498. Susceptibility of Neuroblastoma Primary Tumor-Initiating Cells to Oncolytic Herpes Simplex Virus Is Determined by Nectin-1 Expression

CANCER-ONCOLYTIC VIRUSES II 498. Susceptibility of Neuroblastoma Primary Tumor-Initiating Cells to Oncolytic Herpes Simplex Virus Is Determined by Nectin-1 Expression

Pin-Yi Wang,1 Mark A. Currier,1 Margaret H. Collins,2 Betsy A. DiPasquale,2 Loen Hansford,3 David Kaplan,3,4 Sean Lawler,5 E. Antonio Chiocca,5 Timothy P. Cripe.1 1 Division of Hematology/Oncology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 3 Molecular Genetics, University of Toronto, Toronto, ON, Canada; 4 Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada; 5Department of Neurological Surgery, The Ohio State University, Columbus, OH. Neuroblastoma is the most common extracranial solid tumor in children. Although patients with high-risk disease can usually achieve remission with conventional therapies, disease relapse is common and long-term survival is less than 40%. Bone marrow metastases are enriched for tumor-initiating cells (TICs; Hansford et al., Cancer Research 67:11234, 2007), consistent with the cancer stem cell hypothesis. We previously showed that neuroblastoma cell lines are killed by oncolytic Herpes simplex virus (oHSV) infection (Parikh et al., Ped Blood Cancer 44:469, 2005) and sought to determine the susceptibility of neuroblatoma TICs to oHSV. While NB12 and NB122R cells were relatively susceptible to HSV transduction and cytolysis, NB88R2 cells were resistant. Resistance correlated with low expression of the known major HSV-1 receptor, nectin-1, and could be partially reversed by exogenous nectin-1 expression. Xenograft tumors derived from these TICs retained delity of nectin-1 expression: tumors derived from NB12 cells were positive while those from NB88R2 were negative. Although direct intratumoral oHSV injection efcacy studies are still underway to conrm a differential response to virus therapy, our data suggest that nectin-1 expression may be a predictor of response to oHSV. Using a tissue microarray to screen ∼90 cases of primary neuroblastoma, we found a majority of cases expressed nectin-1, suggesting that overall neuroblastoma is a reasonable target disease for oHSV-based therapy. Interestingly, numerous primary glioblastoma neurosphere cultures were all found to be highly susceptible to oHSV infection but showed very low or absent nectin-1 expression. These cells were positive for nectin-2 and nectin-3, suggesting that HSV-1 might utilize different cellular entry receptors to infect peripheral versus central nervous system tumors. Our novel ndings raise questions about HSV-1 receptor expression on cancer cells that may represent barriers to virus spread within a tumor and may be predictive of susceptibility to oncolytic HSV therapy.

499. Activity of Oncolytic Measles Virus Strains Against Brain Tumor Stem Cells Cory Allen,1 Mark A. Schroeder,1 Jann N. Sarkaria,1 Mark J. Federspiel,1 Stephen J. Russell,1 Evanthia Galanis.1 1 Mayo Clinic College of Medicine, Rochester, MN.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment. Given the resistance of brain tumor stem cells (BTSC) to chemotherapy and radiation therapy, their eradication is thought to be essential for long term glioma remission. We have previously demonstrated that measles virus (MV) derivatives have signicant activity against glioma lines and orthotopic xenografts and are currently being tested in a phase I trial in recurrent GBM patients. We sought to evaluate the antitumor activity of MV derivatives against GBM BTSC. METHODS/RESULTS: Primary GBM xenografts, passaged in the anks of nude mice, were excised, mechanically disrupted with a sterile blade and grown at 37°C in a humidied environment containing 5% CO2 in NeuroCult media (Stemcell Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy

Technologies, Vancouver, BC, CA). GBM6, GBM12 and GBM44 neurospheres were expanded in this media and subsequently half of each were further passaged in DMEM containing 10% FBS to serve as differentiated controls. These were further examined for the expression of the BTSC markers CD133, Nestin and ATF-5, expression of the differention markers GFAP and beta -III-tubulin and expression of the MV receptor CD46, by immunouorescence. Cytopathic effect (CPE) of MV against both BTSC and differentiated controls was examined in vitro using trypan blue exclusion assays. Cells were infected with the MV strains MV-NIS or MV-CEA (MOI 0.1 and 1) and the total number of surviving cells was counted at days 2, 4, 6, 8, &16 post infection and compared against uninfected controls. BTSC expressed high levels of the MV receptor CD46. Both BTSC and corresponding differentiated cells were effectively killed at a comparable rate even at low multiplicity of infection (MOI 0.1). One-step viral growth curves showed similar growth kinetics of oncolytic MV strains in BTSC and their differentiated counterpart. To examine the impact of MV infection on BTSC survival in vivo, GBM 44 BTSC were exposed to MV at an MOI of 10 in vitro and subsequently implanted into the right caudate nucleus of nude mice with uninfected BTSC serving as controls. A signicant prolongation of survival in mice implanted with infected BTSC was observed (p=0.0483). In addition, in vivo therapy experiments were performed in GBM6 and GBM12 BTSC derived orthotopic xenografts in nude mice. Signicant prolongation of survival was observed in animals treated orthotopically with MV (1e6 and 1.8e6 respectively) for both GBM6 and GBM12 BTSC xenografts as compared to UV inactivated virus control treated animals (GBM6 p=0.0021, GBM12 p=0.0416). CONCLUSION: Measles virus derivatives have signicant antitumor activity against brain tumor derived stem cells in vitro and in vivo. Supported by the Mayo Brain SPORE CA 108961

500. Replication-Competent Retrovirus Vector-Mediated Suicide Gene Therapy Achieves Signicant Therapeutic Efcacy Against Human Malignant Mesothelioma Xenografts

Yoshiko Kawasaki,1 Norie Yamaoka,1 Nobuyuki Terada,2 Haruki Okamura,1 Noriyuki Kasahara,3 Shuji Kubo.1 1 Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 2Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; 3Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA. Purpose: Replication-competent retrovirus (RCR) vectors have been shown to achieve signicantly enhanced tumor transduction efciency and therapeutic efcacy in various cancer models. We and others have previously engineered RCR vectors for highly efcient delivery of suicide genes, and as the virus is intrinsically incapable of infecting post-mitotic normal cells, retrovirus spread after intratumoral injection is highly restricted to tumor tissue, particularly in immunocompetent hosts. In the present study, we hypothesized that RCR vector-mediated suicide gene therapy could be effectively applied to the treatment of malignant mesothelioma, a highly aggressive tumor with poor prognosis. Methods: To evaluate the utility and efciency of RCR vectors for gene delivery to mesothelioma cells, RCR-GFP vector, expressing the green uorescent protein (GFP) marker gene, was rst tested on a panel of human malignant mesothelioma and non-malignant transformed mesothelial cell lines in vitro. Transduction efciency and replicative spread of the RCR vector over time was monitored by ow cytometry and in vivo imaging. Next, to evaluate the potential of RCR vector-mediated suicide gene therapy for this malignancy, we employed RCR-yCD, expressing the yeast cytosine deaminase (yCD) suicide gene. Following administration of the prodrug S193