Combination Therapy With Suicide and Cytokine Genes for Hepatic Metastases of Lung Cancer

doi:10.1378/chest.112.5.1332

Chest

Volume 112, Issue 5, November 1997, Pages 1332-1337

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Metastases of lung cancer are a major cause of treatment failure. To evaluate the therapeutic efficacy of gene therapy in metastatic lung cancer, we used adenoviral (ADV) mediated transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene and the cytokine gene interleukin-2 (IL-2) to treat a murine model of metastatic lung cancer in the liver. Hepatic metastases were established by intrahepatic implantation of LL2 cells in syngeneic recipient mice. One week after tumor implantation, various replication defective ADV vectors were injected intratumorally. Treatment with a vector expressing the HSV-tk followed by ganciclovir administration with ADV.tk resulted in significant regression of tumor (p<0.01) as well as prolongation of survival (p<0.001). While a vector expressing mouse IL-2 ADV.IL-2 alone was ineffective, combination therapy with HSV-tk resulted in further tumor regression and improvement of animal survival (p<0.05). These results demonstrate that suicide and cytokine genes can be utilized in combination to treat metastatic lung cancer in vivo.

Section snippets

Recombinant Adenoviral Vector

Recombinant replication defective adenoviral vectors (ElA deleted) containing the gene for β-Galactosidase (ADV.β-Gal), HSV-tk(ADV.tk), and murine IL-2 (ADV.IL-2) under the transcriptional control of the Rous sarcoma virus long terminal repeat were used for gene delivery. Construction and functional characterization of the vectors had been reported in detail before.¹⁴,¹⁶Viral titers (plaque-forming units [pfu]/mL) were determined by plaque-forming assays as described.¹⁴

Cell Line

The Lewis lung cancer

Transduction Efficiency With ADV Vectors in LL2 In Vitro

For the ADV.β-Gal experiments, 100%; transduction of cells as shown by positive staining with X-Gal occurred at an moi of 20,500 without apparent cytotoxicity (results not shown). However, in the ADV.tk experiments, 90%; of the LL2 cells treated with GCV were killed at an moi of only 64 (Fig 1). At an moi of 512, 100%; killing occurred in the ADV.tk+GCV group, while no appreciable cell death was observed in the ADV.tk+PBS group.

Regression of Hepatic Metastases After Suicide Gene Therapy

To define the optimal dose of ADV.tk for tumor regression in vivo,

DISCUSSION

The use of HSV.tk as a suicide gene has been investigated in a number of experimental tumor models.⁸,14, 15, 16, 17, 18, 19, 20 As a first step toward applying this strategy to lung cancer, we tested the efficacy of HSV.tk in a murine model. In the lung cancer cell line LL2, ADV was efficient in the transfer of HSV.tk in vitro, as shown by 100%; cell killing at an moi of 512. This moi was considerably lower than that of 20,500 needed for 100%; transfection by ADV.β-Gal. This might be explained

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Cited by (44)

A herpes oncolytic virus can be delivered via the vasculature to produce biologic changes in human colorectal cancer
2009, Molecular Therapy
Citation Excerpt :
Using such genetic engineering, a number of investigators have attempted to further attenuate this common, naturally well-tolerated virus.3,5 Four such designer viruses specifically made for cancer therapy are currently undergoing clinical investigation.6,7 The origin of the NV1020 virus used for the current trial differs from these agents in that it was originally designed as a vaccine against herpes infections.8
Genetically engineered herpes simplex viruses (HSVs) can selectively infect and replicate in cancer cells, and are candidates for use as oncolytic therapy. This long-term report of a phase I trial examines vascular administration of HSV as therapy for cancer. Twelve subjects with metastatic colorectal cancer within the liver failing first-line chemotherapy were treated in four cohorts with a single dose (3 × 10⁶ to 1 × 10⁸ particles) of NV1020, a multimutated, replication-competent HSV. After hepatic arterial administration, subjects were observed for 4 weeks before starting intra-arterial chemotherapy. All patients exhibited progression of disease before HSV injection. During observation, levels of the tumor marker carcinoembryonic antigen (CEA) decreased (median % drop = 24%; range 13–74%; P < 0.02). One of three individuals at the 10⁸ level showed a 39% radiologic decrease in tumor size by cross-section and 75% by volume. HSV infection was documented from liver tumor biopsies. After beginning regional chemotherapy, all patients demonstrated a further decrease in CEA (median 96%; range 50–98%; P < 0.008) and a radiologic partial response. Median survival for this group was 25 months. During follow-up, no signs of virus reactivation were found. Multimutated HSV can be delivered safely into the human bloodstream to produce selective infection of tumor tissues and biologic effects.
Use of gene therapy in a subcutaneous murine model of lung cancer
2006, Archivos de Bronconeumologia
Demostrar la utilidad del tratamiento génico (TG) in vivo en los tumores subcutáneos de cáncer de pulmón murino.
Se inyectaron a ratones C57BL/6 por vía subcutánea 5 × 10⁵ células de la línea de cáncer de pulmón murino de Lewis. A los 10 días se formaron tumores subcutáneos de unos 5 mm de diámetro. En ese momento se trataron mediante inyección intratumoral con un adenovirus recombinante defectivo portador del gen de la timidincinasa (AdCMV-Tk), o del gen de la interleucina 12 (AdCMV-IL12), o con células dendríticas (CD) singénicas transducidas con el gen de la interleucina 12 (CD-IL12). Como grupos control se incluyeron tumores tratados con suero salino o con un adenovirus con el gen de la ß-galactosidasa (AdCMV-LacZ), que es un gen indicador sin efecto terapéutico. El número de animales por grupo osciló entre 14 y 25 con adenovirus y entre 10 y 12 con CD. A continuación se realizó un seguimiento del tamaño tumoral desde el primer día de tratamiento hasta la tercera (adenovirus) o cuarta (CD) semanas para comparar su evolución.
Se objetivó una disminución significativa del crecimiento de los tumores subcutáneos en los grupos tratados con AdCMV-Tk, AdCMV-IL12 y CD-IL12 comparados con los grupos control tratados con suelo salino y AdCMVLacZ. En el grupo AdCMV-Tk esta diferencia fue estadísticamente significativa desde el día 7, en AdCMV-IL12 desde el día 9 y en CD-IL12 desde el día 10 y se mantuvo hasta el final del seguimiento.
El TG con AdCMV-Tk, AdCMV-IL12 o CD-IL12 es efectivo en nuestro modelo de tumores subcutáneos de células de carcinoma pulmonar de Lewis, ya que es capaz de disminuir su tasa de crecimiento de forma significativa respecto a los grupos de control.
To assess the effectiveness of in vivo gene therapy to treat subcutaneous tumors generated from murine lung cancer cells.
C57BL/6 mice received subcutaneus injections of 5×10⁵ cells from the murine Lewis lung cancer cell line. By 10 days, subcutaneous tumors of approximately 5 mm diameter were formed. At that point, treatment was provided by intratumor injection of a replication-defective recombinant adenovirus carrying the gene for thymidine kinase (AdCMV-Tk) or interleukin (IL) 12 (AdCMV-IL12), or by injection of syngeneic dendritic cells previously transduced with adenovirus containing the IL-12 gene (DC-IL12). Control groups were treated with saline or adenovirus containing the gene for ß-galactosidase (AdCMV-LacZ), which functions as a reporter gene and does not have a therapeutic effect. The number of animals in each group ranged from 14 to 25 in experiments using adenovirus and from 10 to 12 in experiments using dendritic cells. Tumor size was followed for 3 weeks in the case of treatment with adenovirus and 4 weeks for treatment with dendritic cells.
A significant reduction in subcutaneous tumor growth was observed in the groups treated with AdCMVTk, AdCMV-IL12, and DC-IL12 compared with control groups treated with saline or AdCMV-LacZ. The difference was statistically significant from day 7 of treatment in the AdCMV-Tk group, from day 9 in the AdCMV-IL12 group, and from day 10 in the DC-IL12 group, and in all cases it was maintained until the end of the follow-up period.
Gene therapy with AdCMV-Tk, AdCMVIL12, or DC-IL12 is effective in our model of subcutaneous tumors arising from cells of the Lewis lung cancer cell line. The treatment leads to a significant reduction in tumor growth compared with control groups.
Total vascular exclusion of the liver enhances the efficacy of retroviral-mediated associated thymidine kinase and interleukin-2 genes transfer against multiple hepatic tumors in rats
2003, Surgery
Backround. Recent advances in gene transfer technology render gene therapy an attractive treatment of disseminated liver metastases for which other treatments remain disappointing. In this setting, total vascular exclusion of the liver could improve gene transfer to cancer cells and prevent extrahepatic vector spreading during portal infusion of therapeutic genes. We evaluate the efficiency of combined herpes simplex virus type-1 thymidine kinase (HSV1-TK) and interleukin-2 retrovirus-mediated gene transfer through the portal vein, under total vascular exclusion of the liver, in a model of macroscopic multiple liver metastases in rats. Methods. Multifocal liver metastases were established in BDIX rats with intraportal injection of DHDK12 colon cancer cells. On randomization, rats received either vector-producing cells or saline solution under total vascular exclusion of the liver. Vector-producing cells released retroviral vectors encoding Lac-Z in marking studies or HSV1-TK or interleukin-2 in therapeutic studies. Rats were either killed for pathologic studies, or followed for survival. Results. Total vascular exclusion of the liver markedly improved gene transfer efficacy in marking studies. In therapeutic studies we observed a significant reduction in tumor volume of treated rats compared with untreated controls (2170 ± 310 mm³). Although singular HSV1-TK or interleukin-2 gene transfer showed significant efficacy, the greatest tumor volume regression was observed in rats treated with combined HSV1-TK + interleukin-2 gene therapy (145 ± 60 mm³; P =.0001 vs control). This translated into an increased median survival rate compared with either control rats (P =.006) or rats treated with single gene therapy. Conclusion. In a rat model, a significant antitumoral effect against macroscopic multifocal liver metastases can be observed after retrovirus-mediated HSV1-TK and interleukin-2 gene transfer through the portal vein under total vascular exclusion of the liver, followed by ganciclovir administration. We believe that this well-tolerated and efficient therapeutic approach deserves clinical evaluation in patients with disseminated colorectal liver metastases. (Surgery 2003;133:669-77)
An optimal therapeutic expression level is crucial for suicide gene therapy for hepatic metastatic cancer in mice
2003, Hepatology
The most serious problem in current gene therapy is discrepancies between experimental data and actual clinical outcomes, which may be due to insufficient analyses and/or inappropriate animal models. We have explored suicide gene therapy by using various clinically relevant animal models and doubt the clinical use of maximal suicide gene expression, which has been generally recommended. To explore this subject further, we studied what expression level of suicide gene and what promoter led to the maximal clinical benefit in the case of hepatic metastatic cancer in mice. Therapeutic and adverse side effects of 4 adenoviral vectors that express herpes simplex virus thymidine kinase (HSV-tk) under different promoters were scrupulously investigated in 2 mouse models of hepatic metastasis of gastric cancer that possess clinical characteristics. Surprisingly, increases in HSV-tk expression beyond a certain point, achieved by the Rous sarcoma virus long terminal repeat promoter, not only enhanced the adverse side effects of lethal hepatotoxicity and ganciclovir-independent cytotoxicity but also failed to further increase therapeutic potential. Moreover, the carcinoembryonic antigen (CEA) tumor-specific promoter, the therapeutic potential of which had been underestimated, was much more useful—even in the case of low CEA-producing cancer—than had been previously reported. In conclusion, the optimal therapeutic expression level of a suicide gene is a novel concept and a crucial factor for successful cancer gene therapy. The present results, which contradict those of previous studies, alert researchers about possible problems with ongoing and future clinical trials that lack this concept. (HEPATOLOGY 2003;37:155-163.)
Adenoviral herpes simplex virus thymidine kinase gene therapy in an orthotopic lung cancer model
2002, Annals of Thoracic Surgery
Background. Although adenovirus-mediated herpes simplex virus thymidine kinase (HSVtk) gene therapy is a potential candidate for a novel effective therapy for lung cancer, previous studies have been performed only in a subcutaneous tumor model employing nude mice. We studied therapeutic potentials in correlation with accurate adenoviral gene transduction efficiency in a more clinically relevant orthotopic lung cancer model employing imunocompetent mice.
Methods. To analyze the cytotoxicity of adenoviral HSVtk gene transduction and ganciclovir, a cell proliferation assay was performed in vitro. A survival study was carried out in immunocompetent mice with orthotopic lung cancer, which was generated by intrapulmonary inoculation with syngeneic murine lung cancer cells that had been infected beforehand with each adenoviral vector at the predetermined gene transduction efficiencies.
Results. Tumor cells were efficiently killed by infection with adenovirus carrying the HSVtk gene with the addition of ganciclovir in vitro. In the in vivo experiment all control mice died of rapid growth of the primary lung cancer and of metastases to mediastinal lymph nodes within 26 days after tumor inoculation. In contrast 50% and 100% of mice survived more than 40 days after inoculation with adenovirally HSVtk-transfected tumor cells that moderately and highly expressed HSVtk, respectively, when followed by ganciclovir administration. Gene transduction efficiencies were 67%.
Conclusions. Adenovirus-mediated HSVtk gene therapy may be therapeutic for lung cancer when gene transduction efficiencies and sufficient expression levels of HSVtk can be achieved. Moreover, the present findings underscore the importance of the mouse orthotopic lung cancer model for studies of gene therapy.
Gene therapy of metastatic disease: Progress and prospects
2001, Surgical Oncology Clinics of North America
Gene therapy remains a new and exciting therapy that holds the potential to impact the care of many diseases. Cancer gene therapy strategies encompass a major part of this developing field. Initial preclinical and phase I clinical trials have demonstrated the ability to transfer genetic material to cells in vitro and in vivo with resultant expression of biologically active protein. Most of these studies have involved direct injection or local installation of vector. A majority of patients succumbing to cancer do so because of metastatic disease. Clearly, to broaden the impact of cancer gene therapy on these patients’ outcome, new strategies for targeting regional or systemic disease are required. This article offers a review of current vectors and therapeutic strategies along with the application of these in human cancers

View all citing articles on Scopus

: Reprint requests: Savio L. C. Woo, PhD, Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, One Gustave L. Levey Place, New York, NY 10029-6574

^†: Currently at the Department of Medicine, University of Hong Kong.

^‡: Currently at the Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York.

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Chest

Laboratory and Animal InvestigationsCombination Therapy With Suicide and Cytokine Genes for Hepatic Metastases of Lung Cancer

Section snippets

Recombinant Adenoviral Vector

Cell Line

Transduction Efficiency With ADV Vectors in LL2 In Vitro

Regression of Hepatic Metastases After Suicide Gene Therapy

DISCUSSION

Cell

Cancer of the lung

The treatment of non-small cell lung cancer: current perspectives and controversies, future directions

Semin Oncol

Small cell lung cancer

Prognostic factors for survival in advanced non-small-cell lung cancer: univariate and multivariate analyses including recursive partitioning and amalgamation algorithms in 1,052 patients: the European Lung Cancer Working Party

J Clin Oncol

Gene therapy for cancer

Human Gene Ther

The biology of cancer gene therapy

Lab Invest

Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy

Cancer Res

The bystander effect: tumor regression when a fraction of the tumor mass is genetically modified

Cancer Res

In vitro evidence that metabolic cooperation is responsible for the bystander effect observed with HSV tk retroviral gene therapy

Human Gene Ther

Experimental and clinical studies of cytokine gene-modified tumor cells

Human Gene Ther

Laboratory and Animal Investigations
Combination Therapy With Suicide and Cytokine Genes for Hepatic Metastases of Lung Cancer