Complete cure of established murine hepatocellular ... - Nature

Gene Therapy (1999) 6, 525–533  1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt

Complete cure of established murine hepatocellular carcinoma is achievable by repeated injections of retroviruses carrying the herpes simplex virus thymidine kinase gene S Kuriyama1, K Masui1, M Kikukawa1, T Sakamoto1, T Nakatani1, S Nagao1, M Yamazaki1, H Yoshiji1, H Tsujinoue1, H Fukui1, T Yoshimatsu2 and K Ikenaka3 1

Third Department of Internal Medicine, Nara Medical University, Kashihara; 2Institute for Medical Research, Wakunaga Pharm Co, Ltd, Hiroshima; and 3National Institute for Physiological Sciences, Okazaki National Research Institutes, Okazaki, Japan

Although xenotransplantation of retrovirus-producing cells into a tumor has been shown to be effective for the treatment of cancer, injections of recombinant retroviruses are much more feasible for clinical applications. We established a clone producing retroviruses carrying the herpes simplex virus thymidine kinase (HSVtk) gene with titers of up to 4 × 107 colony-forming units/ml, and examined the effectiveness of in vivo gene therapy against cancer. Syngeneic mice were inoculated subcutaneously with murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, and the treatment was initiated after tumors were established. When mice were given an intratumoral injection of HSVtk-carrying retroviruses or their producing cells followed by ganciclovir (GCV) treatment, significantly pro-

longed survival periods were observed. When mice were treated with repeated intratumoral injections of HSVtk-carrying retrovirus-producing cells, significant antitumor responses and some cures were induced by GCV treatment. Furthermore, repeated intratumoral injections of HSVtk-carrying retroviruses and GCV treatment resulted in complete regression of established HCC tumors in all animals used in the experiment. Mice that completely eradicated tumors exhibited protective immunity against wildtype HCC tumors. These results suggest that repeated injections of HSVtk-carrying retroviruses followed by GCV treatment is a potent modality for the treatment of solid tumors.

Keywords: hepatocellular carcinoma; established tumor; retrovirus; in vivo gene therapy; herpes simplex virus thymidine kinase; tumor immunity

Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies, causing an estimated 1 250 000 deaths every year worldwide.1 In the majority of cases (70–90%), HCC is found in conjunction with cirrhosis of the liver.2,3 Radical operation is the only curative modality for HCC but is appropriate in the minority of patients due to their limited hepatic reserves. Therefore, various alternative therapies, such as intra-arterial chemoembolization, percutaneous intratumoral ethanol injection and orthotopic liver transplantation, have been employed, but there is still no satisfactory treatment for HCC.4,5 Novel approaches must be developed if the overall survival rate of patients with HCC is to be significantly improved upon. Gene therapy could provide an innovative therapeutic approach for the treatment of HCC. One of the most promising approaches for the genetic treatment of cancer Correspondence: S Kuriyama, Third Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634–8522, Japan Received 30 September 1998; accepted 18 November 1998

is the use of the so-called suicide genes which render transduced cells sensitive to normally nontoxic prodrugs.6 The herpes simplex virus thymidine kinase (HSVtk) gene is a prototypic suicide gene because it can effectively phosphorylate an antiviral nucleoside analogue ganciclovir (GCV) to its monophosphate form, which is further phosphorylated by cellular kinases to DNA polymerase inhibitors.7 Effectiveness of the HSVtk/GCV system for the treatment of cancer has been demonstrated in animal models carrying various types of cancer and several gene transfer protocols using this system have progressed into human clinical trials for the treatment of cancer.8 Although a major technical impediment to gene transfer is the lack of ideal gene delivery systems, most common vehicles used for clinical trials of gene therapy so far are recombinant virus vectors. Adenoviruses can provide high efficiency of in vivo gene transfer. They, however, induce immune responses in hosts, resulting in severe inflammation and prevention of repeated gene transfer,9,10 although recent data suggest that neutralizing antibodies against adenoviruses do not inhibit efficient intratumoral gene transfer with adenovirus vectors.11 Retrovirus-mediated gene transfer is the method

Complete cure of established HCC S Kuriyama et al

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employed in the majority of approved gene therapy clinical protocols. No serious side-effects have been reported so far in clinical gene therapy trials using retrovirus systems. Importantly, there is no evidence suggesting that contamination of wild-type replication-competent retroviruses (RCRs) has occurred in clinical trial materials. Furthermore, although the numbers are still quite small, to date no evidence supports one of the initial concerns of this vector delivery system, that is, the potential for insertional mutagenesis.12 Some positive attributes of the retrovirus system are that the vectors have sufficient coding capacity for most therapeutic genes, they are relatively easy to produce, gene transfer is stable, and therapeutic levels of gene expression for a variety of genes have been obtained. Thus, it is important to continue studies for improving retrovirus-mediated gene transduction, even as new gene delivery systems are described. Although the efficiency of gene transduction by retroviruses is relatively low, retroviruses do not induce significant host immune responses.13 It is, therefore, possible to repeat gene transfer, which would lead to increased gene transduction efficiency. We previously demonstrated that the bacterial ␤galactosidase gene, lacZ gene, was expressed exclusively in HCC cells not only by in vitro gene transfer but also by in vivo gene transfer, when it was directed by the albumin gene promoter in a retrovirus vector.14 Furthermore, we have shown that the albumin gene promoter can induce much stronger expression of cytokine genes, such as interleukin-2, interleukin-3 and tumor necrosis factor␣ genes, in HCC cells not only by ex vivo gene transfer but also by in vivo gene transfer compared with the simian virus 40 early region promoter and enhancer that is recognized as a generally strong promoter and has been widely used in experimental and clinical gene therapy trials.15,16 We have also demonstrated that HCC cells infected with retroviruses carrying the HSVtk gene under the transcriptional control of the albumin gene promoter were selectively eliminated by GCV treatment, while non-HCC cells infected with the same retroviruses were not.17 Furthermore, we have shown that if HCC cells expressing the HSVtk gene were contained in HCC tumors at very small ratios, their development was substantially suppressed by GCV treatment.18 We report here that established HCC tumors were completely cured by repeated injections of HSVtk-carrying retroviruses followed by GCV treatment in all animals used in the experiment. Furthermore, during the tumor regression, marked infiltration of lymphocytes was induced in tumor sites and animals that completely eradicated established HCC tumors acquired post-treatment immunity to parental HCC tumor.

Results Antitumor effect induced by a single injection of HSVtktransduced cells, HSVtk-carrying retroviruses and HSVtk-carrying retrovirus-producing cells Syngeneic BALB/c mice were inoculated subcutaneously with parental BNL1ME A.7R.1 HCC cells. When HCC tumors reached 5 mm in diameter, animals in the control group received an intratumoral injection of the conditioned medium and those in the treatment groups received an intratumoral injection of HSVtk-transduced

HCC cells, HSVtk-carrying retrovirus-producing cells or HSVtk-carrying retroviruses. Two days later, treatment started and all animals were administered intraperitoneally with GCV for 14 consecutive days. As shown in Figure 1, mice that received an intratumoral injection of the medium followed by GCV treatment developed rapidly growing tumors. Conversely, tumors of animals in all treatment groups did not grow significantly during GCV treatment. Furthermore, tumor volume became smaller compared with that before the treatment at certain timepoints during GCV treatment in two, five and four of eight mice that received an injection of HSVtk-transduced cells, HSVtk-carrying retrovirus-producing cells and HSVtk-carrying retroviruses, respectively. Tumor volume among the three treatment groups was not significantly different during GCV treatment. Survival rates of animals are shown in Figure 2. All animals in the control group died within 35 days after the initiation of GCV treatment. Conversely, survival periods of animals that received an intratumoral injection of HSVtk-transduced cells, HSVtkcarrying retrovirus-producing cells or HSVtk-carrying retroviruses followed by GCV treatment were significantly prolonged, and survival rates on day 45 from the initiation of treatment were 38%, 75% and 50%, respectively. There were, however, no animals that completely eradicated established tumors.

Antitumor effect induced by repeated injections of HSVtk-carrying retrovirus-producing cells The mice bearing established subcutaneous HCC tumors received intratumoral injections of HSVtk-carrying retrovirus-producing cells for 3 consecutive days, followed by intraperitoneal administrations of GCV for 5 consecutive days. The course of the treatment was given twice to seven animals. Mean tumor volume and individual tumor volume were shown in Figure 3. Even after the second course of treatment, tumor volume was not significantly larger compared with that before the treatment.

Figure 1 Antitumor effect on established HCC tumors based on the single treatment protocols. Syngeneic mice were inoculated subcutaneously with 2 × 106 HCC cells suspended in 100 ␮l of PBS. When mice developed established HCC tumors with a diameter of ⬎5 mm, they were given a single intratumoral injection of the conditioned medium (O, n = 8), 2 × 106 HSVtk-transduced cells (쮿, n = 8), 2 × 106 HSVtk-carrying retrovirus-producing cells (쎲, n = 8) or 2 × 106 c.f.u. of HSVtk-carrying retroviruses (쏔, n = 8). Each inoculum volume was 100 ␮l. Two days after the injection, animals were administered intraperitoneally with GCV (50 mg/kg/day) daily for 14 consecutive days. Each data point represents the mean ± s.d. Tumor volume of the control group is significantly larger than that of three treatment groups after day 10, using Student’s t test.


producing cells, no antitumor effects were observed and subcutaneous tumors grew rapidly (data not shown).

Figure 2 Survival rate of mice treated with the single treatment protocols. On day 1, mice bearing established subcutaneous HCC tumors with a diameter of ⬎5 mm were given a single intratumoral injection of the conditioned medium (bold line, n = 8), 2 × 106 HSVtk-transduced cells (dashed line, n = 8), 2 × 106 HSVtk-carrying retrovirus-producing cells (dotted line, n = 8), or 2 × 106 c.f.u. of HSVtk-carrying retroviruses (thin line, n = 8). Each inoculum volume was 100 ␮l. From day 3 to day 16, animals were administered intraperitoneally with GCV (50 mg/kg/day). Survival rates of the control group are significantly lower than those of three treatment groups, using Wilcoxon test.

Antitumor effect induced by repeated injections of HSVtk-carrying or lacZ-carrying retroviruses The mice bearing established subcutaneous HCC tumors received intratumoral injections of HSVtk-carrying or lacZ-carrying retroviruses for 5 consecutive days, followed by intraperitoneal administration of GCV for 5 consecutive days. The course of the treatment was given twice to animals. Repeated intratumoral injections of lacZ-carrying retroviruses followed by GCV treatment did not induce antitumor effects on HCC, and tumors grew rapidly despite the treatment (Figure 4). The first animal died on day 27 after the first injection of the retroviruses. Conversely, as shown in Figure 5, repeated intratumoral injections of HSVtk-carrying retroviruses followed by GCV treatment exhibited profound antitumor effects. After the second course of treatment, tumor volume was reduced significantly compared with that before the treatment. Furthermore, complete tumor regression was observed in two of seven animals. The third course of treatment was given to five mice that still bore tumors and complete cure was achieved in all animals. None of these tumor-free animals experienced tumor recurrence in a 60-day observation period. Histological analysis of tumors treated with HSVtkcarrying retroviruses and GCV The mice bearing established subcutaneous HCC tumors with a diameter of over 5 mm were given an intratumoral injection of HSVtk-carrying or lacZ-carrying retroviruses followed by intraperitoneal administrations of GCV for 5 consecutive days. Two days after the termination of the treatment, tumors were excised and stained with hematoxylin and eosin. There were few accompanying infiltrating cells in the tumors injected with lacZ-carrying

Figure 3 Antitumor effect on established HCC tumors induced by repeated injections of HSVtk-carrying retrovirus-producing cells. Seven syngeneic mice were inoculated subcutaneously with 2 × 106 HCC cells suspended in 100 ␮l of PBS. When mice developed an established HCC tumor with a diameter of ⬎5 mm, 2 × 106 HSVtk-carrying retrovirusproducing cells (100-␮l inoculum volume) were injected intratumorally daily from days 1 to 3, followed by intraperitoneal administration of GCV (100 mg/kg/day) from days 5 to 9. The same treatment was repeated from days 12 to 20. Tumor volume was estimated every 3 or 4 days. Open squares and bars represent the means and s.d., respectively, and black dots represent the individual tumor volume.

Furthermore, tumor volume was smaller compared with that before the treatment in five of seven animals on day 25. Importantly, complete tumor regression was observed in two of seven animals on day 42. These tumor-free animals did not experience tumor recurrence during a 60day observation period. Conversely, when a similar experiment was carried out by intratumorally injecting NIH3T3TK− cells instead of HSVtk-carrying retrovirus-

Figure 4 Antitumor effect on established HCC tumors induced by repeated injections of lacZ-carrying retroviruses. Seven syngeneic mice were inoculated subcutaneously with 2 × 106 HCC cells suspended in 100 ␮l of PBS. When mice developed an established HCC tumor with a diameter of ⬎5 mm, 2 × 106 c.f.u. of lacZ-carrying retroviruses (100-␮l inoculum volume) were injected intratumorally daily from days 1 to 5, followed by intraperitoneal administration of GCV (100 mg/kg/day) from days 6 to 10. The same treatment was repeated from days 16 to 25. Tumor volume was estimated every 3 or 4 days. Each data point represents the mean ± s.d.

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Figure 5 Antitumor effect on established HCC tumors induced by repeated injections of HSVtk-carrying retroviruses. Seven syngeneic mice were inoculated subcutaneously with 2 × 106 HCC cells suspended in 100 ␮l of PBS. When mice developed an established HCC tumor with a diameter of ⬎5 mm, 2 × 106 c.f.u. of HSVtk-carrying retroviruses (100␮l inoculum volume) were injected intratumorally daily from days 1 to 5, followed by intraperitoneal administration of GCV (100 mg/kg/day) from days 6 to 10. The second course of the treatment was given to all animals from days 16 to 25, and the third course was given to five of seven animals from days 37 to 46. Tumor volume was estimated every 3 or 4 days. Each data represents the mean ± s.d.

retroviruses (Figure 6a). Conversely, marked infiltration of lymphocytes was observed throughout the tumor injected with HSVtk-carrying retroviruses and HCC cells were profoundly damaged (Figure 6b). Furthermore, many acidophilic glass-like HCC cells with condensed nuclei were observed in the tumor injected with HSVtkcarrying retroviruses, suggesting that the HSVtk/GCV system induced apoptotic death of HCC cells (Figure 6c).

Tumor immunity induced by HSVtk/GCV system To learn whether the nine tumor-free mice, two treated with injections of HSVtk-carrying retrovirus-producing cells and seven treated with injections of HSVtk-carrying retroviruses, developed immunity to parental HCC tumors, they were rechallenged subcutaneously with a tumorigenic dose of parental HCC cells (1 × 106 cells/100␮l inoculum volume) into contraflank regions and observed for tumor development for another 60 days. All the animals exhibited resistance to parental HCC tumors, with no tumor development. To learn if the immunity is specific to parental HCC tumors, the animals were again inoculated subcutaneously with a tumorigenic dose of colon 26 cells (1 × 106 cells/100 ␮l inoculum volume), ie heterologous but syngeneic murine colonic adenocarcinoma cells. There was no protection against the colonic adenocarcinoma cell challenge, and all mice developed subcutaneous tumors within 7 days with no significant delay of tumor formation compared with control mice.

Figure 6 Histological analysis of tumors injected with HSVtk-carrying or lacZ-carrying retroviruses. The mice bearing established tumors with a diameter of over 5 mm were given an intratumoral injection of 2 × 106 c.f.u. of HSVtk-carrying or lacZ-carrying retroviruses, followed by intraperitoneal administrations of GCV (100 mg/kg/day) for 5 consecutive days. Two days after the termination of the treatment, tumors were excised and stained with hematoxylin and eosin. There were few accompanying infiltrating cells in the tumor injected with lacZ-carrying retroviruses (a). Marked infiltration of lymphocytes with damaged tumor cells was observed in the tumor injected with HSVtk-carrying retroviruses (b). A number of acidophilic glass-like cells with condensed nuclei, indicated by arrowheads, were also observed in the tumor injected with HSVtk-carrying retroviruses (c). The Figure shows representative pictures (magnification, × 200).


Discussion Retrovirus vectors are by far the most frequently used system in clinical trials and this system has been proven so far to be a relatively safe modality with no severe sideeffects. We have demonstrated here that a single intratumoral injection of HSVtk-transduced HCC cells, HSVtkcarrying retroviruses or HSVtk-carrying retrovirus-producing cells followed by GCV treatment inhibited the growth of established HCC tumors, resulting in significantly prolonged survival periods. There were, however, no animals that completely eradicated established tumors. Subsequent studies have demonstrated that repeated intratumoral injections of HSVtk-carrying retroviruses or HSVtk-carrying retrovirus-producing cells followed by GCV treatment could induce profound antitumor effects. Injections of HSVtk-carrying retroviruses proved as effective as those of HSVtk-carrying retrovirusproducing cells. Importantly, complete cures of established tumors were achieved in all animals treated with repeated injections of HSVtk-carrying retroviruses and GCV administration. Furthermore, animals that completely eradicated established HCC tumors acquired immunity against parental HCC tumors. Recently, Tamura et al19 have proposed an ingenious strategy for the treatment of glioma, taking advantage of the behavior of glioma cells. They have demonstrated that glioma cells but not retrovirus-producing fibroblast cells pursued previously injected glioma cells and mixed very well in murine brain and that when HSVtk-transduced glioma cells were injected stereotactically into the previous injection site of wild-type glioma cells, profound antitumor effects were induced by systemic GCV treatment. However, injections of HSVtk-transduced tumor cells are not practical for clinical applications, because it takes a considerable time to establish HSVtktransduced tumor cells from patients. We, therefore, did not examine antitumor effects on established HCC tumors of repeated injections of HSVtk-transduced HCC cells. A major limitation of retrovirus-mediated gene transfer is its low transduction efficiency. To circumvent this, several approaches have been explored. One of them is the injection of retrovirus-producing cells into a tumor. The hope is that retrovirus-producing cells within the tumor would continuously secrete retrovirus vectors for several days at least, increasing the exposure of the tumor cells to retrovirus vector. This approach was validated by experiments in which a producing cell line making retroviruses carrying the lacZ gene was inoculated into gliomas growing in rat brain.20 Histochemical evidence of the lacZ gene transfer was seen in up to 10% of the glioma cells, while normal brain was not transduced. The HSVtk gene was transferred with a similar technique, and efficiencies of 10% to 70% were claimed.21 Culver et al22 have reported the elimination of microscopic brain tumors by stereotactic injections of a producing cell line making HSVtk-carrying retroviruses followed by GCV treatment. We have also shown that when mice with established solid tumors were injected intratumorally with cells making retroviruses carrying the tumor necrosis factor-␣ gene, complete cures were achieved.16 In the present study, it has been demonstrated that repeated injections of HSVtk-carrying retrovirus-producing cells into established HCC tumors could induce profound

antitumor effects, with complete cures in two of seven animals. Injections of retrovirus-producing cells, however, have some regulatory hurdles. Transplantation of cells into humans may be risky due to host inflammatory responses to foreign antigens present in xenogeneic cells. There is also a risk of vascular embolism caused by the injection of a large number of cells. When we injected 2 × 106 HSVtk-carrying retrovirus-producing cells into the portal vein of mice, more than half of the animals died within several hours. Conversely, the injection of 2 × 106 colony-forming units (c.f.u) of HSVtk-carrying retroviruses into the portal vein did not cause any deaths (unpublished data). Furthermore, the time and resources required to manufacture whole-cell therapeutics may render them economically unfeasible. Importantly, it appears impossible to always maintain a sufficient number and excellent quality of retrovirus-producing cells, because the ability of retrovirus-producing cells to make high-titer retroviruses is decreased gradually during cultivation. In the present study, we intended to repeat injections of HSVtk-carrying retrovirus-producing cells, but we could not perform the third course of the treatment due to an insufficient number of retrovirus-producing cells. An alternative approach to increase the transduction efficiency of retrovirus vectors is the use of concentrated retrovirus supernatants. Murine-based retrovirus particles are limited in their ability to be concentrated by ultracentrifugation, because infectibility is severely reduced by the damage to the envelope protein that occurs during ultracentrifugation. Recently, Bowles et al23 developed an efficient technique for the concentration of retrovirus particles through the use of relatively lowspeed centrifugation, and demonstrated that retroviruses can be readily concentrated up to 500-fold. Smiley et al24 have also concentrated retrovirus vectors carrying the HSVtk gene using tangenited flow ultrafiltration, resulting in production of retrovirus vectors with titers of 3 × 107 to 1 × 108 c.f.u./ml. When the high-titer retrovirus preparations were injected directly into established tumors in syngeneic mouse tumor models, significant antitumor responses and some complete cures were observed. We also made efforts to establish clones that can produce high-titer retrovirus vectors. We modified the packaging cell lines, Psi2 and PA317, by stably introducing the polyomavirus early gene and found that the activity of the retrovirus long terminal repeat promoter is stimulated by the polyomavirus early region proteins in these packaging cell lines. Subsequently we have shown that it is possible routinely to establish stable clones producing retrovirus titers over 1 × 107 c.f.u/ml from these packaging cell lines.25 In the present study, we have established a retrovirus-producing clone through repeated subcloning by limiting dilution that can produce high-titer retrovirus vectors. The clone used in the present study could stably produce HSVtk-carrying retroviruses with titers of 2 to 4 × 107 c.f.u./ml at 32°C for several days after reaching confluency. We performed three courses of repeated injections of HSVtk-carrying retroviruses followed by GCV treatment. After the second course of the treatment two of seven animals completely eradicated tumors and after the third course all animals completely eradicated tumors. Tumor recurrence was not observed in any of these cured animals. We previously learned through our

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preliminary experiments that repeated intratumoral injections of retroviruses carrying a lacZ reporter gene with a titer of 1 × 106 c.f.u./ml resulted in very low transduction efficiency, while those of retroviruses with a titer of 1 × 107 c.f.u./ml resulted in 10 to 20% transduction efficiency (data not shown). Smiley et al24 have demonstrated that an intratumoral injection of HSVtk-carrying retroviruses with titers of 1 × 108, 1 × 107 and 1 × 106 c.f.u./ml resulted in transduction frequency of approximately 16%, 11% and 1%, respectively. These results suggest that the use of retroviruses with titers of ⭓1 × 107 c.f.u./ml appears to be critical for achieving effective transduction in vivo. As for the eradication of established tumors using the HSVtk/GCV system, host antitumor immunity appears to play an extremely important role.26 We previously reported that when syngeneic immunocompetent mice were inoculated subcutaneously with mixtures of parental and HSVtk-transduced murine HCC cells at ratios of 75:25 and 50:50, tumor development was substantially inhibited by GCV treatment, with tumor incidences being 11% and 0%, respectively.17 Conversely, when similar experiments were carried out in nude mice, all developed subcutaneous tumors despite GCV treatment (data not shown). In the present study, we realized that regrowth of wild-type HCC tumors treated with HSVtk-carrying retroviruses and GCV was relatively slow after the second course of the treatment. When the tumors started regrowth, the third course of the treatment was performed, resulting in complete tumor regression. These results suggest that host antitumor immunity was elicited potent enough to eradicate tumors completely during the repeated courses of the treatment. Histological analyses may support this possibility because marked infiltration of lymphocytes was induced in tumor sites by the treatment. Furthermore, animals eradicated established HCC tumors exhibited complete resistance to the subsequent rechallenge of wild-type HCC. It is, therefore, suggested that the host immune system plays a crucial role in antitumor effects induced by the HSVtk/GCV system. These results also suggest that repeated injections of HSVtk-carrying retroviruses followed by GCV treatment caused complete tumor regression by eliciting potent tumor immunity in hosts. In the present study, the albumin promoter was used to direct the HSVtk gene contained in a retrovirus vector. It has already been shown that retroviruses can infect only dividing cells, but not nondividing cells.27 Normal hepatocytes, unlike HCC cells, are in a quiescent, nonreceptive stage of cell growth. We have already shown that intraportal injection of retroviruses carrying a lacZ reporter gene under the control of the albumin promoter did not result in lacZ expression in the liver.14 However, the majority of patients with HCC have underlying liver cirrhosis. It should therefore be important to examine whether retroviruses infect cirrhotic liver. Furthermore, although we demonstrated that repeated intratumoral injections of retroviruses resulted in complete cures, the injected volume of retroviruses exceeded the tumor volume, indicating that the injection volume should be reduced for clinical applications. It is, therefore, necessary to increase retrovirus titers further. Thus, although more investigations need to be carried out to prove the usefulness of this strategy, the results demonstrated here suggest that gene therapy for established tumors with

repeated injections of HSVtk-carrying retroviruses followed by GCV treatment is a feasible and promising therapeutic approach. These results also shed light on therapeutically relevant factors that may aid in design of future clinical protocols for retrovirus-based gene therapy against cancer.

Materials and methods Animals Eight-week-old female BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). Animal experiments were performed with approved protocols and in accordance with recommendations for the proper care and use of laboratory animals. Chemicals GCV sodium (GCV) was generously provided by F Hoffmann-La Roche (Basel, Switzerland). Geneticin (G418) was purchased from GIBCO-BRL (Bethesda, MD, USA). Polybrene (hexadimethrine bromide) was obtained from Sigma (St Louis, MO, USA). Cell culture The murine HCC cell line, BNL1ME A.7R.1,28 which was originally established from a BALB/c mouse, murine fibroblast cell line, NIH3T3TK−, and the amphotropic retrovirus packaging cell line, PA317,29 were purchased from the American Type Culture Collection (Rockville, MD, USA). The murine colonic adenocarcinoma cell line, colon 26,30 which was originally established from a BALB/c mouse, was kindly provided by the Chugai Pharmaceutical Company (Tokyo, Japan). All cells except PA317 were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 0.3 mg/ml of l-glutamine, 100 units/ml of ampicillin and 100 ␮g/ml of streptomycin at 37°C in 5% CO2 in air. PA317 cells were grown in conditioned Dulbecco’s modified Eagle’s medium. Retrovirus construct Construction of the Alb e/p-pNT230 and Alb e/p-pIP200 retrovirus vectors, which contain the HSVtk and lacZ gene, respectively, under the transcriptional control of the murine albumin enhancer and promoter, has been described elsewhere,14,31 and diagrams are shown in Figure 7.

Figure 7 Structure of retrovirus vectors. Alb e/p-pNT230 and Alb e/ppIP200 retrovirus vectors contain the neomycin phosphotransferase (neo) gene, which confers G418 resistance on transduced cells, the murine albumin enhancer and promoter (Alb e/p) region, as an internal promoter, and the HSVtk or lacZ gene within two Moloney murine leukemia virus long terminal repeats (LTRs).


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Figure 8 Protocols using retrovirus-mediated in vivo HSVtk gene transfer for the treatment of established HCC tumor. Syngeneic BALB/c mice were inoculated subcutaneously with 2 × 106 murine HCC cells suspended in 100 ␮l of PBS. When mice developed established HCC tumors with a diameter of ⬎5 mm, they were divided randomly into various treatment groups. For single treatment protocols (a), animals received an intratumoral injection of the conditioned medium, 2 × 106 HSVtk-transduced cells, 2 × 106 HSVtk-carrying retrovirus-producing cells or 2 × 106 c.f.u. of HSVtk-carrying retroviruses. Each inoculum volume was 100 ␮l. Each group consisted of eight mice. From 2 days after the injection, animals in all groups were given intraperitoneal administrations of GCV (50 mg/kg/day) daily for 14 consecutive days. For a repeated treatment protocol (b), seven mice bearing established tumors were given intratumoral injections of 2 × 106 HSVtk-carrying retrovirus-producing cells (100-␮l inoculum volume) daily from days 1 to 3, followed by intraperitoneal administrations of GCV (100 mg/kg/day) from days 5 to 9. The same treatment was repeated from days 12 to 20. For other repeated treatment protocols (c), seven mice bearing established tumors were given intratumoral injections of 2 × 106 c.f.u. of HSVtk-carrying or lacZcarrying retrovi¨ruses (100-␮l inoculum volume) daily from days 1 to 5, followed by intraperitoneal administrations of GCV (100 mg/kg/day) from days 6 to 10. The same treatment was repeated to all animals from days 16 to 25. The third course of the treatment was given to five of seven animals in the HSVtk-carrying retrovirus group from days 37 to 46. In all treatment protocols, tumor volume was estimated every 3 or 4 days.

Vector production Production of replication-defective amphotropic retrovirus vectors was accomplished by a calcium phosphate precipitation procedure, as described previously.15 Independent G418-resistant PA317 clones were isolated and expanded as retrovirus-producing ones. Several hightiter retrovirus-producing clones were selected and subsequently subcloned by a limiting dilution technique. Titers of the subclones were assayed by counting G418resistant colonies of retrovirus-infected NIH3T3TK− cells. Subcloning was repeated to establish high-titer retrovirus-producing clones. The highest-titer retrovirus-producing subclone selected for subsequent experiments stably had a titer of 2 to 4 × 107 c.f.u./ml. The culture supernatant of the highest-titer retrovirus-producing subclone was collected, passed through a 0.45-␮m pore filter (Millipore, Bedford, MA,USA), and stored at −80°C. All batches of retrovirus stocks used in subsequent experiments were examined for RCRs by using the S+/L− focus assay (Microbiological Associates, Rockville, MD, USA), and were confirmed to contain no RCRs or at most less than 1 RCR per 4 × 107 c.f.u. or retroviruses (the highest

tested). It served as a source of infectious recombinant retroviruses.

In vitro gene transfer Freshly prepared murine HCC cells were infected with recombinant retroviruses at 37°C, 5% CO2 for 4–6 h in the medium containing 10 ␮g/ml polybrene. The medium was replaced with a fresh virus-free one and the cells were incubated for 2 days. Infected cells were then selected by addition of G418 to a final concentration of 1 mg/ml and the uncloned bulk of G418-resistant cells were expanded as HSVtk transduced cells for subsequent experiments. Single treatment protocols The protocol for the single injection treatment is shown in Figure 8a. Parental murine HCC cells were suspended in phosphate-buffered saline (PBS) at a concentration of 2 × 107 cells/ml, and 100-␮l inoculum volume was injected subcutaneously into the flank regions of syngeneic BALB/c mice. When tumors reached 5 mm in diameter, animals were separated randomly into four groups. Each


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group consisted of eight animals. Animals in the control group received an intratumoral injection of 100 ␮l of the conditioned medium. Animals in the treatment groups were treated by three different protocols. Animals in the first treatment group were injected intratumorally with 2 × 106 HSVtk-transduced HCC cells suspended in 100 ␮l of the conditioned medium. Those in the second treatment group were injected intratumorally with 2 × 106 HSVtkcarrying retrovirus-producing cells suspended in 100 ␮l of the conditioned medium with 10 ␮g/ml polybrene. Those in the third group were injected intratumorally with 2 × 106 c.f.u. of HSVtk-carrying retroviruses contained in 100 ␮l of the conditioned medium with 10 ␮g/ml polybrene. From 2 days after the injection, animals were given GCV at 50 mg/kg body weight per day intraperitoneally for 14 consecutive days. Tumor diameter was measured every 3 or 4 days by a caliper and tumor volume was calculated according to the formula: V (mm3) = A (mm) × B (mm)2/2 (A = largest diameter; B = smallest diameter). Mice that developed excessive size of tumors were killed at proper time-points.

Repeated treatment protocols HCC tumors were developed subcutaneously in syngeneic BALB/c mice as described above. When tumors reached 5 mm in diameter, animals were randomly divided into three treatment groups. Each group consisted of seven animals. The first repeated treatment protocol is shown in Figure 8b. Animals received intratumoral injections of 2 × 106 HSVtk-carrying retrovirus-producing cells suspended in 100 ␮l of the conditioned medium with 10 ␮g/ml polybrene daily from days 1 to 3, followed by intraperitoneal administrations of GCV (100 mg/kg body weight per day) from days 5 to 9. Three days after the termination of the first treatment, animals were given the same treatment. They received intratumoral injections of the same number of retrovirus-producing cells from days 12 to 14, followed by intraperitoneal administration of the same dose of GCV from days 16 to 20. The second and third repeated treatment protocols are shown in Figure 8c. Animals received intratumoral injections of 2 × 106 c.f.u. of HSVtk-carrying or lacZ-carrying retroviruses contained in 100 ␮l of the conditioned medium with 10 ␮g/ml polybrene daily from days 1 to 5, followed by intraperitoneal administrations of GCV (100 mg/kg body weight per day) from days 6 to 10. The same treatment was repeated to all animals from days 16 to 25. The third course of treatment was given to five of seven animals in the HSVtk-carrying retrovirus group from days 37 to 46. Tumor volume was estimated every 3 or 4 days as described above. Histological analysis Mice bearing established subcutaneous HCC tumors with a diameter of over 5 mm were given an intratumoral injection of 2 × 106 c.f.u. of HSVtk-carrying or lacZ-carrying retroviruses contained in 100 ␮l of the conditioned medium with 10 ␮g/ml polybrene, followed by intraperitoneal administration of GCV (100 mg/kg body weight per day) for 5 consecutive days. Two days after the termination of the treatment, animals were killed to excise subcutaneous tumors. Tumors were then fixed in 10% buffered formalin, embedded in paraffin and stained with hematoxylin and eosin.

Statistics Results are expressed as means ± s.d. Standard descriptive statistical tests, ie Student’s t test and Wilcoxon test, were used. A P value ⬍0.05 was considered to indicate a significant difference between groups.

Acknowledgements This work was supported in part by Grant-in-Aid for Scientific Research (B) (07457141, 10470140) from the Ministry of Education, Science, Sports and Culture of Japan.

References 1 Wanebo HJ, Falkson G, Order SE. Cancer of the hepatobiliary system. In: DeVita V, Hellman S, Rosenberg SA (eds). Cancer: Principles and Practice of Oncology. 3rd edn. Lippincott: Philadelphia, 1989, pp 836–874. 2 Kew MC. Clinical, pathologic, and etiologic heterogeneity in hepatocellular carcinoma: evidence from Southern Africa. Hepatology 1981; 1: 366–369. 3 Brotodihardjo AE et al. Hepatocellular carcinoma in western Sydney: aetiology, changes in incidence, and opportunities for better outcomes. Med J Aust 1994; 161: 433–435. 4 Venook AP. Treatment of hepatocellular carcinoma: too many options? J Clin Oncol 1994; 12: 1323–1334. 5 Colleoni M et al. Medical treatment of hepatocellular carcinoma: any progress? Tumori 1994; 80: 315–326. 6 Mullen CA. Metabolic suicide genes in gene therapy. Pharmac Ther 1994; 63: 199–207. 7 Field AK et al. 9-{[2-Hydroxy-1-(hydroxymethyl) ethoxy]methyl其guanine: a selective inhibitor of herpes group virus replication. Proc Natl Acad Sci USA 1983; 80: 4139–4143. 8 Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science 1995; 270: 404–410. 9 Yang Y et al. Cellular immunity to viral antigens limits E1deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA 1994; 91: 4407–4411. 10 Kaplan JM, Smith AE. Transient immunosuppression with deoxyspergualin improves longevity of transgene expression and ability to readminister adenoviral vector to the mouse lung. Hum Gene Ther 1997; 8: 1095–1104. 11 Bramson JL, Hitt M, Gauldie J, Graham FL. Pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing IL-12 but inhibits virus dissemination. Gene Therapy 1997; 4: 1069–1076. 12 Ross G et al. Gene therapy in the United States: a 5-year status report. Hum Gene Ther 1996; 7: 1781–1790. 13 Patijn GA et al. High-efficiency retrovirus-mediated gene transfer into the livers of mice. Hum Gene Ther 1998; 9: 1449–1456. 14 Kuriyama S et al. A potential approach for gene therapy targeting hepatoma using a liver-specific promoter on a retroviral vector. Cell Struct Funct 1991; 16: 503–510. 15 Cao G et al. Construction of retroviral vectors to induce strong hepatoma cell-specific expression of cytokine genes. J Gastroenterol Hepatol 1996; 11: 1053–1061. 16 Cao G et al. Complete regression of established murine hepatocellular carcinoma by in vivo tumor necrosis factor ␣ gene transfer. Gastroenterology 1997; 112: 501–510. 17 Kuriyama S et al. Tissue-specific expression of HSV-tk gene can induce efficient antitumor effect and protective immunity to wild-type hepatocellular carcinoma. Int J Cancer 1997; 71: 470– 475. 18 Kuriyama S et al. Bystander effect caused by suicide gene expression indicates the feasibility of gene therapy for hepatocellular carcinoma. Hepatology 1995; 22: 1838–1846. 19 Tamura M et al. Targeted killing of migrating glioma cells by injection of HTK-modified glioma cells. Hum Gene Ther 1997; 8: 381–391.


20 Short MP et al. Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line. J Neurosci Res 1990; 27: 427–439. 21 Ram Z et al. In situ retrovirus-mediated gene transfer for the treatment of brain tumors in rats. Cancer Res 1993; 53: 83–88. 22 Culver KW et al. In vivo gene transfer with retroviral vectorproducer cells for treatment of experimental brain tumors. Science 1992; 256: 1550–1552. 23 Bowles NE et al. A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepatocyte transduction in vivo. Hum Gene Ther 1996; 7: 1735– 1742. 24 Smiley WR et al. Establishment of parameters for optimal transduction efficiency and antitumor effects with purified high-titer HSV-TK retroviral vector in established solid tumors. Hum Gene Ther 1997; 8: 965–977. 25 Yoshimatsu T, Tamura M, Kuriyama S, Ikenaka K. Improvement of retroviral packaging cell lines by introducing the polyomavirus early region. Hum Gene Ther 1998; 9: 161–172.

26 Vile RG et al. Systemic gene therapy of murine melanoma using tissue specific expression of the HSVtk gene involves an immune component. Cancer Res 1994; 54: 6228–6234. 27 Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol 1990; 10: 4239–4242. 28 Patek PQ, Collins JL, Cohn M. Transformed cell lines susceptible or resistant to in vivo surveillance against tumorigenesis. Nature 1978; 276: 510–511. 29 Miller AD, Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 1986; 6: 2895–2902. 30 Tsuruo T et al. Metastasis after intravenous inoculation of highly metastatic variants of mouse tumors and the effects of several antitumor drugs on the tumors. Gann 1984; 75: 193–198. 31 Kuriyama S et al. Gene therapy for the treatment of hepatoma by retroviral-mediated gene transfer of the herpes simplex virus thymidine kinase. Int Hepatol Commun 1993; 1: 253–259.

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