Electroporation-Enhanced Gene Delivery in Mammary Tumors

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Gene Therapy (2000) 7, 541–547  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt NONVIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Electroporation-enhanced gene delivery in mammary tumors

JM Wells, LH Li, A Sen, GP Jahreis and SW Hui Membrane Biophysics Laboratory, Molecular and Cellular Biophysics Department, Roswell Park Cancer Institute, Buffalo, NY 14263-0001, USA

Electroporation was applied to enhance gene transfer into pulses 1 ms long were applied across tumors, using caliper subcutaneous MC2 murine breast tumors. Cultured MC2 electrodes on the skin surface. Electric field strengths cells were also transfected by electroporation or by cationic ranged from 400–2300 V/cm. Luciferase expression was liposomes in the presence of serum using pSV-luc plasmids. approximately two orders of magnitude higher than controls Electroporation parameters and liposome formulation were in tumors treated with pulses у800 V/cm. Differences optimized to achieve the highest relative levels of transfec- between enhanced relative levels of transfection using tion. An electric field threshold for successful electrotransfec- uncomplexed plasmid and lipoplexes were not statistically tion in cultured cells appeared around 800–900 V/cm. The significant. Distribution of DNA into tumor tissues was moni- liposomes used contained the cationic lipid dioleoyl-3-trime- tored by fluorescence in situ PCR. The highest numbers of thylammonium propane (DOTAP). Multilamellar vesicles fluorescent cells were found in tumors electroporated follow- (MLV) had a 10-fold advantage over small unilamellar ves- ing the injection of plasmid. The significant transfection icles (SUV) in cell culture transfection. For in vivo gene deliv- improvement shows that in vivo electroporation is a powerful ery, the plasmids were injected either alone, or in complex tool for local gene delivery to tumors. Gene Therapy (2000) with MLV or SUV DOTAP liposomes. A series of six electric 7, 541–547.

Keywords: electroporation; breast tumor; gene transfer; liposomes; transfection; mice

Introduction electrodes and create electric field gradients through soft tissues makes electroporation an attractive method for Gene therapy is fast becoming a therapeutic mode for gene delivery to tumors of the head, neck, breasts and cancer therapy. Many new vectors have been designed skin. In vivo electroporation has been applied to introduce for effective gene transfer. Among these newly developed bleomycin to solid tumor cells with significant tumor vectors, nonviral vectors have several advantages over suppressing effects.4,5 This therapeutic mode is at phase retroviral and adenoviral vectors in gene delivery for I/II clinical trial.6 However, the application so far is cancer therapy. For instance, some retroviruses are cap- restricted to membrane transport-limited drugs, (ie able of activating oncogenes or deactivating tumor sup- bleomycin). For cancer gene therapy, a promising pressor genes. In addition, nonviral methods such as cat- approach is the partial transfection of tumors and sur- ionic liposomes, cationic polymers and electroporation rounding cells with genes coding for biological modu- do not usually elicit immune responses. Therefore, they lators (eg cytokines), or the limited transfection of anti- can be repeatedly administered. However, the gene trans- gen-presenting cells for enhancing immunotherapy. fer efficiency of nonviral vectors is still below that of viral Current attempts to inject cytokines have limited success, vectors. Long-term expression is still a challenge for non- because these agents produce strong inflammatory viral methods. With advanced liposome designs and responses and edema. Localized gene therapy allows for improved electroporation technology, the combination of long-term production of these agents in the immediate cationic liposomes and electroporation holds promise for vicinity of tumors. Transfecting a fraction of tumor cells enhanced local gene delivery. with apoptotic (eg Tk), tumor necrosis factor, or tumor Electroporation is a highly efficient method for suppressor genes may also be beneficial, taking advan- delivering exogenous molecules, including DNA, into tage of bystander effects upon neighboring cells. cells. This method has been used widely as means to An earlier electrotransfection experiment, using surface 1 deliver molecules into cells in vitro. Recently, this electrodes on skin, was reported by Titomirov et al,7 method has been applied to permeate the upper layer delivering plasmids coding for a reporter gene into new- (stratum corneum) of the skin for the purpose of drug born mouse skin. They reported transient and long-term 2,3 and gene delivery. The possibility of applying surface transfection of cells isolated and cultured from electroporated skin. Zhang et al8 also reported transfection of skin cells with the LacZ reporter gene, using a combination of Correspondence: SW Hui electric pulse and pressure. Recent studies have shown Received 6 May 1999; accepted 26 November 1999 that electroporation applied in vivo can introduce reporter Gene delivery by electroporation of mammary tumors JM Wells et al 542 genes to murine liver, and indirectly to rat brain Transfection of cultured MC2 tumor cells tumor.9,10 Rols et al11 have demonstrated the possibility In order to establish the transfectability of MC2 cells by of electrotransfection of melanoma in vivo. liposome and electroporation methods, using the same To show that electroporation can significantly enhance plasmid that would be used in in vivo experiments, we gene delivery to tumors, we applied electroporation to a first tested the cells and methods in vitro. The in vitro large number of implanted murine breast tumors. experiments, using cultured MC2 cells, provided us with Because of tumor heterogeneity, studies involving many a starting point in terms of lipoplex types and concen- tumor samples are needed to establish a statistical basis trations, as well as electrical parameters for subsequent for the usefulness of electroporation in gene delivery to in vivo experiments. tumors. Altogether, 105 tumors were used in this experi- Table 1 shows the transfection efficiency using lipo- ment. Variability in tumor size and appearance was plexes made from DOTAP MLV or SUV, as well as MLV examined both visually and by palpation for its effect on or SUV with DOTAP/DOPE at a 1:1 molar ratio. The transfection rates. Furthermore, the effects of different charge ratio for lipid:DNA was 1:2, giving the complex types of liposomes as adjuvant vectors were examined. a net negative charge. As with most other cells we tested, The purpose is to establish the usefulness of electropor- lipoplexes made of MLV were more efficient than those ation with or without liposomes for gene delivery to solid made of SUV.12 With this particular cell line, DOTAP tumors. The efficiencies and conditions for in vitro and in alone was as effective as DOTAP/DOPE mixture. There- vivo transfections, using the same tumor cells and plas- fore, lipoplex of DOTAP alone was used in subsequent mids, are compared. This study illustrates the potential experiments. of local gene delivery to solid tumors by applying electric Electrotransfection results are shown in Figure 2. A pulses with surface electrodes in a way comparable to a pulse field strength threshold is found at 800–900 V/cm, clinical setting. for both plasmid concentrations (162.5 and 650 ␮g/ml) we used. At high pulse field strength of 1600 V/cm, the transfection efficiency drops due to increased cell death. Results Lower transfection efficiency for samples with higher plasmid concentration at the optimal field strength may Tumor classification indicate membrane damage by DNA transport during In order to improve the reproducibility of gene electroporation.13 expression in a heterogeneous population of tumors, we classified tumors into medium–large (8–15 mm wide) and Transfection of tumors without electroporation small (Ͻ8 mm) groupings. Smaller tumors tended to be The relative levels of transfection by injecting naked firm, well vascularized, and fast growing, without areas DNA or lipoplexes made from SUV or MLV are shown of apparent necrosis. Medium-sized tumors were almost in Figure 3. All tumors were injected in the same day, as firm, with slightly uneven regions of growth and little without electroporation, then analyzed 2 days later. A P or no apparent necrosis. Large tumors (Ͼ11 mm) tend to value Ͻ0.05 is considered to be significantly independent be the least firm with variable regions of growth and may of chance for two results to be distinct. The differences contain non-growing regions. Most data presented in our between SUV and naked DNA and between SUV and studies of electroporation are from the medium–large MLV are significant. SUV lipoplexes transfected more group. Differences in luciferase expression between than twice as well as naked DNA or MLV lipoplexes on

medium and large tumors were minor. Data from the average. (Note that the Y-axis of Figure 3 is in log10 scale.) small tumor group are presented separately. The consistency of the results gave a small standard error Gross anatomical features of the tumors were recorded for each. The background level of relative light units at the time of injection. The apparent viability of several (RLU) was subtracted from all data values for this and tumors changed during the period between all other experiments. injection/electroporation and time of death. In few cases, we noted the unambiguous appearance of scabs, or Improving tumor transfection by electroporation eschar-like lesions, and large areas of non-cellular fluid Relative levels of transfection of medium–large tumors in the tumors 2 days after electroporation. This was not with SUV lipoplexes are enhanced by electroporation, as observable in most cases, though. Any apparent necrosis shown in Figure 4. Bars in the middle of each cluster rep- was noted at time of death. Tumors with apparent nec- resent average values of transfection for each applied rotic centers were excluded from the analysis. Sample pulse field strength category. The average values were tumors were cryosectioned for microscopic observation. calculated from the logarithmic values, then converted to When escharosis did appear, it was noted mostly on sur- the antilog value for plotting. From the scatter plot of faces contacting the electrodes. data points in each pulse field category, it is apparent that there is a threshold electric field strength value for In situ PCR successful electroporation enhancement. This threshold Figure 1 shows in situ PCR results from tumors that were occurs at field strengths between 400 and 800 V/cm. injected with no DNA, injected with DNA, but unpulsed, Above the threshold, the levels of transfection do not dif- and tumors injected with DNA and pulsed at 1.2 kV/cm. fer significantly from field value to field value, allowing Sections from control tumors showed very little fluor- for the variability within each category. Their average escence. The tumor with injected plasmids had consider- values are greater than those below the threshold by one ably more fluorescent cells than the control. The highest to two orders of magnitude. The differences are signifi- numbers of fluorescent cells were found in sections from cant, apart from one test pair (400 V/cm against 1600 tumors which had been electroporated following injec- V/cm). At this high end of applied field strength (1600 tion of the plasmid. V/cm), there appears to be a slight decline of transfection

Gene Therapy Gene delivery by electroporation of mammary tumors JM Wells et al 543

Figure 1 Fluorescence in situ PCR micrographs of frozen sections of MC2 tumors. (a) Uninjected tumors, (b) tumors injected with ‘naked’ plasmid DNA, (c) tumors injected with ‘naked’ plasmid DNA and subjected to electroporation (six 1.2 kV/cm pulses). Micrographs were taken with identical exposure and printing process. Bar = 40 ␮m.

Table 1 In vitro transfection with various lipid formulations

Sample Average value P values (RLU) (× 1000) SUV vs MLV

SUV DOTAP 351 ± 72 MLV DOTAP 7781 ± 1078 0.0038 SUV DOPE/DOTAP 790 ± 248 MLV DOPE/DOTAP 7389 ± 1078 0.0057 DNA 0.2

Figure 3 Transfection of MC2 tumors by injection of plasmids, as either uncomplexed DNA or as lipoplexes made from DOTAP SUV or MLV. Transfection is expressed as the logarithmic values of RLU. Control tumors were not injected with DNA. Error bars are for standard error values. For uncomplexed DNA, n = 5; SUV, n = 11; MLV, n = 10. P values using a two-tailed t test with even variance for each category were 0.016 for SUV versus naked DNA, 0.17 for MLV versus naked DNA, and 0.014 for SUV versus MLV.

lipoplexes, which likewise transfected well at 800 V/cm, were not tested with electroporation below that field strength. There are no data for the 1600 V/cm field Figure 2 Electrotransfection of MC2 cells in vitro. 162.5 ␮g/ml (࡯)or 650 ␮g/ml (᭜) of plasmid DNA was used. Six pulses 1 ms long and strength due to loss of mice and tumor necrosis. Transfec- at given field strengths were applied. Transfection was measured by the tion with electroporation at 2300 V/cm had the highest expression of luciferase (in relative luminescence unit RLU). value for any naked DNA injected tumor. The range of transfection values was also the greatest at this field strength. Consequently, the t test of the pair (0 V/cm enhancement. Transfection was much poorer at pulse against 2300 V/cm) is not significant. This is similar to fields as high as 4000 V/cm (data not shown). Transfec- the comparisons involving 1600 V/cm category in SUV tion results from injected but unpulsed samples are again lipoplex results. On an average, those transfections at the significantly higher than those from uninjected (control) 800–1100 V/cm range are again one to two orders of samples. Statistical analysis of SUV lipoplex data is given magnitude greater than that of unpulsed control. Statisti- in Table 2. Since there was no indication that electropor- cal analysis of naked DNA data is given in Table 3. ation with an electric field above 1100 V/cm produced Transfection of small tumors with naked DNA is any advantage, further pulsing at high field strengths shown in Table 4. Data from smaller tumors showed a was discontinued. Because of the spread in data, there trend towards higher relative transfection levels at higher are no statistically significant differences in transfection electric fields. Transfection with SUV lipoplexes showed results between any of the pulse field strengths at 800– the same trend (data not shown). The P values for 0 ver- 1600 V/cm range. sus 1600 V/cm and 0 versus 2300 V/cm were statistically Levels of transfection in medium–large tumors injected significant, 0.012 and 0.0007, respectively. The values for with naked DNA are also enhanced by electroporation, 0 versus 1100 V/cm and 1100 versus 2300 V/cm were as shown in Figure 5. The data show similar trends to 0.096 and 0.056, respectively. SUV lipoplex injected tumors. Naked DNA and MLV Because of the similarity in transfection levels and

Gene Therapy Gene delivery by electroporation of mammary tumors JM Wells et al 544 Table 3 P values of t test analysis for medium–large tumor transfection with naked DNA

t test Control 0 V 800 V 1100 V

0 V 0.034 800 V 0.0005 0.0078 1100 V 0.00065 0.021 0.29 2300 V 0.0028 0.077 0.86 0.72

Table 4 Electrotransfection of small tumors with naked DNA

Sample V/cm Average value of transfection

Control 0 6.533 DNA 1100 84.06 DNA 1600 673.7 Figure 4 Enhancement of transfection of medium–large MC2 tumors by DNA 2300 13304.0 electroporation. Lipoplexes made from DOTAP SUV were injected intratumorally before electroporation at given field strengths. Six pulses 1 ms long were applied across the tumor. Electric field strengths of 800 and 950 V/cm were clustered together for data analysis. Control tumors were not injected with DNA. Bars represent average logarithmic values. pulse field thresholds in naked DNA and SUV lipoplex data sets, data on MLV lipoplex transfection were col- lected only at the three most successful field strengths for Table 2 P values from t test analysis for medium–large tumor the naked DNA and SUV experiments: 800, 1100 and transfection with SUV lipoplexes 1600 V/cm. Again, no significant difference was found between transfection levels of these three field strength Treatment Control 0 V 360–400 V 900–950 V 1100 V settings. It is, therefore, reasonable to pool the transfection data in the 800–1600 V/cm range for each of the 0 V 0.0026 naked DNA, SUV, and MLV lipoplexes treatment groups 360–400 V 0.14 0.24 800–950 V 0.000046 0.0060 0.019 measured at different days. The transfection levels of 1100 V 0.00001 0.00070 0.0067 0.23 these three treatment groups are compared in Figure 6. 1600 V 0.0033 0.039 0.089 0.65 0.21 The difference between MLV and naked DNA was insig- nificant. The two-fold difference in average transfection levels between SUV complexes and DNA and MLVs was not statistically significant, under their most successful electrotransfection conditions. P values for SUV versus DNA and SUV versus MLV were 0.39 and 0.42, respectively.

Figure 5 Enhancement of transfection of medium–large MC2 tumors by electroporation. Uncomplexed (naked) DNA was injected intratumorally before electroporation at given ﬁeld strengths. Six pulses 1 ms long were applied across the tumor. Electric ﬁeld strengths of 800 and 950 V/cm were clustered together for data analysis. Control tumors were not injected with DNA. Bars represent average logarithmic value. Figure 6 Comparison of electroporation-enhanced transfection of MC2 tumors using injected uncomplexed DNA, or lipoplexes made from DOTAP SUV or MLV.

Gene Therapy Gene delivery by electroporation of mammary tumors JM Wells et al 545 Discussion MLV/naked DNA, respectively). Naked DNA is known to be effective in gene delivery to muscles15 and other Although electroporation has been applied successfully tissues, and may contribute to the higher than control in gene delivery to cells in vitro in many cases, reports transfection level in vivo (Figure 3). on gene delivery in vivo, especially to tumor tissues, has The comparison between in vivo and in vitro electro- been scanty. A major difficulty of in vivo gene delivery to transfection experiments is more favorable. The pulse tumors is the tissue heterogeneity. Reliable, quantitative field thresholds for electroporation in vivo and in vitro are conclusion can be derived only if a large number of similar, although the threshold for in vivo electrotransfec- tumors is used. We have not known of any study at tion is more difficult to define. The pulse field distri- this scale. bution through cells in an electroporation chamber in As indicated by the results, the optimal conditions for vitro is well controlled, while the field distribution in in vivo and in vitro experiments are significantly different. tumors is subjected to heterogeneous tissue conductivity, For instance, lipoplexes made of cationic MLV are much not to mention the abrupt drop of the electric field gradi- more effective than SUV in transfecting cultured MC2 ent across the stratum corneum of the skin surrounding cells (Table 1), while naked DNA (without the tumor. It is not surprising that in vivo results are electroporation) is rarely effective. However, the differ- more scattered. ences in relative transfection levels among these prep- For both electroporation and injection alone data, there arations are not very pronounced in vivo, with (Figure 6), is considerable scattering of more than an order of magni- or without electroporation (Figure 3). This is supported tude. The large scattering can be attributed to the biologi- by subsequent control experiments in electroporation cal variations among tumor tissues and from tumor to studies. Injection of naked DNA or SUV lipoplexes with- tumor. Retention of the injected DNA by the tumors adds out electroporation resulted in higher RLU than unin- to the variability. For electroporation data, additional jected tissues (P = 0.0026 and 0.034, respectively in Tables variables such as the effective electrical resistance of 2 and 3). tumors, contact between electrodes and skin surface, site The amount of DNA injected was chosen to be below of DNA injection with respect to the orientation of elec- the quantity which would cause high levels of toxicity to trodes, and extravasation of DNA into surrounding the tumor cells. The 40 ␮l volume injected caused limited tumor tissue differ from case to case, certainly contribu- tumor swelling at the time of injection without significant ting to the data scattering. In general, tumors that leakage of injected DNA from the tumor. This amount is appeared less firm and more scarred usually had low lev- consistent with quantities used by other researchers.5,9 els of relative luciferase expression. SUV lipoplex data The in situ PCR data in Figure 1 shows that in vivo points tend to scatter more than naked DNA data electroporation considerably increases DNA uptake in (Figures 4, 5 and 6), indicating that entrance of larger tumors cells over nonelectroporated tumors. Fluor- lipoplexes into cells demands more critical membrane escence microscopy showed a majority of the tumor cells disruption. The condition to create large membrane dis- were transfected in the vicinity of the needle injection site ruption, as opposed to small pores for DNA molecule in the pulsed tumors, but not in the unpulsed, injected transport, is more affected by tumor structure. tumors. The background fluorescence in the control The skin contributes a significant proportion of the tumor was due to endogenous tumor autofluorescence. electrical resistance during electroporation. In almost all Comparison with pulsed tumors not injected with plas- cases, especially in larger tumors, the electric resistance mid DNA showed comparable minor background fluor- of tumors dropped appreciably after the first few pulses. escence to unpulsed controls. In situ fluorescence was The resistance drop is indicative of the breakdown of the therefore not due to an artifact of the electroporation skin barrier and/or tissue cell membranes. The decrease itself. The in situ PCR results agree well with the trend of resistance leads to increase in current by subsequent detected by transfection with plasmids containing the pulses. Increases in current within a tumor lead to cell luc gene. death. Although cell death within the tumor is beneficial For transfecting cultured cells in vitro, because plasmid in cancer treatment, it defeats the purpose of transfection, DNA is accessible to all cells, the determining factor is and adds to the uncontrolled variability of results. By the transport of plasmid DNA into cells. Endocytosis is microscopy in vitro, we found cell mortality to be low at believed to be the major route of cellular uptake of plas- electric field strengths at or below 900 V/cm. In vivo the mid DNA in the case when lipoplexes are used as applied electric field threshold which produced a similar vehicles.14 The physical state of the lipoplexes is an low level of cell mortality was higher than 900 V/cm. important factor in the uptake process. Because of their Because of pulse-induced cell death in the tumors, fewer size, lipoplexes made from MLV have an advantage over electric pulses might have given more consistent results. those from SUV for uptake by endocytosis, and naked The enhancement of transfection by electroporation is DNA is ineffective because it is not present on the cell significant, given the scattering of data points. For both surface to a significant extent.12 On the other hand, in vivo SUV lipoplexes and naked DNA injections, tumors sub- injected plasmid DNA is accessible only to a portion of jected to electroporation of 800–1100 V/cm resulted in tissue cells. The determining factors could be the readi- significant improvement of transfection over unpulsed ness of naked DNA or lipoplexes to extravasate, pen- controls (P Ͻ 0.01 and ෂ0.02, respectively). No significant etrate, electrophorese and diffuse through tumor tissues. difference is found among results derived from tumors The integrity of the tumor vasculature plays an important pulsed at field strengths greater than 800 kV/cm. Higher role here. In this case, naked DNA and SUV lipoplexes field pulses (Ͼ1600 V/cm) tend to induce more data scat- may have an advantage over MLV because of their tering, especially toward a low transfection range, per- smaller sizes. The difference, however, is not always haps due to increased mortality caused by the electric significant (P = 0.014 and 0.17 for SUV/MLV and field. In the case of SUV lipoplexes, samples pulsed at

Gene Therapy Gene delivery by electroporation of mammary tumors JM Wells et al 546 lower fields (400 V/cm) are no different from unpulsed lipid DOTAP. The amount of DNA injected was always samples (P = 0.24), indicating a threshold pulse field 25 ␮g, in a total volume of 40 ␮l. For naked DNA injec- strength between 400 and 800 V/cm. The exact electric tion, the plasmid was dissolved in TE buffer. For lipo- field threshold probably varies somewhat from tumor to some complex (lipoplex) injection, lipoplexes were made tumor. The variable electrophoretic movement of DNA either with MLVs or with SUVs of DOTAP, with or with- during the pulse time may also account for the uncer- out the addition of a helper lipid, DOPE. All lipids were tainty of the exact threshold value. This threshold value purchased from Avanti Polar Lipids (Alabaster, AL, is comparable to that given by Heller et al9 for the electro- USA). The lipids in chloroform solution were dried in transfection of rat liver, and higher than that reported by vacuo for 4 h, then resuspended as MLVs by vortexing Rols et al11 for electrotransfection of melanomas. for 20 s in PBS. SUVs were made by sonicating MLVs for It appeared from the data (Table 4) that the transfection 10 min in a bath-type sonicator. The initial size range of of smaller tumors increases with stronger electric fields SUV was typically 3 nm in diameter, as measured by within the experimental range above the electric field quasi-elastic light scattering (Nicomp 300 Particle Sizer, threshold of 800 V/cm. Medium–large tumors showed Santa Barbara, CA, USA). Both lipoplexes of SUV/DNA less voltage dependence above the 800 V/cm threshold. and MLV/DNA were formed by mixing DNA and lipid Small tumors were more consistently firm and well vas- at a 1:2 (lipid:DNA) charge ratio. cularized. Their earlier stage of development may render them less susceptible than medium or large tumors to Transfection of MC2 cells in vitro × 6 pulse-induced mortality over the 2-day period between For transfection by lipoplex, 3 10 cells in suspension electroporation and death of the animal. This may result were cultured in 5 ml RPMI 1640 medium with 7% fetal in higher viability and hence transfection levels at higher bovine serum, in each well of six-well plates. Transfection with lipoplexes containing 0.1 ␮mol plasmid DNA and pulse fields. ␮ Our results show that electroporation enhances local 0.05 mol cationic lipid per well was carried out in the gene delivery by about two orders of magnitude (Figures presence of 7% serum for 4 h, after which the cell suspension was centrifuged and resuspended in fresh medium. 4 and 5). The effectiveness is proven through experiments ␮ on a large number of tumor samples. As seen from Figure For electrotransfection, 15 l of cells at a concentration of 5 × 107 cells/ml in B+K buffer were mixed with uncom- 6, the choice of whether to use liposomes or naked DNA 13 with a local injection does not make a statistically signifi- plexed plasmids in a custom made electrode chamber. cant difference. This is our most important finding. The Cells were pulsed six times with 1 ms pulses at specified condition for enhancement of gene delivery is not yet field strengths. Immediately after pulsing, cells were resuspended in 100 ␮l of medium and incubated for 20 optimized. Further experiments on reducing the number ° of pulses and using a series of pulses of decreasing field min at 37 C. Then cells were transferred into 1 ml of strength may further improve the enhancement ratio. fresh medium. Selection of smaller tumors alone could improve the data After 24 h, cells in suspension were centrifuged, supernatant removed and 250 ␮l of cell lysis buffer (Promega) scattering. We believe that, as accuracy and efficiency + improve, in vivo electroporation will become an was added. Lysed cells lysis buffer were centrifuged at important adjuvant therapeutic mode in the future. 11000 g for 10 s to remove cell debris. Extracts were assayed by adding 100 ␮l of luciferin reagent to 10 ␮lof the lysed cell solution. Luciferase activity of the super- Materials and methods natant was measured for 10 s with a Lumat LB 9501 luminometer (Berthold, Dallas, TX, USA). Growth of MC2 mammary tumors In vivo electroporation MC2 murine mammary tumors, which were derived Electric pulses were delivered to tumors via a custom- from inbred C3H/He mice, were propagated in a con- built electrode pair. This parallel pair of stainless steel sanguineous colony. MC2 cells and the initial inbred C3H electrode plates was constructed on calipers to deliver a mice were a gift from Dr Jan Vaage of this institute. 106 uniform pulse across a measured distance (0.3–1.0 cm). cultured MC2 cells were injected subcutaneously into the The design of the calipers permitted direct measurement lateral regions of inbred C3H mice, 4–7 weeks before of the width between the electrodes. The area of the elec- transfection experiments. By then tumors grew to diam- trodes actually in contact with the skin was around 0.5 eters ranging from small (0.4–7 mm) to large (8–15 mm). cm2. Signa electrode gel (Parker Laboratories, Fairfield, Tumor growth between the time of transfection and the NJ, USA) was smeared lightly on the surface of the elec- time of death was generally minor. trodes. The use of electrode gel was necessary to improve consistency of the pulses and to overcome irregularly Preparation of plasmid DNA and lipoplexes ␤ shaped surface features and the resistance barrier of pSV- -galactosidase and pGL3 luciferase plasmids were mouse hair. The gel greatly increased the reproducibility purchased from Promega (Madison, WI, USA). Originally ␤ of the pulse parameters (voltage and current) across the -galactosidase plasmids were used instead of lucifer- tumor tissues. A Velonex (Venus, CA, USA) model 345 ase plasmids, until it was discovered that the endogenous ␤ pulse generator was used to deliver 1 ms square wave -galactosidase levels of the tumors were too high to be pulses. Six consecutive pulses, 2 s apart, were delivered used in vivo. Mammalian ␤-galactosidase genes are not ␤ to each tumor in two different directions normal to the homologous to the bacterial -galactosidase sequences of axis of the injection needle path. the pSV plasmids used, therefore the in situ PCR results were unaffected by mouse ␤-galactosidase genes. The In situ PCR plasmid DNA were injected intratumorally, either alone We developed an in situ PCR protocol for the detection (‘naked DNA’) or in a complex form with the cationic of plasmid ␤-galactosidase DNA uptake into tumor cells

Gene Therapy Gene delivery by electroporation of mammary tumors JM Wells et al 547 transfected in vivo. Control tumors received no injection cols, over a period of 6 months, were pooled. Because the of plasmid DNA. Unpulsed tumors were injected with 25 results differ by several orders of magnitude, and large ␮g pSV ␤-gal plasmids, but were not electroporated. variations exist within the same treatment protocol, the Pulsed samples were injected with 25 ␮g ␤-gal plasmids logarithmic (base 10) values of transfection level (in and pulsed six times at an electric field strength of 1.2 RLU/mg of protein) were analyzed statistically. A Stud- kV/cm. Mice were killed 6 h after injection/ ent’s t test was applied to define the significance of electroporation. Tumors were removed and immediately results between treatment groups. frozen in liquid nitrogen. The frozen tumors were embed- ded and mounted in Cryo-gel embedding medium Acknowledgements (Instrumedics, Hackensack, NJ, USA). Several 4–6-␮m thick sections were cut from the frozen tumors and This work is supported by a grant GM 30969 from the placed on microscope slides, fixed overnight in a 4% for- National Institutes of Health. We thank Dr John Yates, malin, 0.25 m PBS solution and subjected to in situ PCR Department of Cancer Genetics, for the use of the lumino- the next morning. The sections were washed three times meter, and Dorothy Donovan for her help in starting the with PBS and treated with Proteinase K (0.01 mg/ml) at mouse and MC2 cell colonies. Statistical analysis was per- 37°C for 16 min and rinsed with distilled water. The sec- formed with the help of Dr William Greco, Biostatistics tions were then dehydrated through a series of ethanol Facility, which is supported by the CCSR grant CA16056 washes (10, 30, 60 and 100% ethanol in PBS) and air- from the National Cancer Institute. dried. A gasket was placed around each section to form a chamber and 80 ␮l of a PCR reaction mix added to the References sections and covered using a glass cover slip. The PCR 1 Chang BM, Chassy JA, Saunders, Sowers AE (eds). Handbook of reaction mix contained 10 mm Tris, 50 mm KCl, 1.5 mm ␮ Electroporation and Electrofusion. Academic Press: New York, MgCl2, 200 m each of dATP, dCTP, dGTP and dTTP, 10 1992. units TAQ polymerase. 200 pmol of each of the two each 2 Prausnitz MR, Bose VG, Langer R, Weaver JC. Electroporation primers were added to the PCR mix. Fluorescein-12- of mammalian skin: a mechanism to enhance transdermal drug dUTP, 22 pmol (Boehringer Mannheim, Indianapolis, IN, delivery. Proc Natl Acad Sci USA 1993; 90: 10504–10508. USA) was added to the PCR mix to enable fluorescence 3 Gallo SA, Oseroff AR, Johnson PG, Hui SW. Characterization of detection of the PCR product. The primer sequences were the electric pulse induced permeabilization of porcine skin using obtained using the GCG program and the two 18-mer surface electrodes. Biophys J 1997; 72: 2805–2811. primers were synthesized at the Biopolymer Facility of 4 Mir LM, Orlowski S, Belehradek J, Paoletti C. Electrochemother- the institute. The slides with the sections were placed on apy: potentiation of antitumor effect of bleomycin by electric pulses. Eur J Cancer 1991; 27: 68–72. a custom designed copper plate and placed on the heat- 5 Jaroszeski MJ, Gilbert RA, Heller R. In vivo antitumor effects in ing plate of the thermal cycler (Perkin Elmer GeneAmp a hepatoma model. Biochim Biophys Acta 1997; 133: 15–18. PCR 9600). PCR was run for 30 cycles using standard pro- 6 Heller R, Jaroszeski MJ, Glass LF, Messina JL. Phase I/II trial tocol. The sections were then first washed with SSC (300 for the treatment of cutaneous and subcutaneous tumors using mm NaCl, 30 mm Na Citrate, pH 7.0) followed by a final electrochemotherapy. Cancer 1996; 77: 964–971. wash with distilled water and air-dried. The labeled sec- 7 Titomirov AV, Sukharev S, Kistanova E. Electroporation and tions were examined and photographed using an stable transformation of skin cells of newborn mice by plasmid Olympus IMT-2 fluorescence microscope. DNA. Biochim Biophys Acta 1991; 1088: 131–134. 8 Zhang L, Li L, Hoffmann GA, Hoffman RM. Depth targeted efficient gene delivery and expression in the skin by pulsed elec- Transfection assay tric fields: an approach to gene therapy of skin aging and other Forty-eight hours after the injection of DNA or lipoplex, diseases. Biochem Biophys Res Com 1996; 220: 633–636. with or without electroporation, mice were killed and 9 Heller R, Jaroszeski MJ, Atkin A, Moradpour D. In vivo gene tumors removed. Tumors were frozen in liquid nitrogen electroinjection and expression in rat liver. FEBS Lett 1996; 389: until ready for homogenization. PBS without Mg2+ was 225–228. added to tumor extracts at a 9:1 volume ratio. Tumor 10 Nishi T, Yoshizato K, Yamashiro S, Takeshima H. High efficiency in vivo gene transfer using intra-arterial plasmid DNA tissues were thawed, then homogenized on ice for 20 s at injection following in vivo electroporation. Cancer Res 1996; 56: a medium high setting in a Polytron tissue homogenizer 1050–1055. (Brinkmann Instruments, Mississauga, ON). Extracts 11 Rols MP et al. In vivo electrically mediated protein and gene were twice centrifuged for 10 min in a microcentrifuge transfer in murine melanoma. Nat Biotech 1998; 16: 168–171. at 11000 g. The supernatant transferred into new tubes 12 Ross P, Henson ML, Supabhol R, Hui SW. Multilamellar cationic each time. 100 ␮l of extract was mixed with 300 ␮l lucif- liposomes are efficient vectors for in vitro gene transfer in serum. erin assay reagent (Promega). Luciferase activity of the J Liposome Res 1998; 8: 499–520. supernatant was measured for 30 s by luminometer. Rela- 13 Li LH, Ross P, Hui SW. Improving electrotransfection efficiency tive light units (RLU) were normalized to miligrams of by post-pulse centrifugation. Gene Therapy 1999; 6: 364–372. 14 Hui SW, Langner M, Zhao YL, Ross P. The role of helper lipids sample proteins, as determined by a BCA protein assay in cationic liposome mediated gene transfer. Biophys J 1996; 71: (Pierce Chemicals, Rockford, IL, USA). Optical density 590–599. values for the protein assay were measured in a Beckman 15 Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A. Long-term DU-40 spectrophotometer (Fullerton, CA, USA). persistence of plasmid DNA and foreign gene expression in Results from all tumors treated with the same proto- mouse muscle. Hum Mol Genet 1992; 1: 363–369.

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