various types of tissues.

Voluntary Running Suppresses Tumor Growth through Epinephrine- and IL-6-Dependent NK Cell Mobilization and Redistribution

Regular exercise reduces the risk of cancer and disease recurrence. Yet the mechanisms behind this protection remain to be elucidated. In this study, tumor-bearing mice randomized to voluntary wheel running showed over 60% reduction in tumor incidence and growth across five different tumor models. Microarray analysis revealed training-induced upregulation of pathways associated with immune function. NK cell infiltration was significantly increased in tumors from running mice, whereas depletion of NK cells enhanced tumor growth and blunted the beneficial effects of exercise. Mechanistic analyses showed that NK cells were mobilized by epinephrine, and blockade of β-adrenergic signaling blunted training-dependent tumor inhibition. Moreover, epinephrine induced a selective mobilization of IL-6-sensitive NK cells, and IL-6-blocking antibodies blunted training-induced tumor suppression, intratumoral NK cell infiltration, and NK cell activation. Together, these results link exercise, epinephrine, and IL-6 to NK cell mobilization and redistribution, and ultimately to control of tumor growth.