Radiol Oncol 2017; 51(1): 30-39. doi:10.1515/raon-2017-0011 30 research article Electrotransfer of plasmid DNA radiosensitizes B16F10 tumors through activation of immune response Monika Savarin1, Urska Kamensek1, Maja Cemazar1,2, Richard Heller4, Gregor Sersa1,3 1 Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, Slovenia 2 Faculty of Health Sciences, University of Primorska, Izola, Slovenia 3 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia 4 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, USA Radiol Oncol 2017; 51(1): 30-39. Received 20 December 2016 Accepted 18 January 2017 Correspondence to: Prof. Gregor Serša, Department of Experimental Oncology, Institute of Oncology Ljubljana, Zaloška 2, SI-1000 Ljubljana, Slovenia. E-mail: gsersa@onko-i.si Conflict of interest: The authors declare no conflict of interest Background. Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors. Materials and methods. The murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, prolifera- tion, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms. Results. Gene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in ex- cellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response. Conclusions. The results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tu- mor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vascu- lature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response. Key words: gene therapy; electrotransfer; plasmid, irradiation, immune response, melanoma Introduction Electroporation is used as drug delivery system for molecules with hampered transmembrane transport.1 It is effective for delivery of smaller molecules, as chemotherapeutics in electrochemo- therapy (ECT)1-3 and also for larger molecules, as are plasmids in gene electrotransfer (GET). GET is Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA 31 recently getting a lot of scientific consideration and it is used for enhanced DNA delivery into various tissue types (i.e. skin, liver, kidney etc.), as well as into tumors.4-6 Its effectiveness was first demon- strated in a wide range of preclinical studies and has thereafter proceeded to clinical oncology, vet- erinary and human. The results of clinical trials of GET of plasmid encoding human IL-127 and anti- angiogenic plasmid AMEP8, are promising. In addi- tion to GET of therapeutic plasmids, some plasmids devoid of therapeutic genes have also resulted in good antitumor effectiveness.9,10 This phenomenon was observed in different tumors models and by using different plasmids. It was attributed to im- mune sensing of the introduced DNA as a DAMP (Damage Associated Molecular Pattern), which switch leads to activation of an immune response.11 Radiotherapy is one of the principal treatment modalities for primary tumors and their metasta- ses.12 Nowadays, irradiation is widely investigated for its associated effects on priming antitumor im- munity.13 There is growing evidence of irradia- tion’s effect on the antitumor immune response, inducing immunogenic cell death and generating danger signals. An important danger signal after irradiation is DNA released from the nucleus of dy- ing cells. This DNA is recognized by the immune system as a DAMP, and can promote the activation of immune response against irradiated cells.14,15 Studies combining tumor irradiation with GET of different therapeutic plasmid demonstrated tu- mor radiosensitization.16-18 A promising approach to target tumors and its microenvironment with a combined treatment modality is through destruct- ing abnormal tumor blood vessels. One of the promising targets, gaining on its value due to dif- ferent signaling pathways from VEGF, is endoglin. It is a TGF-β coreceptor and has already demon- strated good antitumor and antimetastatic effec- tiveness in different tumor models when targeted with GET of plasmid.18–22 In particular, GET of shRNA for silencing endoglin in B16F10 melanoma mice tumors that express high levels of endoglin, resulted in up to 58% of tumor cures.23 Tissue specific eukaryotic promoters are tightly regulated and mainly drive expression of transgene in specific cell types, although minimal unspecific expression in non-targeted tissue can also occur.24 We constructed a plasmid containing a tissue spe- cific promoter for endothelin and encoding shRNA for silencing endoglin. This plasmid was tested in a previous study, where the effectiveness of the plasmid with tissue specific promoter was com- pared to a plasmid with constitutive promoter in a tumor model that does not express targeted molecule, endoglin. In vivo, the effectiveness of the GET of the plasmids was comparable and resulted in significant radiosensitization, which resulted in prolonged tumor growth delay with nearly 50% of tumor free mice. Thus, the aim of this current study was to determine, whether GET of plasmid, with tissue specific promoter and encoding shRNA for silencing endoglin, can radiosensitize melanoma B16F10 tumors, which express targeted molecule, endoglin, and possibly have also some immu- nomodulatory effectiveness. Materials and methods Cell lines and plasmids Murine melanoma cell line B16F10 (American Type Culture Collection, Manassas, VA, USA) was cultured in advanced minimum essential me- dium (AMEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS, Gibco), 10 mM/L L-glutamine (GlutaMAX, Gibco), 100 U/mL penicillin (Grünenthal, Aachen, Germany) and 50 mg/mL gentamicin (Krka, Novo mesto, Slovenia) in a 5% CO2 humidified incubator at 37°C. The plasmid with tissue specific promoter for endothelial cells, encoding shRNA for silencing endoglin (pET-antiCD105; TS plasmid) was used in experiments as the therapeutic plasmid.18 The con- trol plasmid, encoding shRNA with no homology to any gene in the mouse genome and with constitu- tive CMV promoter, was used as a negative control (pControl).25 Amplification of both plasmids was performed in a competent E.coli (JM107; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). All plasmids were isolated using JETSTAR 2.0 ENDOTOXIN-FREE Plasmid MEGA Kit (Genomed, Löhne, Germany) and diluted in endo- toxin free water to a concentration of 1 µg/µL (in vit- ro experiments) and 4 µg/µL (in vivo experiments). Concentrations of plasmids were measured with a spectrophotometer at 260 nm (Epoch Microplate Spectrophotometer, Take3 Micro-Volume Plate, BioTek, Bad Friedrichshall, Germany) and purity of plasmid was determined by agarose gel elec- trophoresis and measurements of the absorbance ratio at 260 and 280 nm. Experimental animals All animal experiments were conducted in accord- ance with the guidelines for animal experiments of Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA32 the EU Directive and the permission obtained from the Ministry of Agriculture and the Environment of the Republic of Slovenia (Permission No. 34401- 1/2015/16), which was given, based on the approval of the National Ethics Committee for Experiments on Laboratory Animals). Female C57Bl/6 mice, 6-8-week old, purchased from Envigo Laboratories (Udine, Italy), were used in the study. Before the experiment, mice were subjected to an adaptation period of 2 weeks. Animals were maintained under specific patho- gen-free conditions at a constant room tempera- ture, humidity and a 12 h light/dark cycle. Food and water were provided ad libitum. For induction of subcutaneous tumors, a suspension of 1x106 B16F10 cells in 0.1 ml of physiological saline was injected subcutaneously into the back of the mice. The animals bearing tumors of 40 mm3 were ran- domly divided into experimental groups and sub- jected to a specific experimental protocol. The tu- mor measurements were completed when the tu- mors reached 350 mm3, and mice were humanely sacrificed. Experimental groups and the number of animals in each of them were as follows and as described in table Table 1: triple injection of endotoxin-free wa- ter alone (control group; CTRL) or of pControl or TS plasmids alone, or in combination with triple application of electric pulses alone (3 × EP) or com- bined with plasmids (3 × GET (pControl); 3 × GET (TS)). Furthermore, the remaining groups were also the ones in combination with irradiation (IR) and other therapies described above, which are three injections of plasmids (pControl + IR; TS + IR) and in combination with electric pulses (3 × EP + IR; 3 × GET (pControl) + IR; 3 × GET (TS) + IR). In vivo GET In vivo GET of plasmid into subcutaneous tumors was performed 3 times every second day (on days 0, 2 and 4). 12.5 µL (4 µg/µL) of plasmid (150 µg in total) in endotoxin-free H2O was injected intratu- morally 10 min before 8 square electric pulses with a voltage-to-distance ratio of 600 V/cm, a pulse du- ration of 5 ms, and a frequency 1 Hz were applied. Electric pulses were generated by electric pulse generator ELECTRO CELL B10 (Betatech, L’Union, France) and delivered through 2 parallel stainless steel electrodes with 2 or 4 mm distance between them, depending on the tumor volume. After 4 pulses, electrodes were turned for 90° for 4 addi- tional pulses to assure GET to entire tumor. Irradiation of tumors Tumors were locally irradiated with a single dose of 15 Gy on day 1 from the beginning of the experi- ment, at a dose rate of 2.16 Gy/min, using a Darpac 2000 X-ray unit (Gulmay Medical Ltd., Shepperton, UK) operating at 220 kV, 10 mA, with 1.8-mm alu- minum filtration. During irradiation, mice were TABLE 1. Response of B16F10 melanoma to different treatment modalities Therapeutic group DT (days) TGD (days) TF SC n AM SEM AM SEM n % n % CTRL 8 1.2 ± 0.1 0.0 ± 0.1 0 0 - - pControl 8 1.9 ± 0.2 0.7 ± 0.2 0 0 - - TS 8 2.2 ± 0.3 1.0 ± 0.3 0 0 - - 3 × EP 8 3.1 ± 0.3 2.0 ± 0.3 0 0 - - 3 × GET (pControl) 8 9.4 ± 6.5 8.2 ± 2.5 0 0 - - 3 × GET (TS) 9 9.5 ± 3.2 8.6 ± 3.0 4 44 3 75 IR 9 1.8 ± 0.3 0.7 ± 0.3 0 0 - - pControl + IR 9 2.5 ± 0.3 1.3 ± 0.3 1 11 1 100 TS + IR 8 4.0 ± 1.2 2.8 ± 1.2 0 0 - - 3 × EP+IR 9 4.3 ± 1.3 3.2 ± 1.3 2 22 0 0 3 × GET (pControl) + IR 9 36.0 ± n/a 34.9 ± n/a 8 89 5 63 3 × GET (TS) + IR 8 32.0 ± n/a 30.8 ± n/a 7 88 4 57 AM = Arithmetic mean; DT = Tumor doubling time; Groups: (CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET- antiCD105); n = Number of all mice in the group; n/a = Not applicable; SC = Mice resistant to secondary challenge; SEM = Standard error; TF = Tumor free mice; TGD = Tumor growth delay Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA 33 restrained in special lead holders with apertures for irradiation of the tumors. Due to the fixed size of the apertures, some healthy tissue (3 – 5 mm of skin surrounding the tumor) was exposed to the irradiation as well. Tumor growth The therapeutic potential in vivo was assessed by measuring the tumor size every second day and calculating tumor volume according to the for- mula for ellipsoid: V=axbxc π/6, where a, b and c represent perpendicular tumor diameters.21,26 The tumor growth curves were drawn as arithmetic means (AM) with bars representing standard er- rors (SEM). The tumor growth delay for each experimen- tal group was calculated as the difference in tu- mor doubling times of experimental and control groups. Tumor doubling time is the number of days in which the initial tumor volume (40-50 mm3) doubles. Mice that remained tumor free for 70 days were termed tumor free and local tumor control was deemed to have been achieved (Table 1). The weight of the mice was followed as a general in- dex of systemic toxicity, and acute skin reaction in the whole irradiated field around the tumor was evaluated as described elsewhere.17 Tumor challenge of tumor free mice After 70 days, when mice were designated as tu- mor free, they were challenged with a subcuta- neous injection of 1 × 106 B16F10 cells in 0.1 ml of physiological saline in the right flank. Mice that at least 20 days after the challenge remained tumor free were marked as resistant to secondary chal- lenge (Table 1, Figure 2). The growing tumors were measured twice a week and when volume of 150 mm3 was reached mice were sacrificed and tumors were collected for further histological analysis as described below. Histology After therapies, at day 6, three mice from each ex- perimental group were sacrificed. The tumors were excised, fixed in IHC zinc fixative (BD Biosciences, San Diego, CA, USA) and embedded in paraffin. Six consecutive 2-µm thick sections were cut from each paraffin block and stained as followed. To estimate the percent of the area of tumor necro- sis, the first section was stained with hematoxylin and eosin. The other five sections were used for immunohistochemical (IHC) staining to evaluate percentage of hypoxic cells, cells in apoptosis, im- mune cells, proliferating cells and the number of blood vessels. To determine hypoxic cells, rabbit polyclonal antibodies against HIF-1-alpha (ab2185, Abcam, Cambridge, MA, USA) at dilution 1:3500, were used. In addition, apoptosis was evaluated with help of cleaved Caspase-3 (Asp175., Cell sign- aling Technology, Danvers, MA, USA) at dilution 1:1500, whereas immune cells (NK and CTL) were stained with help of Granzyme B (ab4059, Abcam) at dilution 1:1250. For staining proliferating cells, rabbit monoclonal antibodies against Ki-67 (clone SP6, Thermo Fisher Scientific) at dilution 1:1200 were used. The last section was stained for deter- mination of the number of blood vessels, by using primary rabbit polyclonal antibodies against CD31 (ab28364, Abcam) at dilution 1:1000. For these sec- tions, a peroxidase-conjugated streptavidin–biotin system (Rabbit specific HRP/DAB detection IHC kit, ab64261, Abcam) was used as the colorogenic reagent followed by hematoxylin counterstaining. From each slide and each feature (apoptosis, hy- poxia, proliferation, vascularisation and immune cells), five randomly selected viable parts of each tumor were observed and captured under the light FIGURE 1. Histological sections of melanoma tumors on day 6 after the beginning of the therapy. CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET-antiCD105 Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA34 microscopy, by DP72 CCD camera (Olympus, Hamburg, Germany) connected to a BX-51 micro- scope (Olympus) under 40× magnification (numer- ical aperture 0.85). The viable parts were analyzed in blind fashion and the results were presented as the percent (hypoxia, apoptosis, proliferation) of the cells or the number of cells (immune cells) or the structures (vascularization) positive to IHC staining. The percentage of necrosis was contribut- ed to the tumors as whole and was also evaluated in blind fashion, as previously described.18 In addition, tumors that grew up to 150 mm3 after secondary challenge of tumors, were excised, fixed and embedded in paraffin, as described above. Furthermore, from each tumor sections were cut and stained with hematoxylin and eosin to deter- minate morphological changes of tumor cells. Statistical analysis All data were tested for normality of distribu- tion with the Shapiro-Wilk test. The differences between the experimental groups were statisti- cally evaluated by one-way analysis of variance (one-way ANOVA) followed by a Holm-Sidak test for multiple comparison. A P-value of less than 0.05 was considered to be statistically significant. SigmaPlot Software (Systat Software, Chicago, IL, USA) was used for statistical analysis and graphi- cal representation. FIGURE 2. Immune response of melanoma tumors is observed by vitiligo effect. CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET-antiCD105 Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA 35 Results Gene electrotransfer of plasmid silencing endoglin indicates a vascular targeted effects of the therapy GET of either plasmids (pControl, TS) to melanoma tumors had statistically significant antitumor ef- fectiveness compared to untreated tumors, which resulted in 8.2 ± 2.5 and 8.6 ± 3.0 days of tumor growth delay, respectively (Table 1). However, GET (TS) resulted in 44% of tumor free mice and 75% of them were resistant to secondary challenge, whereas in the GET (pControl) group no tumor free mice were obtained. Histological analysis (Table 2) of GET (TS) group demonstrated reduction of vas- cularization (14.9 ± 1.1%) and proliferating cells (49.5 ± 3.8%), whereas hypoxia (46.3 ± 3.0%) lev- els were increased and statistically significant to at least pertinent control groups (CTRL, pControl, TS and 3 × GET (pControl)). The levels of necrosis, apoptosis and number of infiltrating immune cells in the tumors were comparable in both GET treat- ment modalities (Figure 1) and statistically signifi- cant to at least CTRL, pControl and TS. These re- sults indicated the vascular targeted effects of the GET (TS), used for silencing endoglin. Irradiation, alone or combined either with plasmids injection or electric pulses, had minor effect on radioresistant melanoma tumors Irradiation monotherapy with 15 Gy had minor effects on radioresistant melanoma tumor. The tumor growth delay was moderate in the groups of irradiation alone or in combination with injec- tion of plasmids (from 0.7 ± 0.3 to 2.8 ± 1.2 days) and up to 11% of tumor free mice were obtained in the group of irradiation in combination with in- jection of plasmid pControl (Table 1). The results of histological analysis (Table 2, Figure 1) indicates an immunological effect of the irradiation alone or in combination with plasmids injection, since the number of infiltrating immune cells in the tumors was statistically significantly higher in comparison to control groups (CTRL, pControl, TS) and groups applying electric pulses (alone or combined with injection of plasmids (GET (pControl) and GET (TS)), and the tumor free mice were resistant to sec- ondary challenge. Furthermore, irradiation alone caused the reduction in proliferating cell levels (53.3 ± 3.5%) and in tumor vascularization (30.1 ± 2.3%), whereas apoptosis (18.4 ± 1.8%) and hypox- ia (30.2 ± 2.4%) levels were elevated. All of these results were statistically significant compared to CTRL, pControl and TS groups. The application of electric pulses to tumors in combination with tumor irradiation resulted in bet- ter antitumor effectiveness than observed in each of these two treatment modalities alone. The tumor growth delay in combined treatment was 3.2 ± 1.3 days and 22% of tumor free mice were obtained (Table 1). The histological analysis of combined treatment demonstrated statistically significant higher levels of apoptosis (31.0 ± 2.0%) and hypoxia (51.7 ± 2.6%) in comparison to the groups of irradi- ation and electric pulses alone. Among these three groups no statistically significant differences were observed in the levels of necrosis. Furthermore, the analysis of levels of proliferating cells, the number of tumor blood vessels and immune cells infiltrat- ing in the tumors, resulted in similar and moder- ate effectiveness of the combined treatment (3 × EP + IR) and irradiation (alone or combined with injection of plasmids), which statistically signifi- cantly differed from group of electric pulses alone. Nevertheless, in this combined treatment modality (3 × EP + IR) no tumor free mice were resistant to secondary challenge of tumors. Combination of GET and irradiation exerts pronounced antitumor effects The groups combining GET of plasmids (pControl, TS) and irradiation resulted in pronounced thera- peutic effectiveness, with up to 89% and 88% of tumor free mice, respectively, and from those up to 63% and 57% of mice was resistant to second- ary tumor challenge, respectively (Table 1). The histological analysis, of tumors excised 6 days after treatment (Table 2, Figure 1), indicated on similar mode of action of these combined treatment mo- dalities (3 × GET (pControl or TS) + IR), since the elevation of necrosis (65.0 ± 7.6%, 79.2 ± 4.2%), number of immune cells (45.8 ± 3.0%, 46.5 ± 2.1%), reduction of tumor vascularization (18.9 ± 2.0%, 12.8 ± 1.0%) and proliferation (46.0 ± 4.8%, 46.7 ± 4.8%), did not differ between these groups. Only two statistically significant differences between the therapeutic groups combining GET (pControl, TS) and irradiation were observed which were the level of hypoxia (58.0 ± 2.8%, 66.7 ± 1.6%), and ap- optosis (38.7 ± 3.1%, 50.3 ± 2.4%). The high number of infiltrating immune cells (Table 2) in these tumors indicated on important mode of this therapeutic action; the highest and statistically significantly increased number of im- mune cells was observed in the groups combin- Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA36 ing GET of plasmids and irradiation, followed by all the remaining groups that included irradiation (alone, TS, pControl and 3 × EP). In these groups, the immune cells infiltration was comparable and statistically significant to all of the other remaining groups. Furthermore, the immune cells infiltration was the lowest in the groups that included electric pulses (alone or with plasmids alone). Additionally, all of the mice that were tumor free after the thera- pies including irradiation, either alone or in combi- nation with other modalities, had fur discoloration, known as vitiligo, indicating immune response (Figure 2). This was further confirmed with high number of mice that were resistant to secondary challenge in tumor free mice, with the exception of the group combining electric pulses and irradiation (Table 1). In these groups, in which tumors grew af- ter secondary challenge, the growth rate and histol- ogy of the tumors were the same as after the initial induction of tumors (data not shown). In addition, the safety of the treatment (irradia- tion alone or in combination with other modalities) was proven, since no body weight loss over 10%, or any other side effects were observed, except for the temporary hair loss in the irradiated area, without skin desquamation (data not shown). Discussion The results of this study indicate a dual effect of GET of plasmid encoding shRNA for silencing endoglin, the direct and the indirect, both having a radiosensitizing effect. The direct effect was on the tumor vasculature and also on melanoma tu- mor cells, whereas the indirect was observed with the use of plasmid devoid of therapeutic gene, through boosting the immune response in tumors. Furthermore, irradiation had mainly affected mel- anoma cells, although some effect on vasculature could also be noticed. In addition, the higher num- ber of infiltrating immune cells in all of the groups combined with irradiation indicated an important role of the immune system. All of these effects had synergistic action and, in combined treatment mo- dality of GET and irradiation, resulted in increased radiosensitizing effectiveness of melanoma tumors that resulted in prolonged tumor growth delay, which led to 88% of tumor free mice, of which 57% were resistant to secondary challenge of tumors. Dual effectiveness of GET The first direct effect of GET (TS) was on tumor vasculature, which was significantly reduced after the treatment. This vascular effect can be ascribed to the specificity of the plasmid for endothelial cells and to the endoglin silencing. This direct effect on tumor vasculature was also observed in other stud- ies using GET of plasmid for silencing endoglin on melanoma22,23 and other tumor models.18,19,21 The vascular targeted effects in these studies were first confirmed in non-endoglin expressing tumors, i.e. murine mammary adenocarcinoma, by endotheli- TABLE 2. Immunohistological analysis of tumors Therapeutic group Necrosis (%) Apoptosis (%) Hypoxia (%) Proliferation (%) Vascularization (n of structures) Immune cells (n of cells) AM SEM AM SEM AM SEM AM SEM AM SEM AM SEM CTRL 25.0 ± 2.6 11.3 ± 1.6 4.7 ± 1.0 92.2 ± 1.3 51.6 ± 3.2 7.9 ± 0.9 pControl 20.0 ± 2.6 11.6 ± 1.0 5.4 ± 1.0 92.1 ± 1.2 47.1 ± 4.7 8.3 ± 1.1 TS 21.7 ± 2.6 11.4 ± 1.5 5.5 ± 0.7 92.7 ± 1.0 50.5 ± 4.2 7.9 ± 0.7 3 × EP 13.3 ± 3.6 18.7 ± 2.7 7.8 ± 0.8 74.4 ± 1.7 39.9 ± 2.6 14.3 ± 1.7 3 × GET (pControl) 53.3 ± 7.9 19.5 ± 3.0 15.3 ± 2.9 75.6 ± 2.4 35.3 ± 4.2 15.1 ± 1.6 3 × GET (TS) 65.8 ± 7.6 26.7 ± 2.2 46.3 ± 3.0 49.5 ± 3.8 14.9 ± 1.1 19.3 ± 1.7 IR 21.7 ± 4.2 18.4 ± 1.8 30.2 ± 2.4 53.3 ± 3.5 30.1 ± 2.3 26.8 ± 2.6 pControl + IR 37.5 ± 8.0 18.7 ± 1.9 32.3 ± 2.5 54.5 ± 4.7 24.5 ± 1.8 26.4 ± 2.1 TS + IR 43.3 ± 8.0 19.9 ± 1.5 31.6 ± 2.3 52.0 ± 2.9 23.8 ± 1.6 26.4 ± 2.3 3 × EP + IR 25.0 ± 4.8 31.0 ± 2.0 51.7 ± 2.6 59.4 ± 4.0 28.9 ± 2.3 29.3 ± 2.1 3 × GET (pControl) + IR 65.0 ± 7.6 38.7 ± 3.1 58.0 ± 2.8 46.0 ± 4.8 18.9 ± 2.0 45.8 ± 3.0 3 × GET (TS) + IR 79.2 ± 4.2 50.3 ± 2.4 66.7 ± 1.6 46.7 ± 4.8 12.8 ± 1.0 46.5 ± 2.1 AM = Arithmetic mean; Groups: (CTRL = control; EP = electric pulses; GET = gene electrotransfer; IR = irradiation; TS = pET-antiCD105); n = Number of structures or cells; SEM = Standard error Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA 37 al-specific promoter18,21 and non-specific, constitu- tive promoter18–21, that resulted in pronounced an- titumor effectiveness. In melanoma tumor model, B16F10-luc, the GET of plasmids silencing endog- lin resulted in significant antimetastatic effective- ness.22 Furthermore, GET of plasmid silencing endoglin, with constitutive promoter, performed on small melanoma B16F10 tumor model (4 mm3 at the beginning of therapy) confirmed vascular targeted effects, which resulted in prolonged tu- mor growth delay and tumor free mice (58%), also due to nonspecific nature of constitutive plasmid that was used in this study silenced endoglin also in melanoma cells.23 Nevertheless, in the current study, which was done on bigger tumors (40 mm3), in addition to significant increased level of hypox- ia, also decreased number of proliferating tumor cells was observed. This was attributed to the sec- ond direct effect of GET (TS), that is silencing of endoglin also in melanoma cells, since it is known that plasmid with tissue specific promoters can be leaky and can also be transcribed in non-targeted tissue.27 Therefore, GET (TS) has dual direct effects; primarily by targeting vasculature and secondly by inhibition of proliferation of melanoma cells. These two effects together resulted in 44% of tumor free mice, from which 75% were resistant to secondary challenge of tumor cells. Nevertheless, high level of infiltrating immune cells in the tumors, indicated also an indirect ef- fect of this treatment, through the stimulation of immune response. This is also supported by the data obtained with GET of the plasmid devoid of therapeutic gene (pControl). In comparison to GET of therapeutic plasmid, TS, no tumor cures were obtained, however, in other measured parameters, infiltration of immune cells into the tumors, lev- els of necrosis and apoptosis as well as in tumor growth delay no statistically significant differenc- es were obtained between these groups. Thus, to a certain degree, a similar mode of antitumor ac- tion can be ascribed to the GET of plasmid DNA, mainly because the values of these parameters were statistically different from pertinent control groups. Antitumor effectiveness of GET of non- therapeutic (control) plasmids was also observed in other studies9,10, and the authors indicated that effectiveness was due to involvement of immune system. Namely, the plasmid introduced during GET can act as a DAMP that is recognized through different sensors (Pattern Recognition Receptors), leading to the activation of the signal transduction cascade that ultimately triggers the production of type 1 interferons and other cytokines.28 These act as a link between the innate and adaptive im- mune response29 and can induce the adaptive im- mune response against the introduced DNA and consequently the transfected cells. Furthermore, similar immune response can also be triggered by the stress30 that is produced during the transfection procedure, like mechanical stress, heat, and ROS, that have all been previously reported after elec- troporation.31,32 Priming effect of irradiation To target primary tumors, as was done in this study, irradiation is one of the most used treatment modalities.33 This therapeutic approach alone or in combination with injection of plasmids, in ra- dioresistant melanoma tumor model, resulted in moderate tumor growth delay, which was attrib- uted to decreased proliferation of melanoma cells, increased levels of apoptosis and necrosis. Further histological analysis indicated on the radiation damage of the tumor vasculature, as a second ef- fect of the irradiation, as seen in other studies.34,35 This vascular damage was less pronounced than in group of GET (TS), although in comparison to GET (TS), irradiation monotherapy resulted in high- er infiltration of the immune cells in the tumors. Furthermore, in the group combining irradiation and injection of therapeutic plasmid, one mouse was tumor free, which was also resistant to sec- ondary challenge of tumor. This indicates a prim- ing effect of irradiation combined with introduc- tion of foreign DNA, through boosting the immune response. When irradiated cells die, they release their antigens in the context of the danger signal (DAMPS), which result in the priming of a tumor specific immune response against the released an- tigens. Therefore, irradiated tumors can sometimes act as a powerful individualized in situ vaccine13, which is manifested as the abscopal effect of the ir- radiation. This was confirmed in in vivo studies in- dicating that irradiation can induce in vivo priming of T cells to exogenous model antigens engineered to be expressed by tumors.13 In melanoma tumor model, priming of antitumor T cells to the model antigen ovalbumin, was more effective when sin- gle, 15 Gy dose was used, rather than 3 Gy given in 5 consecutive days.36 Further on, another group al- so showed induction of antitumor T-cell responses with other antigen expression, when single 20 Gy dose was applied, but not by 5 Gy given 4 times.37 The combination of electric pulses and irradia- tion exerted moderate antitumor effectiveness that resulted in 22% of tumor free mice. Similar results Radiol Oncol 2017; 51(1): 30-39. Savarin M et al. / Radiosensitization of melanoma by electrotransfer of plasmid DNA38 were obtained in our previous studies on mu- rine mammary adenocarcinoma18, sarcoma17 and Ehrlich-Lettre ascites38, where also 20%, 27% and 54% of tumor free mice were observed, respective- ly. Therefore we can assume that electroporation of tumors contributes to radioresponsiveness of mel- anoma, most probably due to generation of ROS32,39 after application of the electric pules, which also re- sulted in significantly elevated levels of hypoxia in our histological analysis. Immune boosting and radiosensitization by plasmid DNA The combined treatment modalities (combination of GET (TS or pControl) with irradiation) exerted excellent, highly statistically significant antitumor effect in comparison to control groups, which re- sulted in 88% and 89% of tumor free mice, respec- tively, of which 57% and 63% of mice were resist- ant to secondary challenge of tumors, respectively. The results of histological analysis were similar between groups of combined treatment modality (GET + IR), regardless of the applied plasmid. The only two differences were on the levels of apop- tosis and hypoxia. The analysis of the presence of the immune cells showed on the highest number of the immune cells in the groups of GET of plasmids and irradiation, followed by irradiation groups (alone or combined with injections of plasmids or combined with electric pulses). Therefore, it can be presumed that the priming effect of irradiation can be boosted by GET of the plasmid DNA to fully ex- ert vaccinating effect. Additionally, fur discolora- tion, vitiligo, an immune-mediated destruction of normal melanocytes that has also been recognized as a positive prognostic indicator for treatment re- sponse40,41, was also observed in this study in all the groups where tumor free mice were observed (except 3 × GET (TS)). In addition, majority of the tumor free mice were resistant to secondary chal- lenge of tumors, further indicating on the develop- ment of the immune memory. The tumors in exper- imental groups, which were resistant to secondary challenged of tumor, were therefore radiosensibi- lized through GET of plasmid, since in the group combining electric pulses and irradiation, no mice resulted resistant to secondary challenge of tumor. The GET of plasmids in combination with irradia- tion presumably generated many danger signals that collectively define immunogenic cell death.13 Furthermore, the immunogenicity of tumor might also play an important role in combined treatment modality.42 In the current study we ob- tained significant antitumor effectiveness and up to 89% of tumors free mice, that were up to 63% resistant to secondary challenge of tumors, when combining GET and irradiation in melanoma tu- mor model. Furthermore, in our previous study combining the same treatment modality on a dif- ferent tumor model, murine mammary adenocar- cinoma TS/A, we achieved up to 44% of tumors free mice with therapeutic plasmid silencing en- doglin and 20% tumor free mice with the control plasmid.18 We can presume that the boosting of immune response depends on the tumor type, melanoma being more immunogenic that mam- mary adenocarcinoma TS/A. Nevertheless, the mechanisms of these therapies are not fully elu- cidated. Currently, we presume that there is in- volvement of DNA sensors, ROS and specific im- mune response after irradiation, but further stud- ies are needed. Conclusions The results of this study indicate that irradiation can in radioresistant mice tumors, such as mela- noma, by release of tumor associated antigens serve as the target of the immune response. This can be further boosted by GET of plasmid, with or without therapeutic gene, which was confirmed by the equal radiosensitization resulting in prolonged tumor growth delay and up to 89% of tumor free mice, which kept immune memory to melanoma cells. In addition, GET of therapeutic plasmid si- lencing endoglin has also direct effect on vascu- lature and tumors cells; however in combination with radiotherapy this effect was masked by pro- nounced immune response. Acknowledgements The authors would like to acknowledge M. Lavric and S. Kranjc for their help with cell cultures and tumor transplantation, U. Lampreht Tratar and M. Ota for help with IHC, as well as V. Todorovic for useful tips. 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