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Ann Thorac Surg 2002;73:905-910
© 2002 The Society of Thoracic Surgeons
a Department for General Surgery and Thoracic Surgery, University Hospital, Christian-Albrechts-University, Kiel, Germany
Accepted for publication October 18, 2001.
* Address reprint requests to Dr Kurdow, Department for General and Thoracic Surgery, University Hospital, Christian-Albrechts-University, Arnold-Heller Strasse 7, 24105 Kiel, Germany
e-mail: rkurdow{at}surgery.uni-kiel.de
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Abstract |
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Methods. Intrathoracally tumors induced by HSV-TK30 transduced non-small cell lung cancer cell lines Colo 699 (adenocarcinoma) and KNS 62 (squamous cell carcinoma) were monitored for local tumor growth, survival, and metastases. So-called bystander effects were investigated in tumors consisting of as little as 25% TK30 transfected cells and by analysis of gap junctional protein connexin-43 expression.
Results. Survival was significantly prolonged, and tumor growth and pleural metastases were reduced in HSV-TK30-positive tumors of both cell lines. A significant therapeutic effect in bystander experiments was observed in squamous cell carcinoma. This was correlated with higher expression of connexin-43.
Conclusions. Delivery of HSV-TK30 in a VSV-G pseudotyped retroviral vector and subsequent ganciclovir application provided therapeutic efficacy. Despite of low transduction rates achievable in gene transfer in situ, prodrug therapy appears to be feasible in tumor cells with efficient bystander effects.
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Introduction |
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The introduction of herpes simplex virus thymidine kinase (HSV-TK) genes into tumor cells and subsequent systemic ganciclovir (GCV) application offers an effective and valuable approach for the treatment of a variety of tumor types [1–3]. HSV-TK specifically phosphorylates GCV to GCV-phosphate. Cellular kinases provide further phosphorylation to GCV-triphosphate, which inhibits DNA polymerase and induces apoptosis [4]. In addition, activation of a T-cell-mediated immune response during GCV treatment of TK positive tumors causes regression of distant growing TK-negative tumors in immunocompetent hosts [5].
The observation of simultaneous apoptosis of TK-negative tumor cells in line with TK positive neighbor cells during GCV therapy is called "bystander effect." Intercellular transfer of GCV triphosphate through gap junctions consisting of connexins is generally regarded as the major mechanism for bystander effects [6]. There is evidence that transfer of GCV triphosphate in apoptotic vesicles may also contribute [7].
The clinical GCV prodrug approach is limited by unsatisfactory transduction efficiency in vivo [8] and tumor cell-dependent therapy resistance. Mechanisms of resistance are poorly understood but have been attributed to a loss of functional TK protein and to poor bystander effects. These have been described for various tumor entities [9–11].
Previous experiments in our laboratory used a retroviral vector encoding the TK30 thymidine kinase mutant instead of the wild-type gene, and a virus envelope derived from the Vesicular Stomatitis Virus-G (VSV-G) instead of the conventional MoMLV amphotrophic 4070A envelope [12]. This confirmed improved bystander effects and a 40- to 60-fold higher sensitivity of pancreatic tumor cell lines to GCV application. The aim of this study was to evaluate the efficacy of the improved GCV prodrug therapy in NSCLC cell lines in a preclinical murine xenotransplant model [13] with respect to survival, local tumor growth, and metastases.
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Material and methods |
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Cell lines and culture conditions
The 293T (ATCC CRL 1573) cells [15] were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Eggenstein, Germany) supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, UT), 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, and nonessential amino acids (Life Technologies). Colo 699 cells were originally derived from malignant pleural effusion of a patient with adenocarcinoma of the lung. KNS 62 cells were derived from brain metastases of a human squamous cell carcinoma of the lung [13]. Cells were purchased from the German Collection of Microorganisms and Cell Cultures, (Braunschweig, Germany). Cultures were maintained and prepared for xenotransplatation as described previously [13].
Transfection of cells
Twenty-four hours before transfection, 1x106 293T cells were seeded onto primaria plates (Falcon no. 3803, Becton-Dickinson, Heidelberg, Germany). A quantity of 10 µg of each retroviral plasmid vector either rkat EGFP/neo or rkat TK30/neo, 7 µg of the packaging plasmid pKAT1.gag/pol. ATG and 7 µg of pCMV-VSV-G were cotransfected into 293T cells using calcium phosphate coprecipitation [16]. On the next day, fresh media was added and viral supernatants were harvested 24 and 48 hours later, filtered through 0.45-µm low-protein-binding Acrodisc Filters (#4148 Gelmann Sciences, Ann Arbor, MI) and stored at -80°C.
Transduction of human lung carcinoma cell lines
1 x 105 Colo 699 or KNS 62 cells were seeded onto 6-cm2 dishes containing complete medium and 8 mg/mL polybrene. Twenty-four hours later, rkat EGFP/neo or rkat TK30/neo retroviral supernatants containing 1 x 106 colony-forming units were used to transduce lung carcinoma cells. Cells were selected and maintained using 700 mg/mL neomycin (G418).
In vitro cytotoxicity assay
For the in vitro cytotoxicity assay, 1 x 104 rkat TK30/neo rkat cells, were seeded into each well of a 96 well plate. EGFP/neo transduced cells of Colo 699 and KNS 62 served as controls. For bystander assays, 1 x 104 cells of rkat EGFP/neo and rkat TK30/neo cells in a ratio of 90:10, 75:25 and 50:50% were seeded. Various concentrations of GCV (Cymevene, Syntex Pharmaceuticals, Aachen, Germany) were added 24 hours later in volumes of 200 µL to each well. Medium supplemented with GCV was replaced after 3 days. After 5 days, cytotoxicity defined by use of the EZ4U cytotoxicity kit (Bio-Rad Laboratories, Munich, Germany) following the manufacturer‘s instructions. After addition of chromagenic substrate, plates were scanned in an ELISA reader (Anthos, Salzburg, Austria) at 450 nm wavelength. The measurements were evaluated as the percentage of the optical density of untreated control wells (n = 6 for each GCV concentration). The GCV dose effective in 50% cell killing (LD50) was calculated by regression analysis.
Immunofluorescence microscopy
Confluent monolayers of Colo 699 and KNS 62 were mechanically removed from 6-well plates, washed in phosphate-buffered saline (PBS)/Na-acid three times, fixed in 70% methanol, and washed in PBS three times. Cells were incubated with anti-Connexin 43 (Cx43, Chemicon Intl, Temecula, CA), diluted 1:250 for 1 hour on ice, washed three times in PBS and incubated with Cyanin 3-Ak (Dianova GmbH, Hamburg, Germany) 1:200 for another hour on ice. After washing three more times in PBS, cells were fixed in paraformaldehyde 4% on glass slides. Images were obtained by a Zeiss Axioskop 2 photomicrography system.
Immunoblot analysis of Connexin-43 expression
Immunoblot analysis of Connexin-43 (Cx43) expression was carried out as follows. For preparation of protein extracts, TK30 transduced Colo 699 and KNS 62 tumor cells were rinsed twice in cold PBS and lysed in RIPA buffer (0.1% DDS, 1% Nonidte-P40, 0.5% sodiumdeoxycholat in PBS) for 10 minutes on ice. Lysed cells were transfered to microcentrifuge tubes and centrifuged. Equal amounts of total protein (measured with BCA Protein Assay Reagent, [Pierce Chemical Co, Rockford, IL], according to the manufacturer’s recommendations) were loaded onto a standard 12.5% SDS polyacryamide gel. Fractionated proteins were blotted onto a PVDF membrane (Immobilon-P, Millipore, Eschborn, Germany). Cx43 protein was detected by anti-Cx43 (Chemicon Intl) diluted 1:250, and peroxidase conjugated sheep-antimouse antibodies (Amersham life science, Braunschweig, Germany) diluted 1:2000 for 1 hour, respectively. The blot was visualized by the chemoluminescent detection system Western Star Plus (Tropix/Serva, Heidelberg, Germany).
Implantation of cells and GCV treatment of tumors in vivo
Pathogen-free female SCID bg mice (Harlan Winkelmann, Borchen, Germany) were xenotransplanted at the age of 4 to 5 weeks intrapleurally by 2 x 106 cells Colo 699 or KNS 62. They were transduced with either the HSV TK30 or, as a control, the EGFP gene. A mixture of TK30 positive and EGFP cells in a ratio of 25%/75% was employed for in vivo evaluation of bystander effects. Xenotransplantation was performed as described previously [13].
Mice were maintained in sterile microisolator cages under pathogen-free conditions, and fed autoclaved food and water ad libitum. They were handled under sterile conditions in a laminar flow hood. All animals received human care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health in 1996.
Eight days after intrapleural injection, systemic GCV treatment by intraperitoneal injections of 50 mg/kg body weight per day was started. For reasons of pharmakocinetics, daily dose was split in two fractions. In groups of 6 animals each, survival subsequent to intrapleural tumor inoculation and continuous GCV treatment was determined. Groups of 4 animals each were sacrificed after tumor inoculation and 14 days of treatment. Control animals were xenotransplanted by EGFP transduced tumor cells and were treated with GCV as described above.
Clinical and postmortem evaluation
Animals were monitored daily after tumor induction for signs of physical discomfort and tumor growth. They were weighed twice a week. For post mortem evaluation, sternotomy was performed and the intrathoracic situs was documented by photography. Photography was performed using an Olympus Camedia C 2500 L digital camera (Olympus, Tokyo, Japan). Lungs and mediastinum were removed en bloc and weighed, visible metastases of the visceral pleura were counted. If there was a primary tumor in the chest wall, weight and size in two diameters was estimated.
Statistical analysis
Analysis of variance and Scheffé’s analysis were performed using the SPSS software (SPSS Inc, Chicago, IL)
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Results |
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The weight of lungs and mediastinum, serving as a measure of mediastinal and pulmonal metastases, was reduced in TK30 positive Colo 699 tumors after 14 days of GCV therapy (p < 0.01). In KNS 62 TK30- positive tumors, the weights of the lungs and mediastinum were not reduced under GCV treatment in comparison to those in EGFP controls. The number of pleural metastases decreased significantly in TK30-positive KNS 62 and Colo 699 tumors (p < 0.01). KNS 62 TK30 primary tumors at the site of injection were smaller than EGFP controls subsequent to GCV application (p < 0.01, Fig 4). Colo 699 EGFP and TK30-positive tumors did not develop noteworthy tumors at the site of injection but did widely metastasize to the mediastinum and pleura (Fig 4B).
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The source of therapy failure is not completely understood. Golumbek and colleagues [18] reported late recurrences of melanoma after apparent regression, which remained sensitive to further GCV treatment. Zhang and associates [10] observed an increase in size of HSV-TK mRNA in GCV-resistant NSCLC cell populations. Graessmann and coworkers [19] proved a single methylation of HSV-TK gene to be responsible for complete loss of function. Deletion or loss of the HSV-TK gene as a reason for GCV resistance was reported by Yang and colleagues [9] and by Moolten and associates [20].
In addition, the absence of sufficient mechanical bystander effects mediated by the transfer of GCV-3 phosphate through gap junctions from TK-positive to TK-negative cells may be responsible for the development of GCV-resistant populations. As gap junctional intercellular communication (GJIC) differs among various cell lines, differences in susceptibility to GCV therapy can occur even in otherwise equally sensitive cell populations, just differing in the amount of GJIC [9, 10].
The present study demonstrates significant extension of survival of 80% in GCV-treated animals with TK30 positive tumors of two different NSCLC cell lines in comparison to GCV-treated EGFP controls (Fig 3). A significant reduction in local tumor growth and metastases after 14 days of therapy (Fig 4) was demonstrated. Fluorescence-microscopic analysis of EGFP transduced cells (data not shown) proved transduction to be highly efficient in both NSCLC cell lines. This observation is in accordance with previously demonstrated enhanced transduction efficiency and improved GCV sensitivity using VSV-G pseudotyped retroviral vectors [12].
The absence of a T-cell-mediated immune response in SCID bg mice, effecting a distant immunologic bystander effect [5], may be partly responsible for the fact, that long-term survival of animals bearing TK30-positive tumors was not achieved by continuous GCV treatment in this study.
Sufficient bystander effects in vivo were demonstrated for KNS 62 (Figs 3 and 4) in correlation with enhanced Cx43 expression in the cell membranes of KNS 62 in vitro (Fig 2). This supports the generally accepted thesis of GJIC being responsible for bystander effects. As differences in connexin expression cause different quantity of GJIC, this would cause different efficiency in bystander "killing" of TK30 negative cells during GCV therapy of partially TK30-positive tumors. As transduction rates are limited in gene transfer in vivo, bystander effects are required for successful GCV prodrug treatment.
In summary, GCV prodrug therapy using the HSV-TK30 mutant in a VSV-G pseudotyped retroviral vector provides effective treatment of NSCLC tumors in a murine xenotransplant model. The lack of T-cell response and the prolongation of survival by 80% for GCV-treated, TK30-positive tumors of both cell lines is remarkable. The major problem of the prodrug therapy concept remains insufficient gene transfer in vivo. Therefore, GCV prodrug therapy appears to be advantageous in GCV-susceptible tumor cell lines with distinctive mechanical bystander effects as in KNS 62. Stratification of tumors according to their individual pattern of Cx43 expression and a specific improvement of bystander effects must be further approaches in GCV prodrug gene therapy.
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