Direct visualization of electroporation-assisted in vivo gene delivery to tumors using intravital microscopy – spatial and time dependent distribution
© Cemazar et al; licensee BioMed Central Ltd. 2004
Received: 22 September 2004
Accepted: 16 November 2004
Published: 16 November 2004
Electroporation is currently receiving much attention as a way to increase drug and DNA delivery. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal.
Intravital microscopy was used to monitor real time spatial distribution of the green fluorescent protein (GFP) and time dependence of transfection efficiency in syngeneic P22 rat tumor model. DNA alone, liposome-DNA complexes and electroporation-assisted DNA delivery using two different sets of electric pulse parameters were compared.
Electroporation-assisted DNA delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz was superior to other methods and resulted in 22% increase in fluorescence intensity in the tumors up to 6 days post-transfection, compared to the non-transfected area in granulation tissue. Functional GFP was detected within 5 h after transfection. Cells expressing GFP were detected throughout the tumor, but not in the surrounding tissue that was not exposed to electric pulses.
Intravital microscopy was demonstrated to be a suitable method for monitoring time and spatial distribution of gene expression in experimental tumors and provided evidence that electroporation-assisted gene delivery using 8 pulses, 600 V/cm, 5 ms, 1 Hz is an effective method, resulting in early onset and homogenous distribution of gene expression in the syngeneic P22 rat tumor model.
Despite some promising early results, gene therapy does not, as yet, live up to expectation . The main stumbling block remains gene delivery, and all advances in the control of gene expression and selection of therapeutic genes are hampered by inefficient gene transfection. Hence the development of a safe and effective method of gene delivery in vivo is of utmost importance if gene therapy is to move from the experimental to the clinical stage.
Electroporation is currently receiving much attention as a way to increase drug and DNA delivery [2–5]. Electroporation has long been used as an effective in vitro gene delivery system in both prokaryotes and eukaryotic cells. Electroporation is a physical means of importing small molecules and macromolecules into cells via increased cell membrane permeability. Electroporation combined with chemotherapeutic drugs bleomycin and cisplatin (electrochemotherapy) has shown to be very promising antitumor therapy. It was tested on many different tumor types on the preclinical level demonstrating high antitumor effectiveness resulting in tumor cures at very low chemotherapeutic doses. It was also tested in clinical trials for the treatment of accessible cutaneous tumors of different histological types in cancer patients resulting in up to 100% objective responses [4, 6, 7]. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors [4, 8–14]. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach [5, 14, 15]. In studies designed to determine transfection efficiency in tumors, tissue homogenates, tissue sections or measurement of fluorescence in whole-tumor specimens using fluorescence stereomicroscope have been employed. Different plasmids have also been used to analyze transfection efficiencies, such as those encoding the green fluorescence protein (GFP), β-galactosidase or luciferase [5, 14, 15], making direct comparisons difficult.
In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. This information is also important for the timing of gene therapy with other cancer treatment modalities. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal [16, 17]. So far, only one study has used this technique to monitor the activity of an adenovirus in infecting a mammary cell carcinoma over the course of several days . In the present study, we used intravital microscopy to monitor real time spatial distribution of gene transfection using the GFP reporter gene in rat P22 tumors. We followed time dependency of transfection efficiency; compared liposome-DNA complexes and electroporation assisted DNA delivery using two different sets of electrical parameters.
Animals and tumors
All animal experiments were carried out in accordance with the UK animals (Scientific Procedures) Act 1986, following the UKCCCR guidelines and with approval from the local Ethical Review Committee of the Gray Cancer Institute.
Early generations of the P22 transplanted rat carcinosarcoma were used in experiments. Donor tumors were grown subcutaneously in the left flank of 8–10 week old male BD9 rats .
Surgery was carried out under general anesthesia using intraperitoneal injection of fluanisone (10 mg/kg), fentanyl citrate (0.315 mg/kg) ('Hypnorm', Janssen Animal Health, UK) and midazolam (2 mg/kg) ('Hypnovel', Roche Products Ltd., UK) as described previously . Briefly, animals were kept warm using heating pads throughout the surgical procedure and aseptic technique was used throughout. Window chambers, consisting of a double-sided aluminum frame, holding two parallel glass windows approximately 200 μm apart, were surgically implanted into the dorsal skin of male BD9 rats, weighing approximately 200 g. Surgery involved removal of the epidermal and dermal layers of both skin layers of a dorsal skin flap, except for the deepest fascia layer on each side, and then securing the two sides of the chamber to the skin using stainless steel screws and sutures. The two fascia layers moved freely between the two glass windows, following these procedures. Early generation subcutaneous transplants of the P22 rat carcinosarcoma were used as donor tumors, when they reached approximately 0.5 cm in diameter. A tumor fragment (~0.5 mm diameter) was placed onto one of the fascia layers within the window chamber on the day of surgery. Animals were given an intraperitoneal injection of a few milliliters dextrose-saline, immediately following surgery and kept on a warmed pad until recovery from anesthesia. Subsequently, animals were kept in a warm room (30–34°C) until the day of experiment. Three animals were used for each experimental group.
Transfection was carried out 7 to 14 days following surgery, when tumors measured 3–4 mm in diameter, using a plasmid encoding the green fluorescent protein (GFP; p-EGFP-N1, Clontech, Basingstoke, UK). Animals were anaesthetized with Hypnorm and midazolam. The window on the tumor side was removed and 40 μg of DNA alone or encapsulated in lipofectin (30 μl; Life Technologies, Paisley, UK) vesicles was carefully placed on the tumor surface in a total volume of 75 μl in phosphate buffered solution . One minute thereafter, electroporation was carried out by application of 8 square electric pulses generated by an electroporator (built in-house). Pulses were delivered by two flat, parallel stainless steel electrodes (two stainless steel strips: length 15 mm, width 4 mm with rounded corners) 3 mm apart that were placed at the diametrically opposed edges of the tumor. Two different electroporation protocols were used: EP1 – amplitude 180 V (voltage/distance ratio 600 V/cm); pulse length 5 ms; repetition frequency 1 Hz and EP2 – amplitude 390 V (voltage/distance ratio 1300 V/cm); pulse length 0.1 ms; repetition frequency 1 Hz. Immediately after the procedure, the glass window was replaced and GFP fluorescence monitored at selected time points for 6 days.
Intravital microscopy was carried out using an inverted Nikon Diaphot 200 fluorescence microscope, with a stage modified in-house for holding rats. Animals were anaesthetized with Hypnorm and midazolam and placed on the stage, in such a way that the window chamber was located centrally above the objectives using location screws. Rectal temperature was maintained between 34–37°C throughout the experiment, using a thermostatically controlled heating pad beneath the rat and an infrared overhead lamp for maintenance of tumor temperature.
Tumor preparations were alternatively viewed at each time point under transmitted visible light for measurement of tumor diameter and under fluorescence epi-illumination using a 100 W mercury arc lamp, for visualization of GFP fluorescence (excitation filter of 450–490 nm, emission filter of 520 nm) using x1.6, x4, x10 and x20 objective. Prior to transfection, two different regions of interest (ROI) using x10 objective were selected, one in the center of the tumor and one in the tumor periphery. Tumor preparations were monitored at each time point for 15 s using transmitted or epi-illumination.
Observations were recorded in digital format, using a Sony DSR-30P digital videocassette recorder, for off-line analysis. Multiple frames (typically 10) were captured onto computer and the images averaged for the analysis of the fluorescence intensity using the Visilog Image Processing package (Noesis, France). Increase in fluorescence intensity in the tumors was determined by subtracting the values of fluorescence intensity of non-transfected granulation tissue from the values of tumors and normalizing them to the values of non-transfected tissue. The calculations were performed for each animal at all time points.
Statistical analysis was carried out using SigmaStat Statistical software (Systat Software GmbH, Erkrath, Germany). Data were tested for normality using Kolmogorov-Smirnov test and differences between the groups were tested for significance using Holm-Sidak method, after one-way repeated measures analysis of variance was performed. A value of p < 0.05 for the comparisons was considered to represent a significant difference between groups.
Results and discussion
It is worth noting that the fluorescence level remained approximately constant throughout the observation period for all transfection methods. This means that, as the tumors grew during the 6-day observation period (tumor diameter increased approximately 2-fold), plasmid DNA appeared to be present in the progeny cells. In addition, intravital microscopy demonstrated that application of electric pulses to the tumors did not induce major cell damage as no effect on the tumor growth was observed compared to untreated controls. Six days post transfection there was no difference in increase in tumor diameter between the tumors that were transfected with DNA alone (2.2 fold), liposome-DNA complexes (2.3 fold), and electroporation-assisted DNA delivery using EP1 (2.4 fold) or EP2 (2.2 fold).
In conclusion, this study showed that intravital microscopy is useful for monitoring the spatial and temporal efficacy of electroporation methods for gene transfection in animal tumor models. As such, it will be valuable for the evaluation of new methods of optimizing gene delivery. Electroporation-assisted gene delivery using EP1 was found to result in early onset and homogenous distribution of gene expression in the P22 tumor model. Further improvement in transfection efficiency may be gained by optimizing electric pulse parameters.
This work was supported by Cancer Research UK and the Ministry of Education, Science and Sport of the Republic of Slovenia.
- French gene therapy group reports on the adverse event in a clinical trial of gene therapy for X-linked severe combined immune deficiency (X-SCID): Position statement from the European Society of Gene Therapy (ESGT). J Gene Med. 2003, 5: 82-84. 10.1002/jgm.364.
- Neumann E, Rosenheck K: Permeability changes induced by electric impulses in vesicular membranes. J Membr Biol. 1972, 10: 279-290.View ArticlePubMedGoogle Scholar
- Rols MP, Delteil C, Golzio M, Dumond P, Cros S, Teissie J: In vivo electrically mediated protein and gene transfer in murine melanoma. Nat Biotechnol. 1998, 16: 168-171. 10.1038/nbt0298-168.View ArticlePubMedGoogle Scholar
- Mir LM: Therapeutic perspectives of in vivo cell permeabilization. Bioelectrochemistry. 2001, 53: 1-10. 10.1016/S0302-4598(00)00112-4.View ArticlePubMedGoogle Scholar
- Cemazar M, Sersa G, Wilson J, Tozer GM, Hart SL, Grosel A, Dachs GU: Effective gene transfer to solid tumors using different non-viral gene delivery techniques: electroporation, liposomes, and integrin-targeted vector. Cancer Gene Ther. 2002, 9: 399-406. 10.1038/sj.cgt.7700454.View ArticlePubMedGoogle Scholar
- Gothelf A, Mir LM, Gehl J: Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev. 2003, 29: 371-387. 10.1016/S0305-7372(03)00073-2.View ArticlePubMedGoogle Scholar
- Sersa G, Cemazar M, Rudolf Z: Electrochemotherapy: advantages and drawbacks in treatment of cancer patients. Cancer Therapy. 2003, 1: 133-142. [http://www.cancer-therapy.org/index.html]Google Scholar
- Nishi T, Yoshizato K, Yamashiro S, Takeshima H, Sato K, Hamada K, Kitamura I, Yoshimura T, Saya H, Kuratsu J, Ushio Y: High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer Res. 1996, 56: 1050-1055.PubMedGoogle Scholar
- Heller L, Pottinger C, Jaroszeski MJ, Gilbert R, Heller R: In vivo electroporation of plasmids encoding GM-CSF or interleukin-2 into existing B16 melanomas combined with electrochemotherapy induces long-term antitumour immunity. Melanoma Res. 2000, 10: 577-583. 10.1097/00008390-200012000-00010.View ArticlePubMedGoogle Scholar
- Yamashita YI, Shimada M, Hasegawa H, Minagawa R, Rikimaru T, Hamatsu T, Tanaka S, Shirabe K, Miyazaki JI, Sugimachi K: Electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model. Cancer Res. 2001, 61: 1005-1012.PubMedGoogle Scholar
- Lohr F, Lo DY, Zaharoff DA, Hu K, Zhang X, Li Y, Zhao Y, Dewhirst MW, Yuan F, Lee CY: Effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation. Cancer Res. 2001, 61: 3281-3284.PubMedGoogle Scholar
- Cichon T, Jamrozy L, Glogowska J, Missol-Kolka E, Szala S: Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: inhibition of primary tumor growth and metastatic spread. Cancer Gene Ther. 2002, 9: 771-777. 10.1038/sj.cgt.7700497.View ArticlePubMedGoogle Scholar
- Cemazar M, Grosel A, Glavac D, Kotnik V, Skoberne M, Kranjc S, Mir LM, Andre F, Opolon P, Sersa G: Effects of electrogenetherapy with p53 wt combined with cisplatin on survival of human tumor cell lines with different p53 status. DNA Cell Biol. 2003, 22: 765-775. 10.1089/104454903322624975.View ArticlePubMedGoogle Scholar
- Gehl J: Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand. 2003, 177: 437-447.View ArticlePubMedGoogle Scholar
- Bettan M, Ivanov MA, Mir LM, Boissiere F, Dealere P, Scherman D: Efficient DNA electrotransfer into tumors. Bioelectrochemistry. 2000, 52: 83-90. 10.1016/S0302-4598(00)00087-8.View ArticlePubMedGoogle Scholar
- Huang Q, Shan S, Braun RD, Lanzen J, Anyrhambatla G, Kong G, Borelli M, Corry P, Dewhirst MW, Li CY: Noninvasive visualization of tumors in rodent dorsal skin window chambers. Nat Biotechnol. 1999, 17: 1033-1035. 10.1038/13736.View ArticlePubMedGoogle Scholar
- Jain RK, Munn LL, Fukumura D: Dissecting tumour patophysiology using intravital microscopy. Nat Rev Cancer. 2002, 2: 266-276. 10.1038/nrc778.View ArticlePubMedGoogle Scholar
- Tozer GM, Prise VE, Wilson J, Cemazar M, Shan S, Dewhirst MW, Barber PR, Vojnovic B, Chaplin DJ: Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: Intravital microscopy and measurement of vascular permeability. Cancer Res. 2001, 61: 6413-6422.PubMedGoogle Scholar
- Golzio M, Teissie J, Rols MP: Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci USA. 2002, 99: 1292-1297. 10.1073/pnas.022646499.View ArticlePubMedPubMed CentralGoogle Scholar
- Miller AD: The problem with cationic liposome/micelle-based non-viral vector systems for gene therapy. Curr Med Chem. 2003, 14: 1195-1211.View ArticleGoogle Scholar
- Yoo GH, Hung MC, Lopez-Berenstein G, LaFollette S, Ensley JF, Carey M, Batson E, Reynolds TC, Murray JL: Phase I trial of intratumoral liposome EiA gene therapy in patients with recurrent breast and head and neck cancer. Clin Cancer Res. 2001, 7: 1237-1245.PubMedGoogle Scholar
- Sersa G, Cemazar M, Semrov D, Miklavcic D: Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice. Bioelectrochem Bioenerg. 1996, 39: 61-66. 10.1016/0302-4598(95)01866-2.View ArticleGoogle Scholar
- Gehl J, Sorensen TH, Nielsen K, Raskmark P, Nielsen SL, Skovsgaard T, Mir LM: In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. Biochim Biophys Acta. 1999, 1428: 233-240.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/4/81/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.