Neural stem cell-mediated enzyme/prodrug therapy for glioma: preclinical studies.
ABSTRACT: High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)-expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non-tumor-bearing and orthotopic glioma-bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.
Project description:Medulloblastoma is a heterogeneous diffuse neoplasm that can be highly disseminated, and is the most common malignant childhood brain tumor. Although multimodal treatments have improved survival rates for patients with medulloblastoma, these tumors are associated with high morbidity and mortality. New treatment strategies are urgently needed to improve cure rates and, importantly, to spare normal brain tissue from neurotoxicity and patients from life-long cognitive and functional deficits associated with current therapies. In numerous preclinical brain tumor models, neural stem cells (NSCs) have shown great promise as delivery vehicles for therapeutic genes. Here, we have used an established, genetically modified human NSC line (HB1.F3.CD) to deliver carboxylesterase (CE) to cerebellar tumor foci and locally activate the prodrug camptothecin-11 (CPT-11) (Irinotecan) to the potent topoisomerase I inhibitor SN-38. HB1.F3.CD NSC tumor tropism, intratumoral distribution and therapeutic efficacy were investigated in clinically relevant experimental models. Magnetic resonance imaging was used for in vivo tracking of iron nanoparticle-labeled NSCs, and to assess the therapeutic efficacy of CE-expressing HB1.F3.CD cells. As compared with controls, a significant decrease in tumor growth rate was seen in mice that received both NSCs and CPT-11 as their treatment regimen. Thus, this study provides proof-of-concept for NSC-mediated CE/CPT-11 treatment of medulloblastoma, and serves as a foundation for further studies toward potential clinical application.
Project description: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.
Project description:<h4>Background</h4>Immortalized, clonal HB1.F3.CD 21 human neural stem/progenitor cells (NSCs), loaded with therapeutic cargo prior to intraperitoneal (IP) injection, have been shown to improve the delivery and efficacy of therapeutic agents in pre-clinical models of stage III ovarian cancer. In previous studies, the distribution and efficacy of the NSC-delivered cargo has been examined; however, the fate of the NSCs has not yet been explored.<h4>Methods</h4>To monitor NSC tropism, we used an unconventional method of quantifying endocytosed gold nanorods to overcome the weaknesses of existing cell-tracking technologies.<h4>Results</h4>Here, we report efficient tumor tropism of HB1.F3.CD 21 NSCs, showing that they primarily distribute to the tumor stroma surrounding individual tumor foci within 3 h after injection, reaching up to 95% of IP metastases without localizing to healthy tissue. Furthermore, we demonstrate that these NSCs are non-tumorigenic and non-immunogenic within the peritoneal setting.<h4>Conclusions</h4>Their efficient tropism, combined with their promising clinical safety features and potential for cost-effective scale-up, positions this NSC line as a practical, off-the-shelf platform to improve the delivery of a myriad of peritoneal cancer therapeutics.
Project description:Neural stem cells (NSCs) are inherently tumor-tropic, which allows them to migrate through normal tissue and selectively localize to invasive tumor sites in the brain. We have engineered a clonal, immortalized allogeneic NSC line (HB1.F3.CD21; CD-NSCs) that maintains its stem-like properties, a normal karyotype and is HLA Class II negative. It is genetically and functionally stable over time and multiple passages, and has demonstrated safety in phase I glioma trials. These properties enable the production of an "off-the-shelf" therapy that can be readily available for patient treatment. There are multiple factors contributing to stem cell tumor-tropism, and much remains to be elucidated. The route of NSC delivery and the distribution of NSCs at tumor sites are key factors in the development of effective cell-based therapies. Stem cells can be engineered to deliver and/or produce many different therapeutic agents, including prodrug activating enzymes (which locally convert systemically administered prodrugs to active chemotherapeutic agents); oncolytic viruses; tumor-targeted antibodies; therapeutic nanoparticles; and extracellular vesicles that contain therapeutic oligonucleotides. By targeting these therapeutics selectively to tumor foci, we aim to minimize toxicity to normal tissues and maximize therapeutic benefits. In this manuscript, we demonstrate that NSCs administered via intracerebral/ventricular (IVEN) routes can migrate efficiently toward single or multiple tumor foci. IVEN delivery will enable repeat administrations for patients through an Ommaya reservoir, potentially resulting in improved therapeutic outcomes. In our preclinical studies using various glioma lines, we have quantified NSC migration and distribution in mouse brains and have found robust migration of our clinically relevant HB1.F3.CD21 NSC line toward invasive tumor foci, irrespective of their origin. These results establish proof-of-concept and demonstrate the potential of developing a multitude of therapeutic options using modified NSCs.
Project description:Abstract Human neural stem cells (NSCs) are inherently tumor-tropic, localizing to invasive brain tumor foci following intracerebral administration in preclinical models of primary and secondary brain tumors, thus overcoming obstacles limiting current therapeutic modalities. These NSCs can be engineered to express numerous anti-cancer agents, making them attractive drug delivery vehicles. By concentrating therapy selectively at the tumor sites, toxicity to normal tissues and associated side effects are minimized, improving quality of life. A first-in-human pilot safety/feasibility clinical trial for recurrent glioma patients using a clonal, allogeneic NSC line (HB1.F3.CD21) for cytosine deaminase/5-fluorocytosine (5-FC) enzyme/prodrug gene therapy demonstrated safety, proof of concept for brain tumor localized NSC-mediated conversion of prodrug to active chemotherapeutic agent, non-tumorigenicity, and non-immunogenicity with a single round of treatment (Portnow et al, 2016). HB1.F3.CD21 NSCs were further engineered to secrete a modified human carboxylesterase (hCE1m6) that converts irinotecan (IRN) to the potent topoisomerase-1 inhibitor SN-38. Presented are the preclinical in vitro and in vivo pharmacokinetic studies optimizing dose and scheduling of hCE1m6-NSCs and a clinically equivalent bolus dose of IRN. Efficacy studies in orthotopic glioma models with multiple treatment cycles demonstrated a highly significant increase in the long-term survival distribution in mice treated with hCE1m6-NSCs + IRN as compared to IRN alone (Holm-Bonferroni adjusted p-value <0.0001). Results of IND-enabling safety/toxicity studies showed no significant increase in toxicity, as determined by daily clinical observations, blood chemistries, CBCs, and histopathology, in mice treated with hCE1m6-NSCs + IRN as compared to IRN alone. These results led to FDA approval for a recently initiated first-in-human phase I dose escalation, multiple treatment cycle study of intracranially administered carboxylesterase expressing NSCs in combination with intravenous irinotecan for the treatment of recurrent high grade gliomas (IND 16265; clincialtrial.gov ID NCT02192359).
Project description:Oncolytic adenoviral virotherapy (OV) is a highly promising approach for the treatment of glioblastoma multiforme (GBM). In practice, however, the approach is limited by poor viral distribution and spread throughout the tumor mass.To enhance viral delivery, replication, and spread, we used a US Food and Drug Administration-approved neural stem cell line (NSC), HB1.F3.CD, which is currently employed in human clinical trials. HB1.F3.CD cells were loaded with an oncolytic adenovirus, CRAd-Survivin-pk7, and mice bearing various human-derived GBMs were assessed with regard to NSC migration, viral replication, and therapeutic efficacy. Survival curves were evaluated with Kaplan-Meier methods. All statistical tests were two-sided.Antiglioma activity of OV-loaded HB1.F3.CD cells was effective against clinically relevant human-derived glioma models as well as a glioma stem cell-enriched xenograft model. Median survival was prolonged by 34% to 50% compared with mice treated with OV alone (GBM43FL model median survival = 19.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.26, 95% confidence interval [CI] = 1.21 to 12.23, P = .02; GBM12 model median survival = 43.5 days, OV alone vs NSC + OV, hazard ratio of survival = 2.53, 95% CI = 1.21 to 10.38, P = .02). OV-loaded HB1.F3.CD cells were shown to effectively migrate to the contralateral hemisphere and hand off the therapeutic payload of OV to targeted glioma cells. In vivo distribution and migratory kinetics of the OV-loaded HB1.F3.CD cells were successfully monitored in real time by magnetic resonance imaging. OV-loaded NSCs retained their differentiation fate and were nontumorigenic in vivo.HB1.F3.CD NSCs loaded with CRAd-Survivin-pk7 overcome major limitations of OV in vivo and warrant translation in a phase I human clinical trial for patients with GBM.
Project description:The tumor-tropic properties of neural stem cells (NSCs) led to the development of a novel strategy for delivering therapeutic genes to tumors in the brain. To apply this strategy to the treatment of brain metastases, we made a human NSC line expressing cytosine deaminase (F3.CD), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil, an anticancer agent. In vitro, the F3.CD cells significantly inhibited the growth of tumor cell lines in the presence of the prodrug 5-FC. In vivo, MDA-MB-435 human breast cancer cells were implanted into the brain of immune-deficient mouse stereotactically, and F3.CD cells were injected into the contralateral hemisphere followed by systemic 5-FC administration. The F3.CD cells migrated selectively into the brain metastases located in the opposite hemisphere and resulted in significantly reduced volumes. The F3.CD and 5-FC treatment also decreased both tumor volume and number of tumor mass significantly, when immune-deficient mouse had MDA-MB-435 cells injected into the internal carotid artery and F3.CD cells were transplanted into the contralateral brain hemisphere stereotactically. Taken together, brain transplantation of human NSCs, encoding the suicide enzyme CD, combined with systemic administration of the prodrug 5-FC, is an effective treatment regimen for brain metastases of tumors.
Project description:CPT-11 (irinotecan) has been investigated as a treatment for malignant brain tumors. However, limitations of CPT-11 therapy include low levels of the drug entering brain tumor sites and systemic toxicities associated with higher doses. Neural stem cells (NSCs) offer a novel way to overcome these obstacles because of their inherent tumor tropism and ability to cross the blood-brain barrier, which enables them to selectively target brain tumor sites. Carboxylesterases (CEs) are enzymes that can convert the prodrug CPT-11 (irinotecan) to its active metabolite SN-38, a potent topoisomerase I inhibitor. We have adenovirally transduced an established clonal human NSC line (HB1.F3.CD) to express a rabbit carboxylesterase (rCE) or a modified human CE (hCE1m6), which are more effective at converting CPT-11 to SN-38 than endogenous human CE. We hypothesized that NSC-mediated CE/CPT-11 therapy would allow tumor-localized production of SN-38 and significantly increase the therapeutic efficacy of irinotecan. Here, we report that transduced NSCs transiently expressed high levels of active CE enzymes, retained their tumor-tropic properties, and mediated an increase in the cytotoxicity of CPT-11 toward glioma cells. CE-expressing NSCs (NSC.CEs), whether administered intracranially or intravenously, delivered CE to orthotopic human glioma xenografts in mice. NSC-delivered CE catalyzed conversion of CPT-11 to SN-38 locally at tumor sites. These studies demonstrate the feasibility of NSC-mediated delivery of CE to glioma and lay the foundation for translational studies of this therapeutic paradigm to improve clinical outcome and quality of life in patients with malignant brain tumors.
Project description:Pathotropic neural stem and/or progenitor cells (NSCs) can potentially deliver therapeutic agents to otherwise inaccessible cancers. In glioma, NSCs are found in close contact with tumor cells, raising the possibility that specificity of NSC contact with glioma targets originates in the tumor cells themselves. Alternatively, target preferences may originate, at least in part, in the tumor microenvironment. To better understand mechanisms underlying NSC interactions with glioma cells, we examined NSC-target cell contacts in a highly simplified 3-dimensional peptide hydrogel (Puramatrix) in which cell behaviors can be studied in the relative absence of external cues. HB1.F3 is an immortalized clonal human NSC line extensively characterized in preclinical investigations. To study contact formation between HB1.F3 NSCs and glioma cells, we first examined co-cultures of eGFP-expressing HB1.F3 (HB1.F3.eGFP) NSCs and dsRed-expressing U251 glioma (U251.dsRed) cells. Using confocal microscopy, HB1.F3.eGFP cells were observed contacting or encircling U251.dsRed glioma cells, but never the reverse. Next, examining specificity of these contacts, no significant quantitative differences in either percentages of HB1.F3 NSCs contacting targets, or in the extent of target cell encirclement, were observed when HB1.F3.eGFP cells were presented with various potential target cells (human glioma and breast cancer cell lines, patient-derived brain tumor lines, non-tumor fibroblasts, primary mouse and human astroglial cells, and primary adult and newborn human dermal fibroblasts) except that interactions between HB1.F3 cells did not progress beyond establishing contacts. Finally cytoskeletal mechanisms employed by HB1.F3.eGFP cells varied with the substrate. When migrating in Puramatrix, HB1.F3 NSCs exhibited intermittent process extension followed by soma translocation, while during encirclement their movements were more amoeboid. We conclude that formation of contacts and subsequent encirclement of target cells by HB1.F3 NSCs is an intrinsic property of these NSCs, and that preferential contact formation with tumor cells in vivo must therefore be highly dependent on microenvironmental cues.
Project description:Glioblastoma multiforme (GBM) remains fatal despite intensive surgical, radiotherapeutic, and chemotherapeutic interventions. Neural stem cells (NSCs) have been used as cellular vehicles for the transportation of oncolytic virus (OV) to therapeutically resistant and infiltrative tumor burdens throughout the brain. The HB1.F3-CD human NSC line has demonstrated efficacy as a cell carrier for the delivery of a glioma tropic OV CRAd-Survivin-pk7 (CRAd-S-pk7) in vitro and in animal models of glioma. At this juncture, no study has investigated the effectiveness of OV-loaded NSCs when applied in conjunction with the standard of care for GBM treatment, and therefore this study was designed to fill this void. Here, we show that CRAd-S-pk7-loaded HB1.F3-CD cells retain their tumor-tropic properties and capacity to function as in situ viral manufacturers in the presence of ionizing radiation (XRT) and temozolomide (TMZ). Furthermore, for the first time, we establish a logical experimental model that aims to recapitulate the complex clinical scenario for the treatment of GBM and tests the compatibility of NSCs loaded with OV. We report that applying OV-loaded NSCs together with XRT and TMZ can increase the median survival of glioma bearing mice by approximately 46%. Most importantly, the timing and order of therapeutic implementation impact therapeutic outcome. When OV-loaded NSCs are delivered prior to rather than after XRT and TMZ treatment, the median survival of mice bearing patient-derived GBM43 glioma xenografts is extended by 30%. Together, data from this report support the testing of CRAd-S-pk7-loaded HB1.F3-CD cells in the clinical setting and argue in favor of a multimodality approach for the treatment of patients with GBM.