Suppression of murine mammary carcinoma growth and ... - Nature

Cancer Gene Therapy (2002) 9, 16 –27 D 2002 Nature Publishing Group All rights reserved 0929-1903 / 02 $25.00 www.nature.com/cgt

Suppression of murine mammary carcinoma growth and metastasis by HSVtk/GCV gene therapy using in vivo electroporation Masa-Aki Shibata,1 Junji Morimoto,2 and Yoshinori Otsuki 1 1

Department of Anatomy and Biology; and 2Laboratory Animal Center, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan.

The effectiveness of electroporation as a means of gene transfection, both in vitro and in vivo, was tested using the herpes simplex virus 1 thymidine kinase ( HSVtk ) gene in combination with ganciclovir ( GCV ) administration as therapy against murine mammary cancer. Approximately 80% of BJMC3879 metastatic mammary carcinoma cells, derived from MMTV - infected BALB / c mice, died as a result of HSVtk / GCV treatment 72 hours after the transfection; decreased DNA synthesis was also seen. Mammary tumors induced by inoculation of syngeneic mice with BJMC3879 cells were subsequently treated by direct injection of vector containing HSVtk ( pHSVtk ) alone, empty vector or saline alone twice a week. After each injection, the tumors were subjected to in vivo electroporation. Mice treated with pHSVtk or saline were intraperitoneally injected with GCV at 40 mg / kg five times a week. Significantly reduced tumor volumes were observed for the pHSVtk + GCV group in experimental week 2 and thereafter throughout the 2 - month study. DNA synthesis was significantly decreased as well in the pHSVtk + GCV group compared with all other groups. Furthermore, metastasis to lymph nodes and lungs was significantly suppressed by HSVtk / GCV treatment. Expression of HSVtk in the tumors was confirmed by RT - PCR. Macrophage accumulations were frequently observed in the peripheries of necrotic regions in HSVtk / GCV - treated tumors, where levels of apoptosis were significantly higher than those observed in other groups. We therefore conclude that in vivo electroporation can result in efficient gene transfer and that the HSVtk / GCV prodrug system strongly suppresses tumor growth and metastases in this model. Cancer Gene Therapy ( 2002 ) 9, 16 – 27 DOI: 10.1038 / sj / cgt / 7700415 Keywords: HSVtk; electroporation; gene therapy; mammary cancer; mouse

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reast cancer is one of the most frequent female cancers and the incidence has increased dramatically during the past decade.1 In the United States, breast cancer ranks second only to lung cancer as a cause of death among women,2 77% of those deaths occurring in patients aged 55 years and older.3 Although breast cancer remains only the fifth leading cause of cancer deaths in women in Japan, the number of breast cancer deaths in this country has risen 2.6 -fold between 1975 and 1998.4 The phenomenon of increasing incidence is observed throughout many countries worldwide. As this trend is predicted to continue into the future, therapies aimed at controlling tumor growth and metastasis have earned serious attention. Conventional therapies used in breast cancer have yielded positive results; nevertheless, the rates of the cure and survival of this disease remain unsatisfactory. There is a need for the development of more effective and less toxic therapies that would reduce morbidity and mortality. Because the majority of patients with breast cancer die from disseminated metastases,5 such therapies Received October 9, 2001. Address correspondence and reprint requests to: Prof Yoshinori Otsuki, Department of Anatomy and Biology, Osaka Medical College, 2 - 7 Daigaku - machi, Takatsuki, Osaka 569 - 8686, Japan. E - mail: [email protected] - med.ac.jp

should address not only suppression of primary lesions but also suppression and/ or eradication of metastatic growth. Gene therapy is a rapidly evolving field with many applications and approaches. Breast cancer is one area where basic research for gene therapy is under way using local delivery methods. The introduction of suicide genes seems very promising among the various approaches being investigated in gene therapy. One such strategy involves the insertion of herpes simplex virus 1 thymidine kinase ( HSVtk ) into the tumor cells, which then converts a nontoxic prodrug into a lethal compound. So far, the most successful enzyme /prodrug combination tested both in vitro and in vivo is HSVtk and ganciclovir ( GCV ).6 It specifically targets the cell division processes crucial to tumor growth and survival.7 One key feature of this system is a ‘‘bystander effect’’ by which complete tumor regression can occur after GCV administration, even when only 10% of tumor cells have been transduced with HSVtk.8 Gene delivery in current gene therapy studies rely largely on recombinant viral vectors and adenoviruses have an advantage in that they show a high transient efficiency of gene transduction. However, serious concerns about the use of viral vectors have been raised. Most of the adenoviral vectors used are rendered replication deficient; however, it has been reported that new unwanted variants, including

HSVtk gene therapy in mouse mammary neoplasia Shibata et al

those that regain replication competency, can develop as a result of recombination during the production process.9 In addition, host immunogenicity may prevent their repeated use. Retroviral vectors can integrate into host chromosomes in dividing cells of the tumor and result in chronic expression for cancer therapy, but they usually give rise to low titers. It is also possible for methylation to occur10 and induce production of replication- competent retrovirus.11 Thus, there are still significant hurdles in vector selection and manipulation that must be overcome before large clinical trials for cancer therapy can be initiated.11 Electroporation has long been used to effectively transport molecules into living cells in vitro. Recent reports now demonstrate that it can transfect DNA into the cells of rodent skeletal muscle,12,13 skin,14 and liver15 in vivo. Although in vivo electroporation has disadvantages in common with other nonviral gene transfer methods, e.g., transfection efficiency is very low compared with that for viral methods, it has some attractive advantages over using viral vectors. First, because immunogenicity is not expected, repeat transfections are possible and may compensate for low initial transfection efficiency. Secondly, it is very easy to perform and can accommodate vectors with large DNA sizes. Third, it is a safe procedure in vivo. As mentioned above, transfection efficiency of in vivo electroporation is low, but repeated application can result in the accumulation of transduced cells. Moreover, because the HSVtk /GCV system has shown the bystander effect, it is possible that other gene candidates and target tissues may show enhanced effectiveness with in vivo electroporation as a method of delivery. Goto et al16 have demonstrated that HSVtk /GCV gene therapy using electroporation successfully inhibits solid tumor growth in an animal model. Subsequently, therapeutic studies of HSVtk / GCV using in vivo electroporation have reported appreciable results in other animal tumor models.17,18 Therefore, the present experiment was conducted to evaluate whether HSVtk / GCV treatment by in vivo electroporation is an effective therapy for metastatic murine mammary cancer. Materials and methods Vectors

The plasmid pGT60mcs, which encodes for HSVtk, was obtained from InvivoGen (San Diego, CA ) and heretofore referred to as ‘‘pHSVtk’’ in this manuscript. To produce the empty control vector, the HSVtk gene was deleted from pGT60mcs via digestion with SmaI/ MluI. The lacZ reporter gene is encoded within the pCMV ( Clontech Laboratories, Palo Alto, CA ), and the pGL3 (Promega, Madison, WI ) contains the luciferase reporter gene. All vectors are regulated by the CMV enhancer /promoter. These plasmids were extracted from Escherichia coli (DH5 strain ) and purified by means of a modified alkaline lysis procedure using Quiagen Plasmid Maxi Kit (Quiagen, Valencia, CA ). Mammary carcinoma cells and animals

A mammary carcinoma cell line, Jyg -MC, was established from mammary tumors of the Chinese wild mouse M. m.

musculus Sub -Jyg, which carries the mouse mammary tumor virus ( MMTV ). MMTV, purified from medium in which Jyg -MC cells were grown, was inoculation into the inguinal mammary glands of female BALB / c mice and resulted in the development of mammary carcinomas.19 The BJMC3879 cell line was subsequently derived from the metastatic focus within a lymph node in one of the inoculated mice and shows a high metastatic propensity, especially to lymph nodes and lungs ( Morimoto et al, in preparation). The BJMC3879 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM ) containing 10% fetal bovine serum (FBS ) with streptomycin / penicillin in an incubator under 5% CO2. A total of 62 female 6- week -old BALB / c mice were used in this study (Japan SLC, Hamamatsu, Japan ). The animals were housed five to a plastic cage with wood chip bedding and free access to water and food under conditions of controlled temperature ( 21± 28C ), humidity (50 ± 10% ) and lighting (12 –12 hour light– dark cycle ). All were held for a 1 -week acclimatization period before test initiation. Animals were euthanized by diethyl ether. All manipulations of mice were performed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals in Osaka Medical College. In vitro transfection experiments

The BJMC3879 mammary carcinoma cells were subjected to cationic lipid transfection ( Lipofectamine 2000, Life Technologies, Rockville, MD ) with pGL3 vector and maximum transfection efficiency was determined by luciferase activity ( Luminoskan Ascent, A Thermo BioAnalysis, Helsinki, Finland ). The maximum transfection was obtained with 0.5 g plasmid and 1.5 l cationic lipid reagent. To examine killing effects of HSVtk /GCV on BJMC3879 cells, cells were harvested into six - well plates in DMEM containing 10% FBS without antibiotics. The cells were transfected with pCMV or pHSVtk under the optimal condition mentioned above and treated with GCV ( Wako Pure Chemicals, Osaka, Japan ): pCMV alone, pHSVtk alone, GCV alone, and pHSVtk with GCV. GCV at a final concentration of 20 M in the medium was added after the incubation with the plasmid and the cationic lipid reagent for 15 minutes. Seventy - two hours after the transfection, the numbers of viable cells per well were counted using a hemocytometer with trypan blue exclusion. The concentration of GCV used in this study was determined based on a preliminary study with concentrations of 0.1– 100 M. Nontransfected cells were completely resistant to even high concentrations of GCV at 100 M ( data not shown ). In vitro DNA synthesis analysis

DNA synthesis was evaluated 48 hours after transfection. Cells were incubated for 30 minutes in culture medium containing 50 M 5 - bromo -20 -deoxyuridine (BrdU; Sigma, St. Louis, MO ) and trypsinized. Cells were then counted using a hemocytometer, fixed with 70% ethanol, and distributed 1105 cells/well in a 96 -well opaque plate. The plates were dried using a hair dryer. Cells within each well were incubated with 1 N HCl for 20 minutes at 378C, rinsed with PBS, and incubated with an anti -BrdU

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antibody /peroxidase conjugate ( Fab fragment, Roche Diagnostics, Indianapolis, IN ) for 60 minutes, rinsed, and reacted with luminol substrate ( Chemiluminescence, Roche Diagnostics ) for 5 minutes. The light emissions of the samples were then measured using a Luminoskan (A Thermo BioAnalysis ). In vivo electroporation of reporter gene into tumors

BJMC3879 cells (5106 cells /0.3 mL in PBS ) were inoculated subcutaneously into the right inguinal region of 27 female BALB / c mice. Three weeks later, when tumors had developed to approximately 0.5 cm in diameter, 50 g of pCMV ( 0.5 g/ l saline ) encoding galactosidase or saline alone was injected slowly into the implanted tumors using a 27 - gauge needle at a total volume of 100 l while the animals were under diethyl ether anesthesia. Electroporation was performed about 5 minutes after the injection by applying a conductive gel ( Aquasonic 100, Parker Lab., NJ ) topically to the unshaved skin over the tumor. Electric pulses were then delivered directly to the tumor via ‘‘clothespin’’ electrodes using a square - wave electropulser BTX T820 (BTX, San Diego, CA ) monitored by a graphic pulse analyzer ( Enhancer 400, BTX ). The electrodes (BTX ) consisted of a pair of stainless steel disks of 0.5 cm in diameter. The optimal level of eight electric pulses with a pulse length of 20 milliseconds was determined based on a previous report.13 Three mice each received 0, 50, 100, 150, and 200 V per treatment. Mice were sacrificed by exsanguination 3 days after the electroporation; tumors

were immediately excised and frozen in liquid nitrogen and stored at 808C for analyses. To check levels of -galactosidase, tumors were thawed, minced into fine pieces using a scalpel, and homogenized in lysing buffer (100 mM potassium phosphate / 1 mM DL dithiothretiol /0.1% Triton X -100 ) containing 100 mg /mL PMSF and 1 mg / mL aprotinin, and incubated on ice for 45 minutes. Extracts were then cleared by centrifugation and total protein concentrations were determined by the Bradford assay ( Bio - Rad, Hercules, CA ). Levels of - galactosidase in 500 g protein from each sample were determined using the Luminescent - galactosidase Genetic Reporter System II (ClonTech ) with the Luminoskan (A Thermo BioAnalysis ). The results indicated that - galactosidase activity peaked at 100 V (data not shown); therefore, electroporation conditions for further vector studies were set at eight pulses of 20 milliseconds each at 100 V. For sequential analyses of -galactosidase expression, tumors from each of three mice receiving pCMV or saline and electroporation at 100 V were excised after 1, 2, and 3 days of treatment. Each tumor was cut in half and embedded in Tissue -Tek OCT compound ( Miles, Elkhart, IN ) and frozen in liquid nitrogen. Expression of galactosidase was visualized by X - gal reaction on the frozen sections according to manufacture’s protocol (Roche Diagnostics ). In vivo electroporation of HSV tk / GCV to tumors

BJMC3879 cells (5106 cells/0.3 mL in PBS ) were inoculated into the right inguinal region of 35 female

Figure 1 Cell viability ( A ) and BrdU incorporation ( B ) in the murine mammary carcinoma cell line BJMC3879 transfected with HSVtk and exposed to 20 M GCV. Cell counts were taken 72 hours and BrdU labeling indices 48 hours after transfection with GCV treatment. A: Approximately 80% of the cells in the pHSVtk + GCV cultures died ( **P < .01 ). B: BrdU incorporation into S - phase cells was significantly decreased in pHSVtk + GCV cultures compared with cultures receiving other treatments, indicating suppressed DNA synthesis ( **P < .01 ). rlu: relative light unit. Data presented as means ± SD.

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BALB / c mice. The animals were then randomly allocated to four groups comprised of either 10 or 5 mice each. Two weeks post inoculation, when tumors had reached a size of 0.1 –0.2 cm in diameter, vector treatment and electroporation were conducted same as above. In vivo electroporation was conducted using the BTX T820 electropulser in eight pulses with a pulse length of 20 milliseconds at 100 V by topically applying a conductive gel (Aquasonic 100 ) determined previously. Vector injection and electrogene transfer were performed two times a week ( Monday and Thursday ). GCV at 40 mg /kg body weight or saline was injected intraperitoneally (i.p. ) five times a week ( Monday through Friday ). Groups were defined by the following treatments: empty vector, pHSVtk alone, GCV alone, and pHSVtk +GCV. The size of each treated mammary tumor was measured weekly using a caliper and tumor volume was calculated using the following formula: maximum diameter (minimum diameter )20.4.20 Individual body weights were recorded weekly. After 8 weeks of treatment, subcutaneous masses and lymph nodes ( axillary and femoral regions and any appearing abnormal ) were removed, immediately fixed in 10% phosphate - buffered formalin, and embedded in paraffin. A portion of each tumor was also immediately frozen in liquid nitrogen for molecular analyses. Lungs were inflated with the fixative, excised, and immersed in the fixative; all lobes were trimmed and examined for metastatic foci and processed for histopathology. They were cut at a thickness of 4 m, and stained with hematoxylin and eosin for histopathological examination.

PBS and exposed to anti – FITC -peroxidase ( 1:100 ) for 30 minutes at 378C. Apoptotic cells were visualized using diaminobentidine / H2O2 and 0.1% methyl green staining. TUNEL - positive cells were separately counted in both viable and necrotic regions of the tumors. The numbers of TUNEL - positive cells in viable regions per 5000 cells were counted in five random high power fields ( 400) and expressed as a percentage of the total cells counted. Areas in

DNA synthesis

All animals in the treatment groups were injected i.p. with 100 mg /kg body weight BrdU ( Sigma ) 1 hour before being exsanguinated under ether anesthesia. Within each treatment group, five animals were selected for evaluation of DNA synthesis rates as inferred by BrdU incorporation. Using the paraffin- embedded tissue sections, DNA was denatured by a 20 -minute incubation in 4 N HCl solution for 20 minutes at 378C. The incorporated BrdU was then revealed by an anti -BrdU mouse monoclonal antibody ( Clone Bu20a, Dako, Glostrup, Denmark ). The number of S -phase cells incorporating BrdU into DNA per 5,000 cells was counted in five random high power fields (400 ). BrdU labeling indices were expressed as a percentage of total cells counted. Determination of apoptotic indices

For the quantitative analyses of apoptosis, 4- m sections from paraffin- embedded tumor sections were assayed by the terminal deoxynucleotidyl transferase –mediated dUTP FITC nick end –labeling (TUNEL ) method using an apoptosis in situ detection kit (Wako ) with modifications to the manufacturer’s protocol. Briefly, tissue sections were deparaffinized, hydrated through graded ethanol, and incubated with 60 g /mL proteinase K for 30 minutes at 378C. After washing with PBS, the tumor sections were incubated with terminal deoxynucleotidyl transferase and FITC -dUTP ( 1:00 ) for 60 minutes at 378C, rinsed with

Figure 2 Sequential in vivo expression of - galactosidase in murine mammary carcinoma BJMC3879 cells after electroporation of the reporter gene. A: At day 1 post electroporation, no expression of galactosidase was detected. B: Weak expression was observed by day 2 ( arrows ). C: Markedly elevated expression of - galactosidase was observed by day 3. Histochemistry for - gal reaction, 100.

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necrotic regions were determined using a color image processor (MCID, Imaging Research, Ontario, Canada ) and expressed as average numbers of TUNEL- positive cells per square millimeter. Sections from testes were used as positive controls. Detection of HSV tk with RT - PCR

Total RNA were isolated from tumor tissues and incubated with DNase I at 258C for 20 minutes. The reaction was then stopped by addition of 2.5 mM EDTA at 658C for 15 minutes and RNA were reverse transcribed using oligo(dT )12 – 18 at 428C for 50 minutes. The mixture was then incubated with RNase H ( Superscript, Gibco BRL, Gaithersburg, MD ) at 378C for 20 minutes.

cDNA ( 200 ng) was amplified using a thermal cycler ( GeneAmp PCR System 2400, Perkin -Elmer, Foster, CA ) at 35 cycles of 948C for 1 minute, 608C for 1 minute, and 728C for 1 minute. Primer sequences were: HSVtk, 50 -AGC AAG AAG CCA CGG AAG TC -30 and 50 -TCC CGG AGG TAA GTT GCA GCA -30. The primer sequences for GAPDH, which was used as an internal control, were 50 -TGA AGG TCG GTG TGA ACG GAT TTG GC -30 and 50 - CAT GTA GGC CAT GAG GTC CAC CAC -30. Statistical analyses

The significance of differences between groups in body weights, quantitative histopathology data, levels of DNA synthesis and of apoptosis were analyzed using the two -

Figure 3 Body weight curves ( A ) tumor volume ( B ) and macrosopic appearances of tumors ( C ) in mice receiving HSVtk / GCV therapy. A: Groups consisted of 10 mice each in the empty vector, pHSVtk + GCV, and GCV groups, whereas the pHSVtk group contained five mice. Mean body weight curves did not show statistical difference among groups with exception of the pHSVtk + GCV group at week 6 ( *P < .05 ). B: Tumor volume was significantly reduced in the pHSVtk + GCV group from week 2 on ( **P < .01 at each time point ). Data expressed as means ± SD. C: The relative effect of HSVtk / GCV therapy on clinical tumor appearance. Syngeneic BALB / c mice bearing BJMC3879 mammary carcinomas were treated two times a week with intratumoral injection of 50 g empty vector or 50 g pHSVtk vector followed by electroporation. GCV at 40 mg / kg body weight was injected i.p. five times a week. Large tumors with ulceration and scarring developed in mice treated with either the empty vector or GCV or pHSVtk alone; much smaller tumors without ulcer / scar formation were characteristic of mice treated with pHSVtk + GCV.

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Figure 4 Histopathological analysis of tumors in mice treated with HSVtk / GCV therapy. A: Massive cell death in a mammary tumor. Macrophages ( arrows ) were especially numerous in the peripheral zones of necrotic regions in these tumors. B: Higher magnification of macrophage accumulations in the periphery of a necrotic area. C: Metastasis to lymph node, a frequent occurrence in mice receiving the empty vector. D: Lymph node from a mouse in the pHSVtk + GCV group. The majority of mice in this group did not show metastases to lymph nodes. E: Metastases in the lung of a mouse receiving the empty vector. F: Metastasis in the lung of a mouse receiving pHSVtk + GCV. Metastatic lung foci were much smaller in the pHSVtk + GCV group than in other treatment groups. H&E staining; A, E, and F 100; B – D 200.

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Figure 5 The effect of HSVtk / GCV therapy on metastasis to lymph nodes ( A, B ) and lungs ( C, D ). A: A significant decrease in the incidence of lymph node metastasis was noted in the pHSVtk + GCV group compared with all other groups ( **P < .01 ). B: The number of lymph nodes with metastasis / mouse was also significantly reduced in this group ( **P < .01 ). C: HSVtk / GCV therapy had little effect on the incidence of lung metastasis overall. D: Lung metastases were categorized as microscopic and / or macroscopic foci comprised of >30 cells ( open columns ) and macroscopic nodules >1 mm diameter ( closed columns ). The difference between the numbers of foci comprised of more than 30 cells in the pHSVtk + GCV group versus the numbers in the empty vector group were statistically significant ( *P < .05 ), but this significance was not apparent in comparisons between pHSVtk + GCV and other groups ( open columns ). However, the numbers of nodules >1 mm were significantly reduced in the pHSVtk + GCV group ( *P < .05 ) compared with all other groups ( closed columns ). Data presented as means ± SD.

sided Student’s t test via the method of Welch, a method that provides for insufficient homogeneity of variance. The

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differences in metastatic incidence were examined by Fisher’s exact probability test.


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Figure 6 RT - PCR analysis of HSVtk mRNA levels ( A ), labeling indices of BrdU ( B ) and apoptosis ( C – E ) in mammary tumors. A: Tumor RNA was used as the negative control, N, and pHSVtk plasmid was used as a positive, P. As can be seen, expression of HSVtk was confirmed only in tumors treated with vectors containing HSVtk. GAPDH was used as an internal control. B: BrdU labeling indices were significantly decreased in HSVtk / GCV - treated tumors compared with all other groups ( **P < .01 compared with empty vector or GCV alone; yP < .05 compared with pHSVtk alone ). C: Percentages of TUNEL - positive cells in non - necrotic regions were similar between groups ( open columns ) ( C ), whereas the numbers of TUNEL - positive cells were significantly elevated in necrotic regions ( closed columns ) ( C ). Data presented as means ± SD. D, E: In situ TUNEL staining in tumors. Whereas TUNEL - positive cells are observed in the empty vector group ( D ), many more TUNEL - positive cells are seen in the tissues peripheral to necrotic regions in tumors receiving HSVtk / GCV therapy ( E ), indicating a higher apoptotic rate. Note also the frequent macrophages containing TUNEL - positive particles in the HSVtk / GCV - treated tumor ( E ) compared with the tumor receiving empty vector only ( D ). TUNEL staining with methyl green. 200.

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Results Effects of HSV tk on cell proliferation of mammary carcinomas in vitro

HSVtk -transfected mammary carcinoma cells treated with GCV showed a significant reduction of 82 –88% in numbers of viable cells compared with all other groups (Fig 1A ). DNA synthesis, as assessed by BrdU incorporation, was also tremendously decreased in HSVtk -transfected cells treated with GCV compared with all other groups ( Fig 1B ). Gene transfer efficiency of in vivo electroporation

Mammary carcinomas were subjected to a single intratumoral injection with pCMV encoding -galactosidase followed by in vivo electroporation and sampled for transfection efficiency over time. In sequential analysis of -galactosidase expression, although the expression was barely detected in the mammary carcinomas at day 1 after the injection, weak expression at day 2 and strong expression at day 3 were observed ( Fig 2A –C ). Body weights and general condition of mice given HSV tk / GCV electrogene therapy

Body weights of mice receiving therapeutic treatments are shown in Figure 3A. With the exception of the pHSVtk +GCV group in week 6, there were no significant differences in body weights among treated groups compared with the empty vector group. Body weight curves are similar between the pHSVtk +GCV group and the groups receiving pHSVtk or GCV alone. The general condition of all animals was good throughout the study. Inhibition of tumor growth and metastasis

Tumor volumes in mice receiving therapeutic treatments are presented in Figure 3B. Tumor growth was significantly suppressed in the pHSVtk +GCV group from week 2 on compared with all other groups. As can be seen in Figure 3C, large tumors developed in mice treated with the empty vector (tumor volume 2331 ± 885 mm3 ), GCV ( 1783 ± 264 mm3 ) and pHSVtk ( 2247 ± 550 mm3 ) alone. In contrast, tumors in the pHSVtk + GCV group were much smaller (562± 249 mm3; P < .01). In addition, the surface of the tumors treated with the empty vector, GCV or pHSVtk showed ulceration and scarring, which was not a feature of tumors in the pHSVtk +GCV group. Histopathologically, the implanted mammary carcinomas proved to be moderately differentiated adenocarcinomas. Tumors were accompanied by central necrosis and some inflammatory reactions independent of treatments. However, massive necrotic areas with infiltrations of imflamatory cells were observed in the pHSVtk +GCV group (Fig 4A ). Macrophage accumulations in necrotic regions were much stronger in the pHSVtk + GCV group than in those observed in the empty vector or in the GCV alone group (Figs 4A and B, 6D and E ). Metastases to lymph nodes and lungs were also evaluated histopathologically. A high incidence of metastasis to lymph nodes was frequently observed in the left femoral ( opposite side

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to the implanted site ), bilateral in the axillary (Fig 4C ) and occasionally in the iliac nodes in all groups with exception of the pHSVtk + GCV group (Fig 4D ). As shown in Figure 5A, lymph node metastasis was found in mice treated with empty vector and with both pHSVtk and GCV alone at incidences of 80% to 100%. However, the incidence of lymph node involvement in the pHSVtk + GCV group reached only 20%. Furthermore, the overall numbers of lymph node with metastasis were significantly reduced in the pHSVtk +GCV group compared with all other groups (Fig 5B ). In contrast, the incidence of lung metastasis was high and not significantly different among groups (Fig 5C ). Multiplicity of the metastatic lung foci is illustrated in Figure 5D. Metastatic foci were divided into two categories: foci consisting of more than 30 cells (all metastatic foci; microscopic and macroscopic size ) and those measuring more than 1 mm (macroscopic size only ). In the pHSVtk + GCV group, there was a significant decrease in the numbers of metastatic foci greater than 1 mm (P < .05 compared with all groups ) as well as a tendency toward fewer metastases of any size ( P< .05 compared with empty vector, but not compared to other groups ) (Figs 4E and F, 5D). Detection of HSV tk expression by RT - PCR

As shown in Figure 6A, HSVtk was expressed in tumors transfected with pHSVtk by electroporation. No expression of HSVtk was observed in tumors transfected with empty vector or treated with GCV alone. DNA synthesis and apoptosis

Levels of DNA synthesis, as evaluated by BrdU labeling, were significantly decreased in mammary tumors receiving pHSVtk + GCV treatment compared with all other groups ( Fig 6B ). Although levels of TUNEL -positive cells in viable regions of tumors were comparable among groups, the levels of TUNEL - positive cells in necrotic regions were significantly elevated with pHSVtk + GCV exposure compared with any other treatment ( Fig 6C– E ).

Discussion

In this study, we have determined that in vivo electroporation of pHSVtk is not only effective, but also that transduction of HSVtk with GCV treatment suppresses the growth and metastasis of mammary cancers derived from a mammary tumor cell line. The results suggest the feasibility of using in vivo electroporation as a tool for vector transduction in gene therapy as well as the efficacy of the HSVtk / GVC treatment itself. Recombinant viral vectors account for the majority of gene delivery approaches used in current cancer gene therapy studies because of their high efficiency of transduction as a main advantage. However, they may have potential risks for production of replication competent virus.9 However, none of the available vectors satisfies all the criteria of an ideal gene delivery system. Although the big disadvantage of nonviral gene therapy methods is their low


efficiency of transduction, they are still attractive. It was recently reported that, under suitable conditions, transduction by in vivo electroporation was approximately equivalent to using an adenoviral vector concentration of 106 transduction units/mL, but not as effective when compared to higher viral concentrations.21 More recently, experimental gene therapy by in vivo electroporation gene transfer was demonstrated to be effective in murine transplanted tumor models, including colon adenocarcinoma,16 melanoma,17 and hepatocellular carcinoma.18 In confirmation, our study demonstrated that a single electroporation, performed under appropriate conditions, provided good transduction in mammary tumors using the - galactosidase reporter gene. There are several other incentives to add in vivo electroporation to the gene therapy arsenal. Increased transduction efficiency appears to be achievable by manipulation of voltage, pulse length, and frequency of treatment rather than by increasing the vector concentrations. The problems associated with immunogenicity may be avoided, even with repeated treatments, and the method is relatively easy and well tolerated. Perhaps the most significant benefit is that, because the procedure is precisely targeted, destruction of tumor without injury to neighboring normal tissues would be possible. When considering specific clinical applications, external neoplasias and organs such as the breast are obvious choices for electroporation. One of the more attractive candidates for gene therapy against various types of cancer is the introduction of HSVtk to tumor cells followed by GCV administration; GCV is then converted by the transfected HSVtk into highly toxic GCV phosphates.7 Phase I /II clinical trials of HSVtk /GCV therapy report that some patients have achieved partial and complete clinical remission of their tumors.22 - 24 Many studies in animal models have demonstrated that HSVtk /GCV treatment exerts antitumor effects in various cancers16,25 – 27 including mammary cancer. To corroborate this, we first confirmed whether HSVtk /GCV treatment would kill murine mammary carcinoma cells BJMC3879 in vitro. Whereas BJMC3879 cells were resistant to GCV at 100 M, 80% of HSVtk -transduced BJMC3879 cells died when 20 M of GCV was added to the culture medium. DNA synthesis, as measured by BrdU incorporation, was also decreased in the HSVtk + GCV –treated cells compared with other nontransfected cultures. The majority of the gene therapy studies using animals have been completed within 12 to 15 days after the experimental treatment;25,27,28 some others have run 25 days or slightly longer.26,29 Although a massive necrotic region containing numerous apoptotic cell death was observed in tumors due to HSVtk / GCV treatment, active tumor cells in small area of the necrotic regions were noted in our study, as was recently reported in another investigation.16 These cells can be a source of regrowth after cessation of the HSVtk / GCV treatment. Therefore, we followed the treatment effects over a relatively longer experimental period of 56 days. In addition, because we wished to evaluate any antimetastatic ability of the HSVtk /GCV therapy, a longer experimental period was required. We found that tumor growth was significantly suppressed by HSVtk / GCV treatment throughout the study. Furthermore, the incidence and severity of

metastasis was reduced by HSVtk /GCV treatment compared to controls. DNA synthesis, assessed by BrdU incorporation, was also significantly decreased in the HSVtk /GCV treatment in vivo as well as in vitro. This is associated with retardation of tumor growth and inhibition of metastasis. HSVtk /GCV treatment has been reported to induce cell cycle arrest in S and G2 /M phases.30 - 32 Although the inhibition of metastasis may simply be a reflection of suppression of tumor growth and decreased DNA synthesis, this therapeutic benefit is apparent because patients presenting with metastatic disease are frequently incurable using conventional therapies. It is reported that once breast cancers reach 4 cm or larger, the chances of tumor recurrence or metastases increase dramatically;5 therefore, treatments that offer suppression of both tumor growth and metastasis have significant clinical implications. HSVtk / GCV therapy has an additional advantage of eliciting a bystander effect in which the neighboring untransduced tumor cells become susceptible to GCV killing after HSVtk treatment. Experimental data from human and mouse tumor models suggest different, but not mutually exclusive, mechanisms that explain the bystander effect. Both activation of the immune system and transfer of the toxic GCV phosphate form to adjacent untransduced tumor cells via either gap junctions or phagocytosis of apoptotic particles might precipitate the effect.7 Whereas HSVtk /GCV therapy does suppress transplanted murine hepatocellular carcinoma in immunocompetent syngeneic mice, the efficacy is decreased in athymic nude mice that do not have an intact T-cell system, suggesting that T cell – mediated immune responses may be a critical factor for the bystander phenomenon.26 In our study, macrophage accumulations were frequently observed in tissues peripheral to the central necrosis in the mammary tumors and these accumulations were much more prominent in the HSVtk + GCV group than those observed in other groups. Furthermore, TUNEL assays demonstrated that the macrophages contained apoptotic cell debris and levels of apoptosis in the peripheral zones were significantly elevated in the HSVtk + GCV group. Phagocytosis of material from dying HSVtk - expressing cells has previously been suggested as the mechanism for the bystander effect induced by HSVtk /GCV.33 This effect is probably mediated by multiple cascades of immune responses generated by the delivery of HSVtk. Although it has also been shown that gap junctions play an essential role in peripheral HSVtk /GCV toxicity,6 because the capacity for gap junctional intercellular communication seems to be lost in most cancer cells,34 we tend to believe that a T cell – mediating immune response may be a crucial factor in the present study, as previously suggested by Kuriyama et al.26 Regardless of mechanism, it appears that as few as 10% of cells need to express HSVtk to produce this phenomenon.8 The tumor growth was apparently suppressed by electrogene therapy conducted two times a week in the present study, but complete regression was not noted. It was reported that electrogene therapy administered three times a week suppressed tumor growth much more stronger than it does two times a week.16 A dosage effect may exist in electrogene therapy.

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In conclusion, we demonstrate that HSVtk /GCV gene therapy, using electroporation as the method of in vivo transduction, suppresses tumor growth and reduces metastasis in a mouse mammary cancer model. Growth suppression is associated with decreased DNA synthesis and increased cell death, including an increase in apoptosis. Transfection of HSVtk by in vivo electroporation may serve as a method for gene therapy for mammary cancer models and in other cancers as well.

Acknowledgments

We thank Deborah E Devor-Henneman (National Cancer Institute - FCRDC, MD ) for critical review of the manuscript. This investigation was supported by the Development of Characteristic Education Grant from the Japan Private School Promotion Foundation and from the High -Tech Research Center Grant at Osaka Medical College from the Ministry of Education, Science Culture of Japan.

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