Stem Cells Expressing Cytosine Deaminase Research Articles

Genetically engineered neural stem cells expressing cytosine deaminase and interferon-beta enhanced T cell-mediated antitumor immunity against gastric cancer in a humanized mouse model

AimsGastric cancer (GC) is an invasive, fatal disease with a poor prognosis. Gene-directed enzyme prodrug therapy via genetically engineered neural stem cells (GENSTECs) has been widely studied in various malignancies, such as breast, ovarian, and renal cancer. In this study, the human neural stem cells expressing cytosine deaminase and interferon beta (HB1.F3.CD.IFN-β) cells were applied to convert non-toxic 5-fluorocytosine to cytotoxic 5-fluorouracil and secrete IFN-β. Materials and methodsHuman lymphokine-activated killer cells (LAKs) were generated by stimulating human peripheral blood mononuclear cells (PBMCs) by interleukin-2, and we evaluated the cytotoxic activity and migratory ability of LAKs co-cultured with GNESTECs or their conditioned media in vitro. A GC-bearing human immune system (HIS) mouse model was generated by transplanting human PBMCs followed by subcutaneous engraftment of MKN45 cells in NSG-B2m mice to evaluate the involvement of T cell-mediated anti-cancer immune activity of GENSTECs. Key findingsIn vitro studies showed the presence of HB1.F3.CD.IFN-β cells facilitated the migration ability of LAKs to MKN45 cells and activated their cytotoxic potential. In MKN45-xenografted HIS mice, treatment with HB1.F3.CD.IFN-β cells resulted in increased cytotoxic T lymphocyte (CTL) infiltration throughout the tumor, including the central area. Moreover, the group treated to HB1.F3.CD.IFN-β showed increased granzyme B expression in the tumor, eventually enhancing the tumor-killing potential of CTLs and significantly delaying tumor growth. SignificanceThese results indicate that the HB1.F3.CD.IFN-β cells exert anti-cancer effects on GC by facilitating the T cell-mediated immune response, and GENSTECs are a promising therapeutic strategy for GC.

Neural stem cells expressing cytosine deaminase and interferon-[beta] suppressed the growth and metastasis of anaplastic thyroid cancer cells

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Cancer-Specific Inhibitory Effects of Genetically Engineered Stem Cells Expressing Cytosine Deaminase and Interferon-β Against Choriocarcinoma in Xenografted Metastatic Mouse Models

Cancer treatments using stem cells expressing therapeutic genes have been identified for various types of cancers. In this study, we investigated inhibitory effects of HB1.F3.CD and HB1.F3.CD.IFN-β cells expressing Escherichia coli cytosine deaminase (CD) and human interferon-β (IFN-β) genes in intravenously (i.v.) injected mice with a metastasis model. In this treatment, pro-drug 5-fluorocytosine (5-FC) is converted to cytotoxic drug 5-fluorouracil by hNSCs expressing the CD gene, which inhibits DNA synthesis in cancer cells. Moreover, IFN-β induces apoptosis and reduces the growth of cancer cells. Upon MTT assay, proliferation of choriocarcinoma (JEG-3) cells decreased when co-cultured with hNSCs expressing CD and IFN-β genes. To confirm the cancer-tropic effect of these stem cells, chemoattractant factors (VEGF, CXCR4, and C-kit) secreted from JEG-3 cells were identified by polymerase chain reaction. hNSCs migrate toward JEG-3 cells due to ligand-receptor interactions of these factors. Accordingly, the migration capability of hNSCs toward JEG-3 cells was confirmed using an in vitro Trans-well assay, in vivo subcutaneously (s.c.) injected mice groups (xenograft model), and metastasis model. Intravenously injected hNSCs migrated freely to other organs when compared to s.c. injected hNSCs. Thus, we confirmed the inhibition of lung and ovarian metastasis of choriocarcinoma by i.v. injected HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. Treatment of these stem cells also increased the survival rates of mice. In conclusion, this study showed that metastatic cancer was diminished by genetically engineered hNSCs and noncytotoxic drug 5-FC. This is the first report of the therapeutic potential of i.v. injected hNSCs in a metastasis model; therefore, the results indicate that this stem cell therapy can be used as an alternative novel tool to treat metastatic choriocarcinoma.

Abstract B13: A growth of human choriocarcinoma cells was selectively inhibited by therapeutic neural stem cells expressing cytosine deaminase and interferon-β in cellular and xenograft models

Abstract In this study, human neural stem cells (hNSCs) expressing Escherichia coli cytosine deaminase (CD) and human interferon-β (IFN-β) was used to cancer treatment strategy for human choriocarcinoma cancer. Prodrug 5-fluorocytosine (5-FC) is converted to 5-fluorouacil (5-FU) by CD, which induces an effect of killing the tumor cells through DNA synthesis inhibition. Also, IFN-β suppresses tumor growth through the apoptotic process. To manufacture animal models xenografted with choriocarcinoma, CMFDA pre-labeled JEG-3 cells (2×106) were injected intravenously (i.v.) into the tail vein of athymic nude mice (group A) and injected subcutaneously (s.c.) into the back of the mice (group B). Subcutaneously injected cancer cells were mixed with Matrigel (BD Biosciences, Bedford, MA, USA) at 1:1 volume ratio of Matrigel to PBS in 100 μl. On group A, CM-dil pre-labeled hNSCs (2×106) were injected intravenously after a week. On group B, when the tumor volume reached at 100mm3, CM-Dil pre-labeled hNSCs (2×106) were injected subcutaneously adjacent to the tumor mass. Every mice received i.p. injections of 5-FC (500 mg/kg/day) every day for 21 days. In a modified trans-well migration assay, NSCs selectively migrated to JEG-3 choriocarcinoma cells. It was revealed that the tumor-tropic properties of engineered NSCs were attributed to chemoattractant factors (CXCR4, SCF, SDF-1a, VEGF) secreted by JEG-3 cells. When JEG-3 cells co-cultured with engineered NSCs in the presence of 5-FC, the viability of JEG-3 cells was markedly decreased. The present results suggested that NSCs expressing CD gene and IFN-β gene have a potential therapeutic effect against choriocarcinoma. Consequently, hNSC therapy may be a clinically effective tool for the treatment of human choriocarcinoma cancer. In addition, we are currently studying on an in vivo model to demonstrate that selective anti-tumor effect of NSCs. Note: This abstract was not presented at the conference. Citation Format: Gyu-Sik Kim, Kyung-Chul Choi. A growth of human choriocarcinoma cells was selectively inhibited by therapeutic neural stem cells expressing cytosine deaminase and interferon-β in cellular and xenograft models [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B13.

Abstract B14: Injection of therapeutic stem cells expressing cytosine deaminase and interferon-beta resulted in the inhibition of K562 human chronic myeloid leukemia cells in a xenograft model

Abstract Gene-directed enzyme/prodrug therapies have been found to be more advantageous compared to conventional cancer treatment method. One of these, a cytosine deaminase (CD)/5-fluorocytosine (5-FC) system, is known to induce apoptosis of cancer cells by converting 5-FC, a prodrug, to its metabolically active form, 5-fluorouracil. In this study, human neural stem cells (hNSCs) derived enzyme/prodrug therapy was used to treat leukemia. The parental hNSCs, HB1.F3, were engineered to express E. coli CD and/or human interferon-β. To manufacture animal models xenografted with leukemia, K-562 cells (1×106) were mixed with Matrigel (BD Biosciences, Bedford, MA, USA) at 1:1 volume ratio of Matrigel to PBS in 100 μl and injected subcutaneously (s.c.) into the back of athymic nude mice. This animal study was performed for 24 days after hNSCs injections. When tumor volume reached 500 mm3, CM-Dil pre-labeled hNSCs (4 x 106 cells per mouse) were injected subcutaneously adjacent to the tumor mass. Another group, CM-dil pre-labeled hNSCs (4 x 106 cells per mouse) was injected intravenously. hNSCs were injected on the first day of each week. Two days after the injection of hNSCs, all mice received i.p. injections of 5-FC (500 mg/kg/day in 100 μl PBS) every day for 24 days. At 24 h after the last 5-FC treatment, the mice were euthanized and tumor masses were harvested for molecular analysis. In a xenografted mouse model administered with hNSCs intravenously or subcutaneously, hNSC significantly inhibits the growth of tumor mass and extends survival date in the presence of a prodrug. In addition, HB1.F3.CD.IFN-β treatment group showed more antitumor effect compared with HB1.F3.CD treatment group, indicating that IFN- β may have a synergistic effect for directly killing leukemia tumors. The present results represent that engineered hNSCs and prodrug treatment inhibited the proliferation of leukemia. These results suggest that gene therapy employing genetically engineered stem cells expressing CD and IFN-β may be effective for treating leukemia. Note: This abstract was not presented at the conference. Citation Format: Geon-Tae Park, Kyung-Chul Choi. Injection of therapeutic stem cells expressing cytosine deaminase and interferon-beta resulted in the inhibition of K562 human chronic myeloid leukemia cells in a xenograft model [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B14.

Combined treatment, based on lysomustine administration with mesenchymal stem cells expressing cytosine deaminase therapy, leads to pronounced murine Lewis lung carcinoma growth inhibition.

The combination of stem cell-based gene therapy with chemotherapy comprises an advantageous strategy that results in a reduction of system toxicity effects and an improvement in the general efficacy of treatment. In the present study, we estimated the efficacy of adipose tissue-derived mesenchymal stem cells (AT-MSCs) expressing cytosine deaminase (CDA) combined with lysomustine chemotherapy in mice bearing late stage Lewis lung carcinoma (LLC). Adipose tissue-derived mesenchymal stem cells were transfected with non-insert plasmid construct transiently expressing fused cytosine deaminase-uracil phosphoribosyltransferase protein (CDA/UPRT) or the same construct fused with Herpes Simplex Virus Type1 tegument protein VP22 (CDA/UPRT/VP22). Systemic administration of 5-fluorocytosine (5FC) and lysomustine was implemented after a single intratumoral injection of transfected AT-MSCs. We demonstrated that direct intratumoral transplantation of AT-MSCs expressing CDA/UPRT or CDA/UPRT/VP22 followed by systemic administration of 5FC resulted in a significant tumor growth inhibition. There was a 56% reduction in tumor volume in mice treated by AT-MSCs-CDA/UPRT + 5FC or with AT-MSCs-CDA/UPRT/VP22 + 5FC compared to control animals grafted with lung carcinoma alone. Transplantation of AT-MSCs-CDA/UPRT + 5FC and AT-MSCs-CDA/UPRT/VP22 + 5FC prolonged the life span of mice bearing LLC by 27% and 31%, respectively. Co-administration of lysomustine and AT-MSCs-CDA/UPRT + 5FC led to tumor growth inhibition (by 86%) and life span extension (by 60%) compared to the control group. Our data indicate that a combination CDA/UPRT-expressing AT-MSCs with lysomustine has a superior antitumor effect in the murine lung carcinoma model compared to monotherapies with transfected AT-MSCs or lysomustine alone, possibly because of a synergistic effect of the combination therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of genetically engineered human neural stem cells expressing cytosine deaminase and interferon-beta on the growth of lymph node metastatic colorectal adenocarcinoma

Effects of genetically engineered human neural stem cells expressing cytosine deaminase and interferon-beta on the growth of lymph node metastatic colorectal adenocarcinoma

Anti-proliferative Effect of Engineered Neural Stem Cells Expressing Cytosine Deaminase and Interferon-β against Lymph Node-Derived Metastatic Colorectal Adenocarcinoma in Cellular and Xenograft Mouse Models.

PurposeGenetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma.Materials and MethodsCD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes.ResultsIn results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals.ConclusionThe current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer.

Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer

As an approach to improve treatment of breast cancer metastasis to the brain, we employed genetically engineered stem cells (GESTECs, HB1.F3 cells) consisting of neural stem cells (NSCs) expressing cytosine deaminase and the interferon-beta genes, HB1.F3.CD and HB1.F3.CD.IFN-β. In this model, MDA-MB-231/Luc breast cancer cells were implanted in the right hemisphere of the mouse brain, while pre-stained GESTECs with redfluorescence were implanted in the contralateral brain. Two days after stem cells injection, 5-fluorocytosine (5-FC) was administrated via intraperitoneal injection. Histological analysis of extracted brain confirmed the therapeutic efficacy of GESTECs in the presence of 5-FC based on reductions in density and aggressive tendency of breast cancer cells, as well as pyknosis, karyorrhexis, and karyolysis relative to a negative control. Additionally, expression of PCNA decreased in the stem cells treated group. Treatment of breast cancer cells with 5-fluorouracil (5-FU) increased the expression of pro-apoptotic and anti-proliferative factor, BAX and p21 protein through phosphorylation of p53 and p38. Moreover, analysis of stem cell migratory ability revealed that MDA-MB-231 cells endogenously secreted VEGF, and stem cells expressed their receptor (VEGFR2). To confirm the role of VEGF/VEGFR2 signaling in tumor tropism of stem cells, samples were treated with the VEGFR2 inhibitor, KRN633. The number of migrated stem cells decreased significantly in response to KRN633 due to Erk1/2 activation and PI3K/Akt inhibition. Taken together, these results indicate that treatment with GESTECs, particularly HB1.F3.CD.IFN-β co-expressing CD.IFN-β, may be a useful strategy for treating breast cancer metastasis to the brain in the presence of a prodrug.

Selective antitumor effect of neural stem cells expressing cytosine deaminase and interferon-beta against ductal breast cancer cells in cellular and xenograft models

Due to their inherent tumor-tropic properties, genetically engineered stem cells may be advantageous for gene therapy treatment of various human cancers, including brain, liver, ovarian, and prostate malignancies. In this study, we employed human neural stem cells (HB1.F3; hNSCs) transduced with genes expressing Escherichia coli cytosine deaminase (HB1.F3.CD) and human interferon-beta (HB1.F3.CD.IFN-β) as a treatment strategy for ductal breast cancer. CD can convert the prodrug 5-fluorocytosine (5-FC) to its active chemotherapeutic form, 5-fluorouracil (5-FU), which induces a tumor-killing effect through DNA synthesis inhibition. IFN-β also strongly inhibits tumor growth by the apoptotic process. RT-PCR confirmed that HB1.F3.CD cells expressed CD and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β. A modified transwell migration assay showed that HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward MCF-7 and MDA-MB-231 human breast cancer cells. In hNSC-breast cancer co-cultures the viability of breast cancer cells which were significantly reduced by HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. The tumor inhibitory effect was greater with the HB1.F3.CD.IFN-β cells, indicating an additional effect of IFN-β to 5-FU. In addition, the tumor-tropic properties of these hNSCs were found to be attributed to chemoattractant molecules secreted by breast cancer cells, including stem cell factor (SCF), c-kit, vascular endothelial growth factor (VEGF), and VEGF receptor 2. An in vivo assay performed using MDA-MB-231/luc breast cancer mammary fat pad xenografts in immunodeficient mice resulted in 50% reduced tumor growth and increased long-term survival in HB1.F3.CD and HB1.F3.CD.IFN-β plus 5-FC treated mice relative to controls. Our results suggest that hNSCs genetically modified to express CD and/or IFN-β genes can be used as a novel targeted cancer gene therapy.

Adenosine potentiates the therapeutic effects of neural stem cells expressing cytosine deaminase against metastatic brain tumors

Tumor-tropic properties of neural stem cells (NSCs) provide a novel approach with which to deliver targeting therapeutic genes to brain tumors. Previously, we developed a therapeutic strategy against metastatic brain tumors using a human NSC line (F3) expressing cytosine deaminase (F3.CD). F3.CD converts systemically administered 5-fluorocytosine (5-FC), a blood-brain barrier permeable nontoxic prodrug, into the anticancer agent 5-fluorouracil (5-FU). In this study, we potentiated a therapeutic strategy of treatment with nucleosides in order to chemically facilitate the endogenous conversion of 5-FU to its toxic metabolite 5-FU ribonucleoside (5-FUR). In vitro, 5-FUR showed superior cytotoxic activity against MDA-MB-435 cancer cells when compared to 5-FU. Although adenosine had little cytotoxic activity, the addition of adenosine significantly potentiated the in vitro cytotoxicity of 5-FU. When MDA-MB‑435 cells were co-cultured with F3.CD cells, F3.CD cells and 5-FC inhibited the growth of MDA-MB-435 cells more significantly in the presence of adenosine. Facilitated 5-FUR production by F3.CD was confirmed by an HPLC analysis of the conditioned media derived from F3.CD cells treated with 5-FC and adenosine. In vivo systemic adenosine treatment also significantly potentiated the therapeutic effects of F3.CD cells and 5-FC in an MDA-MB-435 metastatic brain tumor model. Simple adenosine addition improved the antitumor activity of the NSCs carrying the therapeutic gene. Our results demonstrated an increased therapeutic potential, and thereby, clinical applicability of NSC-based gene therapy.

Pancreatic tumor mass in a xenograft mouse model is decreased by treatment with therapeutic stem cells following introduction of therapeutic genes

Pancreatic cancer is the fourth most common cause of cancer-related mortality. In the present study, we employed 2 types of therapeutic stem cells expressing cytosine deaminase (CD) with or without human interferon-β (IFN‑β), HB1.F3.CD and HB1.F3.CD.IFN-β cells, respectively, to selectively treat pancreatic cancer. The CD gene converts the non-toxic prodrug, 5-flurorocytosine (5-FC), into the toxic agent, 5-fluorouracil (5-FU). In addition, human IFN-β is a potent cytokine that has antitumor effects. To generate a xenograft mouse model, PANC-1 cells (2x10(6)/mouse) cultured in DMEM containing 10% FBS were mixed with Matrigel and were subcutaneously injected into Balb/c nu/nu mice. In the migration assay, the stem cells expressing the CD or IFN-β gene effectively migrated toward the pancreatic cancer cells, suggesting the presence of chemoattractant factors secreted by the pancreatic tumors. In the co-culture and MTT assay, antitumor activity of the therapeutic stem cells was observed in the presence of 5-FC was shown that the growth of PANC-1 cells was inhibited. Furthermore, these effects were confirmed in the xenograft mouse model bearing tumors originating from PANC-1 cells. Analyses by histological and fluorescence microscopy showed that treatment with the stem cells resulted in the inhibition of pancreatic cancer growth in the presence of 5-FC. Taken together, these results indicate that stem cells expressing the CD and/or IFN-β gene can be used to effectively treat pancreatic cancer and reduce the side-effects associated with conventional therapies.

Suppression of the growth of human colorectal cancer cells by therapeutic stem cells expressing cytosine deaminase and interferon-β via their tumor-tropic effect in cellular and xenograft mouse models

Genetically engineered stem cells (GESTECs) exhibit a potent therapeutic efficacy via their strong tumor tropism toward cancer cells. In this study, we introduced the human parental neural stem cells, HB1.F3, with the human interferon beta (IFN-β) gene which is a typical cytokine gene that has an antitumor effect and the cytosine deaminase (CD) gene from Escherichia coli (E. coli) that could convert the non-toxic prodrug, 5-fluorocytosine (5-FC), to a toxic metabolite, 5-fluorouracil (5-FU). Two types of stem cells expressing the CD gene (HB1.F3.CD cells) and both the CD and human IFN-β genes (HB1.F3.CD.IFN-β) were generated. The present study was performed to examine the migratory and therapeutic effects of these GESTECs against the colorectal cancer cell line, HT-29. When co-cultured with colorectal cancer cells in the presence of 5-FC, HB1.F3.CD and HB1.F3.CD.IFN-β cells exhibited the cytotoxicity on HT-29 cells via the bystander effect. In particular, HB1.F3.CD.IFN-β cells showed the synergistic cytotoxic activity of 5-FU and IFN-β. We also confirmed the migration ability of HB1.F3.CD and HB1.F3.CD.IFN-β cells toward HT-29 cells by a modified migration assay in vitro, where chemoattractant factors secreted by HT-29 cells attracted the GESTECs. In a xenograft mouse model, the volume of tumor mass was decreased up to 56% in HB1.F3.CD injected mice while the tumor mass was greatly inhibited about 76% in HB1.F3.CD.IFN-β injected mice. The therapeutic treatment by these GESTECs is a novel strategy where the combination of the migration capacity of stem cells as a vector for therapeutic genes towards colorectal cancer and a synergistic antitumor effect of CD and IFN-β genes can selectively target this type of cancer.

Effects of Genetically Engineered Stem Cells Expressing Cytosine Deaminase and Interferon-Beta or Carboxyl Esterase on the Growth of LNCaP Prostate Cancer Cells

The risk of prostate cancer has been increasing in men by degrees. To develop a new prostate cancer therapy, we used a stem cell-derived gene directed prodrug enzyme system using human neural stem cells (hNSCs) that have a tumor-tropic effect. These hNSCs were transduced with the therapeutic genes for bacterial cytosine deaminase (CD), alone or in combination with the one encoding human interferon-beta (IFN-β) or rabbit carboxyl esterase (CE) to generate HB1.F3.CD, HB1.F3.CD.IFN-β, and HB1.F3.CE cells, respectively. CD enzyme can convert the prodrug 5-fluorocytosine (5-FC) into the activated form 5-fluorouracil (5-FU). In addition, CE enzyme can convert the prodrug CPT-11 into a toxic agent, SN-38. In our study, the human stem cells were found to migrate toward LNCaP human prostate cancer cells rather than primary cells. This phenomenon may be due to interactions between chemoattractant ligands and receptors, such as VEGF/VEGFR2 and SCF/c-Kit, expressed as cancer and stem cells, respectively. The HB1.F3.CE, HB.F3.CD, or HB1.F3.CD.IFN-β cells significantly reduced the LNCaP cell viability in the presence of the prodrugs 5-FC or CPT-11. These results indicate that stem cells expressing therapeutic genes can be used to develop a new strategy for selectively treating human prostate cancer.

Synergistic effects of genetically engineered stem cells expressing cytosine deaminase and interferon-β via their tumor tropism to selectively target human hepatocarcinoma cells

Stem cells have received a great deal of attention for their clinical and therapeutic potential for treating human diseases and disorders. Recent studies have shown that it is possible to genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non-toxic prodrugs to toxic metabolites, selectively migrate toward tumor sites and reduce tumor growth. In this study, we evaluated whether these GESTECs are capable of migrating to hepatocarcinoma cells and examined the potential therapeutic efficacy of gene-directed enzyme prodrug therapy against liver cancer cells in cellular and animal models. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to Hep3B hepatocarcinoma cells. GESTECs, that is, HB1.F3.CD or HB1.F3.CD.interferon-β (IFN-β) cells, engineered to express a suicide gene, cytosine deaminase (CD), selectively migrated toward liver cancer cells. Treatment of Hep3B, human liver cancer cells, with the prodrug 5-fluorocytosine (5-FC) in the presence of HB1.F3.CD or HB1.F3.CD.IFN-β cells resulted in the inhibition of Hep3B cell growth. In a xenografted mouse model injected with hepatocarcinoma, we investigated the therapeutic effect of these stem cells. For 9 weeks, the xenografted mice were treated with HB1.F3.CD or HB1.F3.CD.IFN-β in the presence of 5-FC. A growth of tumor mass was inhibited about 40-50% in the mice treated with GESTECs and a prodrug. In addition, we further confirmed the cytotoxic effect on tumor cells by histological analysis and migratory effect of therapeutic stem cells. Taken together, GESTECs expressing a fusion gene encoding CD and IFN-β may exert a synergistic antitumor effect on this type of tumor.

Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models

As human amniotic fluid-derived stem cells (hAFSCs) are capable of multiple lineage differentiation, extensive self-renewal and tumor targeting, they may be valuable for clinical anticancer therapies. In this study, we used hAFSCs as vehicles for targeted delivery of therapeutic suicide genes to breast cancer cells. hAFSCs were engineered to produce AF2.CD-TK cells in order to express two suicide genes encoding bacterial cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) that convert non-toxic prodrugs, 5-fluorocytosine (5-FC) and mono-phosphorylate ganciclovir (GCV-MP), into cytotoxic metabolites, 5-fluorouracil (5-FU) and triphosphate ganciclovir (GCV-TP), respectively. In cell viability test in vitro, AF2.CD-TK cells inhibited the growth of MDA-MB-231 human breast cancer cells in the presence of the 5-FC or GCV prodrugs, or a combination of these two reagents. When the mixture of 5-FC and GCV was treated together, an additive cytotoxic effect was observed in the cell viability. In animal experiments using female BALB/c nude mouse xenografts, which developed by injecting MDA-MB-231 cells, treatment with AF2.CD-TK cells in the presence of 5-FC and GCV significantly reduced tumor volume and weight to the same extent seen in the mice treated with 5-FU. Histopathological and fluorescent staining assays further showed that AF2.CD-TK cells were located exactly at the site of tumor formation. Furthermore, breast tissues treated with AF2.CD-TK cells and two prodrugs maintained their normal structures (for example, the epidermis and reticular layers) while breast tissue structures in 5-FU-treated mice were almost destroyed by the potent cytotoxicity of the drug. Taken together, these results indicate that AF2.CD-TK cells can serve as excellent vehicles in a novel therapeutic cell-based gene-directed prodrug system to selectively target breast malignancies.

Genetically engineered stem cells expressing cytosine deaminase and interferon-β migrate to human lung cancer cells and have potentially therapeutic anti-tumor effects

Recent studies have shown that genetically engineered stem cells (GESTECs) produce suicide enzymes that convert non-toxic pro-drugs to toxic metabolites which selectively migrate toward tumor sites and reduce tumor growth. In the present study, we evaluated whether these GESTECs are capable of migrating to lung cancer cells and examined the potential therapeutic efficacy of gene-directed enzyme pro-drug therapy against lung cancer cells in vitro. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to lung cancer cells. GESTECs [i.e., HB1.F3.CD or HB1.F3.CD.interferon-β (IFN-β)] engineered to express a suicide gene, cytosine deaminase (CD), selectively migrated toward lung cancer cells. Treatment of a human non-small cell lung carcinoma cell line (A549, a lung carcinoma derived from human lung epithelial cells) with the pro-drug 5-fluorocytosine (5-FC) in the presence of HB1.F3.CD or HB1.F3.CD.IFN-β cells resulted in the inhibition of lung cancer cell growth. Based on the data presented herein, we suggest that GESTECs expressing CD may have a potent advantage for selective treatment of lung cancers. Furthermore, GESTECs expressing fusion genes (i.e., CD and IFN-β) may have a synergic antitumor effect on lung cancer cells.

Co-Culture of Breast Cancer Cells with Genetically Engineered Stem Cells Expressing Cytosine Deaminase and Interferon-Beta Resulted in the Migration and Their Growth Inhibition In Vitro.

Co-Culture of Breast Cancer Cells with Genetically Engineered Stem Cells Expressing Cytosine Deaminase and Interferon-Beta Resulted in the Migration and Their Growth Inhibition In Vitro.

The growth of brain tumors can be suppressed by multiple transplantation of mesenchymal stem cells expressing cytosine deaminase

Suicide genes have recently emerged as an attractive alternative therapy for the treatment of various types of intractable cancers. The efficacy of suicide gene therapy relies on efficient gene delivery to target tissues and the localized concentration of final gene products. Here, we showed a potential ex vivo therapy that used mesenchymal stem cells (MSCs) as cellular vehicles to deliver a bacterial suicide gene, cytosine deaminase (CD) to brain tumors. MSCs were engineered to produce CD enzymes at various levels using different promoters. When co-cultured, CD-expressing MSCs had a bystander, anti-cancer effect on neighboring C6 glioma cells in proportion to the levels of CD enzymes that could convert a nontoxic prodrug, 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) in vitro. Consistent with the in vitro results, for early stage brain tumors induced by intracranial inoculation of C6 cells, transplantation of CD-expressing MSCs reduced tumor mass in proportion to 5-FC dosages. However, for later stage, established tumors, a single treatment was insufficient, but only multiple transplantations were able to successfully repress tumor growth. Our findings indicate that the level of total CD enzyme activity is a critical parameter that is likely to affect the clinical efficacy for CD gene therapy. Our results also highlight the potential advantages of autograftable MSCs compared with other types of allogeneic stem cells for the treatment of recurrent glioblastomas through repetitive treatments.