Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease. Most KS tumor cells are latently infected with KSHV and are of endothelial origin. While PEL-derived cell lines maintain KSHV indefinitely, all KS tumor-derived cells to date have lost viral genomes upon ex vivo cultivation. To study KSHV latency and tumorigenesis in endothelial cells, we generated telomerase-immortalized human umbilical vein endothelial (TIVE) cells. TIVE cells express all KSHV latent genes 48 h postinfection, and productive lytic replication could be induced by RTA/Orf50. Similar to prior models, infected cultures gradually lost viral episomes. However, we also obtained, for the first time, two endothelial cell lines in which KSHV episomes were maintained indefinitely in the absence of selection. Long-term KSHV maintenance correlated with loss of reactivation in response to RTA/Orf50 and complete oncogenic transformation. Long-term-infected TIVE cells (LTC) grew in soft agar and proliferated under reduced-serum conditions. LTC, but not parental TIVE cells, formed tumors in nude mice. These tumors expressed high levels of the latency-associated nuclear antigen (LANA) and expressed lymphatic endothelial specific antigens as found in KS (LYVE-1). Furthermore, host genes, like those encoding interleukin 6, vascular endothelial growth factor, and basic fibroblast growth factor, known to be highly expressed in KS lesions were also induced in LTC-derived tumors. KSHV-infected LTCs represent the first xenograft model for KS and should be of use to study KS pathogenesis and for the validation of anti-KS drug candidates.Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is believed to be the causative agent for Kaposi's sarcoma (KS) (for a review, see references 1 and 27). Within KS tumor lesions, the majority of cells express endothelial markers and are latently infected with KSHV, as defined by the presence of the circular viral genome and limited viral-gene expression. Studying KSHV's role in KS is complicated by the fact that cells explanted from KS lesions lose the KSHV genome after several cell divisions in culture (2, 43). In addition to KS, KSHV is associated with two lymphoproliferative diseases: primary effusion lymphomas (PEL) and multicentric Castleman's disease (MCD) (11, 59). In contrast to KS lesions, PEL-derived cell lines that are latently infected with KSHV are readily established in culture. These cells maintain viral episomes indefinitely and remain dependent on KSHV for survival (31,33). Therefore, many aspects of KSHV biology have been studied in PEL-derived cell lines rather than in endothelial cells (56; for a review, see reference 1).Several endothelial-cell-derived tissue culture models have been described. Common to these models, which are based on dermal microvascular endothelial cells (DMVEC), are their susceptibility to cell-free infection with PEL-derived KSHV and their capabili...

Adenoviral oncolytic virotherapy represents an attractive treatment modality for central nervous system (CNS) neoplasms. However, successful application of virotherapy in clinical trials has been hampered by inadequate distribution of oncolytic vectors. Neural stem cells (NSCs) have been shown as suitable vehicles for gene delivery because they track tumor foci. In this study, we evaluated the capability of NSCs to deliver a conditionally replicating adenovirus (CRAd) to glioma. We examined NSC specificity with respect to viral transduction, migration and capacity to deliver a CRAd to tumor cells. Fluorescence-activated cell sorter (FACS) analysis of NSC shows that these cells express a variety of surface receptors that make them amenable to entry by recombinant adenoviruses. Luciferase assays with replication-deficient vectors possessing a variety of transductional modifications targeted to these receptors confirm these results. Real-time PCR analysis of the replication profiles of different CRAds in NSCs and a representative glioma cell line, U87MG, identified the CRAd-Survivin (S)-pk7 virus as optimal vector for further delivery studies. Using in vitro and in vivo migration studies, we show that NSCs infected with CRAd-S-pk7 virus migrate and preferentially deliver CRAd to U87MG glioma. These results suggest that NSCs mediate an enhanced intratumoral distribution of an oncolytic vector in malignant glioma when compared with virus injection alone.

Chemoresistance due to heterogeneity of the tumor microenvironment (TME) hampers the long-term efficacy of frontline therapies for lung cancer. Current combination therapies for lung cancer provide only modest improvement in survival, implicating necessity for novel approaches that suppress malignant growth and stimulate long-term anti-tumor immunity. Oxidative stress in the TME promotes immunosuppression by tumor infiltrating myeloid-derived suppressor cells (MDSC), which inhibit host protective anti-tumor immunity. Using a murine model of lung cancer, we demonstrate that a combination treatment with gemcitabine and a superoxide dismutase mimetic targets immunosuppressive MDSC in the TME and enhances the quantity and quality of both effector and memory CD8+ T cell responses. At the effector cell function level, the unique combination therapy targeting MDSC and redox signaling greatly enhanced cytolytic CD8+ T cell response and further decreased T regulatory cell infiltration. For long-term anti-tumor effects, this therapy altered the metabolism of memory cells with self-renewing phenotype and provided a preferential advantage for survival of memory subsets with long-term efficacy and persistence. Adoptive transfer of memory cells from this combination therapy prolonged survival of tumor-bearing recipients. Furthermore, the adoptively-transferred memory cells responded to tumor re-challenge exerting long-term persistence. This approach offers a new paradigm to inhibit immunosuppression by direct targeting of MDSC function, generate effector and persistent memory cells for tumor eradication, and prevent lung cancer relapse.

Use of cells as therapeutic carriers has increased in the past few years and has developed as a distinct concept and delivery method. Cell-based vehicles are particularly attractive for delivery of biotherapeutic agents that are difficult to synthesize, have reduced half-lives, limited tissue penetrance or are rapidly inactivated upon direct in vivo introduction. Initial studies using cell-based approaches served to identify some of the key factors for the success of this type of therapeutic delivery. These factors include the efficiency of cell loading with a therapeutic payload, the means of cell loading and the nature of therapeutics that cells can carry. However, one important aspect of cell-based delivery yet to be fully investigated is the process of actual delivery of the cell payload in vivo. In this regard, the potential ability of cell carriers to provide site-specific or targeted delivery of therapeutics deserves special attention. The present review focuses on a variety of targeting approaches that may be utilized to improve cell-based therapeutic delivery strategies. The different aspects of targeting that can be applied to cell vehicles will be discussed, including physical methods for directing cell distribution, intrinsic cell-mediated homing mechanisms and the feasibility of engineering cells with novel targeting mechanisms. Development of cell targeting strategies will further advance cell vehicle applications, broaden the applicability of this delivery approach and potentiate therapeutic outcomes.

Herpes simplex virus type 1 (HSV-1) mutants lacking the c 1 34.5 neurovirulence loci are promising agents for treating malignant glioma. Arming oncolytic HSV-1 to express immunostimulatory genes may potentiate therapeutic efficacy. We have previously demonstrated improved preclinical efficacy, biodistribution, and safety of M002, a c 1 34.5-deleted HSV-1 engineered to express murine IL-12. Herein, we describe the safety and biodistribution of M032, a c 1 34.5-deleted HSV-1 virus that expresses human IL-12 after intracerebral administration to nonhuman primates, Aotus nancymae. Cohorts were administered vehicle, 10 6 , or 10 8 pfu of M032 on day 1 and subjected to detailed clinical observations performed serially over a 92-day trial. Animals were sacrificed on days 3, 31, and 91 for detailed histopathologic assessments of all organs and to isolate and quantify virus in all organs. With the possible exception of one animal euthanized on day 16, neither adverse clinical signs nor sex-or dose-related differences were attributed to M032. Elevated white blood cell and neutrophil counts were observed in virus-injected groups on day 3, but no other significant changes were noted in clinical chemistry or coagulation parameters. Minimal to mild inflammation and fibrosis detected, primarily in meningeal tissues, in M032-injected animals on days 3 and 31 had mostly resolved by day 91. The highest viral DNA levels were detected at the injection site and motor cortex on day 3 but decreased in central nervous system tissues over time. These data demonstrate the requisite safety of intracerebral M032 administration for consideration as a therapeutic for treating malignant brain tumors.