HSV-tk expressing mesenchymal stem cells exert bystander effect on human glioblastoma cells
Introduction
Combination of Herpes simplex virus thymidine kinase (HSV-tk) with prodrug ganciclovir (GCV) and the variations thereof remain still the most widely used approach to gene directed enzyme prodrug therapy. Viral thymidine kinase exhibits high efficiency in monophosphorylating ganciclovir, which is a first step in GCV conversion into toxic metabolites. Further phosphorylation by endogenous enzymes produces GCV-triphosphate (GCV-TP) as most active metabolite to inhibit the target cellular DNA polymerases [1]. Although positively charged cytotoxic GCV-triphosphate cannot passively diffuse to exert cytotoxicity in neighboring cells, HSV-tk/GCV combination produces significant bystander effect both in vivo and in vitro[2]. Major mechanism responsible for the GCV-TP transfer into neighboring cells is gap junctions’ formation between the cells in close contact. Strategies that increase gap junction formation generally improve the therapeutic efficiency [3], [4]. Bystander effect can be also facilitated by phagocytosis of apoptotic vesicles containing GCV-TP by non-TK-expressing cells [5]. Host immune system plays a role in mediating distant bystander effect and vaccination effect in terms of therapeutic outcome in vivo (reviewed in [6]).
HSV-tk/GCV enzyme/prodrug combination was proposed for suicide gene therapy approaches and proven to be safe in preclinical trials. Significant increase in patient survival was achieved in glioblastoma patients treated with HSV-tk/GCV mediated by adenoviral vector application into the resected tumor site [7]. However therapeutic efficiency in clinical trials was limited mostly due to the poor efficiency of target cell infection.
There were several attempts to circumvent the problem of low therapeutic efficiency. One of the possible solutions to improve the extent of tumor-selective targeting might be to exploit inherent tumor-tropic properties of stem/progenitor cells. Several groups have reported capability of stem cells to serve as cellular delivery vehicles for the enzyme prodrug therapy approaches (as reviewed in [8], [9]). Danks et al. [10], have recently demonstrated long term disease-free survival in 90% neuroblastoma bearing mice mediated by neural stem cell delivery of rabbit carboxylesterase and prodrug CPT-11 (irinotecan). Combination of cytosine deaminase with prodrug 5-fluorocytosine has proven efficacious in several models of tumor therapy, when either endothelial progenitor cells [11], neural progenitor cells [12], [13] or mesenchymal stem cells were used as cellular delivery [14], [15].
Cellular delivery of HSV-tk combined with GCV treatment was demonstrated to exert bystander killing effect on glioblastoma in rat animal model [16]. Suicide HSV-tk gene expression in rat bone marrow-derived tumor infiltrating cells upon intratumoral delivery followed by GCV administration resulted in highly significant prolongation of tumor-free-survival. Another group using same prodrug/enzyme combination with rat neural stem cells could demonstrate inhibitory effect on glioblastoma growth in both ipsilateral and contralateral setting [17].
The capability of human adipose-tissue derived mesenchymal stem cells to serve as delivery vehicles for cytosine deaminase/5-fluorocytosine was recently reported [14], [15]. Transgene expressing cells in combination with prodrug were able to mediate antitumor effect upon systemic administration on mouse xenotransplant model. In present study we aimed to evaluate whether human mesenchymal stem cells derived from adipose tissue (AT-MSC) could be exploited as cellular vehicles for HSV-tk/GCV enzyme/prodrug combination to mediate cytotoxic effect on human tumor cells. We demonstrate AT-MSC capability of the gap junction intercellular communication (GJIC) with glioblastoma cells as a mechanism contributing to bystander killing by TK-MSC/GCV therapeutic paradigm.
Section snippets
Cell lines and chemicals
Human glioblastoma multiforme 8-MG-BA and 42-MG-BA cell lines (kindly provided by Dr. A. Perzelova, Medical Faculty, Comenius University, Bratislava), U-118 MG (ATCC Number HTB-15™), breast adenocarcinoma MCF7 (ECACC cat. No. 86012803), cervical epitheloid carcinoma HeLa (ECACC Cat. No. 93021013), retroviral packaging mouse cell lines GP+E−86 and GP+envAm12 (kindly provided by Dr. J. Bies NCI NIH, Bethesda) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FCS and
Results
HSV-tk bearing plasmid was transfected into mouse packaging cell line. G418-resistant cells GP+envAm12/pAPtk were found to be highly sensitive to GCV with LD50 = 0.5 μM GCV and viral titers in retrovirus-containing medium were 1.75 × 106 cfu per ml. In order to prepare therapeutic cells, two isolates of human AT-MSC were transduced with retrovirus-containing medium produced from packaging cell line and G418-selected to achieve stable transduction. Both AT-MSC isolates could express HSV-tk transgene
Discussion
As previously reported [14], [15], we were able to achieve stable transgene expression in human mesenchymal stem cells by means of retroviral transduction. HSV-tk transgene was expressed to similar level in two different isolates with no significant difference in GCV sensitivity. TK-MSC did not exhibit any changes in morphological, proliferative or biological properties in response to transgene modification in our hands so far. This notion is well consistent with report from the Morizono et al.
Conflict of interest
None declared.
Acknowledgements
We thank M. Dubrovcakova, V. Frivalska and L. Baranovicova for excellent technical assistance, D.Guba, M.D., Institute of Medical Cosmetics, Bratislava, for providing us with material for AT-MSC isolation.
The study was supported by the VEGA Grant No. 2/7060/27 and 2/0119/08, APVV Grant No. APVV-0260-07, financial support from FIDURA Capital Consult GmbH, Munich, SPP Foundation and grant support awarded by the League against Cancer.
References (33)
- et al.
Suicide genes for cancer therapy
Mol. Aspects Med.
(2007) - et al.
AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study
Mol. Ther.
(2004) Prodrug cancer gene therapy
Cancer Lett.
(2008)- et al.
Embryonic endothelial progenitor cells armed with a suicide gene target hypoxic lung metastases after intravenous delivery
Cancer Cell
(2004) - et al.
Bystander killing of malignant glioma by bone marrow-derived tumor-infiltrating progenitor cells expressing a suicide gene
Mol. Ther.
(2007) - et al.
Genetically engineered neural stem cells migrate and suppress glioma cell growth at distant intracranial sites
Cancer Lett.
(2007) - et al.
Connexins as targets for cancer chemoprevention and chemotherapy
Biochim. Biophys. Acta
(2005) - et al.
Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction
J. Am. Coll. Cardiol.
(2008) - et al.
Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclovir system: fifteen years of application
Curr. Gene Ther.
(2003) - et al.
Influence of the bystander effect on HSV-tk/GCV gene therapy
Curr. Gene Ther.
(2002)
The extent of heterocellular communication mediated by gap junctions is predictive of bystander tumor cytotoxicity in vitro
Proc. Natl. Acad. Sci. USA
Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve gap junctions and bystander effects
Histol. Histopathol.
The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified
Cancer Res.
Stem and progenitor cell-mediated tumor selective gene therapy
Gene Ther.
Cancer Res.
Targeting of melanoma brain metastases using engineered neural stem/progenitor cells
Neuro Oncol.
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