STEM-24. THERAPEUTIC APPROACH OF STEM CELL CARRYING RETROVIRAL REPLICATING VECTORS IN HUMAN GLIOMA MODEL

Abstract Toca 511, an improved retroviral replicating vectors (RRVs) expressing a codon-optimized cytosine deaminase, has shown highly promising evidence of therapeutic benefit in preclinical and clinical studies for gene therapy of glioma. In the present study, we engineered human mesenchymal stem cells (MSC) as tumor-homing cellular carriers that produce and release RRV, and evaluated the effect of this mode of virus delivery on the time course of intratumoral RRV dissemination. Human MSC isolates were engineered to produce RRV (MSC-RRV). Cytotoxicity assays confirmed efficient prodrug activator function in U-87 glioma cells transduced with vectors produced from MSC-RRV. To evaluate intratumoral migration activity and tumor-homing migration activity in vivo, individual MSC isolates were injected either directly into human glioma xenografts, or into the contralateral brain hemisphere. MSC-RRV isolates showing the highest levels of intratumoral migration activity were selected, and the efficiency of intratumoral dissemination and tumoricidal activity achieved by these isolates was compared against injection of RRV virus preparations in subcutaneous glioma models. Compared to virus injection, MSC-RRV achieved 1.6x-higher levels of tumor transduction (p< 0.05), and 2x-reduced tumor growth (p=0.018) at earlier time points. In short-term survival studies using lower doses of MSC-RRV cells vs. RRV virus injected 14 days post-establishment of intracranial U-87 gliomas, a small but statistically significant prolongation of median survival was seen with intracranial tumors treated with MSC-RRV as compared to RRV (26 vs. 20 days, p< 0.05) after only a single cycle of 5-FC prodrug. Thus, MSC can be employed as mobile tumor-homing RRV-producer cells, which release RRV as they migrate to tumor foci in vivo and actively penetrate into individual tumor masses, resulting in more rapid tumor transduction and earlier therapeutic efficacy, which may be advantageous particularly for multi-focal and metastatic disease. This study was funded by the California Institute for Regenerative Medicine (TR2-01791)

Factors in the Selection of Surface Disinfectants for Use in a Laboratory Animal Setting.

Because surface disinfectants are an important means of pathogen control within laboratory animal facilities, these products must have an appropriate spectrum of antimicrobial activity. However, many other factors must also be considered, including effects on human health, environmental safety, and animal behavior. Aqueous solutions of sodium hypochlorite often are considered to be the 'gold standard' for surface disinfection, but these products can be corrosive, caustic, and aversive in odor. This study was designed to identify disinfectants that are as effective as hypochlorite solutions but more acceptable for use in a laboratory animal setting. An antiviral disinfectant-efficacy assay was developed by using viral vectors that expressed green fluorescence protein as surrogates for wild-type viruses of concern in laboratory animals. Efficacy testing revealed that most of the products were highly effective when used against viral vectors in suspension. However, when the disinfectants were challenged by buffering virus in protein or drying virus on nonporous surfaces, the hypochlorite and peroxymonosulfate products performed the best. Review of safety data sheets for the agents indicated that a peroxide-based product was considerably safer than the other products tested and that the pH of most products was not conducive to disposal down a drain. Behavioral testing of Swiss Webster, C57Bl/6, and BALB/c mice showed that the hypochlorite- and peroxide-based products were clearly aversive, given that the mice consistently avoided these products. All of these factors must be considered when choosing the appropriate disinfectant

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed

The Phosphatase PHLPP1 Regulates Akt2, Promotes Pancreatic Cancer Cell Death, and Inhibits Tumor Formation

The kinase Akt mediates resistance of pancreatic cancer (PaCa) cells to death and is constitutively active (phosphorylated) in cancer cells. Whereas the kinases that activate Akt are well characterized, less is known about phosphatases that dephosporylate and thereby inactivate it. We investigated regulation of Akt activity and cell death by the phosphatases PHLPP1 and PHLPP2 in PaCa cells, mouse models of PaCa, and human pancreatic ductal adenocarcinoma (PDAC). We measured the effects of PHLPP overexpression or knockdown with small interfering RNAs on Akt activation and cell death. We examined regulation of PHLPPs by growth factors and reactive oxygen species, as well as associations between PHLPPs and tumorigenesis. PHLPP overexpression inactivated Akt, whereas PHLPP knockdown increased phosphorylation of Akt in PaCa cells. Levels of PHLPPs were greatly reduced in human PDAC and in mouse genetic and xenograft models of PaCa. PHLPP activities in PaCa cells were down-regulated by growth factors and Nox4 reduced nicotinamide adenine dinucleotide phosphate oxidase. PHLPP1 selectively dephosphorylated Akt2, whereas PHLPP2 selectively dephosphorylated Akt1. Akt2, but not Akt1, was up-regulated in PDAC, and Akt2 levels correlated with mortality. Consistent with these results, high levels of PHLPP1, which dephosphorylates Akt2 (but not PHLPP2, which dephosphorylates Akt1), correlated with longer survival times of patients with PDAC. In mice, xenograft tumors derived from PaCa cells that overexpress PHLPP1 (but not PHLPP2) had inactivated Akt, greater extent of apoptosis, and smaller size. PHLPP1 has tumor suppressive activity and might represent a therapeutic or diagnostic tool for PDAC

Immunosuppressive tumor-infiltrating myeloid cells mediate adaptive immune resistance via a PD-1/PD-L1 mechanism in glioblastoma

BackgroundAdaptive immune resistance in the tumor microenvironment appears to attenuate the immunotherapeutic targeting of glioblastoma (GBM). In this study, we identified a tumor-infiltrating myeloid cell (TIM) population that expands in response to dendritic cell (DC) vaccine treatment. The aim of this study was to understand how this programmed death ligand 1 (PD-L1)-expressing population restricts activation and tumor-cytolytic function of vaccine-induced tumor-infiltrating lymphocytes (TILs).MethodsTo test this hypothesis in our in vivo preclinical model, we treated mice bearing intracranial gliomas with DC vaccination ± murine anti-PD-1 monoclonal antibody (mAb) blockade or a colony stimulating factor 1 receptor inhibitor (CSF-1Ri) (PLX3397) and measured overall survival. We then harvested and characterized the PD-L1+ TIM population and its role in TIL activation and tumor cytolysis in vitro.ResultsOur data indicated that the majority of PD-L1 expression in the GBM environment is contributed by TIMs rather than by tumor cells themselves. While PD-1 blockade partially reversed the TIL dysfunction, targeting TIMs directly with CSF-1Ri altered TIM expression of key chemotactic factors associated with promoting increased TIL infiltration after vaccination. Neither PD-1 mAb nor CSF-1Ri had a demonstrable therapeutic benefit alone, but when combined with DC vaccination, a significant survival benefit was observed. When the tripartite regimen was given (DC vaccine, PD-1 mAb, PLX3397), long-term survival was noted together with an increase in the number of TILs and TIL activation.ConclusionTogether, these studies elucidate the role that TIMs play in mediating adaptive immune resistance in the GBM microenvironment and provide evidence that they can be manipulated pharmacologically with agents that are clinically available. Development of immune resistance in response to active vaccination in GBM can be reversed with dual administration of CSF-1Ri and PD-1 mAb

Expression of PD-1 by T Cells in Malignant Glioma Patients Reflects Exhaustion and Activation

PurposeGlioblastoma (GBM) is the most common primary malignant tumor in the central nervous system. Our recent preclinical work has suggested that PD-1/PD-L1 plays an important immunoregulatory role to limit effective antitumor T-cell responses induced by active immunotherapy. However, little is known about the functional role that PD-1 plays on human T lymphocytes in patients with malignant glioma.Experimental Design: In this study, we examined the immune landscape and function of PD-1 expression by T cells from tumor and peripheral blood in patients with malignant glioma.ResultsWe found several differences between PD-1+ tumor-infiltrating lymphocytes (TIL) and patient-matched PD-1+ peripheral blood T lymphocytes. Phenotypically, PD-1+ TILs exhibited higher expression of markers of activation and exhaustion than peripheral blood PD-1+ T cells, which instead had increased markers of memory. A comparison of the T-cell receptor variable chain populations revealed decreased diversity in T cells that expressed PD-1, regardless of the location obtained. Functionally, peripheral blood PD-1+ T cells had a significantly increased proliferative capacity upon activation compared with PD-1- T cells.ConclusionsOur evidence suggests that PD-1 expression in patients with glioma reflects chronically activated effector T cells that display hallmarks of memory and exhaustion depending on its anatomic location. The decreased diversity in PD-1+ T cells suggests that the PD-1-expressing population has a narrower range of cognate antigen targets compared with the PD-1 nonexpression population. This information can be used to inform how we interpret immune responses to PD-1-blocking therapies or other immunotherapies

The Phosphatase PHLPP1 Regulates Akt2, Promotes Pancreatic Cancer Cell Death, and Inhibits Tumor Formation

BACKGROUND & AIMS: The kinase Akt mediates resistance of pancreatic cancer (PaCa) cells to death and is constitutively active (phosphorylated) in cancer cells. Whereas the kinases that activate Akt are well characterized, less is known about phosphatases that dephosporylate and thereby inactivate it. We investigated regulation of Akt activity and cell death by the phosphatases PHLPP1 and PHLPP2 in PaCa cells, mouse models of PaCa, and human pancreatic ductal adenocarcinoma (PDAC). METHODS: We measured the effects of PHLPP overexpression or knockdown with small interfering RNAs on Akt activation and cell death. We examined regulation of PHLPPs by growth factors and reactive oxygen species, as well as associations between PHLPPs and tumorigenesis. RESULTS: PHLPP overexpression inactivated Akt, whereas PHLPP knockdown increased phosphorylation of Akt in PaCa cells. Levels of PHLPPs were greatly reduced in human PDAC and in mouse genetic and xenograft models of PaCa. PHLPP activities in PaCa cells were down-regulated by growth factors and Nox4 reduced nicotinamide adenine dinucleotide phosphate oxidase. PHLPP1 selectively dephosphorylated Akt2, whereas PHLPP2 selectively dephosphorylated Akt1. Akt2, but not Akt1, was up-regulated in PDAC, and Akt2 levels correlated with mortality. Consistent with these results, high levels of PHLPP1, which dephosphorylates Akt2 (but not PHLPP2, which dephosphorylates Akt1), correlated with longer survival times of patients with PDAC. In mice, xenograft tumors derived from PaCa cells that overexpress PHLPP1 (but not PHLPP2) had inactivated Akt, greater extent of apoptosis, and smaller size. CONCLUSIONS: PHLPP1 has tumor suppressive activity and might represent a therapeutic or diagnostic tool for PDAC