Complete Regression of Established Subcutaneous B16 ... - Cell Press

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doi:10.1016/j.ymthe.2006.06.010

Complete Regression of Established Subcutaneous B16 Murine Melanoma Tumors after Delivery of an HIV-1 Vpr-Expressing Plasmid by in Vivo Electroporation Andrea N. McCray,1,* Kenneth E. Ugen,1,2,*,y Karuppiah Muthumani,3 J. J. Kim,4 David B. Weiner,3 and Richard Heller1,2 1

Department of Medical Microbiology and Immunology and 2Center for Molecular Delivery, University of South Florida College of Medicine, Tampa, FL 33612, USA 3 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA 4 VGX Pharmaceuticals, Blue Bell, PA 19422, USA *These authors contributed equally to this study. y

To whom correspondence and reprint requests should be addressed at the Department of Medical Microbiology and Immunology, MDC 10, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, USA. Fax: +1 813 974 4151. E-mail: [email protected].

Available online 1 September 2006

Novel therapies and delivery methods directed against malignancies such as melanoma, and particularly metastatic melanoma, are needed. The HIV-1 accessory protein Vpr (viral protein R) has previously been demonstrated to induce G2 cell cycle arrest as well as in vitro growth inhibition/ killing of a number of tumor cells by apoptosis. In vivo electroporation has been utilized as an effective delivery method for pharmacologic agents and DNA plasmids that express ‘‘therapeutic’’ proteins and has been targeted to various tissues, including malignant tumors. For the study reported here, we hypothesized that intratumoral delivery of a Vpr expression plasmid through in vivo electroporation would induce apoptosis and growth attenuation or regression of melanoma tumors. Established subcutaneous B16.F10 melanoma tumors were injected intratumorally with a Vpr-expressing (either 25 or 100 Mg) plasmid, followed by electroporation, on day 0 (i.e., when tumors had attained an appropriate size) and day 4. Treatment with 25 or 100 Mg of the Vprexpressing plasmid resulted in complete tumor regression with long-term survival in 14.3 and 7.1% of the mice, respectively. In addition, electroporative delivery of the Vpr-expressing plasmid was shown to induce apoptosis in tumors after intratumoral injection. This is the first report demonstrating the ability of Vpr, when delivered as a DNA expression plasmid with in vivo electroporation, to attenuate melanoma lesion growth and induce complete tumor regression coupled with long-term survival of mice in a highly aggressive and metastatic solid tumor model. Key Words: In vivo electroporation, HIV-1 Vpr, B16.F10 melanoma, metastasis, tumor regression, long-term survival

INTRODUCTION Melanoma accounts for nearly 80% of the mortality due to skin cancers, predominantly because of metastatic spread [1]. Treatment options for melanoma, particularly metastatic melanoma, other than surgery are relatively limited and underscore the need for the development of novel efficacious therapies. Novel therapies against malignant tumors should ideally induce cytotoxicity against tumor cells without affecting normal cells and provide an efficient level of tumor cell killing to overcome resistance to apoptosis. One such novel therapy that fulfills these requirements is the

MOLECULAR THERAPY Vol. 14, No. 5, November 2006 Copyright C The American Society of Gene Therapy 1525-0016/$30.00

approximately 15-kDa HIV-1 accessory protein Vpr (viral protein R) [2,3]. Previous studies have demonstrated that Vpr, in addition to its accessory and regulatory functions as a protein of HIV-1, has various other activities including induction of G2 cell cycle arrest [4,5] and apoptosis [6]. In fact, it has been demonstrated that Vpr elicits in vitro antiproliferative effects in a number of tumor cell lines, including melanoma, and induces p53-independent apoptosis [7–9]. Also, an important characteristic of Vpr that makes it an attractive putative anti-cancer agent is its ability to target, inhibit, and kill almost exclusively proliferating cells, such as tumor cells [6,8]. Therefore,

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current evidence indicates that normal (i.e., nonmalignant) cells are not significantly inhibited nor killed by Vpr. As well, we previously demonstrated that Vpr can suppress melanoma growth (i.e., lung colonization) in a mouse model when tumor cells were transfected in vitro with Vpr and subsequently injected intravenously [10]. Likewise, investigations from other groups have also demonstrated an anti-cancer activity for Vpr [6,8,11–14]. Based upon these provocative findings it was hypothesized that Vpr would have anti-tumor activity when delivered in vivo into established murine melanoma tumors. In vivo electroporation has been demonstrated to be a safe and efficient method for gene delivery that increases membrane permeability by exposing cells to electric fields and effectively enhances expression of protein from relevant DNA plasmid vectors [15–17]. The technique of administering a DNA plasmid that expresses immunomodulatory molecules through electroporation has been applied to numerous types of tumors such as colon and renal [18,19], mammary [20], esophageal [21], and liver [22] malignancies. There are several advantages associated with using DNA expression plasmids to deliver therapeutic proteins of choice, including the apparent superior safety profile for this nonviral delivery modality compared to the administration of agents/proteins through attenuated viral vectors [23–25]. The efforts, however, to use DNA expression plasmids therapeutically have often been limited most likely by ineffective/inefficient delivery methods. To this end, electroporation should have potential for enhancing the in vivo delivery and expression of therapeutic DNA expression plasmids. Our group has previously used various reagents/molecules successfully to demonstrate the use of in vivo electroporation as a delivery method to obtain complete regression of these murine melanoma lesions [26,27]. A number of recent studies have focused on the electrically mediated delivery of cytokine plasmids [26,28–31]. However, the development of plasmid-based reagents that encode other therapeutic proteins is relevant to determine and characterize. Therefore, based upon the previously determined in vitro anti-tumor activity of Vpr we hypothesized that intratumoral gene delivery of a plasmid expressing this protein, through in vivo electroporation, would result in tumor growth attenuation or complete regression of established B16 melanoma tumors in mice. We selected the B16.F10 murine melanoma model for these studies since this tumor cell line has been extensively characterized and is a very good model for many human malignancies due to its highly invasive and metastatic nature. In addition, this tumor is nonimmunogenic or poorly immunogenic and is characteristically difficult to treat or bcureQ once visible tumors have developed. These qualities further contribute to its attractiveness as a model for human tumors [32,33]. In fact, typically in this model any therapeutic intervention resulting in only

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tumor growth delay is considered to be significant and potentially translatable to the treatment of human melanomas. In this report, we demonstrate the therapeutic activity of in vivo electrically mediated delivery of a Vprexpressing DNA plasmid against established subcutaneous B16.F10 melanoma tumors in syngeneic C57BL/6 mice. This is the first description of the ability of a plasmid expressing this HIV-1 accessory protein to cause complete regression, when delivered by in vivo electroporation, of an aggressive metastatic tumor such as melanoma.

RESULTS Construction and Confirmation of Expression of pVpr The construction of the Vpr plasmid used in this study is depicted in Fig. 1A. We used the DNA fragment containing the HIV-1 strain NL4-3 as a template for construction of the Vpr plasmid (pVpr). A bbackboneQ control plasmid (pcDNA3.1) in which the Vpr gene was absent was used as a negative control. Briefly, after PCR amplification using specific primers the product was purified and digested with HindIII and BamHI restriction enzymes and the fragments were ligated into the pcDNA3.1 vector. Positive clones were identified by restriction digestion and the correct clones were confirmed by DNA sequencing. To confirm and examine the expression of Vpr, we performed a standard in vitro transcription/translation assay with [35S] methionine followed by an immunoprecipitation analysis using an anti-Vpr antibody. Fig. 1B demonstrates well-defined bands at an approximate size of 14–15 kDa in the pVpr lane. The pcDNA3.1 vector backbone was used as a negative control. To test further for expression of pVpr in a relevant cell line, we transfected HeLa cells with the vector control or pVpr. We extracted total protein 72 h posttransfection and immunoblotted it with the anti-Vpr antibody. As indicated in Fig. 1C, the Vpr construct expressed a detectable Vpr protein at the size of 14–15 kDa, indicating the effectiveness and ability of the construct to be used in the subsequent in vivo experiments. In Vitro Vpr-Induced Apoptosis A previous study demonstrated that Vpr activates apoptotic events through a caspase 9-mediated mechanism [34]. To investigate the potential ability of Vpr to induce apoptosis in B16.F10 cells, we utilized flow-cytometric methods to measure annexin V binding as an apoptosisassociated specific event. In this experiment, we transfected B16.F10 cells, as indicated, with 10 Ag of pcDNA3.1, pGag, or pVpr. We analyzed the cells at 72 h posttransfection for apoptosis by annexin V staining depicted in a histogram (Fig. 2). A histogram of annexin V staining shows that 48.9% of the cells were induced to undergo apoptosis in Vpr-transfected cells compared to 0% in the backbone vector or 6.9% with a control pGag construct. These results indicated the ability of Vpr to

MOLECULAR THERAPY Vol. 14, No. 5, November 2006 Copyright C The American Society of Gene Therapy

doi:10.1016/j.ymthe.2006.06.010

FIG. 1. Construction and expression of the Vpr plasmid. (A) Schematic representation of Vpr plasmid construction. (B) Expression from the Vpr plasmid. Expression plasmids (2 Ag) were used for coupled in vitro transcription/translation reactions carried out as described under Materials and Methods. Immunoprecipitation of the in vitro-translated proteins was performed with an anti-Vpr polyclonal antibody. The immunoprecipitated proteins were eluted from the Sepharose beads and subjected to SDS–PAGE using 12% gels and processed for fluorography as described under Materials and Methods. The expression of the Vpr protein is indicated by the arrow. (C) Western blot analysis of Vpr protein expression. Protein extracted from HeLa cells transfected with 5 Ag of vector control or pVpr plasmids was prepared as described under Materials and Methods and resolved on a 12% SDS–PAGE gel. Gels were transferred to PVDF membranes and immunoblotted with antiVpr (1:500)-specific antibodies as indicated. The arrowheads indicate the molecular size markers.

stimulate apoptosis specifically in B16.F10 melanoma cells after in vitro transfection. Vpr Effects on Melanoma Growth When Delivered With in Vivo Electroporation To examine any therapeutic effect of in vivo electroporative delivery of pVpr, we performed posttreatment tumor volume measurements to assess any attenuation of tumor growth as well as the induction of complete tumor regression coupled with long-term mouse survival. As


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indicated, for these experiments, we treated the appropriately sized tumors among the various groups with either 25 or 100 Ag of pVpr on days 0 and 4 with or without in vivo electroporation and measured the lesions at different posttreatment time points. Fig. 3 summarizes melanoma tumor growth in the various treatment groups during the initial 25 days of the experiment. Day 25 posttreatment was chosen as the endpoint for the mean fold increase curve because this was the last time point at which a majority of the treated mice were still alive for accurate assessment of growth attenuation for each group. The absence of a mean fold tumor volume increase value at day 25 for a particular group indicates that all of the mice had expired or had been sacrificed by that time point. During the first week following treatment, tumor growth was retarded to some extent in all the treatment groups compared to the untreated control group (i.e., Figs. 3B, C, and D versus 3A). Values for the mean fold increase in tumor volume, compared to day 0, are indicated for all the treatment groups through day 18 since at this time point at least 50% of all of the mice in each of the treatment groups were still alive, with the exception of three (21.4%) mice alive in the untreated group. At day 18 posttreatment the mean fold tumor volume increase was significantly less ( P b 0.05, Student’s t test) for the P+E+ 25 Ag pVpr, P+E 100 Ag pVpr, and P+E+ 100 Ag pVpr groups, with values of 11.3, 21.2, and 7.6, respectively, compared to the no-treatment group (mean fold tumor volume increase of 45.4). The value for the P+E 25 Ag pVpr group was not significantly different from that of the no-treatment group. Likewise, at day 18 the V+E 100 Ag pcDNA3.1 and V+E+ 100 Ag pcDNA3.1 control groups (i.e., backbone vector delivered without or with electroporation), but not the saline plus electroporation control, were significantly decreased compared to the no-treatment group. For the groups with mean fold tumor volume increase values at day 25, both the P+E+ 25 Ag and P+E+ 100 Ag pVpr groups had significantly decreased values (i.e., 16.2 and 12.4, respectively) compared to the V+E+ 100 Ag pcDNA3.1 group (i.e., 29) and the P+E 100 Ag pVpr group (i.e., 74.4). These results indicated an initial backbone vector plus electroporation effect, in terms of slowing of tumor growth. However, at day 25, the mean fold tumor volume increases for the pVpr plus electroporation treatment groups were significantly suppressed compared to the values measured in the pcDNA3.1 control plus electroporation group. While tumor growth rate reduction is an important evaluative criterion for assessing therapeutic potential, the critical parameter is the regression of existing tumors, i.e., a bcure.Q We observed stable tumor regression only in the groups that received the pVpr plus electroporation treatment regimens (Table 1). The 100 Ag pVpr+ E+ group had the highest percentage of mice

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FIG. 2. Vpr-induced in vitro apoptosis. B16 murine melanoma tumor cells (1.5–2 106 cells) were transfected with pcDNA3.1 (control vector), pGag (coding negative control), or pVpr. Cells were collected 72 h posttransfection and stained with annexin V–FITC and PI. Vpr-induced apoptosis analysis was performed on gated low forward scatter and side scatter cells. The values indicated in the histograms are the frequencies of annexin V-positive cells. Data are representative of two independent experiments.

with complete tumor regression (42.9%) on day 14 posttreatment. In addition, on day 14 posttreatment, mice that received 25 Ag pVpr+ E+ or 100 Ag pcDNA3.1+ E+ had complete tumor regression in 14.3 and 7.1% of mice, respectively. Importantly, the pcDNA3.1+ E+ treated mouse that experienced tumor regression remained tumor free for only 4 days before recurrence of the tumor. At day 28 posttreatment, 14.3% of mice treated with 25 Ag pVpr plus electroporation and 21.4% of mice treated with 100 Ag pVpr plus electroporation had complete tumor regression. It is important and relevant to indicate that none of the mice in the pcDNA3.1+ E+ treatment group underwent prolonged complete tumor regression nor did they survive long term. This finding is further presented in the results summarized in the survival curve shown in Fig. 4. As indicated, the ultimately relevant end-point indicating the anti-tumor therapeutic potential of the pVpr plus electroporation treatment would be the ability of this regimen to induce complete tumor regression coupled with long-term survival of the mice, i.e., a bcure.Q This would be an obvious requisite for any future meaningful translation of this treatment to the clinic. Sustained tumor regression and survival for 100 days posttreatment is considered the benchmark for cure in mice in this model. To that end we examined, after the initiation of the treatment regimens, the overall survival and complete tumor regression among the various groups through this temporal benchmark. These data are presented in the Kaplan–Meier survival curve in Fig. 4. Mice in the untreated group as well as in groups receiving 25 or 100 Ag pVpr without electroporation, pcDNA3.1 with or with-

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out electroporation, or saline plus electroporation all succumbed to tumor burden, with none surviving to the day 100 benchmark. The mean survival time for untreated mice was 13.6 days. Mice in the untreated group had a significantly shorter survival (i.e., at P b 0.05) compared to all groups that received either dose of pVpr with or without electroporation, the high dose pcDNA3.1 group with or without electroporation, and the saline plus electroporation group. The mean survival times for mice treated with 25 or 100 Ag pVpr without electroporation, 100 Ag pcDNA3.1 without electroporation, 100 Ag pcDNA3.1 with electroporation or saline plus electroporation (i.e., electric pulses alone) were 17.8, 19.4, 19.3, 24.2 and 17.3 days, respectively. The mean survival times for the mouse groups treated with 25 or 100 Ag pVpr plus electroporation were 40.5 and 43.2 days, respectively. There was no statistical difference between the mean survival times for mice treated with 25 Ag and with 100 Ag pVpr plus electroporation. Delivery of the plasmid with electroporation in the 25 or 100 Ag pVpr treatment regimen resulted in complete tumor regression coupled with long-term survival for at least 100 days in 14.3 and 7.1% of the mice, respectively. Likewise, there was no statistically significant difference ( P b 0.05) between the regression/long-term survival rates in the 25 Ag versus 100 Ag pVpr plus electroporation treatment groups. In Vivo Analysis of Vpr-Induced Cell Death It was important to determine the in vivo Vpr expression efficiency and any Vpr-induced apoptosis after intratumoral injection of pVpr into established subcutaneous


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FIG. 3. B16 melanoma tumor growth responses to pVpr delivered with or without in vivo electroporation. Mean fold increase in tumor volume (compared to day 0) at various posttreatment time points is shown for the treatment groups: (A) no treatment, (B) 25 Ag pVpr with or without electroporation, (C) 100 Ag pVpr with or without electroporation, and (D) 100 Ag pcDNA3.1 with or without electroporation and saline plus electroporation. Key: P, pVpr; V, pcDNA3.1; E+ and E indicate with or without electroporation, respectively.

B16 tumors with in vivo electroporation. To address this issue, we excised melanoma tumors 48 h after a single intratumoral treatment with 100 Ag of pVpr delivered through electroporation. After appropriate sectioning and

TABLE 1: Percent of mice with complete tumor regressions Groups No Treatment P+E 25 Ag pVpr P+E+ 25 Ag pVpr P+E 100 Ag pVpr P+E+ 100 Ag pVpr V+E 100 Ag pcDNA3.1 V+E+ 100 Ag pcDNA3.1 Saline+E+

Day 14

Day 28

0 0 14.29% (2 out of 14) 0 42.86% (6 out of 14) 0 7.14% (1 out of 14)* 0

N/A N/A 14.29% (2 out of 14) N/A 21.43% (3 out of 14) N/A 0 N/A

N/A indicates that all mice in this group are dead at this time point. * Indicates that this mouse only had tumor regression for 4 days before tumor recurred.


tissue fixation, we stained tumor sections with a rabbit polyclonal anti-Vpr antibody and a TUNEL reaction mixture for measurement of protein expression and apoptosis, respectively (Fig. 5). After examination of the sections by fluorescence microscopy under dual filters we assessed the areas of fluorescence, indicative of expression of Vpr and apoptosis. Figs. 5A and B show tumor sections from pVpr plus electroporation treatment and pcDNA3.1 plus electroporation treatment, which were stained with Hoechst reagent, which is a positive control that stains nuclei. Figs. 5C and D represent PE-conjugated anti-Vpr antibody staining for the pVpr plus electroporation and pcDNA3.1 control plus electroporation groups, respectively. Although some background staining is present in the pcDNA3.1 section, staining in the pVpr section is considerably more extensive and intense, indicating expression of Vpr. Figs. 5E and F show overlays of the tumor sections indicating Vpr and Hoechst staining of pVpr plus electroporation and pcDNA3.1 plus electroporation treatment groups, respectively. Figs. 5G and H show Hoechst staining for consecutive sections from the

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pVpr- and pcDNA3.1-treated tumors and serves as the positive control for nuclei in the TUNEL staining. Finally, Figs. 5I and J show TUNEL staining (specifically nuclear staining) of sections from tumors treated with pVpr plus electroporation and pcDNA3.1 plus electroporation, respectively. This result demonstrated specific TUNEL staining (fluorescein), indicative of apoptosis, only in the pVpr plus electroporation treatment group. In summary, the results demonstrate specific Vpr expression and apoptosis induced by the Vpr DNA construct when delivered intratumorally, with electroporation, into these aggressive and established subcutaneous B16.F10 tumors. Overall, the data demonstrate the ability of a plasmid expressing Vpr, when delivered electroporatively to B16.F10 tumors, to result in effective long-term survival of treated mice coupled with complete tumor regression.

DISCUSSION

FIG. 4. Kaplan–Meier survival curve of mice in different treatment groups up to 100 days posttreatment. See definitions and the key provided in the legend for Fig. 3.

As indicated, a number of investigations have determined that the HIV-1 accessory protein Vpr induces cell cycle arrest at the G2/M phase of the cell cycle as well as stimulation of apoptosis in a large variety of tumor cells [4–6]. In addition, some of us have previously reported that B16.F10 melanoma cells, when

FIG. 5. In vivo expression of Vpr and induction of apoptosis after pVpr treatment with electroporation. For localization of Vpr in vivo, tumor sections were frozen in OCT medium and cryopreserved and 5-Am sections were prepared for viewing. Sections were stained with the TUNEL reaction mixture and anti-Vpr antibody (1:2000). The left represents tissue sections from tumors injected with pVpr plus in vivo electroporation and the right from tumors injected with the control plasmid plus in vivo electroporation. (A and B) Nuclear staining (Hoechst) for the sections stained with anti-Vpr. (C and D) Sections stained with antiVpr antibody (PE conjugated, red). (E and F) Overlays of the Hoechst and anti-Vpr antibody for the sections stained with anti-Vpr antibody. (G and H) Nuclear staining (Hoechst) of the sections for the TUNEL assay staining. (I and J) TUNEL assay staining (fluorescein conjugated, green) for apoptosis. The data presented are from one of two independent assays that produced similar results. The images are shown at 20 original magnification.

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transfected with a plasmid expressing Vpr, are significantly less efficient in inducing tumor colonies in the lungs of C57BL/6 mice following intravenous injection [10]. We therefore hypothesized that Vpr, when delivered to established murine melanoma tumors, will mediate a significant anti-tumor effect with potential clinical utility. Therefore, based on the rationale presented in the introduction, Vpr was delivered as a DNA expression plasmid. The successful use of electroporation to transfer plasmid to cells in vitro and the concept that electric fields can be safely and efficiently offered a basis for electrically mediated delivery of plasmid in vivo [16,17]. Several studies have demonstrated that electrically mediated delivery of plasmids that encode and express therapeutic molecules can be directed efficiently to tumors. As indicated above, the delivery of therapeutic plasmids has been demonstrated in many tumor types. Examples involving experimental melanoma treatment indicated that delivery of plasmids encoding GM-CSF and IL-2 [35], IL-12 [26,28,36,37], IL-15 [31], tumor antigens [38,39], dominant-negative Stat3 [40], and IFN-a [41] elicit an anti-tumor effect. Importantly, electrically mediated delivery of molecules has been associated with minimal adverse effects and can safely be administered multiple times. Therefore, in vivo electroporation was utilized as a delivery method to maximize the potential for a therapeutic effect of Vpr when administered as a DNA expression plasmid. The data presented here indicate that pVpr, when delivered through electroporation, was expressed intratumorally in subcutaneous melanomas at presumably sufficiently high levels to induce complete tumor regression coupled with survival in some of the treated mice. In addition, attenuation of tumor growth was noted in a number of the groups, which was clearly enhanced when the plasmid was delivered by electroporation. Also, there appeared to be evidence of an initial and temporary bnonspecificQ plasmid plus electroporation or electroporation alone effect in terms of effects on tumor growth. That is, the vector backbone plus electroporation treatment regimen attenuated tumor growth. The effects of noncoding DNA and electroporation on attenuating tumor growth have previously been reported in other studies and have been attributed to the putative immunostimulatory activity of CpG motifs within these plasmids [29,42]. Some of these previous studies, by other investigators, also demonstrated tumor regression; however, the electroporation conditions used in these investigations were considerably more stringent than the conditions used in the study reported here. Likewise, an initial and temporary mild tumor growth attenuation effect was noted in the electroporation alone control group. This may be attributable to an initial inflammatory effect or necrosis effect due to electroporation. These observations underscore the necessity to utilize the


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bminimalQ electroporative conditions for the elicitation of bgene productQ-specific therapeutic effects. However, it should be pointed out that in the study reported here the control treatment groups (i.e., pcDNA3.1 control vector plus electroporation or electroporation alone) did not result in long-lasting complete tumor regression with long-term mouse survival. The pcDNA3.1+ E+ treatment induced tumor regression in only one mouse and was very short-lived (i.e., 4 days). Therefore, only the pVpr plus electroporation treatment regimen resulted in bcuresQ in any of the mice. As indicated, B16.F10 melanoma tumors are characteristically highly invasive and metastatic as well as being poorly immunogenic [32,33]. In addition, complete regression of established B16.F10 tumors, coupled with longterm survival, is extremely difficult to attain through therapeutic interventions. In addition, it is significant that in this study the treatment regimen was delivered to established tumors rather than administered either concomitantly with initial tumor cell challenge or before tumors had visibly formed. At early time points in the study, the 100 Ag pVpr plus electroporation treatment group had high percentages of mice with complete tumor regression. However, in a number of these animals the tumors recurred, presumably due to residual viable cells from this aggressive tumor. In sum, the electroporative delivery of 100 Ag pVpr was more effective than the 25 Ag dose at the early time points in terms of tumor growth attenuation and regression. However, by day 100 posttreatment more mice ultimately underwent tumor regression in the 25 Ag treatment group than in the 100 Ag treatment group, even though this difference was not statistically significant. As such, these findings demonstrated the clinical potential for this treatment strategy and suggest that to increase the percentage of mice undergoing long-term tumor regression additional treatments as well as modulation of the intervals of treatment may be necessary. The ultimate therapeutic end-point was complete tumor regression and long-term survival rather than simply attenuation of tumor growth, which has been the benchmark of most other studies. Therefore, we conclude that the percentages of the complete tumor regression coupled with long-term survival attained in our study with pVpr plus electroporation treatment are biologically significant and warrant further mechanistic studies as well as the assessment of methods to maximize the anti-tumor effect through the use of different plasmid doses and Vpr treatment regimens. In addition, once the anti-tumor effect can be maximized for the pVpr+ E+ treated mice, potential immune mechanisms, such as presentation of specific melanoma tumor antigens released by the apoptotic process, could be further investigated. Overall, the data presented underscore the therapeutic anti-tumor activity of both Vpr and the

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electroporation delivery method and suggest a potential clinical utility for this treatment modality against melanomas as well as possibly other tumor types. In sum, the data suggest that gene delivery using Vpr provides a way to harness the cell killing potential of Vpr in HIV-infected hosts and utilize it against malignant tumors such as melanomas.

MATERIALS

AND

METHODS

Construction and confirmation of expression of the Vpr plasmid. The expression plasmid for wild-type Vpr (NL4-3) was amplified by singleround PCR using Vpr-specific primers and subcloned into the pcDNA3.1 vector (Invitrogen, San Diego, CA, USA) as previously described [7]. The quantities of the Vpr (pVpr) and control plasmids required for these experiments were generated using endotoxin-free Clontech Giga kits (Clontech, Palo Alto, CA, USA). Using the TNT-coupled in vitro transcription/translation system (Promega Corp., Madison, WI, USA) 35Slabeled protein products were generated from plasmids containing pVpr or control vector (i.e., devoid of the Vpr gene). The reaction mixture was prepared according to the instructions supplied by the manufacturer and the reaction was carried out at 308C for 1 h as described previously [10]. Proteins were immunoprecipitated using an anti-Vpr polyclonal antibody (diluted to 1:1000 from an antibody stock obtained from the NIH AIDS Research and Reference Reagent Program, Bethesda, MD, USA). The immunoprecipitated protein complexes were eluted from the Sepharose beads by being boiled briefly followed by electrophoresis and resolution on SDS–PAGE gels. The gel was fixed, treated with a 1 M sodium salicylate solution, and dried in a gel drier (Bio-Rad, Hercules, CA, USA). The dried gel was exposed overnight to X-ray film and developed using the automated developer (Kodak, Rochester, NY, USA). For Western blot analysis proteins were separated by SDS–PAGE and blotted onto a PVDF transfer membrane using standard methods [7]. Blots were blocked with 5% BSA in TBS-T20 (10 mM Tris–HCl, pH 8, 150 mM NaCl, 0.05% Tween 20) for 1–2 h followed by detection of proteins with specific anti-Vpr polyclonal antibody described above in 3% BSA in TBS-T20 and with HRPcoupled goat anti-mouse or anti-rabbit IgG (Sigma) in 1:5000 dilution. Bands were subsequently visualized by autoradiography using the Amersham ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). B16 melanoma tumor cells and susceptible syngeneic mice. C57BL/6 mice, the murine strain syngeneic for the B16.F10 melanoma tumor cell line, were used in this study and were purchased from the National Cancer Institute. Mice were housed and maintained during this study in accordance with AALAM guidelines. The B16.F10 murine melanoma cell line (CRL 6475) was originally purchased from ATCC and was maintained for studies as monolayers in culture in 90% McCoy’s medium supplemented with 10% fetal bovine serum. For the preparation of single-cell suspensions, a monolayer of cells was detached from flasks using 0.05% trypsin–0.53 mM EDTA, centrifuged, washed, and resuspended at the proper concentration for injection in the tumor induction experiments described below. Fluorescence-activated cell sorting (FACS)-based in vitro apoptosis Assay. Exponentially growing B16.F10 cells (1.5–2 106 total number) were seeded onto 60-mm plates 24 h before transfection with the backbone control vector (pcDNA3.1), a coding vector negative control expressing HIV-1 gag (pGag), or pVpr using the FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN, USA). Cells were washed twice with complete RPMI 1640 and incubated for 48 h and FACS analysis was performed to identify cells undergoing apoptosis. Equal numbers of cells from each experimental group were collected. Apoptosis was evaluated using an annexin V assay kit (PharMingen, San Diego, CA, USA) and analyzed directly on a Coulter EPICS flow cytometry system (Coulter, Hialeah, FL, USA) using FlowJo software (Tree Star, USA).

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Tunel apoptosis assay and Vpr staining of B16.F10 melanoma tumors in vivo. Potential Vpr-mediated cell death in in vivo-treated B16.F10 melanoma tumors was assessed. Established melanoma lesions were treated with 100 Ag of pVpr or backbone control plasmids plus electroporation. Tumors were removed 48 h after treatment and sectioned. For each treatment group, consecutive 5-Am tumor sections were prepared from the tumor sample to be used in the Vpr antibody staining and the TUNEL assay. Sections were fixed with ice-cold methanol for 10 min. Sections were blocked in 3% BSA in PBS/Triton X-100 for 1 h at room temperature. Fc receptors were blocked with mouse BD Fc Block in 3% BSA in PBS/Triton X-100 (3 Ag/ml) for 1 h at room temperature (BD Pharmingen). All corresponding tumor sections were counterstained with the polyclonal Vpr antibody (diluted 1:2000) for 1 h at room temperature and were incubated with PE-conjugated secondary antibody, Alexa Fluor 594 (diluted 1:8000) (Molecular Probes, Inc., Eugene, OR, USA). The sections were stained with Hoechst, which stains DNA and serves as the positive control for the tissue. Apoptosis was measured using the TACS in situ kit TdT-Fluorescein (R&D Systems, Minneapolis, MN, USA) as per the manufacturer’s instructions. In short, tumor sections were treated with proteinase K (R&D Systems) for 1 h at room temperature. A TUNEL reaction mixture (biotinylated nucleotides integrated into DNA fragments by TdT) was added to each tissue sample and incubated in a humidified chamber for 1 h at 378C in the dark. The biotinylated nucleotides were identified by a streptavidin–fluorescein conjugate (R&D Systems). The sections were also stained with Hoechst to detect nuclei. To correlate apoptosis with expression of Vpr-specific staining, all of the tumor sections were assessed using a Genus 2.81 imaging system equipped and connected to a microscope (Nikon Model E1000). Tumor induction and measurements. Tumors were induced by subcutaneous injection of 106 B16.F10 cells, which were prepared as described above (viability of cells was greater than 90% as measured by trypan blue exclusion), into the left flanks of C57BL/6 mice. Tumors were allowed to grow to the appropriate size (average tumor volumes from each group ranged from 38 to 74 mm3) before commencement of the treatment regimen. This approximate tumor volume has been determined to be an ideal minimal size for intratumoral injection since the administered treatment volume is retained effectively within the lesion with no significant leakage, providing confidence that the entire dose has been administered. Tumor volumes were measured before and at periodic intervals following treatment using a digital caliper by measuring the longest diameter (a) and the next longest diameter (b) perpendicular to (a). Using those measurements the tumor volume was calculated by the formula V = ab 2 k/6. The mice were followed in the experiments for 100 days or until tumor volume was determined to be 1300 mm3 (at which point any mice had usually succumbed to tumor burden or were requisitely and appropriately euthanized due to the size of the tumor). Intratumoral plasmid treatment and in vivo electroporation. Female C57BL/6 mice at 6–7 weeks of age were injected with B16.F10 melanoma cells as indicated above and tumors were allowed to grow to the required size as stated above. Tumors were then treated intratumorally with either 25 or 100 Ag of the Vpr plasmid (pVpr) or control/backbone plasmid vector (pcDNA3.1). Subsequently (i.e., within 1 min) tumors from the appropriate groups were subjected to in vivo electroporation using a custom-made applicator containing six penetrating electrodes that was inserted into the tissue around the tumor. Six pulses that were 100 As long at a field strength of 1500 V/cm were administered using a BTX T820 pulse generator (BTX/Harvard Apparatus, Hollister, MA, USA) and autoswitcher (Genetronics, San Diego, CA, USA). These electroporation conditions have been previously described in other studies by the group and were selected because of their less stringent parameters coupled with the ability to elicit a significant biological effect. Treatments were administered on days 0 and 4 with pVpr doses of either 25 or 100 Ag. For the treatment groups, P+ or P indicates with or without treatment with the pVpr plasmid and E+ or E indicates with or without electroporation, respectively. V+ designates the control bbackboneQ vector (pcDNA3.1), at a dose of 100 Ag, which was delivered with electroporation (E+). The treatment groups were as follows: PE


doi:10.1016/j.ymthe.2006.06.010

(no treatment), P+E 25 Ag, P+E+ 25 Ag, P+E 100 Ag, P+E+ 100 Ag, V+E,V+E+ and saline plus E+. The in vivo experiments were conducted twice with a total n = 14, with the exception of the groups V+E and Saline+ E+, which had n = 7. The mean tumor volume was calculated for each group at selected time points after the treatment regimen. Additional quantitative measurements made during the study were fold increase in tumor volume compared to day 0 as well as percentage of mice undergoing complete tumor regression coupled with long-term survival. Statistical analysis. Among the various treatment groups the mean survival time was calculated. Statistical analysis of any treatment differences was assessed by using the Student t test method. Significance was determined to be P b 0.05.

ACKNOWLEDGMENTS The work was supported in part by an F31 Individual NRSA NIH/NCI Fellowship to A. N. McCray. The following reagent, used in this study, was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 NL4-3 Vpr antiserum (1-46) from Dr. Jeffrey Kopp. We also acknowledge Dr. Chuanhai Cao for assistance in producing the Vpr-expressing plasmids for this study. This work has been supported in part by the Pathology Core Facility, Histology Laboratory, at the University of South Florida College of Medicine and at the H. Lee Moffitt Cancer Center & Research Institute. We also acknowledge Dr. Jaya Padmanabhan for technical assistance with the immunohistochemistry. RECEIVED FOR PUBLICATION FEBRUARY 13, 2006; REVISED JUNE 6, 2006; ACCEPTED JUNE 13, 2006.

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