uracil phosphoribosyltransferase inhibit - Wiley Online Library

IJC International Journal of Cancer

Human adipose tissue-derived mesenchymal stem cells expressing yeast cytosinedeaminase::uracil phosphoribosyltransferase inhibit intracerebral rat glioblastoma Veronika Altanerova1,2, Marina Cihova1, Michal Babic3, Boris Rychly4, Katarina Ondicova5, Boris Mravec5,6 and Cestmir Altaner1,2 1

Cancer Research Institute, Slovak Academy of Sciences, Bratislava, Slovakia St. Elisabeth Cancer Institute, Bratislava, Slovakia 3 Institute of Macromolecular Chemistry of the Academy of Sciences of the Czech Republic, Prague, Czech Republic 4 Cytopathos Ltd., Bratislava, Slovakia 5 Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia 6 Institute of Experimental Endocrinology, Slovak Academy of Sciences, Bratislava, Slovakia 2

Glioblastoma is an aggressive cancer with dim prognosis for glioblastoma patients. The median survival time for patients treated with conventional therapies like surgery, radiotherapy plus concomitant and adjuvant temozolomide chemotherapy is only 14.6 months.1 The highly invasive character of glioblastoma cells together with extensive neovascularization of tumor tissue and dispersion of tumor cells deeply into surrounding normal tissue can be partially related to the failure of standard glioblastoma therapy modalities. In addition, the presence of cancer stem cells (CSCs) in the main tumor mass Key words: glioblastoma, mesenchymal stem cells, suicide gene therapy, CDy::UPRT, continuous intracerebro-ventricular 5-FC delivery Grant sponsors: Slovak League against Cancer, SPP Foundation, FIDURA Capital Consult GmbH, Munich, Germany DOI: 10.1002/ijc.26278 History: Received 7 Mar 2011; Accepted 14 Jun 2011; Online 5 Jul 2011 Correspondence to: Cestmir Altaner, Laboratory of Molecular Oncology, Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 833 91, Bratislava, Slovakia, Tel.: þ421-2-59327426; Fax: þ421-2-59327250, E-mail: [email protected]

C 2011 UICC Int. J. Cancer: 130, 2455–2463 (2012) V

can contribute to the therapeutic failure. It is believed that CSCs, residing within a perivascular niche, are initiating glioblastoma growth.2,3 CSCs possess stem cells characteristics, i.e., they multiply very rarely by asymmetric cell division, are resistant to toxic agents and are therefore resistant to cytotoxic therapies requiring cell division for their effect.4 Therefore, it is obvious that effective treatment of glioblastoma must include killing of tumor cells present in the tumor mass, of disseminated tumor cells in brain tissue and of tumor initiating CSCs, which is a rather challenging task. Earlier we have shown that human adipose tissue-derived mesenchymal stem cells (AT-MSCs) engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CDy::UPRT)—therapeutic stem cells (CDy-ATMSC) are able to convert the relatively non-toxic 5-fluorocytosine (5-FC) to the active toxic drug 5-fluorouracil (5-FU). CDy-AT-MSCs have the ability to track and engraft into tumors.5 Prodrug cancer gene therapy driven by MSCs represents an attractive tool to activate the prodrug directly within the tumor mass, thus avoiding systemic toxicity.6 For a comprehensive review see the work by Bexell et al.7 In addition, MSCs lack major histocompatibility complex (MHC-II) and show only minimal MHC-I expression.8–10 Therefore, their

Cancer Therapy

Prodrug cancer gene therapy by mesenchymal stem cells (MSCs) targeted to tumors represents an attractive tool to activate prodrugs directly within the tumor mass, thus avoiding systemic toxicity. In this study, we tested the feasibility and efficacy of human adipose tissue-derived MSCs, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracranial rat C6 glioblastoma. Experiments were designed to simulate conditions of future clinical application for high-grade glioblastoma therapy by direct injections of therapeutic stem cells into tumor. We demonstrated that genetically modified therapeutic stem cells still have the tumor tropism when injected to a distant intracranial site and effectively inhibited glioblastoma growth after 5-fluorocytosine (5-FC) therapy. Coadministration of C6 cells and therapeutic stem cells with delayed 5-FC therapy improved the survival in a therapeutic stem cell dose-dependent manner and induced complete tumor regression in a significant number of animals. Continuous intracerebroventricular delivery of 5-FC using osmotic pump reduced the dose of prodrug required for the same therapeutic effect, and along with repeated administration of therapeutic stem cells increased the survival time. Intracerebral injection of therapeutic stem cells and treatment with 5-FC did not show any detectable adverse effects. Results support the arguments to begin clinical studies for treatment of high-grade brain tumors.

2456

therapeutic potential can be tested in animal models. Earlier our results from in vivo experiments showed that CDy-ATMSCs administered subcutaneously as a mixture with tumor cells, or intravenously, significantly inhibited the growth of human colon adenocarcinoma cells HT-295 and of xenografts in nude mice of human melanoma and glioma treated with 5-FC.11 The preclinical study has proven efficacious in the treatment of human tumor cells derived from bone metastases of prostate carcinoma.12 At first, neural stem cells (NSCs) had emerged as a promising treatment modality for malignant brain tumors. Tumor tropism of NSCs is of great advantage especially in treatment of high grade brain tumors, having neovascularization capability and ability to infiltrate deeply into brain.13,14 Aboody et al.15 demonstrated that NSCs administered intracranially possess extensive tropism for experimental glioma. Immortalized NSCs engineered to express bacterial cytosine deaminase have been exploited as a tumor-targeting strategy for glioma gene therapy.16 Bone marrow mesenchymal stem cells (BMMSCs) have been found to have similar properties of NSCs, such as extensive migratory capability and tropism for brain tumors.17,18 In this study, we explored, by in vitro and in vivo investigations, the feasibility and efficacy of human adipose tissue derived MSCs engineered to express yeast CDy::UPRT to treat intracranial rat glioblastoma established by intracerebral application of C6 rat glioblastoma cells. The cell population of C6 rat glioblastoma has been shown to be composed mostly of CSCs.19–21 Experiments were designed to partially simulate conditions of future clinical application for glioblastoma therapy. We tested continuous intracerebroventricular delivery of 5-FC and its influence on the survival time of treated animals. We demonstrated that CDy-AT-MSCs/5-FC system of suicide gene therapy significantly inhibited glioblastoma growth.

Material and Methods

Cancer Therapy

Cells and chemicals

Rat glioblastoma C6 cells and C6 cells selected for resistance to 5-FU designated C6r5-FU were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 5% fetal calf serum (FCS) and an Antibiotic–Antimycotic mix (Life Technologies, Corporation, Carlsbad, California, USA) in humidified atmosphere and 5% CO2 at 37 C. Human ATMSCs were isolated from lipoaspirates using a collagenase type VII digestion and plastic adherence technique as described previously.5 The material was obtained from healthy individuals undergoing elective lipoaspiration. All donors of adipose tissue and blood platelets provided an informed written consent. Therapeutic stem cells CDy-ATMSCs were prepared by transduction of AT-MSCs with CDy::UPRT gene as described previously.5 Briefly, AT-MSCs were infected with retrovirus prepared on helper cells and transduced cells were selected for resistance to G418 (0.6 mg/ mL). AT-MSCs were expanded in low glucose (1,000 mg/L) DMEM supplemented with 5% human platelet extract.

CDy-AT-MSCs/5-FC inhibit intracerebral glioblastoma

Platelet extract preparations

Platelets from healthy blood donors prepared by apheresis in bag contained 5 1011/L platelets in blood plasma. Bags were irradiated by 30 Gy to deplete leucocytes. To release platelet-derived growth factors, trombocytes in the bag were frozen at 80 C and subsequently thawed at 37 C. The procedure was repeated twice, tissue culture tested heparin (20 units per mL) was added to lysed platelets and platelets bodies were eliminated by centrifugation at 27,200g for 45 min. The supernatant was filtered through a 0.22 lm GP Millipore Express Plus Membrane Stericup and designated as platelet extract (PE). For cell cultivation, several preparations were pooled to avoid individual donor variations, and the PE aliquots were stored at 20 C. Tests for bacterial, fungal and mycoplasma contaminations were in all isolates negative. Stem cell labeling with poly(L-lysine)-modified iron oxide nanoparticles

Preparation of poly(L-lysine)-modified iron oxide nanoparticles was described previously.22 Semiconfluent culture of CDy-AT-MSCs plated the day before was labeled by addition of poly(L-lysine)-modified nanoparticles to tissue culture medium (20 lg Fe/mL). Cells were cultivated for 4 days, washed with phosphate buffered saline (PBS) (three times) and dissociated by trypsin-EDTA. A cell suspension of 106/5 lL in PBS was used for brain injection. Direct co-culture of C6 cells and CDy-AT-MSC in vitro

Quadruplicates of C6 cells and C6r5-FU (2.6 103) were plated in 96-well plates. Next day CDy-AT-MSCs were added to wells to form increasing ratio of therapeutic stem cells to tumor cells as indicated in Figure 1b. The concentration of 5FC in the medium of control cells and in mixed cultures was adjusted to 100 lg/mL. Medium with 5-FC was changed twice during the experiment (8 days). Cell survival was measured by the standard 3-(4-5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS assay) and values obtained in wells containing tumor cells only were set to 100%. Experimental values are expressed as mean percentage of control survival 6 SE. Cell proliferation assays

Eight duplicates of C6 cells (2,500 cells per well) for each treatment were plated into 96-well plates and incubated overnight at 37 C. Culture medium was replaced with medium containing 100 lg/mL 5-FC or 100 lg/mL 5-FU 24 h later. Each subsequent day plates were subjected to the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, Wisconsin, USA) and read out spectrophotometrically at 490 nm. Results were expressed as the percentage of proliferation, where the proliferation of cells in culture medium without 5-FC or 5-FU was set to100%. Similar results were obtained in three independent experiments. C 2011 UICC Int. J. Cancer: 130, 2455–2463 (2012) V

Altanerova et al.

2457

Figure 1. Sensitivity of rat glioblastoma cells C6 and C6r5-FU to the cytotoxic effect of CDy-AT-MSCs in presence of 5-FC in vitro. (a) Sensitivity of glioblastoma C6 cells to 5-FU and to 5-FC. C6 cells were exposed for 7 days to 100 lg/mL of 5-FU and 5-FC, respectively. Cell viability was quantified by the MTS assay and compared to the viability in absence of the drugs. (b) Cytotoxic effect mediated by CDy-AT-MSCs in the presence of 5-FC on C6 and C6r5-FU cells. In direct co-culture experiments C6 cells were seeded in the same wells with therapeutic stem cells at increasing ratios. Relative cell viability mean values 6 SD were evaluated spectrophotometrically by MTS assay and expressed as

Animal experiments

Adult male Sprague-Dawley rats were used for in vivo experiments in accordance with institutional guidelines under the approved protocols. Rats were anesthetized with intramuscular injection of a mixture of 5.5 mg Ketamine hydrochloride and 0.5 mg Xylazine in 100 lL of PBS/100 g of body weight. Suspension of C6 cells or mixture of C6 cells with either ATMSCs or CDy-AT-MSCs in 5 lL PBS was slowly injected intracerebrally for 5 min period using Hamilton syringe. The needle was left in the place for 2 min and then slowly elevated for 3 min. The application was performed with a Kopf digital stereotaxic instrument (coordinates: AP: 0.0 mm; L: 2.5 mm; V: 6.0 mm). Scheme of localization of injections was according to Paxinos and Watson.23 In separate experiments, groups of rats inoculated with C6 cells alone or with mixtures of C6 cells either with AT-MSCs or CDy-AT-MSCs were treated intracerebrally with 5-FC. A brain’s cannula was surgically implanted into the left lateral ventricle of the rats, connected by a tube with the ALZET osmotic pump Model 2ML2 filled with 2 mL of 5-FC solution C 2011 UICC Int. J. Cancer: 130, 2455–2463 (2012) V

(Ancotil). The osmotic pump was inserted under the back skin of rats. The osmotic pump was replaced by a new one 11 days later. Animals were inspected every day for their behavior and body weights. Animals with excessive weight loss and uncoordinated behavior were sacrificed by decapitation and their brains were analyzed histopathologically. Statistical analysis

Values were calculated as mean 6 SD or expressed as mean percentage of control 6 SE. The Kaplan–Meier survival curves and statistically significant differences were determined using GraphPad Prism Version 4.0 software.

Results Sensitivity of rat glioblastoma cells C6 to the cytotoxic effect of 5-FU and 5-FC

Our objective was to verify the feasibility and efficacy of CDy::UPRT-expressing AT-MSCs as vehicles for intracranial enzyme prodrug therapy for glioblastoma. To accomplish this, we initially tested the sensitivity of C6 cells in in vitro

Cancer Therapy

percentage of cell viability measured in the absence of therapeutic stem cells (*p < 0.01; **p < 0.001 compared to control).

2458


Figure 2. Ability of C6 cells to form spheroids and tumor tropic properties of CDy-AT-MSCs. (a) C6 glioblastoma cells grown in neurobasal medium supplemented with growth factors form spheroids. (b) The CDy-AT-MSCs labeled with poly(L-lysine)-modified superparamagnetic iron oxide nanoparticles (106/5 lL) were injected into the left brain hemisphere opposite to injected C6 glioblastoma cells (105/5 lL). Six

Cancer Therapy

days later the slices of brain were histologically analyzed by Prussian blue iron staining procedure.

experiments. Cytotoxicity was quantified by the MTS assay after direct exposure of the cells to 5-FU and 5-FC for several time periods. The results showed that the population of glioblastoma C6 cells is composed mostly from 5-FU sensitive cells, but a small cell proportion possesses 5-FU resistance at given drug concentration. Growth of C6 cells in the absence of tested drug and in the presence of the same concentration of 5-FC was not influenced (Fig. 1a). We did experiments to isolate less sensitive C6 cells from the cell population by exposing them repeatedly to 5-FU and multiplying the surviving cells. After many repeated cycles of this treatment, we obtained cell population of C6 cells enriched for 5-FU resistant cells (C6r5-FU). In separate experiments, the 5-FU dose that decreased cell viability of C6 cells by 50% (IC50) was found to be 2 lg/mL in 4 days and IC50 of C6r5-FU cells was 4.8 lg/mL.

of 1 therapeutic cell per 1260 tumor cells, cell viability decreased to 80% in 8 days in both cell lines. Killing effect of therapeutic stem cells on C6 and C6r5-FU cells was not significantly different. With increasing numbers of therapeutic stem cells the cytotoxic effect increased in both kinds of cells reaching about 2% of surviving cells at the end of the experiment as detected by the MTS assay (Fig. 1b). The surviving cells morphologically looked as dead cells. Attempts to grow these cells failed, neither glioma cells nor CDy-AT-MSCs were able to divide under growth stimulation conditions. The results show that therapeutic stem cells can kill the C6 glioma cells and also cells being enriched for C6 5-FU resistance. Co-cultures of C6 cells with CDy-AT-MSCs in the absence of 5-FC as well as in the presence of AT-MSCs did not show any cytotoxic effect. The C6 cells in neurobasal medium form spheroids

Rat glioblastoma cells C6 and C6r5-FU are sensitive to the cytotoxic effect of CDy-AT-MSCs in presence of 5-FC in vitro

In order to compare the cytotoxic effect of therapeutic stem cells CDy-AT-MSCs on the C6 glioblastoma cells and on C6 cells enriched for 5-FU resistant cells, direct co-culture experiments were performed. Toxicity of CDy-AT-MSCs in vitro relies on the capacity of these cells to convert the prodrug in situ, on the ability of the formed 5-FU to pass from the therapeutic stem cells to the neighboring target cells (bystander effect), and on induction of tumor cell apoptosis by therapeutic stem cells. The killing effect of therapeutic stem cells in the presence of 5-FC is quite effective. At a ratio

The C6 cells are highly aggressive tumor cells, what can be attributed to the presence of CSCs.20,21 Indeed, when we plated C6 cells into neurobasal medium supplemented with growth factors, all cells formed spheroids (Fig. 2a). Low dose of C6 cells able to induce tumor and formation of spheroids support the presence of tumor initiating cells in this glioblastoma cell line. CDy-AT-MSCs migrate to glioblastoma cells in vivo

In order to find whether the genetically modified MSCs are able to migrate to glioblastoma cells when applied to the brain, the following experiments were performed. CDy-AT-MSCs were labeled with poly(L-lysine)-modified superparamagnetic C 2011 UICC Int. J. Cancer: 130, 2455–2463 (2012) V

Altanerova et al.

CDy-AT-MSCs mediate inhibition of glioblastoma growth in vivo

To test the described experimental approach under conditions providing statistically significant data and to avoid stress due to repeated stereotaxic brain inoculations, mixtures of 5 105 of C6 tumor cells either with the same number of AT-MSCs or at various CDy-AT-MSC/C6 ratios were injected stereotaxically into the brain of each rat (n ¼ 8 in each group) (Fig. 3b). In separate experiments, we tested the dose of C6 cells able to induce lethal tumor growth. The dose as low as 105 cells injected intracerebrally induced a tumor leading to death in all animals during 22 days. Therefore, a dose of 5 105 C6 cells was chosen for all experiments to guarantee quick onset of tumor growth in coinjection experiments. Animals were treated with daily dose of 500 mg/kg of 5-FC intraperitoneally starting on the third day after tumor cell and therapeutic cell injection. On day 75, the experiments were ended and animals were inspected for tumors by biopsy and histopathology. Kaplan–Meier survival graphs revealed that the entire control group, inoculated with C6 cells alone and group inoculated with a mixture of C6 cells and the same number of AT-MSCs died between 25th and 31st day after tumor implantation. Within the therapeutic group of animals injected with mixtures of tumor cells with 5% of therapeutic stem cells (ratio 1:0.05) 12.5% of animals survived. In the animal group that received 10% of therapeutic stem cells the survival reached 25%. In the group that received 50% of therapeutic stem cells the survival was 37%. In the group of animals that received the same number of therapeutic stem cells as tumor cells, only three animals out of eight died after a double survival time. Sixty-three percent of animals survived up to the C 2011 UICC Int. J. Cancer: 130, 2455–2463 (2012) V

end of the experiment without any signs of tumor development or brain damage. Animal body weights correlated well with therapeutic effects. No tumor cells were found in brains of survived treated animals as histopathology examination showed. The results clearly show that the tumor-free survival of animals depends on the therapeutic stem cell dose. Intracerebro-ventricularly administered 5-FC improves the therapeutic outcome

The 5-fluorocytosine (an anti-mycosis drug) has a high clearance rate as established from studies in humans.24–26 In order to test whether the delivery of prodrug directly into the brain would improve tumor free survival, we designed experiments, in which groups of rats inoculated with C6 cells alone or with mixtures of C6 cells either with AT-MSCs or CDy-ATMSCs were treated intracerebrally with 5-FC. The cannula was surgically implanted into the left lateral brain ventricle of each rat and afterwards connected through a tube with an ALZET osmotic pump filled with 2 mL of a 5-FC solution (Ancotil). In this way, the rats received continuously 5-FC in a dose of 5 lL per hour for 11 days when we replaced the osmotic pump with a new one simultaneously with the repeated therapeutic stem cells application. In addition, we tested whether repeated and increased application of therapeutic stem cells (2 106/5 lL) would prolong the survival of animals inoculated with glioblastoma cells. Kaplan–Meier survival graphs have shown the same therapeutic effect and along with repeated administration of therapeutic stem cells increased the survival time (Fig. 3c). Results revealed that animal control group inoculated with a mixture of C6 and AT-MSCs (1:0.5) died in average on the 24thday after implantation. Within the therapeutic group of animals injected with therapeutic stem cells once, 63% of animals were tumor-free up to 124 days when the experiment ended. The absence of tumor in brains was verified by histopathology. On the site of the tumor inoculation regressive changes, calcifications and presence of macrophages were detected. Tumor-free survival in the group treated with therapeutic stem cells twice reached 88%. The experiment has clearly shown that tumor-free survival depends on the dose of therapeutic stem cells. In addition, continuous delivery of 5-FC into the brain decreased the therapeutic dose of 5-FC substantially. Rats (average body weight ¼ 250 g) received 125 mg of 5-FC per day by the intraperitoneal route, while the daily dose of 5-FC delivered by osmotic pump intracerebroventricularly was only 0.144 mg/day. Intracerebroventricular routes of 5-FC administration and repeated application of therapeutic stem cells improved the survival time in a therapeutic stem cell dose-dependent manner significantly.

Discussion In this study, we tested the feasibility and efficacy of yeast CD::UPRT expressing human AT-MSCs to target and inhibit glioblastoma in vitro and in vivo using C6 rat glioblastoma cells. The immunologically privileged nature of the brain and

Cancer Therapy

iron oxide nanoparticles (SPIO) and stereotaxically injected into the brain of rats contralaterally to inoculated C6 glioblastoma cells. The SPIO labeled therapeutic stem cells were allowed to migrate for 6 days. After this period the brain slices of two animals were examined by histological staining for iron. All remaining rats started to be treated with 5-FC (Ancotil) intraperitoneally (500 mg/kg of body weight/per day). As shown in Figure 2b, the iron-labeled therapeutic stem cells migrated from the opposite hemisphere and infiltrated the tumor. Thus, the genetically modified MSCs did not lose the tumor tropic properties. In addition, the therapeutic effect of labeled therapeutic stem cells was observed. While all animals injected with C6 tumor cells died in an interval of 20–23 days, a portion of contralaterally treated animals with therapeutic stem cells in two different doses survived up to the end of the experiment (90 days; Fig. 3a). Histopathological brain examination revealed no tumor presence in all survived animals. Despite the results that treated rats are not statistically significant because of the low number of animals in the groups, absence of tumor cells in the brains, 90 days survival and vitality of animals expressed by increasing body weight suggest that they were completely cured.

2459

Cancer Therapy

2460


Figure 3. Glioblastoma growth inhibition mediated by CDy-AT-MSCs in vivo. (a) Contralaterally administered CDy-AT-MSCs labeled with poly(L-lysine)-modified superparamagnetic iron oxide nanoparticles exert an anti-tumor effect in the presence of 5-FC. Tumors were induced by 5 105 C6 cells inoculated intracerebrally. Animals in the second group (n ¼ 2) and third group (n ¼ 3) received contralaterally 106 or 105 labeled therapeutic stem cells, respectively. The SPIO labeled therapeutic stem cells were allowed to migrate for 6 days and rats started to be treated with 5-FC intraperitoneally (Ancotil 500 mg/kg of body) for 20 days. On day 90 animals were sacrificed and brains inspected by histopathology. (b) Tumors in all groups were induced with 5 105 C6 cells alone or mixed with different numbers of therapeutic stem cells in ratios indicated in the figure. On day 74 animals were killed and brains inspected by histopathology. Statistical significance was calculated by comparison to C6þAT-MSC group. (c) Glioblastoma growth inhibition mediated by CDy-AT-MSCs with continuous administration of 5-FC into the brain in vivo. All animals including control group were treated with Ancotil by means of an ALZET osmotic pump Model 2ML2 connected by tube with cannula implanted into the brain ventricle. Statistical significance was calculated by comparison to C6þAT-MSC group.


the absence of MHC-II on human MSCs provide favorable conditions for using human MSCs for experiments on animals without immunological problems. Results of stem celldirected cancer suicide gene therapies from our laboratory5,11,12,27 and from others28–30 have shown that mesenchymal stem cell-based approaches are very promising. Tumor tropism of MSCs is of great advantage especially in treatment of high-grade brain tumors with inherent neovascularization capability and ability to infiltrate deeply into brain.18 Beside stem cell-directed prodrug gene therapies, other stem cellsderived therapeutic systems were found effective. The gene for interleukin-4 transferred into neural progenitor cells derived from Sprague-Dawley rats led to the progressive disappearance of large C6 glioblastomas.14 Strong anti-tumor effects have been reported following intracranial administration of gene modified NSC expressing IL-1228 or the tumor necrosis factor-related apoptosis inducing ligand.29 MSCs from bone marrow or from adipose tissue have the tumor migratory ability as well. It has been shown that bone marrow derived MSCs share some characteristics with pericytes.30,31 This property might facilitate the migration of MSCs to highly vascularized glioblastomas. Indeed, it was shown in a rat cerebral ischemia model that intracranially implanted MSCs, without changing morphologically to a neural phenotype, can migrate towards an infarction.32 Therefore, NSCs could be substituted with MSCs as a therapeutic vehicle for gene therapy against glioma.33 In addition, BMMSCs or AT-MSCs have the advantage of ease of isolation and expansion in vitro for clinical use.34 From an ethical point of view, injection of autologous MSCs into patients with malignant brain tumors is more acceptable compared to neural cells derived from embryonic stem cells. Recently, the first clinical study entitled ‘‘A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically-Modified Neural Stem Cells Expressing E. Coli Cytosine Deaminase for Treatment of Recurrent High Grade Gliomas’’ has started.35 We observed strong inhibition of tumor growth in several independent experiments when therapeutic stem cells were injected intracerebrally to C6 glioblastoma in rats receiving the prodrug 5-FC. The number of tumor-free animals was proportional to the percentage of the therapeutic stem cells injected, showing that the outcome is not casual. Interestingly, even 5–10% of therapeutic stem cells were able to induce a total tumor regression in 12% or 25% of treated animals, respectively. High percentage of animals, which survived after the therapeutic stem cells treatment could be regarded as indirect evidence that also CSCs were destroyed. To simulate potential delivery of 5-FC to patients by means of an intraventricular catheter system (Ommaya reservoir), osmotic pumps for 5-FC delivery were used. To the best of our knowledge, intracerebro-ventricular infusion of 5-FC was used for the first time in CD/5-FC system. Repeated application of therapeutic stem cells and intracerebro-ventricular infusions of 5-FC increased the number of tumor-free animals and their survival. Similar observations were recently C 2011 UICC Int. J. Cancer: 130, 2455–2463 (2012) V

2461

reported.36 We have not observed the acceleration of tumor formation in animals co-injected with AT-MSCs, which might be due to human origin of cells. This observation is in a disagreement with our previous results obtained from human colon carcinoma xenografts on nude mice5 and human prostate carcinoma cells derived from metastases.12 It is known that MSC cells may support subcutaneous tumor growth when co-injected with tumor cells.37,38 Tumor growth support by AT-MSCs is caused by several factors such as growth factor production, immunosuppressive character of MSCs and by formation of a favorable microenvironment for cancer cells.39 On the contrary, other studies have observed that MSCs may inhibit tumor growth in animal models40 and possess anti-tumorigenic effects on a model of Kaposi’s sarcoma.41 CDy-AT-MSCs used in this study have the advantage of being safe because of a suicide gene presence. The administration of 5-FC eliminates not only tumor cells but also therapeutic stem cells, as we have previously reported.5 When we compared the efficacy of cytosine deaminase transduced adipose tissue-derived MSCs with bone marrow-derived MSCs, we did not see any significant difference. Therefore, the use of either BM-MSCs or AT-MSCs for this type of stem cell-targeted gene therapy for cancer in future clinical trials will depend on the ease of obtaining the cells. Both types of cells could be derived from the same patient thus further augmenting the safety of their use for therapy. The efficiency of stem cell-targeted therapy for glioblastoma may in part be explained by pericyte-like properties of adipose-derived MSCs. It has been shown that targeting of both tumor endothelium and tumor pericytes synergistically affects tumor vascularization and tumor growth.42 Implantation of CDy-AT-MSCs could attack also glioblastoma stem cells known to reside within a perivascular niche.43 Tumor initiating cells in rat glioma models expressed apart from the neural lineage marker glial fibrillary acidic protein also stem/progenitor markers such as CD133 and nestin.44 The observed high efficacy of the yeast cytosine deaminase fused with uracil phoshoribosyltransferase can be assigned to ability of the enzyme to convert formed 5-FU to 5-fluorouridine monophosphate, which directly kills cytosine deaminase expressing cells and surrounding cells via the bystander effect.45 Recently, the therapeutic efficacy and safety of human AT-MSCs engineered with the tumor necrosis factorrelated apoptosis-inducing ligand against brain stem gliomas in animal model was showed.46,47 Thus the stem cell-based targeted gene therapy opens the possibility for potential treatment of inoperable diffuse intristic pontine gliomas, tumors with a dismal prognosis. In conclusion, the results achieved demonstrated that stem cell-targeted tumor prodrug gene therapy is regarded as a promising treatment modality for glioblastoma. The results are supporting arguments to begin clinical studies for treatment of high grade brain tumors. Mesenchymal stem celldirected suicide gene therapy could offer a hope for patients with high-grade glioblastoma.

Cancer Therapy

Altanerova et al.


2462

Acknowledgements Authors thank Dusan Guba from the Institute of Medical Cosmetics, Bratislava, Slovakia for providing them with material for AT-MSCs isolation, A. Robert Neurath from Virotech, USA and to Jarmila

Cihova for reading the manuscript and comments. Authors also thank Viera Frivalska and Maria Dubrovcakova for technical assistance and Vladimir Geczi for animal maintenance. The authors indicate no potential conflict of interest.

References

Cancer Therapy

1.

Stupp R, Hegi ME, van den Bent MJ, Mason WP, Weller M, Mirimanoff RO, Cairncross JG. Changing paradigms—an update on the multidisciplinary management of malignant glioma. Oncologist 2006;11:165–80. 2. Charles N, Holland EC. The perivascular niche microenvironment in brain tumor progression. Cell Cycle 2010;9:3012–21. 3. Becher OJ, Hambardzumyan D, Fomchenko EI, Momota H, Mainwaring L, Bleau AM, Katz AM, Edgar M, Kenney AM, CordonCardo C, Blasberg RG, Holland EC. Gli activity correlates with tumor grade in platelet-derived growth factor-induced gliomas. Cancer Res 2008;68:2241–9. 4. Soltysova A, Altanerova V, Altaner C. Cancer stem cells. Minireview. Neoplasma 2005;52:435–40. 5. Kucerova L, Altanerova V, Matuskova M, Tyciakova S, Altaner C. Adipose tissuederived human mesenchymal stem cells mediated prodrug cancer gene therapy. Cancer Res 2007;67:6304–13. 6. Altaner C. Prodrug cancer gene therapy. Cancer Lett 2008;270:191–201. 7. Bexell D, Gunnarsson S, Tormin A, Darabi A, Gisselsson D, Roybon L, Scheding S, Bengzon J. Bone marrow multipotent mesenchymal stroma cells act as pericytelike migratory vehicles in experimental gliomas. Mol Ther 2009;17:183–190. 8. Le Blanc K. Immunomodulatory effects of fetal and adult mesenchymal stem cells. Cytotherapy 2003;5:485–9. 9. Koppula PR, Chelluri LK, Polisetti N, Vemuganti GK. Histocompatibility testing of cultivated human bone marrow stromal cells—a promising step towards pre-clinical screening for allogeneic stem cell therapy. Cell Immunol 2009;259:61–5. 10. Griffin MD, Ritter T, Mahon BP. Immunological aspects of allogeneic mesenchymal stem cell therapies. Hum Gene Ther 2010;21:1641–55. 11. Kucerova L, Matuskova M, Pastorakova A, Tyciakova S, Jakubikova J, Bohovic R, Altanerova V, Altaner C. Cytosine deaminase expressing human mesenchymal stem cells mediated tumour regression in melanoma bearing mice. J Gene Med 2008; 10:1071–82. 12. Cavarretta IT, Altanerova V, Matuskova M, Kucerova L, Culig Z Altaner C. Adipose tissue-derived mesenchymal stem cells expressing prodrug-converting enzyme inhibit human prostate tumor growth. Mol Ther 2010;18:223–31.

13. Aboody KS, Brown A, Rainov NG, Bower KA, Liu S, Yang W, Small JE, Herrlinger U, Ourednik V, Black PM, Breakefield XO, Snyder EY. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000;97:12846–51. 14. Benedetti S, Pirola B, Pollo B, Magrassi L, Bruzzone MG, Rigamonti D, Galli R, Selleri S, Di Meco F, De Fraja C, Vescovi A, Cattaneo E, et al. Gene therapy of experimental brain tumors using neural progenitor cells. Nat Med 2000;6:447–50. 15. Aboody KS, Najbauer J, Schmidt NO, Yang W, Wu JK, Zhuge Y, Przylecki W, Carroll R, Black PM, Perides G. Targeting of melanoma brain metastases using engineered neural stem/progenitor cells. Neuro-oncology 2006;8:119–26. 16. Aboody KS, Najbauer J Danks MK. Stem and progenitor cell-mediated tumor selective gene therapy. Gene Ther 2008;15:739–52. 17. Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol 2002;174:11–20. 18. Au P, Tam J, Fukumura D, Jain RK. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood 2008;111:4551–58. 19. Kondo T, Setoguchi T, Taga T. Persistence of a small subpopulation of cancer stemlike cells in the C6 glioma cell line. Proc Natl Acad Sci USA 2004;101:781–86. 20. Zheng X, Shen G, Yang X, Liu W. Most C6 cells are cancer stem cells: evidence from clonal and population analyses. Cancer Res 2007;67:3691–97. 21. Zhou XD, Wang XY, Qu FJ, Zhong YH, Lu XD, Zhao P, Wang DH, Huang QB, Zhang L, Li XG. Detection of cancer stem cells from the C6 glioma cell line. J Int Med Res 2009;37:503–10. 22. Babic M, Hora´k D, Trchova´ M, Jendelova´ P, Glogarova´ K, Lesny´ P, Herynek V, Ha´jek M, Sykova´ E. Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labelling. Bioconjug Chem 2008;19: 740–50. 23. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press, 1997. 256 p. 24. Huber BE, Austin EA, Good SS, Knick VC, Tibbels S, Richards CA. In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

modified to express cytosine deaminase. Cancer Res 1993;53:4619–26. Trinh QT, Austin EA, Murray DM, Knick VC, Huber BE. Enzyme/prodrug gene therapy: comparison of cytosine deaminase/5fluorocytosine versus thymidine kinase/ ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line. Cancer Res 1995;55:4808–12. Dawborn JK, Page MD, Schiavone DJ. Use of 5-fluorocytosine in patients with impaired renal function. Br Med J 1997;4:382–84. Matuskova M, Hlubinova K, Pastorakova A, Hunakova L, Altanerova V, Altaner C, Kucerova L. HSV-tk expressing mesenchymal stem cells exert bystander effect on human glioblastoma cells. Cancer Lett 2010;290:58–67. Ehtesham M, Kabos P, Kabosova A, Neuman T, Black KL, Yu JS. The use of interleukin 12-secreting neural stem cells for the treatment of intracranial glioma. Cancer Res 2002;62:5657–63. Ehtesham M, Kabos P, Gutierrez MA, Chung NH, Griffith TS, Black KL, Yu JS. Induction of glioblastoma apoptosis using neural stem cellmediated delivery of tumor necrosis factorrelated apoptosis-inducing ligand. Cancer Res 2002;62:7170–4. Bexell D, Scheding S, Bengzon J. Toward brain tumor gene therapy using multipotent mesenchymal stromal cell vectors. Mol Ther 2010;18:1067–75. Bexell D, Gunnarsson S, Tormin A, Darabi A, Gisselsson D, Roybon L, Scheding S, Bengzon J. Bone marrow multipotent mesenchymal stroma cells act as pericytelike migratory vehicles in experimental gliomas. Mol Ther 2009;17:183–90. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang L, Norotte C, Teng PN, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301–13. Zhao LR, Duan WM, Reyes M, Keene CD, Verfaillie CM, Low WC. Human bone marrow stem cells exhibit neural phenotypes and ameliorate neurological deficits after grafting into the ischemic brain of rats. Exp Neurol 2002;174:11–20. Schaffler A, Buchler C. Concise review: adipose tissue-derived stromal cells—basic and clinical implications for novel cellbased therapies. Stem Cells 2007;25:818–26. http://www.clinicaltrials.gov/ct2/show/ NCT01172964?term¼ stemþcellþANDþcancerþþANDþ gene&rank¼6.


Altanerova et al.

41.

42.

43.

44.

progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatine matrix. Exp Mol Pathol 2003;75: 248–55. Khakoo AY, Pati S, Anderson SA, Reid W, Elshal MF, Rovira II, Nguyen AT, Malide D, Combs CA, Hall G, Zhang J, Raffeld M, et al. Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med 2006;203: 1235–47. Bergers G, Song S, Meyer-Morse N, Bergsland E, Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 2003;111:1287–95. Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C, Hamner B, Oh EY, Gaber MW, Finklestein D, Allen M, Frank A, Bayazitov IT, et al. A perivascular niche for brain tumor stem cells. Cancer Cell 2007; 11:69–82. Bexell D, Gunnarsson S, Siesjo¨ P, Bengzon J, Darabi A. CD133þ and nestinþ tumor-

initiating cells dominate in N29 and N32 experimental gliomas. Int J Cancer 2009; 125:15–22. 45. Shi DZ, Hu WX, Li LX, Chen G, Wei D, Gu PY. Pharmacokinetics and the bystander effect in CD::UPRT/5-FC bi-gene therapy of glioma. Chin Med J (Engl) 2009 122: 1267–72. 46. Lee DH, Ahn Y, Kim SU, Wang KC, Cho BK, Phi JH, Park IH, Black PM, Carroll RS, Lee J, Kim SK. Targeting rat brain stem glioma using human neural stem cells and human mesenchymal stem cells. Clin Cancer Res 2009;15: 4925–34. 47. Choi SA, Hwang SK, Wang KC, Cho BK, Phi JH, Lee JY, Jung HW, Lee DH, Kim SK. Therapeutic efficacy and safety of TRAIL-producing human adipose tissue-derived mesenchymal stem cells against experimental brainstem glioma. Neuro Oncol 2011; 13:61–9.

Cancer Therapy

36. Chang DY, Yoo SW, Hong Y, Kim S, Kim SJ, Yoon SH, Cho KG, Paek SH, Lee YD, Kim SS, Suh-Kim H. The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase. Int J Cancer 2010;127:1975–83. 37. Zhu W, Xu W, Jiang R, Qian H, Chen M, Hu J, Cao W, Han C, Chen Y. Mesenchymal stem cells derived from bone marrow favor tumor cell growth in vivo. Exp Mol Pathol 2006;80 267–74. 38. Hall B, Andreeff M Marini F. The participation of mesenchymal stem cells in tumor stroma formation and their application as targeted-gene delivery vehicles. Handb Exp Pharmacol 2007;180: 263–83. 39. Kucerova L, Matuskova M, Hlubinova K, Altanerova V, Altaner C. Tumor cell behaviour modulation by mesenchymal stromal cells. Mol Cancer 2010;9:129. 40. Ohlsson LB, Varas L, Kjellman C, Edvardsen K, Lindvall M. Mesenchymal

2463
