Maja Čemažar

The importance of electric field distribution for effective in vivo electroporation of tissues.

Cells exposed to short and intense electric pulses become permeable to a number of various ionic molecules. This phenomenon was termed electroporation or electropermeabilization and is widely used for in vitro drug delivery into the cells and gene transfection. Tissues can also be permeabilized. These new approaches based on electroporation are used for cancer treatment, i.e., electrochemotherapy, and in vivo gene transfection. In vivo electroporation is thus gaining even wider interest. However, electrode geometry and distribution were not yet adequately addressed. Most of the electrodes used so far were determined empirically. In our study we 1) designed two electrode sets that produce notably different distribution of electric field in tumor, 2) qualitatively evaluated current density distribution for both electrode sets by means of magnetic resonance current density imaging, 3) used three-dimensional finite element model to calculate values of electric field for both electrode sets, and 4) demonstrated the difference in electrochemotherapy effectiveness in mouse tumor model between the two electrode sets. The results of our study clearly demonstrate that numerical model is reliable and can be very useful in the additional search for electrodes that would make electrochemotherapy and in vivo electroporation in general more efficient. Our study also shows that better coverage of tumors with sufficiently high electric field is necessary for improved effectiveness of electrochemotherapy.

Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice

Electrochemotherapy is a new and promising approach in treatment of tumors employing locally applied high voltage d.c. electric pulses in combination with chemotherapeutic drugs. Increased permeability of the plasma membrane, induced by electric pulses, enables the chemotherapeutic drugs to enter into the cytosol, thus potentiating their anti-tumor effectiveness. In most of the studies published until now, trains of pulses were delivered via percutaneous parallel plate electrodes, so that the tumor was situated in between the electrodes. In our study the train of electric pulses was divided into two trains, the second one oriented perpendicularly to the first one. This changing of the electrode orientation resulted in improved anti-tumor efficacy of the electrochemotherapy: prolonged tumor growth delay and higher percentage of short and long term complete responses of the tumors. In this paper we also suggest a possible explanation for the observed effect, based on the knowledge of electric field distribution in the tissue and induced transmembrane potential.

Electrochemotherapy: variable anti-tumor effect on different tumor models

Abstract Electrochemotherapy is a new approach in the treatment of tumors that takes advantage of the permeabilization of the cell membrane by electric pulses to facilitate the delivery of chemotherapeutic drugs into the cells. According to the procedures described previously, the anti-tumor effectiveness of electrochemotherapy with bleomycin (BLM) was tested on three different murine tumor models, with different biological characteristics, to determine the variability in anti-tumor response. An arrest of growth of fibrosarcoma SA-1, malignant melanoma B-16 and Ehrlich ascites tumor (EAT) was observed in all mice subjected to electrochemotherapy, whereas neither BLM nor electric pulses had an effect on tumor growth when compared with controls. Partial and complete responses were also observed. The best anti-tumor response was observed for the SA-1 tumor model, where tumor growth delay was 31 days and 62% of the animals were free of tumor 100 days after the treatment. Side-effects of electrochemotherapy were demonstrated by body weight loss of the treated animals as well as some animal mortality recorded up to 7 days after the treatment, especially in the animals where tumors were located close to the spine. Our results are in accordance with previous results, and prove that use of high voltage electric pulses is promising for potentiation of BLM anti-tumor effectiveness.

Improvement of combined modality therapy with cisplatin and radiation using electroporation of tumors

PURPOSE To evaluate whether a local drug delivery method, i.e., electroporation of tumors, increases the radiosensitizing effect of cisplatin. METHODS AND MATERIALS Subcutaneous Ehrlich-Lettre ascites (EAT) tumors in CBA mice were treated either by cisplatin, electric pulses, or ionizing radiation. In electrochemotherapy protocol, electric pulses were given to the tumor 3 min after intravenous injection of cisplatin. The interval between electrochemotherapy and irradiation was 20 min. Treatment effectiveness was evaluated by tumor growth delay and local tumor curability. RESULTS Electrochemotherapy of EAT tumors proved to be effective treatment, resulting in 12% tumor cures, whereas treatment with cisplatin or electric pulses alone did not yield any tumor cures. As expected, injection of cisplatin 20 min prior to irradiation, increased radioresponse of tumors from 27% to 73% tumor cures. Electroporation of tumors also increased radiation response of tumors to 54% tumor cures. Electrochemotherapy given prior to irradiation increased radioresponsiveness of tumors, resulting in 92% tumor cures. CONCLUSIONS This study shows that delivery of cisplatin into the cells by electroporation of tumors increases the radiosensitizing effect of cisplatin. However, some effect may also be ascribed to application of electric pulses to the tumors that in our study also predisposed tumor cells to radiation damage.

Anti-tumor effectiveness of electrochemotherapy with bleomycin is increased by TNF-α on SA-1 tumors in mice

With the aim to increase anti-tumor effectiveness of electrochemotherapy, adjuvant immunotherapy with tumor necrosis factor-alpha (TNF-alpha) was tested on tumors in mice. Increased anti-tumor effectiveness on SA-1 tumors was observed after combining TNF-alpha, injected either intratumorally or peritumorally, with electrochemotherapy using suboptimal dose of bleomycin (BLM). The increased anti-tumor effectiveness was neither the result of potentiated anti-tumor effectiveness of TNF-alpha due to exposure of tumors to electric pulses, nor due to interaction with BLM. Therefore, the effect of adjuvant TNF-alpha treatment might be immunomodulatory, augmenting the anti-tumor activity of electrochemotherapy, and possibly adding a systemic component to the localized electrochemotherapy treatment.

In Situ Monitoring of Electric Field Distribution in Mouse Tumor during Electroporation

PURPOSE To investigate the feasibility of magnetic resonance (MR) electric impedance tomography ( EIT electric impedance tomography ) technique for in situ monitoring of electric field distribution during in vivo electroporation of mouse tumors to predict reversibly electroporated tumor areas. MATERIALS AND METHODS All experiments received institutional animal care and use committee approval. Group 1 consisted of eight tumors that were used for determination of predicted area of reversibly electroporated tumor cells with MR EIT electric impedance tomography by using a 2.35-T MR imager. In addition, T1-weighted images of tumors were acquired to determine entrapment of contrast agent within the reversibly electroporated area. A correlation between predicted reversible electroporated tumor areas as determined with MR EIT electric impedance tomography and areas of entrapped MR contrast agent was evaluated to verify the accuracy of the prediction. Group 2 consisted of seven tumors that were used for validation of radiologic imaging with histopathologic staining. Histologic analysis results were then compared with predicted reversible electroporated tumor areas from group 1. Results were analyzed with Pearson correlation analysis and one-way analysis of variance. RESULTS Mean coverage ± standard deviation of tumors with electric field that leads to reversible electroporation of tumor cells obtained with MR EIT electric impedance tomography (38% ± 9) and mean fraction of tumors with entrapped MR contrast agent (41% ± 13) were correlated (Pearson analysis, r = 0.956, P = .005) and were not statistically different (analysis of variance, P = .11) from mean fraction of tumors from group 2 with entrapped fluorescent dye (39% ± 12). CONCLUSION MR EIT electric impedance tomography can be used for determining electric field distribution in situ during electroporation of tissue. Implementation of MR EIT electric impedance tomography in electroporation-based applications, such as electrochemotherapy and irreversible electroporation tissue ablation, would enable corrective interventions before the end of the procedure and would additionally improve the treatment outcome.

MELANOMA METASTASES TO THE NECK NODES: ROLE OF ADJUVANT IRRADIATION

PURPOSE To review experiences in the treatment of regionally advanced melanoma to the neck and/or parotid with emphasis on the role of adjuvant radiotherapy. PATIENTS AND METHODS Clinical and histopathologic data, treatment details, and outcomes in patients treated during the period 2000-2006 at the Institute of Oncology, Ljubljana, Slovenia, were reviewed. RESULTS A total of 40 patients with 42 dissections underwent surgery, and 43 patients with 45 dissections received irradiation postoperatively to a median equivalent dose (eqTD(2): 2 Gy/fraction, 1 fraction/day, 5 fractions/week) of 60 Gy (range, 47.8-78.8). Regional control 2 years after surgery was 56% (95% confidence interval [CI] 40-72%) and after postoperative radiotherapy 78% (CI 63-92%) (p = 0.015). On multivariate analysis, postoperative radiotherapy (yes vs. no: hazard ratio [HR] 6.3, CI 2.0-20.6) and sum of the risk factors present (i.e., risk factor score; HR 1.7 per score point, CI 1.2-2.6) were predictive for regional control. On logistic regression testing, the number of involved nodes was associated with the probability of distant metastases (p = 0.021). The incidence of late toxicity did not correlate with the mode of therapy, eqTD(2), or fractionation pattern. CONCLUSIONS Adjuvant radiotherapy has the potential to compensate effectively for the negative impact of adverse histopatologic features to disease control in a dissected nodal basin. More conventionally fractionated radiotherapy regimens using fraction doses of 2-2.5 Gy, with cumulative eqTD(2)> or =60 Gy, are recommended. The number of involved lymph nodes is proposed as an additional criterion for limiting the implementation of adjuvant irradiation.

Determination of platinum in tumour tissues after cisplatin therapy by electrothermal atomic absorption spectrometry

A simple procedure is described for the determination of platinum in tumours after cisplatin therapy. Tumours were digested in 65% nitric acid by incubation at 37 degrees C for 2 days and platinum analysed under optimum conditions by electrothermal atomic absorption spectrometry with Zeeman background correction. The reproducibility of measurements in general was better than +/- 2%. The calibration graph was linear from 30.0 up to 1000 micrograms l-1 of platinum, while the limit of detection (3 sigma) was found to be 3.0 micrograms l-1 (sample volume 20 microliters). Aqueous standard solutions and the standard addition method were applied in the calibration procedure. Under the recommended analytical conditions, the sample matrix did not influence the determination of platinum significantly. In 72% of samples analysed the differences between results obtained by the two calibration procedures did not exceed +/- 5%.

Use of nitric acid in sample pretreatment for determination of trace elements in various biological samples by ETAAS

Trace elements in liquid biological samples may be determined by direct electrothermal atomic absorption spectrometry (ETAAS). In our previous work it was found that samples containing proteins or DNA may leak out of the graphite tube before the drying step, despite the addition of various modifiers. In order to keep the sample to the graphite tube, samples were diluted before analysis 1 + 1 with 32% v/v nitric acid, or 5 microl of 32% v/v nitric acid was added to the graphite tube before ETAAS determination. Applying the proposed procedure, the concentrations of lead in eluted fractions after gel chromatographic separation of human cerebellar nucleus dentatus supernatant and platinum in isolated DNA samples were determined. The use of nitric acid in sample pretreatment prevent sample leakage out of the graphite tube, provided for even drying and considerably reduced nonspecific absorption in lead determination. The repeatability of measurements was better than + 6%. The accuracy of the procedure was checked by spiking samples. The recoveries for both elements lay between 93--104%. Nitric acid was found to be a better modifier than TRITON X-100.

Determination of vinblastine in tumour tissue with liquid chromatography–high resolution mass spectrometry

There are virtually no analytical methods that describe determination of vinblastine and other vinca alkaloids in tumour tissue, albeit quantitative data on tumour drug amount is essential for maximal benefit of a particular anticancer treatment. The analytical method presented herein uses state-of-the-art sample preparation, separation and detection techniques to allow sensitive and selective determination of vinblastine in tumour tissue. After cryogenic grinding and sonication, tumour suspensions were extracted by Oasis MAX solid phase extraction and analysed for vinblastine with ultra-high performance liquid chromatography coupled to positive electrospray ionisation-high resolution mass spectrometric detection. The developed analytical method quantifies vinblastine down to 23 ng/g tumour tissue and shows satisfactory linearity (r(2)>0.99), precision (1.1-8.2%), accuracy (98%) and high selectivity with almost complete absence of matrix effects. The proposed method was found suitable to follow vinblastine levels in mice tumours and could be used to support preclinical pharmacologic studies.