Radiol Oncol 2003; 37(1): 43-8. Tumor blood flow modifying effects of electrochemotherapy: a potential vascular targeted mechanism Gregor Serša1, Maja Čemažar1 and Damijan Miklavčič2 1Institute of Oncology Ljubljana, 2University of Ljubljana, Faculty of Electrical Engineering, Ljubljana, Slovenia Background. The aim of this study was to determine the tumor blood flow modifying, and potential vas-cular targeted effect of electrochemotherapy with bleomycin or cisplatin. Materials and methods. Electrochemotherapy was performed by application of short intense electric pulses to the tumors after systemic administration of bleomycin or cisplatin. Evaluated were antitumor effec-tiveness of electrochemotherapy by tumor measurement, tumor blood flow modifying effect by Patent blue staining technique, and sensitivity of endothelial and tumor cells to the drugs and electrochemotherapy by clonogenicity assay. Results. Electrochemotherapy was effective in treatment of SA-1 tumors in A/J mice resulting in substan-tial tumor growth delay and also tumor cures. Tumor blood flow reduction following electrochemotherapy correlated well with its antitumor effectiveness. Virtually complete shut down of the tumor blood flow was observed already at 24 h after electrochemotherapy with bleomycin whereas only 50% reduction was ob-served after electrochemotherapy with cisplatin. Sensitivity of human endothelial HMEC-1 cells to elec-trochemotherapy suggests a vascular targeted effect for electrochemotherapy in vivo with bleomycin as well as with cisplatin. Conclusion. These results show that, in addition to direct electroporation of tumor cells, other vascular tar-geted mechanisms are involved in electrochemotherapy with bleomycin or cisplatin, potentially mediated by tumor blood flow reduction, and enhanced tumor cell death as a result of endothelial damage by elec-trochemotherapy. Key words: sarcoma experimental - drug therapy - blood supply; bleomycin; cisplatin; electroporation; drug delivery systems Received 1 October 2002 Accepted 15 November 2002 Correspondence to: Gregor Serša, Ph.D., Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia; Phone/Fax: +386 1 433 74 10; E-mail: gser-sa@onko-i.si Introduction Enhanced delivery of chemotherapeutic drugs into tumor cells by electroporation is termed electrochemotherapy.1 A local in-crease in plasma membrane permeability, after exposure of tumor nodules to electric pulses (electroporation), results in increased 44 Serša G et al. / Tumor blood flow modifying effects of electrochemotherapy uptake of chemotherapeutic drugs into the tumor cells. Electrochemotherapy has been shown to be successful for drugs such as bleomycin and cisplatin, which normally ex-hibit impeded transport through the plasma membrane. The increased antitumor effec-tiveness of bleomycin and cisplatin combined with electroporation has already been demonstrated in experimental and clinical studies although the underlying mechanisms remain to be clarified. 1-4 In addition to increased drug delivery into the cells, application of electric pulses to the tumors was found to exert tumor blood flow modifying effect.5,6 Electric pulses, as used in preclinical and clinical studies were found to reduce tumor blood flow. Transient reduction in tumor blood flow down to 30% of control was found, but recovered to almost pre-treat-ment level within 24 hours.5 Application of electric pulses to solid tumors would not be expected to selectively electroporate tumor cells alone. All cells in all areas where electric field exceeds the critical threshold level would be electroporated.1,7 Therefore endothelial cells are also potential targets for electroporation. Since the initial concentration of the drug is the highest in tumor blood vessels, during electroporation, electrochemotherapy is probably effective on endothelial cells in the tumor blood vessels. This may lead to severe damage to the vascu-lature of the tumors and consequently induce a secondary cascade of tumor cell death, e.g. by abrogating oxygen supply to the cells. This phenomenon, described as vascular targeted therapy, has been exploited in several stud-ies.8 The aim of this study was to elucidate tumor blood flow modifying and vascular tar-geted effects of electrochemotherapy with bleomycin or cisplatin by measuring tumor perfusion, and cells survival of endothelial cells in relation to their antitumor effective-ness. Radiol Oncol 2003; 37(1): 43-8. Materials and methods Animals, tumors and cell lines A/J mice of both sexes, purchased from the Institute Rudjer Bošković, Zagreb, Croatia, were used. Subcutaneous murine fibrosarco-ma SA-1 tumors (The Jackson Laboratory, Bar Harbour, ME) were implanted, by injecting 0.1 ml NaCl (0.9%) containing 5 x 105 viable tumor cells under the skin on the rear dor-sum. Six to 8 days after implantation, when tumors reached approximately 40 mm3 in volume (6 mm in diameter) mice were randomly divided into experimental groups, consisting of at least 6 mice. Treatment protocols were approved by the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia No. 323-02-237/01. Human dermal microvascular endothelial cells (HMEC-1) cells were generously provid-ed by Dr. F.J. Candal (Center for Disease Control, Atlanta, USA). Cells were grown as monolayer in D-MEM supplemented with 10% fetal calf serum (FCS, Sigma, USA) in a humidified incubator at atmospheric oxygen supplemented with 5% CO2 at 37°C. They were routinely subcultured twice per week. Electrochemotherapy protocol Bleomycin (Bleomycin, Mack, Germany) was dissolved in phosphate buffered saline and the dose of 5mg/kg in 0.2 ml volume was in-jected intravenously. Bleomycin solution was prepared freshly for daily injections. cis-Diamminedichloroplatinum (II) (Cispla-tin) was obtained from Bristol Myers Squibb (Austria) as a crystalline powder and a stock solution prepared in sterile H2O at a concen-tration of 1 mg/ml. The final cisplatin solu-tion was freshly prepared in 0.9% NaCl each day. Cisplatin at a dose of 4 mg/kg in 0.2 ml volume was injected intravenously. Electric pulses were delivered by two flat, parallel stainless steel electrodes 8 mm apart (two stainless steel strips: length 35 mm, width 7 mm with rounded corners), which Serša G et al. / Tumor blood flow modifying effects of electrochemotherapy 45 were placed percutaneously at the opposite margins of the tumor. Good contact between the electrodes and the skin was assured by means of conductive gel (Parker Laboratories, New York, USA). Eight square-wave pulses of 1040 V amplitude (amplitude to distance ratio 1300 V/cm), with a pulse width of 100 m s and repetition frequency of 1 Hz were generated by electroporator Jouan GHT 1287 (Saint Herblaine, France). In the electrochemothera-py protocol, tumors were exposed to electric pulses 3 minutes after bleomycin or cisplatin injection. Treatments were performed with-out anesthesia and were well tolerated by the animals. Tumor growth was followed by measuring three mutually orthogonal tumor diameters (e1, e2 and e3) using a vernier caliper on each consecutive day following treatment. Tumor volumes were calculated by the formula P x e1 x e2 x e3 /6. From the calculated volumes the arithmetic mean and SE were calculated for each experimental group. Tumor growth delay was calculated for each individual tumor by subtracting the doubling time of each tumor from the mean doubling time of the control group and then averaged for each ex-perimental group. Assessment of tumor staining by Patent blue Patent blue (Byk Gulden, Switzerland) was used to estimate tumor perfusion. Patent blue (1.25%), diluted in 0.2 ml 0.9% NaCl, was in-jected into tail vein of animals after tumor treatment. The dye was distributed evenly through the blood at approximately 1 minute, thereafter animals were sacrificed and tumors were carefully dissected. Tumors were cut along their largest diameter and the stained versus non-stained tissue per cross-section was immediately estimated visually by two persons. The mean of both estima-tions was used as an indicator of tumor perfusion. The results of individual experiments were pooled and presented as arithmetic mean and SE for each experimental group. Cytotoxicity assay for SA-1 and HMEC-1 cells treated by electrochemotherapy The sensitivity of the SA-1 and HMEC-1 cells to combined treatment with bleomycin or cis-platin and electric pulses (electrochemothera-py) was determined by in vitro colony forming assay. The cells (2.2 x 107 cells/ml) were mixed with bleomycin or cisplatin. One half of the mixture was exposed to 8 electric pulses (electric field intensity 1400 V/cm, pulse duration 100 m s, frequency 1 Hz) and the oth-er half served as a control for bleomycin or cisplatin treatment alone. The bleomycin con-centrations used ranged from 0.1 nM to 100 m M and the cisplatin concentrations from 16.7 to 670 m M. The cells were incubated with each drug for 5 min. The survival of cells treated with electrochemotherapy was nor-malized to electric pulses treatment alone. The IC50 values (drug concentration that re-duced cell survival to 50% of control) were de-termined for each treatment group. Statistical analysis The significance of differences between the mean values of the groups was evaluated by modified t-test (Newman Keuls test) after a one way analysis of variance was performed and fulfilled. Sigma Stat statistical package (SPSS, USA) was used for statistical analysis. P levels less than 0.05 were taken as statisti-cally significant. Results Antitumor effectiveness Electrochemotherapy with either bleomycin or cisplatin was effective in inducing cytotox-icity in subcutaneous SA-1 tumors (Table 1). Treatment of tumors with electric pulses alone had a minor effect on tumor growth, re-sulting in only 1.3 days tumor growth delay. Treatment of mice with bleomycin or cis-platin alone had also minor effects on tumor Radiol Oncol 2003; 37(1): 43-8. 46 Serša G et al. / Tumor blood flow modifying effects of electrochemotherapy Table 1. Antitumor effectiveness of electrochemotherapy on SA-1 tumors in mice (* P<0.05) Therapy n Tumor doubling time (Days, AM± SE) Tumor growth delay (Days, AM± SE) Cures Control 20 1.8 ± 0.05 0 % Electric pulses 17 3.1 ± 0.2* 1.3 ± 0.2 0 % Bleomycin (5 mg/kg) 20 1.9 ± 0.1 0.1 ± 0.1 0 % Electrochemotherapy with bleomycin 17 34.5 ± 2.9* 32.7 ± 2.9 70 % Cisplatin (4 mg/kg) 10 3.7 ± 0.4* 1.9 ± 0.4 0 % Electrochemotherapy with cisplatin 10 12.1 ± 1.6* 10.3 ± 1.6 0 % growth; bleomycin having none, whereas cis-platin inducing 1.9 days tumor growth delay. When using bleomycin in electrochemothera-py, a highly significant growth delay of 32.7 days was achieved and 70% of the animals were cured (tumor free 100 days after the treatment). The animals tolerated the treat-ment well without scaring of the treatment area. Electrochemotherapy with cisplatin also resulted in good antitumor effect with reduc-tion in tumor size at three days after the treat-ment, regrowth after 8 days, however no tumor cures were achieved. Tumor growth de-lay was 10.3 days, which was highly signifi-cant compared to the antitumor effectiveness of either single modality. Figure 1. Changes in tumor blood flow at 1, 8 or 24 h after electrochemotherapy (ECT) with bleomycin (BLM) or cisplatin (CDDP), measured by Patent blue staining. Eight electric pulses (EP) were applied to the tumor (amplitude to distance ratio 1300 V/cm, pulse width 100 Ks, repetition frequency 1 Hz) 3 minutes after intravenous injection of 5 mg/kg of bleomycin or 4 mg/kg of cisplatin. Mean values ± SE of the mean of at least 6 mice per point. Tumor blood flow changes Electrochemotherapy, either with bleomy-cin or cisplatin, induced substantial reduc-tion of tumor blood flow. Untreated SA-1 tumors showed very low incidence of necrosis with approx. 90% of the tumor area stained with Patent blue. When electric pulses were applied to a tumor reduction in tumor stain-ing was observed (Figure 1). By 1 hour after the application of electric pulses the percent-age of unstained tumor section had increased to 45% and after 8 hours further increased to 65%, however tumor blood flow recovered almost completely within 24 hours after this treatment. Treatment with bleomycin alone did not induce changes in tumor blood flow. However, electrochemotherapy with bleomy-cin demonstrated substantial increase in un-stained tumor area at 8 hours after treatment, and virtually complete shut down of tumor perfusion at 24 hours after therapy compared to electric pulses alone (Figure 1). Treatment with cisplatin alone had minimal tumor blood flow modifying effect. However, electrochemotherapy with cisplatin demonstrated greatly increased unstained tumor area at 8 hours after treatment which re-mained significantly higher at 24 hours after the treatment compared to treatment with electric pulses alone (Figure 1). Cytotoxicity of electrochemotherapy to tumor and endothelial cells The sensitivity of SA-1tumor cells and human endothelial cells HMEC-1 to bleomycin and Radiol Oncol 2003; 37(1): 43-8. Serša G et al. / Tumor blood flow modifying effects of electrochemotherapy 47 cisplatin as well as to electrochemotherapy was evaluated by in vitro colony forming as-say (Table 2). Endothelial cells were more sensitive to bleomycin than tumor cells. The potentiation of bleomycin cytotoxicity by electroporation was 5000-fold for endothe-~ lial cells and 2400-fold for tumor cells. ~ Electrochemotherapy with cisplatin was less effective on endothelial as on tumor cells, but potentiation of cisplatin cytotoxicity by elec-troporation was bigger for endothelial cells ( 10-fold), as for tumor cells ( 8-fold). ~~ Discussion This study shows tumor blood flow modify-ing and vascular targeted effect of elec-trochemotherapy with bleomycin as well as with cisplatin. The sensitivity of endothelial cells to electrochemotherapy with either, bleomycin or cisplatin correlates well with the enhanced reduction of tumor blood flow induced by electrochemotherapy in vivo and its antitumor effectiveness. As many preclinical and clinical studies have shown, electrochemotherapy either with bleomycin or cisplatin leads to high percent-age of tumor cures, on many tumor types test-ed so far.1,3,4,9,10 Electroporation was shown to significantly increase drug accumulation in the tumor cells.1,11 In electrochemotherapy treated tumors more than twice the amount of platinum was determined in whole tumors, Table 2. Cytotoxicity of electrochemotherapy to human endothelial HMEC-1 and mouse tumor SA-1 cells in vitro Cell line / Group HMEC-1 SA-1 (IC50; m M) (IC50; m M) Bleomycin 20.0 60.0 Electrochemotherapy with bleomycin 0.004 0.025 Cisplatin 380.0 166.0 Electrochemotherapy with cisplatin 40.0 20.0 as well as bound to DNA compared with cis-platin treatment alone.11 In view of our previ-ous study observing that electrochemothera-py with cisplatin induced more than 20-fold increase in cell kill compared with cisplatin treatment alone, we proposed that, in addition to direct electroporation of tumor cells, other mechanisms may be involved in antitumor effectiveness of electrochemotherapy.11 The direct blood flow modifying effect of electric pulses applied to the tumors has now been established. Application of electric pulses reduces blood flow selectively at the site of its application, i.e. within the tumor site, with-out modifying flow in normal tissues.5 Recently, a new method by staining of tumors with Patent blue was evaluated, giving data on tumor blood flow, in support of that found using the 86RbCl extraction technique.12 Sin-ce the two methods correlated well, Patent blue staining technique was preferred in this study, because of its simplicity. The present study confirms the results of our previous study that application of electric pulses to the tumors induces transient reduction in tumor blood flow. Tumor blood flow modifying effect of elec-trochemotherapy was greater than after application of electric pulses alone. This effect was especially dramatic in electrochemother-apy with bleomycin, but in lesser extent after electrochemotherapy with cisplatin. Tumor blood flow after electrochemotherapy with bleomycin was completely shut down already at 24 hours after therapy, indicating that tumor vasculature was irreversibly damaged.12 Since HMEC-1 endothelial cells were more sensitive to electrochemotherapy with bleo-mycin in vitro than SA-1 tumor cells, this vas-cular shut down may be ascribed in large part to the death of endothelial cells. In contrast, endothelial cells were less sensitive to elec-trochemotherapy with cisplatin in vitro, which was also reflected in less severe tumor blood flow changes induced by this therapy with flow partly restored after 24 hours.13 Radiol Oncol 2003; 37(1): 43-8. 48 Serša G et al. / Tumor blood flow modifying effects of electrochemotherapy In summary, several mechanisms are in-volved in antitumor effectiveness of elec-trochemotherapy. Electroporation of the tumors increases delivery of cytotoxic drugs in-to the tumor cells, potentiating their cytotoxi-city. Additionally, the current study demon-strates that nonselective electroporation of solid tumors enables cytotoxic action of elec-trochemotherapy to endothelial cells and en-hanced tumor cytotoxicity by a vascular tar-geted mechanism. Acknowledgment This work was supported by the Ministry of Education, Science and Sport of the Republic of Slovenia. References 1. Mir LM, Orlowski S. Mechanisms of elec-trochemotherapy. Adv Drug Deliver Rev 1999; 35: 107-18. 2. Serša G, Čemažar M, Miklavčič D. Antitumor ef-fectiveness of electrochemotherapy with cis-di-amminedichloroplatinum(II) in mice. Cancer Res 1995; 55: 3450-5. 3. Heller R, Gilbert R, Jaroszeski MJ. Clinical application of electrochemotherapy. Adv Drug Deliver 1999; 35: 119-29. 4. Serša G, Štabuc B, Čemažar M, Miklavčič D, Rudolf Z. Electrochemotherapy with cisplatin: clinical experience in malignant melanoma pa-tients. Clin Cancer Res 2000; 6: 863-7. 5. Serša G, Čemažar M, Parkins CS, Chaplin DJ. Tumour blood flow changes induced by application of electric pulses. Eur J Cancer 1999; 35: 672-7. 6. Gehl J, Skovsgaard T, Mir LM. Vascular reactions to in vivo electroporation: characterization and consequences for drug and gene delivery. Biochim Biophys Acta 2002; 1569: 51-8. 7. Neumann E, Kakorin S. Digression on membrane electroporation and electroporative delivery of drugs and genes. Radiol Oncol 1998; 32: 7-17. 8. Chaplin DJ, Hill SA, Bell KM, Tozer GM. Modification of tumor blood flow: Current status and future direction. Sem Radiat Oncol 1998; 8: 151-63. 9. Mir LM, Glass LF, Serša G, Teissie J, Domenge C, Miklavčič D, Jaroszeski MJ, Orlowski S, Reintgen DS, Rudolf Z, Belehradek M, Gilbert R, Rols MP, Belehradek JJr, Bachaud JM, DeConti R, Štabuc B, Čemažar M, Coninx P, Heller R. Effective treat-ment of cutaneous and subcutaneous malignant tumours by electrochemotherapy. Br J Cancer 1998; 77: 2336-42. 10. Serša G. Electrochemotherapy: Animal work review. 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