Tumor Blood Flow Modifying Effect of Electrochemotherapy with Bleomycin

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1 ANTICANCER RESEARCH 19: (1999) Tumor Blood Flow Modifying Effet of Eletrohemotherapy with Bleomyin GREGOR SERSAl, MAJA CEMAZARl, DAMIJAN MIKLAVCIC 2 and DAVID J. CHAPLIN 3 l/nstitute of Onology Ljubljana, Department of Tumor Biology, ZaloSka 2, S/- / Ljubljana, Slovenia; 2 University of Ljubljana, Faulty of Eletrial Engineering, TriasKa 25, S/- / Ljubljana, Slovenia; 3Tumour Miroirulation Group, Gray Laboratory Caner Researh Trust, Mount Vernon Hospital, Northwood HA6 21R, Middlesex, u.k. Abstrat. Bakground: Eletrohemotherapy ombines administration of the hemotherapeuti drug, followed by appliation of eletri pulses in order to inrease drug delivery into the ells. The aim of this study was to determine the tumor blood flow modifying effet of eletrohemotherapy with bleomyin and orrelate it with its antitumor effetiveness and extent of tumor nerosis. Materials and methods: Eletrohemotherapy of SA-1 subutaneous tumors in A/l mie was performed by appliation of eletri pulses to the tumors, following administration of bleomyin, and antitumor effetiveness determined by tumor growth delay and tumor ures as well as extent of tumor nerosis. Tumor blood modifying effet of therapy was evaluated by Patent blue staining tehnique and 86 RbCI extration tehnique. Results: A good orrelation of the two methods evaluating tumor blood flow, Patent blue staining and the established 86 RbCI extration tehnique was found (r=.944). Eletrohemotherapy resulted in omplete and permanent shut down of tumor blood flow within 12 hours, whih lasted for at least 5 days. The results on tumor blood flow redution orrelated well with the good antitumor effetiveness of eletrohemotherapy and with the extent of the nerosis in the tumors. Conlusions: The results indiate that Patent blue staining tehnique is a simple and reliable method for estimation of tumor blood flow and that antitumor effetiveness of eletrohemotherapy with bleomyin ould be partly attributed to its tumor blood modifying and anti-vasular effet. In eletrohemotherapy, eletri pulses are used as a means of inreasing hemotherapeuti drug delivery into Correspondene 1: Gregor Sersa, Ph.D., Institute of Onology Ljubljana, Zaloska 2, SI-1 Ljubljana, Slovenia. Tel.!Fax: , gsersa@onko-i.si. Key Words: Tumor blood flow, eletroporation, eletrohemotherapy, bleomyin. the ells [1]. Exposure of ells to eletri pulses under speifi onditions, inreases plasma membrane permeability, temporally and reversibly, without affeting ell viability [2,3]. This tehnique, termed eletroporation, has been used for insertion of various moleules into the ells, inluding hemotherapeuti drugs and genes [1,4,5]. Among several hemotherapeuti drugs tested for potentiation of their ytotoxiity by eletroporation, bleomyin and isplatin have been found to be very suitable, due to their limited transport through the plasma membrane [6,7]. The inreased antitumor effetiveness of bleomyin and isplatin, ombined with eletri pulses has been demonstrated in experimental and linial studies [6-16]. In addition to inreased drug delivery into the ells, eletri pulses were found to exert tumor blood modifying effet. In our previous studies we demonstrated that appliation of eletri pulses to the tumors, redues tumor blood flow, whih was dependent on the amplitude of the applied eletri pulses [17,18]. Eletri pulses, as used in our standard in vivo experimental onditions in eletrohemotherapy, redued tumor blood flow up to 3% of the pre-treatment value. This redution of tumor blood flow ould ontribute to the antitumor effetiveness of eletrohemotherapy, by prolonging exposure of tumor ells to the hemotherapeuti drug [19,2]. The measurements of tumor blood flow hanges after appliation of eletri pulses were made by reliable, but laborious tehniques suh as 86RbCl extration tehnique and ontrast enhaned magneti resonane imaging [17,18]. In searh for an easier approah, whih would be simple, quik and indiate of tumor blood flow hanges, tumor staining with Patent blue was introdued. In addition, we examined the tumor blood flow modifying effet of eletrohemotherapy with bleomyin, whih has not been yet evaluated. Therefore, the aim of our study was to determine the tumor blood flow modifying effet of eletri pulses by 25-75/99 $

2 ANTICANCER RESEARCH 19: (1999) Patent blue staining, and the results obtained with this tehnique were orrelated with the measurements obtained by the 86RbCI extration tehnique. In addition, tumor blood flow modifiation was evaluated on tumors treated by eletrohemotherapy with bleomyin and was orrelated with antitumor effetiveness and extent of tumor nerosis, after eletrohemotherapy. Materials and Methods Animals and tumors. A/J mie of both sexes, purhased from the Institute Rudjer Boskovi, Zagreb, Croatia, were used. They were maintained at 22 C with a natural day/night light yle in a onventional animal olony. The mie were 8-12 weeks old at the beginning of the experiments. The tumor used was fibrosaroma SA I (The Jakson Laboratory, Bar Harbour, ME). SA-I ells were obtained from the asiti form of the tumors in mie, whih were serially transplanted twie per week. Subutaneous tumors were implanted, by injeting.1 ml NaCI (.9%) ontaining 5 x 1 5 viable tumor ells under the skin on the rear dorsum. Six to 8 days after implantation, tumors reahed approximately 4 mm 3 in volume (6 mm in diameter) and mie were randomly divided into experimental groups, onsisting of at least 6 mie. Experiments were repeated twie. Appliation of eletri pulses. Eletri pulses were delivered as previously desribed [7]. Briefly, the appliator onsisted of two flat parallel eletrodes 8 mm apart (two stainless-steel strips, width 7 mm, with rounded orners). Eletrodes were plaed perutaneously at the opposing margins of the tumor. Good ontat between the eletrodes and the overlying skin was assured by means of ondutive gel (Parker Laboratories, in., N.Y., USA). Square wave high voltage (diret urrent) pulses of 14 V (\3 V/m - voltage to distane ratio), with pulse width \ ms at repetition frequeny I Hz were generated by eletropulsator Jouan GHT 1287 (Jouan, Frane). The number of eletri pulses applied was ranging from I to \ during eah treatment. When more than one eletri pulse was applied, the number of eletri pulses was divided into two sets, the seond set oriented perpendiularly to the first, with a time interval of 1 seond in order to over the whole tumor with suffiiently high eletri field [21-23]. Treatment with eletri pulses was performed without anaesthesia and was well tolerated by the mie. Treatment protools were approved by the Medial Ethial Committee of the Ministry 'of Health of the Republi of Slovenia No /97. Eletrohemotherapy. Eletrohemotherapy onsisted of bleomyin (BLM) injetion followed by the appliation of 8 eletri pulses. BLM (Bleomyin, Mak, Germany) was dissolved in phosphate buffered saline and the dose of 1 I-lg/mouse in.2 ml volume was injeted into lateral tail vein of the preheated mie. BLM solution was prepared freshly for daily injetions. The interval between the BLM injetion and appliation of eletri pulses was 3 minutes. Pertinent ontrol groups were inluding untreated, eletri pulses only, and bleomyin only treated tumors. Antitumor effetiveness of eletrohemotherapy was assessed by measurements of tumor diameters in three orthogonal diretions using Vernier alliper on onseutive days following treatment. Arithmeti mean and standard error of the mean was alulated for eah experimental group. Tumor doubling time was alulated from the growth urve of individual tumors. Tumor growth delay was alulated from the mean tumor doubling time of the experimental groups ompared to untreated tumors [7]. Measurement of relative tumor blood flow. Relative tumor perfusion was measured by 86RbCI extration tehnique at various time ~oints following appliation of eletri pulses [17,24,25]. Briefly, 8 RbCI tissue radioativity measured I minute after an intravenous injetion, via the tail, was used to alulate relative blood flow as a proportion of ardia output. A minimum of 6 mie per grouft were injeted via the tail vein with 185 kbq (37 MBq/ml) 6RbCI (Amersham PLC, Little Chalfont, Buks., UK) and sarified by ervial disloation following I minute to allow for irulation of the traer. Immediatel~ thereafter the tumors were disseted, weighed and ounted for 6Rb radioativity by gamma ounter (Institute Jozef Stefan, Ljubljana, Slovenia). The tails of injeted mie were also removed and ounted to measure residual ativity at the site of injetion. Results were rejeted if the tail ounts were greater than 1% of the ativity of the injeted solution. Relative tissue perfusion was alulated as follows; radioativity in the tumors was expressed as a perentage of the total ativity injeted (minus that remaining in the tail) per gram. This gives a measure of perfusion as a funtion of ardia output and is expressed as % injeted ativity per gram or relative uptake (% ontrol). Assessment of tumor staining by Patent blue. Patent blue (Byk Gulden, Switzerland) was used to estimate tumor perfusion. Patent blue (1.25%) diluted in.2 ml physiologial saline was injeted into tail vein of animals after they were subjeted to speifi treatment. After the dye was evenly distributed through the tissue for approximately I minute, animals were sarified and tumors were arefully disseted. Tumors were ut along their largest diameter and the perentage of stained versus non-stained ross-setion was immediately estimated visually by two persons. The mean of both estimations was used as an indiator of tumor perfusion. The results of the experiments were pooled together and were presented as mean and standard error of the mean for eah experimental group [26]. Histology of tumors. Tumour histology was performed on the same tumors as were used for the Patent blue staining. The speimen were fixed in 1% buffered neutral formalin. One tissue blok ut through the largest diameter of the tumor was embedded in paraffin and stained with haematoxylin-eosin by standard method. Slides of 6 tumors per group were examined in a blind fashion. Tumor nerosis was determined in a whole mount tumor setion through the largest tumor diameter [19J. Statistial analysis. Statistial signifiane was evaluated by modified t-test (Bonferroni t-test) after one-way ANOVA was performed and fulfilled. Pearson orrelation oeffiient was alulated to determine the orrelation between Patent blue staining method and 86RbCI extration tehnique. Sigmastat statistial software (SPSS in.) was used for statistial alulations. Results Correlation between 86 RbCl extration and Patent blue staining tehnique. To determine the orrelation between 86RbCI extration tehnique and 'Patent blue staining of the tumors, two sets of experiments were onduted evaluating effet of eletri pulses appliation on tumor blood flow. Eah method measures different parameter, 86RbCI extration tehnique measures flow of plasma through the tumor, whereas the Patent blue staining measures the olletion of the dye in vasular and interstitial spae. In the first set of experiments the hanges in tumor 418

3 Sersa et al: Tumor Blood Flow Modifying Effet of Eletrohemotherapy blood flow were determined by both methods. A series of 8 eletri pulses (14Y) that were applied to the tumors dereased tumor blood flow to approximately 3% of the pre-treatment value (Figure 1). This redution, as measured by the two tehniques, was maximal at about 3-6 minutes, thereafter slowly inreasing to almost reahing pre-treatment level by about 24 hours. The data obtained by the two tehniques orrelated (r=.944; p<o.ool). In the seond set of experiments dependene of tumor blood flow hanges with respet to the number of eletri pulses applied was established. To denote the time of the minimum blood flow, tumor blood flow hanges were determined 3 minutes after appliation of eletri pulses ~ 1 4Y), by measurement of relative tumor blood flow by 6RbCl extration tehnique and tumor staining by Patent blue. The redution of tumor blood flow was dependent on the number of applied eletri pulses, reahing the minimum value of approximately 3% of the pretreatment value at 8 and 1 eletri pulses applied (Figure 2). Again, a very good result orrelation was ootained by the two tehniques (r=.962; p<o.ooi). These results demonstrate that Patent blue staining of the SA-1 tumors is reliable tehnique for estimation of tumor blood flow, is simf,le and quik, and provides similar information as the 8 RbCl extration tehnique. Antitumor effetiveness, tumor blood flow hanges and extent of nerosis in eletrohemotherapy treated tumors. Sine the Patent blue staining provided good estimation of blood flow hanges, this method was used for evaluation of blood flow hanges in tumors treated with eletrohemotherapy and pertinent ontrol groups. Eletrohemotherapy with bleomyin was found to have very good antitumor effet. When using the bleomyin dose of 1 Ilg/mouse 7% of eletrohemotherapy treated animals were tumour free 1 days after the treatment. In ontrast, treatment with eletri pulses and bleomyin as single treatment indued only moderate tumor growth delay inspite of onsiderable blood flow redution after appliation of eletri pulses (Table I, Figure 1,2). Evaluation of tumor blood flow in the tumors treated by eletrohemotherapy demonstrated that tumor perfusion was steadily dereasing up to 12 hours after the treatment, reahing almost omplete shut down of tumor perfusion. These tumors did not restore blood flow up to 5 days after the treatment (Figure 3). Appliation of eletri pulses only indued the same hanges in tumor perfusion as demonstrated in previous experiments (Figure 1,3). However, bleomyin treatment alone did not indue hanges in tumor blood flow; tumor staining after bleomyin treatment was not signifiantly different ompared to ontrol. The tumors in the ontrol group were stained up to 8% after 5 days. Data on extent on tumor nerosis after eletrohemotherapy with bleomyin orrelated well with data on Table I. Tumor growth after eletrohemotherapy with bleomyin. Group n DT/days P ompared GD/days Cures (average to DT of (average ± SE) ontrol ± SE) Control 2 1.8±.5 % Bleomyin IOOIlg!mouse 2 1.9±.1 >.5.1 ±.1 % Eletri pulses ±.2 <.5 1.3±.2 % Eletrohemotherapy ± 2.9 < ±2.9 7 % tumor blood flow hanges, however with few hours delay (Figure 4). Extensive nerosis of tumors treated with eletrohemotherapy was observed already 24 h after treatment and tumors remained fully neroti thereafter. Also, appliation of eletri pulses indued destrution of the tumors, extent of nerosis was transient up to 55%, and orrelated with tumor blood flow hanges and moderate antitumor effetiveness (Figure 4, Table I). The treatment with bleomyin did not result in tumor nerosis being in aomparable range as that in the untreated tumors. Disussion This study shows that measurement of tumor blood flow hanges an be adequately measured by Patent blue staining tehnique as with 86RbCl extration tehnique. We also found that eletrohemotherapy with bleomyin ompletely and permanently shuts down tumor blood flow, and that this observation orrelates well with antitumor effetiveness, as well as with the extent of tumor nerosis. Results of this study show that Patent blue staining of the tumors orrelate with measurements of relative blood perfusion measured by 86RbCl extration tehnique. The finding was onfirmed when transient hanges in tumor blood perfusion were evaluated after appliation of eletri pulses, as well in measurements when tumors were exposed to different number of eletri pulses. Although the two tehniques evaluate different parameters results were very similar. Namely, Patent blue staining shows distribution of the dye in the vasular and interstitial spae, whereas 86RbCl extration tehni~ue measures flow of plasma in the tumors. Sine the 6RbCl extration tehnique is a time onsuming and laborious tehnique [24,25], staining of tumours with Patent blue ould be used as an estimate of the tumor blood flow hanges, predominantly in the situations when a quik and simple tehnique is desired. Patent blue is a non-toxi dye that is used in lymphography, therefore there is no limitation for its use in patients. In eletrohemotherapy, eletri pulses are used as a 419

4 ANTICANCER RESEARCH 19: (1999) g 6 u SO 'Ii., "Rb extration tehnique Time after appliation of eletri PUIMS (hours) ~ 7 (; E 6 i!! e SO 4 'Ii " Control EIe1rI pulses --6- Bleomyin. Eletrohemotherapy Time after therapy (days) Figure 1. Time ourse of hanges in tumor blood flow after appliation of 8 eletri pulses (14 V. pulse width 1 fls, rr;getition frequeny I Hz). Tumor blood flow hanges were measured by 6RbCI extration of SA-I tumors and by tumor staining by Patent blue. Mean values ± standard error of the mean of at least 6 mie per point. Figure 3. Time ourse of hanges in tumor blood flow after eletrohemotherapy with bleomyin, measured by Patent blue staining. Eight eletri pulses were applied to the tumor ( 14 V. pulse width 1 fls, repetition frequeny I Hz) 3 minutes after intravenous injetion of 1 flg of bleomyin. Mean values ± standard error of the mean of at least 6 mie per point.! (; E i!." C 'Ii 11 SO ~ ::J ~ SO '!; O~ :l.5 Control Eletri pulses --'- Bleomyin -- Eletrohemotherapy Number of eletri pulses applied Time after therapy (days) Figure 2. Tumor blood flow hanges as a funtion of number of eletri pulses applied (15 V. purse width 1 fls, rr;getition frequeny I Hz). Tumor blood flow hanges were measured by 6RbCl extration of SA-I tumors and by tumor staining by Patent blue. Mean values ± standard error of the mean of at least 6 mie per point. Figure 4. Time ourse of hanges in tumor nerosis after eletrohemotherapy with bleomyin. Eight eletri pulses were applied 1 the tumour (15 V. pulse width 1 fls, repetition frequeny I Hz) 3 minutes after intravenous injetion of 1 flg of bleomyin. Mean values ± standard error of the mean of at least 6 mie per point. means to deliver suffiient amount of hemotherapeuti drug into the ells for effetive ytotoxi ation [1], This method is aimed at hemotherapeuti drugs that have hampered transport through the plasma membrane, though one inside the ell they are highly ytotoxi. This mehanism has been demonstrated to be effetive for the hemotherapeuti drugs, bleomyin as well as isplatin, in different tumor models [6,7,9]. However, our reent studies have demonstrated, that appliation of eletri pulses to the tumors an modify tumor blood flow [17,18]. As demonstrated in this study, appliation of eletri pulses that are used in eletrohemotherapy, results in substantial redution of tumor blood flow, whih is restored within 24 hours. This effet may ontribute to the 42

5 Sersa et at: Tumor Blood Flow Modifying Effet of Eletrohemotherapy antitumor effetiveness of eletrohemotherapy, by entrapment of the drug within the tumor, and thus by prolonging its antitumor ation. As demonstrated in this study, blood flow in the tumors treated by eletrohemotherapy with 1 fag bleomyin is abrogated within 12 hours after the treatment and is not restored thereafter. The results of antitumor effetiveness of eletrohemotherapy with bleomyin and extent of tumor nerosis orrelate well with the tumor blood flow modifying effet. The quik. and dramati effet of eletrohemotherapy an be attributed to the high ytotoxiity of bleomyin after eletroporation of tumor ells. However, to some extent the antitumor effetiveness may be asribed also to ytotoxi effet of eletrohemotherapy to endothelial ells in tumor blood vessels. Bleomyin, after eletroporation of endothelial ells, may severely damage the vasulature of the tumors and onsequently indue a asade of tumor ell death by abrogating oxygen supply of the ells. This phenomenon, desribed as vasular targeted therapy, reently gained high interest and has been exploited in several studies [27]. Abnormal physiology of the tumor blood vessels has been used as a target, to obtain antitumor effet [28]. Several drugs have been used till now, suh as ombrestatin A4 and others [27]. However, further studies are needed to determine sensitivity of the endothelial ells to eletrohemotherapy and to establish the differential sensitivity between the tumor and endothelial ells. As our preliminary study shows, endothelial ells are at least as sensitive as tumor ells to eletrohemotherapy with bleomyin (unpublished data). Therefore, when analysing antitumor effetiveness of eletrohemotherapy, several mehanisms an be identified; eletroporation that failitates drug aumulation in the ells [19]; redued tumor blood flow after appliation of eletri pulses only that prolongs exposure of the ells to the drug [17], immune system that inreases antitumor effetiveness of eletrohemotherapy [29,3] and as demonstrated in this study, effetiveness of eletrohemotherapy. with bleomyin ould be partly attributed to its tumor blood modifying and anti-vasular effet. Aknowledgements This work was supported by the Ministry of Siene and Tehnology of the Republi of Slovenia. DJC is supported by the Caner Researh Campaign of United Kingdom. Help of Prof. Dr. Rastko Golouh and tehnial assistane of Alenka Kljun is greatly aknowledged. Referenes Mir LM, Orlowski S, Belehradek JrJ, Teissie J, Rols MP, Sersa G, Miklavi, Gilbert R, Heller R: Biomedial appliation of eletri pulses with speial emphasis on antitumor eletrohemotherapy. 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6 ANTICANCER RESEARCH 19: (1999) 19 Cemazar M, Miklavi, Sanar J, Oolzan V, Golouh R, Sersa G: Inreased platinum aumulation in SA-I tumour ells after in vivo eletrohemotherapy with isplatin. Br J Caner 79: , Cemazar M, Milai R, Miklavi D, Dolzan V, Sersa G: Intratumoral isplatin administration in eletrohemotherapy: antitumor effetiveness, sequene dependene and platinum ontent. Anti-Caner Drugs 9: , Sersa G, Cemazar M, Miklavi D: Changing eletrode orientation improves the effiay of eletrohemotherapy of solid tumors in mie. Bioeletroh Bioener 39: 61-66, Miklavi D, Beravs K, Semrov D, Cemazar M, Demsar F, Sersa G: The importane of eletri field distribution for effetive in vivo eletroporation of tissues. Biophys J 74: , Semrov D, Miklavi D: Calulation of the eletrial parameters in eletrohemotherapy of solid tumors in mie. Com put Bioi Med 28: , Sapirstein LA: Regional blood flow by frational distribution of indiators. Am J Physiol 193: , Hill SA, Denekamp J: Site dependent response of tumours to ombined heat and radiation. Br J Radiol 55: , Miklavi D, Jarm T, Cemazar M, Sersa G, An DJ, Belehradek JrJ, Mir LM: Tumor treatment by diret eletri urrent. Tumor perfusion hanges. Bioeletroh Bioenerg 43: , Chaplin DJ, Hill SA, Bell KM, Tozer GM: Modifiation of tumor blood flow: Current status and future diretion. Sem Radiat Onol8: , Denekamp J, Hill SA, Hobson B: Vasular olusion and tumour ell death. Eur J Caner Clin Onol 19: , Mir LM, Roth C, Orlowski S, Quintin-Colona F, Fradelizi D, Belehradek JJr, Kourilsky P: Systemi antitumor effet of eletrohemotherapy ombined with histoinompatible ells sereting interleukin-2. J Immunother 17: 3-8, Sersa G, Miklavi D, Cemazar M, Belehradek JJr, Jarm T, Mir LM: Eletrohemotherapy with CDDP on LPB saroma: omparison of the antitumor effetiveness in immunoompetent and immunodefiient mie. Bioeletroh Bioenerg 43: , Reeived Marh 22, 1999 Aepted May 25,