A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth ...factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells.
Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has ...so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (
), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.
Prodrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. ...Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects.
Here we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts.
In both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1-2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells.
These results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity.
Toca 511, a retroviral replicating vector (RRV) encoding the yeast cytosine deaminase (yCD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anticancer ...drug 5-fluorouracil (5-FU), is currently being evaluated in Phase II/III clinical trials for glioma, and showing highly promising evidence of therapeutic activity. Here we evaluated RRV-mediated prodrug activator gene therapy as a new therapeutic approach for pancreatic ductal adenocarcinoma (PDAC). RRV spread rapidly and conferred significant cytotoxicity with prodrug in a panel of PDAC cells. Efficient intratumoral replication and complete inhibition of tumor growth upon 5-FC administration were observed in both immunodeficient and immunocompetent subcutaneous PDAC models. Biodistribution of RRV was highly restricted in normal tissues, especially in immunocompetent hosts. Tumor growth inhibition by Toca 511 followed by 5-FC was also confirmed in the orthotopic PDAC model. This study provides the first proof-of-concept for application of Toca 511 and Toca FC (extended release 5-FC) to the treatment of human PDAC, and provided support for inclusion of PDAC in a Phase I study evaluating Toca 511 in various systemic malignancies, (NCT02576665), which has recently been initiated.
Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, ...we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvβ3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.
EMP2 is a tetraspan protein linked with aggressive disease.
EMP2 correlates with activated Src in patients with GBM. Using intracranial mouse models, EMP2 promotes tumor cell invasiveness. Antibodies to EMP2 reduce GBM tumor load
EMP2 is a novel therapeutic target in GBM.
The clinical outcome for patients with GBM remains poor, and thus new targeted therapies are needed.
There is increasing evidence for the presence of cancer stem-like progenitors in malignant brain tumors. This subpopulation of progenitor cells, the so-called “cancer stem cells (CSCs)”, may play a ...pivotal role in brain tumor initiation, growth, and recurrence. Here we describe the establishment of one permanent brain tumor stem cell line that able to form new spheres after culture under adherent monolayer conditions and to recapitulate the properties of the original tumor upon transplantation into immunodeficient mice. Re-formed spheres retained their stem cell properties and isolated single CSCs from these spheres formed spheres/tumors even after long-term cultures (over 2 years). These data suggested that a small population of CSCs preserved its stem cell properties even after serial passages under non-adherent/adherent culture conditions. Evaluation of underlying metabolic events and assessment of the biological features of these viable cell lines will yield useful knowledge on the
in situ behavior of brain tumors.
PET imaging for gene & cell therapy Collins, Sara A; Hiraoka, Kei; Inagaki, Akihito ...
Current gene therapy,
02/2012, Letnik:
12, Številka:
1
Journal Article
Recenzirano
As the interest in gene therapy increases, the development of an efficient and reliable means to monitor gene delivery and expression in patients is becoming more important. An ideal imaging modality ...would be non-invasive, allowing for repeated imaging, thus validating stages subsequent to vector administration and allowing for the improvement of clinical protocols. Positron Emission Tomography (PET) has been employed for some time in clinical imaging and has in more recent years been adapted to enable imaging in small animal models, including gene therapy models for a range of diseases. PET imaging is based on the detection of trace quantities of positron-emitting molecular probe within cells postadministration, permitting imaging of target molecules in vivo, and numerous tracers have been developed for a wide range of applications, including imaging of reporter gene activity. Use of radiolabelled substrates that interact with specific transgene proteins, has identified a number of reporter genes that are suitable for imaging vector mediated gene delivery and expression in both pre-clinical and clinical situations. These reporter genes enable non-invasive analysis of the location, level and kinetics of transgene activity. Among the various imaging modalities in existence, the PET approach displays arguably the optimum characteristics in terms of sensitivity and quantitation for in vivo gene expression measurements. Given the existing availability of PET scanning equipment and expertise in hospitals, this imaging modality represents the most clinically applicable means of analysing gene therapy in patients. This review outlines the principles of PET imaging in the context of gene and cell therapy at both pre-clinical and clinical levels, comparing PET with other relevant modalities, and describes the progress to date in this field.
Abstract
Amphotropic retroviral replicating vector (RRV) Toca 511, expressing the yeast cytosine deaminase (CD) prodrug activator gene, showed promising evidence of therapeutic benefit and increased ...survival in early-phase trials for recurrent high-grade glioma. While a multi-center Phase 3 trial did not meet its overall endpoints, highly statistically significant survival was observed within predetermined patient subgroups compared to matched randomized control patients, and clinical investigation is on-going. Hence it is worthwhile to consider strategies aimed at enhancing therapeutic efficacy, such as delivering combinations of multiple transgenes. However, RRVs encoated with the same envelope compete for the same cancer cell surface receptors. We have now developed novel RRV encoated (‘pseudotyped’) with a heterologous envelope derived from Gibbon ape leukemia virus (GALV), which utilizes a different cell surface receptor from the native amphotropic retrovirus envelope for cellular entry. RRV(GALV) vectors expressing either GFP or HSV thymidine kinase (TK) were constructed, and efficient replication and transgene expression was observed in > 90% of both established and primary human glioblastoma cells within 14 days after initial infection at 0.01 (1%) multiplicity of infection (MOI). Genomic stability of RRV(GALV) vectors was also confirmed over prolonged propagation. Established and primary human glioblastoma cells infected with RRV(GALV)-TK vector showed ≥ 50%-90% reduction in cell viability after exposure to Ganciclovir prodrug in the range of 1µM-100µM for 5 days, as compared to uninfected control cells or cells infected with RRV(GALV)-GFP control vector. Furthermore, dual infection with RRV(GALV)-TK and amphotropic RRV-CD (Toca 511) resulted in synergistic cytotoxicity upon simultaneous exposure to their respective prodrugs. Further data will be presented from on-going studies evaluating these vectors in intracerebral glioblastoma models. These results indicate that GALV envelope-pseudotyped RRV can efficiently deliver prodrug activator gene therapy in experimental glioma models, and open the door to combinatorial gene therapy regimens with this vector platform.
Abstract
Early-phase clinical trials of Toca 511, a retroviral replicating vector (RRV) expressing an optimized form of yeast cytosine deaminase for prodrug activator gene therapy, showed highly ...promising evidence of therapeutic benefit and increased survival in recurrent high-grade glioma, but an international Phase 3 trial failed to meet primary and secondary endpoints. Nonetheless, promising results observed within predetermined subgroups, combined with the highly favorable safety profile of this approach, make it worthwhile to consider alternative prodrug-activating enzymes in order to increase therapeutic efficacy. In this study, we employed RRV to deliver modified versions of another prodrug activator gene, E.coli nitroreductase (NTR), which activates the prodrug CB1954 to generate a potent bifunctional alkylating agent. We constructed RRV encoding two different isoforms of wild-type E.coli NTR genes (NfsA, NfsB) as well as variants of both isoforms extensively modified to optimize human codon usage and vector stability. Further efforts to improve the potency of these prodrug activator enzymes included the construction of additional new vectors incorporating point mutations at the active site which have previously been demonstrated to improve prodrug conversion efficiency. NTR transgene insertion did not affect RRV replication, which resulted in increasing NTR expression over time in human glioma cells in culture. Sequence optimization resulted in enhanced genomic stability of the vectors upon serial passage and also in significantly reduced cell viability in vitro on treatment of fully transduced U87 and U87-EGFRviii glioma cells with CB1954 prodrug. In orthotopic U87-EGFRviii intracerebral tumor models, stereotactic intratumoral injection of sequence-optimized RRV-NTR and repeated cycles of prodrug treatment resulted in rapid and significant reduction of tumor burden as monitored by bioluminescence imaging, leading to prolonged survival benefit. These data indicate that improved therapeutic efficacy can be achieved with optimized NTR prodrug activator gene therapy delivered by RRV in experimental glioma models.
Abstract
Oncolytic virus (OV) elicits an antitumor immune response in addition to tumor-cell killing. However, it is unknown if a pre-existing antitumor immune response aids or hinders OV therapy. To ...study the interplay between the endogenous immune response and OV we profiled tumors from immunocompetent intracranial models of medulloblastoma and glioblastoma treated with measles virus (MV) and retrovirus (Toca-511), using single-cell and bulk-RNA sequencing. For both models, we assayed specimens from two treatment groups, a mid-treatment group where mice were sacrificed upon observation of treatment response, and a post-treatment group where treatment-resistant mice were sacrificed when they had succumbed to their disease. As controls, we included a cohort treated with heat-inactivated virus and a cohort treated with PBS. Both MV and Toca-511 OV therapies significantly improve survival compared to controls. From each of the MV and Toca-511 treatment/control groups, 3 mice per group were profiled via RNA sequencing. Additionally, tumors from 5 mice treated with MV alone and 4 mice treated with MV and anti-PD1 immune-checkpoint blockade (ICB) were subjected to single-cell RNA sequencing (scRNA-seq). ScRNA-seq generated 38,491 cells after filtering for quality control and successfully captured both endogenous and viral RNAs. We found that in samples treated with both OV and ICB there were higher rates of virally infected cells, depletion of M2-polarized myeloid infiltrates, and depletion of endothelial cells, compared to controls. Comparing virally infected tumor cells to non-infected controls we observed a down-regulation of translation initiation-complex genes and an upregulation of markers of oxidative phosphorylation. Infected cells from treatment-resistant tumors upregulate antigen presentation and myeloid chemotaxis genes. These studies show that OV enhances survival and anti-brain-tumor immunity. They support MV and ICB as potentially being synergistic. They suggest translation control and perturbation of the myeloid compartment as strategies for enhancing intracellular MV proliferation and infection efficiency, respectively.
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