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. Author manuscript; available in PMC: 2018 Mar 20.
Published in final edited form as: Int J Hyperthermia. 2016 Sep 20;33(2):170–177. doi: 10.1080/02656736.2016.1229045

Radiofrequency Hyperthermia-Enhanced Herpes Simplex Virus-Thymidine Kinase/Ganciclovir Direct Intratumoral Gene Therapy of Hepatocellular Carcinoma

Jianfeng Wang 1,2,†, Yaoping Shi 1,†, Zhibin Bai 1, Yonggang Li, Longhua Qiu 1, Guy Johnson 1, Feng Zhang 1,*, Xiaoming Yang 1,*
PMCID: PMC5572512  NIHMSID: NIHMS885610  PMID: 27569361

Abstract

Purpose

To determine the feasibility of using radiofrequency hyperthermia (RFH) to enhance the therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) for the treatment of hepatocellular carcinoma (HCC).

Materials and Methods

Human HCC cells (HepG2) were first transfected with lentivirus/luciferase. For both in-vitro confirmation and in-vivo validation, luciferase-labeled HCC cells and HCC tumor xenografts on mice received different treatments: (i) combination therapy of intratumoral HSV-TK/GCV-mediated gene therapy plus MR imaging-heating-guidewire (MRIHG)–mediated RFH; (ii) gene therapy only; (iii) RFH only; and (iv) phosphate-buffered saline (PBS) as control. Cell proliferation was quantified. Tumor changes were monitored by ultrasound imaging and bioluminescence optical imaging before and at days 7 and 14 after treatments, which were correlated with subsequent histology.

Results

In vitro, the lowest cell proliferation was seen in the combination therapy group compared with control groups (29±6% VS 56±9%, 93±4%, and 100±5%, P < 0.05). Ultrasound imaging of treated animal xenografts showed smaller relative tumor volume in combination therapy group than those in three control groups (0.74±0.19 VS 1.79±0.24, 3.14±0.49 and 3.22±0.52, P < 0.05). Optical imaging demonstrated significant decrease of bioluminescence signals of tumors in the combination therapy group, compared to those in three control groups (1.2±0.1 VS 1.9±0.2% VS 3.3±0.6% VS 3.5±0.4%, P < 0.05). These imaging findings were correlated well with histologic confirmation.

Conclusion

RF-hyperthermia can enhance HSV-TK/GCV-mediated gene therapy of HepG2 cell line and mice human HCC xenografts, which may open new avenues for effective management of HCC using MR/RF-hyperthermia integrated interventional gene therapy.

Keywords: Hepatocellular Carcinoma, radiofrequency hyperthermia, gene therapy, herpes simplex virus thymidine kinase

1. Introduction

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with an annual incidence of more than 1 million worldwide, and is the third leading cause of cancer-related death (1–4). Surgical resection or orthotopic liver transplantation has been considered as the first option for patients with very early stage HCCs (2, 5, 6). However, these therapies are often precluded, because of poor hepatic reserve due to liver cirrhosis, a shortage of donors or diseases at advanced tumor stages (3, 6–8).

Radiofrequency ablation (RFA) is accepted as a safe and effective first-line treatment modality for the patients with early stages of primary HCC, who are not suitable for hepatic resection or liver transplantation (2, 6, 8–11). However, inadequate or sublethal RFA treatment may accelerate both malignant transformation and tumor progression in HCC (9, 12), which may in part contribute to significantly higher incidence of local recurrence (up to 53%) than surgical resection (13). Large tumor size is also a major risk factor of local recurrence because of poorly defined ablative margins (2, 14, 15). It is necessary to apply sufficiently high temperatures and secure wide therapeutic margins, in an attempt to completely eradicate all residual HCC within the treated focal lesion.

However, a critical clinical problem with the current RFA techniques lies in its inconsistency in achieving complete response with large or irregular tumors, or when there is a “heat-sink” effect caused by peritumoral blood vessels (16). To overcome this problem, new strategies have been explored to extend the area of RFA-induced necrosis, such as RFA combined with transarterial chemoembolization (17), use of adjuvant sorafenib (18) or temporary occlusion of hepatic vessels(19, 20). Compared with RFA alone, combination therapies may improve outcomes in patients with HCC, but tumor recurrence rates remain unacceptably high (21).

Gene therapy is a frontier of modern medicine. To date, nearly 1,000 gene therapy trials have been initiated or completed worldwide, with most of these trials focusing on gene-based treatments of cancers. Transfection of cancer cells with the herpes simplex virus thymidine kinase gene followed by ganciclovir administration (HSV-TK/GCV) represents a promising example for treating malignancies. In comparison to conventional chemotherapies, tumor cells transfected with the HSV-TK gene can not only convert a prodrug into a toxic metabolite and thereby selectively eliminate tumor cells, but also produce a “bystander effect” that induces the death of neighboring untransfected cancer cells(22). However, a critical weakness with systemic HSV-TK/GCV therapy is its low rate of gene transfection (less than 10%) and consequent low tumoricidal effect. Attempts to solve this problem have been made by increasing the concentration of HSV-TK genes, however this may lead to substantial toxicities to other organs via a systemic gene delivery approach.

A previous study showed that radiofrequency (RF) heating can enhance Lentivirus-mediated gene transfection and transduction efficiency in arterial atherosclerotic tissues, which involved the use of a magnetic resonance imaging heating guidewire (MRIHG) to induce a local hyperthermia in the target tissue (7, 23). Based on the concept of using RF energy to enhance gene transduction, the purpose of this study was to establish the feasibility of use of MRIHG-mediated RF hyperthermia (RFH) to augment the benefit of HSV-TK/GCV-mediated gene therapy on HCCs.

2. Materials and Methods

2.1 Study Design

This study was divided into two phases: (a) in-vitro experiments to confirm RFH-enhanced therapeutic efficacy of HSV-TK/GCV on human HCC cells; and (b) in-vivo experiments to validate the feasibility of using optical and ultrasound imaging to monitor RFH-enhanced HSV-TK/GCV gene therapeutic effect on mouse models with HCC xenografts.

2.2 Production of HSV-TK/lentivirus

HSV-TK lentiviral vector and 3rd generation lenti-combo packing mix (Applied Biological Materials Inc., Richmond, BC, Canada) and a HSV-TK plasmid vector, pORF9-HSV1tk (InvivoGen, San Diego, CA) were obtained commercially. All plasmids were amplified and extracted from E. coli and purified using the Qiagen Hispeed Plasmid Max Kit (Qiagen,Inc.,Valencia, CA). The final concentration of the plasmids in stock was adjusted to 2.0 mg/mL by dilution with phosphate-buffered saline (PBS).

Recombinant lentivirus-carrying HSV-TK was produced by transient transfection of T293 cells, according to the protocol provided by the manufacturer (Applied Biological Materials Inc). Briefly, 10 µg of HSV-TK lentiviral expression vector, 10 µg of 3rd generation packaging system mix and 80 µL of transfection reagent were used to transfect T293 cells. The viral supernatant was concentrated using an Amicon Ultra-15 centrifugal filter units (Ultracel 100K, Millipore, Billerica, MA). Viral titer was determined based on the expression of green fluorescence protein (GFP) in transduced T293 cells as previously described (24).

2.3 In Vitro Experiments

2.3.1 Cell Culture and RFH-Enhanced Gene Therapy

For the purpose of using molecular imaging to evaluate the therapeutic effect, human HCC cells (HepG2) were first transfected with luciferase/red fluorescence protein (Luc/RFP) gene/lentivirus, to create Luc/RFP-positive HepG2 cells according to the protocol provided by the manufacturer (GeneCopoeia Inc., Rockville, MD). Luc/RFP-positive cells were sorted out using fluorescence-activated cell sorting (Aria II, Becton Dickinson, Franklin Lakes, NJ). Cells were then cultured in DMEM medium (Hyclone, South Logan, UT) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA) and incubated with a humidified 5% CO2 atmosphere at 37 °C.

HepG2 cells were cultured in 4-chamber cell culture slides (Nalge Nunc International, Rochester, NY). RFH was performed by attaching a 0.022-inch MRIHG under the bottom of chamber 4 of the chamber slide. The MRIHG was connected to a custom RF generator for heating. A sterile 1.1-mm fiber optic temperature sensor was placed at the bottom of each chamber and connected to a temperature thermometer (PhotonControl, Burnaby BC, Canada) for real-time monitoring of the temperature change during RFH (23, 25).

Cells in different groups were treated by (a) 30-min RFH at approximately 42 °C with HSV-TK/lentivirus (106 IU/well), followed by exposure to 50 µg/mL GCV one day after gene transduction; (b) 30-min RFH at approximately 42 °C with HSV-TK/plasmid, followed by exposure to 50 µg/mL GCV one day after gene transduction; (c) 30-min RFH-only; (d) HSV-TK/lentivirus+GCV therapy only (without RFH); (e) HSV-TK/plasmid+GCV therapy only (without RFH) and (f) PBS to serve as a control.

To prepare the HSV-TK/plasmid gene transfection group cells, 0.020-µg/µL plasmid solution in sterile deionized water was combined with the addition of transfection reagent (FuGENE® HD, Promega, Madison, WI) with a FuGENE:DNA ratio of 4.5:1. Twenty-four hours after cell seeding, 0.50-µg HSV-TK/plasmid was added to the chamber. The cells were then exposed to 50 µg/mL GCV for 72 hours (26). The HSV-TK/lentivirus gene transduction group cells were transfected with lentivirus supernatant at a dose of 106 IU/cell in the presence of 8 µg/mL hexadimethrine bromide solution (Polybrene, Abbott Laboratories, Chicago, Il). Cells were then incubated for 24 hours, followed by an exposure of cells to 50 µg/ml GCV for 72 hours.

2.3.2 Cell proliferation assay

Cell proliferation was evaluated by MTS assay 72 hours after the GCV treatment. Briefly, MTS (3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) agent was added to the cell chambers and incubated for 4 h. The absorbance was measured using a microplate reader at 490 nm. The relative cell proliferations of the different cell groups were evaluated using the equation Atreated-Ablank/Acontrol-Ablank, where A is the absorbance. Cells on slides were subsequently washed twice with PBS, fixed in 4% paraformaldehyde, counterstained with 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA), and then imaged with a laser confocal microscope (A1R; Nikon, Tokyo, Japan). The experiments for each cell group were repeated six times.

2.3.3 Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

The total RNA was extracted using All Prep DNA/RNA Mini kit (Qiagen, Inc., Valencia, CA). Quantitative RT-PCR was performed using an Mx3005PTM real time PCR system (Stratagene, La Jolla, CA, USA) with the TaqMan EZ RT-PCR kit, according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). We used forward primer 5′-GGTGATGACCTCTGCCCAGAT-3′, reverse primer 5′-TGTGAGGAGCCAGAACAGCAT-3′ and TaqMan(R) MGB Probe 6FAM-TGG GAA TGC CCT ATG C-MGBNFQ.

2.4 In Vivo Experiments

2.4.1 Animal model

The animal protocol was approved by the Institutional Animal Care and Use Committee. Thirty-six nu/nu mice (Charles River Laboratories, Wilmington, MA) aged 4–6 weeks were included in this study. 5×106 – 1×107 Luc/RFP-positive HCC (HepG2) cells in 100-µl Matrigel (Corning Life Sciences, Corning, NY) were subcutaneously injected into the left back of each mouse for tumor creation. Once the tumor size reached 5–8 mm in diameter, we initiated the treatment procedures.

2.4.2 DNA/liposome complex preparation

FuGENE /DNA complexes were prepared by adding a solution of 50-µg plasmid DNA and FuGENE® HD reagent in 50-µl sterile deionized water. This DNA-liposome mixture was then incubated for 45 min at room temperature before intratumoral injection. The mixture was directly injected into the subcutaneous HCC xenografts on mice under ultrasound guidance.

2.4.3 RF heating and gene therapy

The animals were anesthetized with 1–3% isoflurane in 100% oxygen. Six mice in each of six groups were treated by (a) RFH plus HSV-TK/lentivirus therapy, which was carried out by intratumoral injections of 108 HSV-tk/lentivirus in a total of 60-µL PBS, followed by RFH at approximately 42°C for 30 mins; (b) RFH plus HSV-TK/plasmid therapy, which was carried out by intratumoral injection of 50-µg plasmid in a total of 50-µL sterile deionized water, followed by RFH at approximately 42°C for 30 mins; (c) 30-min RFH alone; (d) HSV-TK/lentivirus therapy without RFH; (e) HSV-TK/plasmid therapy without RFH; and (f) PBS to serve as a control. RFH was performed by inserting a 0.022-inch MRIHG into the tumor with its hot spot at the center of each tumor, which was precisely positioned under real-time ultrasound imaging guidance. A 400-µm micro-fiber optical thermal sensor was subcutaneously placed in parallel to the MRIHG, so that the temperature at the RF-heated tumor could be measured instantaneously. One day after the gene injection, GCV at a daily dose of 5 mg/kg was intraperitoneally administered for 14 consecutive days.

2.4.4 Optical Imaging Follow-up

Optical imaging was performed on an In-Vivo Optical Imaging Systems (In-vivo Extreme, Rheinstetten, Germany). Each animal was imaged the day before treatment and days 7 and 14 after the treatment. Animals received an intraperitoneal injection of d-luciferin 150 mg/kg (Interchim, France). Twenty minutes later, the bioluminescence images were acquired using the in-vivo imaging system. Bioluminescent signal intensity of each tumor was quantified by summing detected photon counts using Bruker MI software (Brucker, Billerica, MA). Data were normalized to relative signal intensity (RSI) by using following equation: RSI =SIDn/SID0, where SI is signal intensity, Dn represents days after treatment and D0 is the day before treatment.

2.4.5 Ultrasound Imaging Follow-up

Ultrasound imaging of each tumor was performed the day before treatment and days 7 and 14 after the treatment using a 6–13 MHz linear transducer (Sonosite Inc, Bothell, WA). The longest dimensions of each tumor (in three orthogonal axes, X, Y and Z) were measured and used to calculate the volume of each tumor (volume = X*Y*Z*π/6). Data were normalized to relative tumor volume (RTV) by using the following equation: RTV = VDn/VD0where V is tumor volume, Dn represents days after treatment, and D0 is the day before treatment.

2.4.6 Histologic correlation/confirmation

Tumors were harvested at day 14 after treatments. Tumor tissues were cryosectioned at 8-µm slices for apoptosis staining. Level of apoptosis was determined with a terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) using TACS XL Blue Label kit (Trevigen, Gaithersburg, MD). On one slide, six fields were randomly photographed using an Olympus DP72 digital camera. Apoptosis results were analyzed as the apoptotic index, defined as the number of apoptotic cells / total number of cells ×100%.

2.4.7 RNA preparation and RT-qPCR

Tumor tissues from the HSV-TK/GCV-treated groups and the HSV-TK/GCV/RFH-treated group were collected. HSV-TK gene expression level in tumor tissues was quantified by qRT-PCR as described in the in-vitro experiment.

2.5 Statistical Analysis

Statistical software (SPSS, Version 19.0; Chicago, Ill) was used for all data analyses. Non-parametric Mann-Whitney U test was used to compare (i) relative proliferation rates among different cell groups; (ii) relative signal intensities measured by optical images; (iii) relative tumor volumes measured by ultrasound images; and (iv) HSV-TK gene expression rates at end points among various animal groups. A P value of less than 0.05 was considered significant difference.

3. Results

Radiofrequency hyperthermia enhanced the therapeutic effect of HSV-TK/GCV on hepatocellular cancer cell line HepG2

The in-vitro experiments showed the lowest viability in the cell group treated with HSV-TK/lentivirus+GCV plus RFH, compared to those of groups with HSV-TK/plasmid+GCV plus RFH, HSV-TK/lentivirus+GCV alone, HSV-TK/ plasmid+GCV alone, RFH alone and the control group with PBS (29.5±6.6% vs 52.1±3.1% vs 55.9±8.9% vs 75.9±4.3% vs 93.4±3.6% vs 100%, p < 0.05) (Figure 1A). The results of confocal microscopy are consistent with the MTS assay results, showing much less cells survived with the treatment of HSV-TK/lentivirus+GCV plus RFH than other groups (Figure 1B).

Figure 1.

Figure 1

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Therapeutic effect on hepatocellular cancer cells. (A) Confocal microscopy shows a decreased numbers of cells in the group with combination therapy of RFH plus HSV-TK/plasmid and HSV-TK/lentivirus, compared with the control, RFH-alone and gene alone groups. The red florescence represents the m-Cherry protein in the cytoplasm of cells and blue fluorescence emitted from DAPI which bound to the double-stranded DNA in the nuclei.(B) MTS assay was performed for six samples of each cell group, which demonstrated the lowest cell proliferation in the combination therapy group with RFH plus HSV-TK/lentivirus compared with those in other five groups (* P < 0.05).

The synergistic therapeutic effect of RFH combined with HSV-TK/GCV on mice subcutaneous hepatocellular cancers

Hepatocellular cancers were transfected with luciferase/m-cherry/lentivirus gene before being subcutaneously inoculated into the back of the mouse for creating tumor xenografts. All the mice tumors carrying the luciferase gene have the capability of catalyzing the oxidation of luciferin to emit bioluminescence (Figure 2A). The signal intensity is well correlated with the bioactivities of the tumors. This therapeutic effect was well mirrored with in-vivo follow-up optical imaging that showed that only in the tumor group treated with HSV-TK/lentivirus+GCV plus RFH demonstrated decreased relative photon counts after the treatment. Compared with the control group, tumors in the combination therapy group have more than 80% decrease of bioluminescence signal intensity (Figure 2B). Corresponding to the change of the bioluminescence signal intensity, follow-up ultrasound imaging also demonstrated that the treatment of HSV-TK/lentivirus+GCV plus RFH significantly inhibited the growth of tumors manifesting as the lowest average relative tumor volume. Compared with the control group, the average relative tumor volume decreased 79.6% in the group with combination therapy (Figure 3). qRT-PCR quantitative analysis of HSV-TK gene expression showed that RFH could enhance HSV-TK gene expression level in both HCC cells and mice tumors with one-fold increase of HSV-TK gene expression in cells transfected with HSV-TK/plasmid and 71% increase of gene expression in the cells HSV-TK/lentivirus. The increase of gene expression was consistent with the lowest relative photons and tumor volumes in the group treated with HSV-TK/lentivirus+GCV plus RFH, compared with the group treated by HSV-TK/GCV and RFH alone (Figure 4).

Figure 2.

Figure 2

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Optical/x-ray images of tumors (red/orange color) in six animal groups at days 0, 7 and 14 after different treatments. (A) The optical images demonstrate a significant decrease of photon signals of tumors in the animal group treated by combined therapy (gene+RFH), compared with RFH-alone, gene-alone and control animal groups, with the lentivirus/HSV-TK+RFH group showing the lowest signal. (B) Quantitative analysis of relative photon counts in each group shows that the lowest relative photon counts were seen in the group treated with the combination therapy of lentivirus/HSV-TK+GCV plus RFH (* P < 0.05).

Figure 3.

Figure 3

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(A) Ultrasound images of tumors in six animal groups (arrows). The tumor size in the group with combination therapy of HSV-TK/lentivirus+GCV plus RFH is the smallest, compared with other six groups. (B) Quantitative analysis of tumor volumes in six mice of each group shows that the lowest relative tumor volumes were seen in the group treated with the combination therapy of lentivirus/HSV-TK+GCV plus RFH (* P < 0.05).

Figure 4.

Figure 4

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qRT-PCR quantitative analysis of HSV-TK gene expression in cells and tumor tissues. RFH could enhance HSV-TK gene expression level in both HCC cells and mice tumors.

Measurements of the harvested tumors show that the tumor size in the combination therapy group is the smallest, compared with the other three groups (Figure 5). Histologic analysis of tumor apoptosis further displayed a higher apoptotic index in the group treated with HSV-TK/lentivirus+GCV plus RFH than in the groups treated with HSV-TK/plasmid+GCV plus RFH or RF hyperthermia alone (41.77%±13.5 % vs 23.3%±10.4%, P < 0.05; and 41.77%±13.5 % vs 4.57% ±1.71 %, P < 0.05) (Figures 6).

Figure 5.

Figure 5

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Histology of tumors. Representative tumors harvested from six different animal groups, showing the smallest tumor size treated by combination therapy (lentivirus/HSV-TK+GCV plus RFH), compared with other treatments.

Figure 6.

Figure 6

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Apoptosis with TUNEL staining of tumor tissues. (A) Apoptotic staining shows more apoptotic cells (blue dots) in the combination therapy group (gene+RFH) in comparison to the control groups. (B) Quantitative analysis of apoptosis in tumor tissues for six mice in each group shows that RFH combined with either plasmid/HSV-TK+GCV or lentivirus/HSV-TK+GCV could induce significantly more apoptosis than the therapy of plasmid/HSV-TK+GCV, lentivirus/HSV-TK or RFH alone (* P < 0.05).

4. Discussion

Radiofrequency ablation (RFA) is widely accepted as the first alternative for patients with early-stage HCC who are not suitable for surgical resection or liver transplantation (9, 27). RFA can effectively induce the necrosis of HCC measuring 3 cm or smaller because the ablative hyperthermia can yield a necrotic area of 5 cm or larger in diameter in one session, which can secure the complete ablation of a 3-cm HCC with a margin of at least 0.5 cm. However, routine HCC screening is not frequently carried out in detection of early-stage HCC for patients with underlying high risks of developing HCC, such as chronic hepatitis B and hepatic cirrhosis. This leads to the reality that a vast majority of HCCs are diagnosed at advanced stage, often with tumor sizes exceeding 5 cm. With the recent technical advances, such as the cluster electrode and the use of a generator that can deliver more thermal energy to tissues, attempts has been made to treat HCCs larger than 3–5 cm in diameter (2), but the local recurrence rate from such attempts is still much higher than that from RFA of the tumor lesions with a diameter less than 3 cm. Studies reported that tumor size is the only significant predictor for local tumor progression (2, 18). For the larger tumors, the tumor cells in the tumor margin can’t be eradicated by the insufficient hyperthermia due to heat sink effect of large vessels around the tumors margins (2, 18).

In this study, we investigated the possibility of using non-ablative hyperthermia at a temperature of 42 °C to enhance intratumoral HSV-TK/GCV-mediated gene therapy of HCCs. Findings of the study demonstrated that MRIHG-induced RF hyperthermia can significantly enhance HSV-TK gene expression in HCC cells and HCC tumors; which thereby resulted in a low survival of HCC cells in in-vitro experiments as well as shrunken tumor volumes and decreased optical signal intensities of treated tumors in the in-vivo experiments. In addition, our study provides evidence that both optical imaging and ultrasound imaging offer useful tools to assess the response of HCC to gene therapeutic regimens for such basic studies.

Given the paucity of systemic therapies available, gene therapy may become an attractive therapeutic alternative for HCC. Among the strategies, transfer of the HSV-TK gene, followed by GCV therapy has been extensively studied (28–31). GCV is phosphorylated by HSV-TK to be di- and tri-phosphorylated molecules by endogenous enzymes in the target cell, which then induces death of the tumor cell by inhibiting cellular DNA polymerase activity(22). Gene transfer relies on the use of genetically engineered viral or non-viral vectors that promote incorporation into cells and allows its transient or stable expression. Sufficient transduction efficiency and expression of the transgene are two key components to be considered when promoting a therapeutic effect on genetically manipulated cells (31). Among various vectors, lentiviral vectors exhibit the unique advantages over adenoviral vectors and adeno-associated viral (AAV) vectors. Lentiviral vector can yield the much higher transduction and transfection efficiency than plasmid vector and AAV vector but less hepatic toxicity and systemic immunologic response than adenoviral vectors (29–31). However, one study revealed that after intratumoral injection of lentivirus/LacZ gene, only 5 and 20% of tumor cells exhibited the LacZ gene expression (22), which hints the necessity of developing strategies to increase the gene transduction and transfection in tumor cells. This study establishes the proof of principle that RF hyperthermia can enhance the gene expression in HCC cells and boost the therapeutic effect of HSV-TK/GCV on both HCC cells in vitro and HCC tumors in vivo. Although unknown, the mechanism of hyperthermia-enhanced gene therapy may be associated with the increased permeability of cell membranes, which allows for more influx of genetic material. Ultimately, one application of this effect would be to take advantage of the RF hyperthermia at the margin of RF ablate tumors, which can specifically enhance HSV-TK/GCV-mediated gene therapy, and eradicate potential residual non-ablated tumor cells at the tumor margin. The current study has confirmed the feasibility that along with the session of RF ablation, HSV-TK/lentivirus genetic material can be directly delivered into the margins of the tumors through a transarterial catheter or a multichannel needle for direct intratumoral administration. Targeted vector introduction into the tumor via hyperthermia may prove beneficial in avoiding the toxicity of systemic exposure while achieving higher rates of gene expression and an improved therapeutic benefit.

In this study, we used a hyperthermia temperature of 42.0°C only, additional experiments are warranted to select the optimal temperature and to maximize the enhancing effect of RF hyperthermia on gene therapy. Because our study focused on establishing the proof of principle of the technique, we only explored the feasibility of this technique on mouse tumors. A further study of technique validation should be carried out in middle-to-large animals with orthotopic liver cancers, such as rabbits bearing hepatic VX2 tumors, which is a necessary step for clinical translation of the technique. In addition, in the present study we had to limit our follow-up time up to two weeks after the treatments. This was because longer follow-up period would result in the xenograft tumor masses, especially in the control animal group, becoming more than ten percent of the body weight, which was not approved by our Institutional Animal Care and Use Committee. Thus, as a limitation, this study did not allow us to evaluate the long-term therapeutic effects with follow-up optical imaging and ultrasound imaging.

In conclusion, local non-ablative RF hyperthermia can enhance HSV-TK/GCV-mediated therapeutic effect on human HCC cell line (HepG2) and mice HCC xenografts, which may help to decrease marginal tumor recurrence in RF-ablated hepatic malignancies by innovative integration of RF technology, interventional oncology, and direct intratumoral gene therapy, rather than systemic gene therapy.

Acknowledgments

This study was supported by an NIH RO1EBO12467 grant and a SIR foundation pilot research grant.

Grant support: This study was supported by an NIH RO1EBO12467 grant and Society of Interventional Radiology Pilot Research Grant.

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