TY - THES A1 - Worschech, Andrea T1 - Oncolytic Therapy with Vaccinia Virus GLV-1h68 - Comparative Microarray Analysis of Infected Xenografts and Human Tumor Cell Lines - T1 - Onkolytische Therapy mit Vaccinia Virus GLV-1h68 - Vergleichende Mikroarray Analyse von infizierten Xenografts und humanen Tumorzelllinien - N2 - Aim of this thesis was to study the contribution of the hosts immune system during tumor regression. A wild-type rejection model was studied in which tumor regression is mediated through an adaptive, T cell host response (Research article 1). Additionally, the relationship between VACV infection and cancer rejection was assessed by applying organism-specific microarray platforms to infected and non-infected xenografts. It could be shown that tumor rejection in this nude mouse model was orchestrated solely by the hosts innate immune system without help of the adaptive immunity. In a third study the inflammatory baseline status of 75 human cancer cell lines was tested in vitro which was correlated with the susceptibility to VACV and Adenovirus 5 (Ad5) replication of the respective cell line (Manuscript for Research article 3). Although xenografts by themselves lack the ability to signal danger and do not provide sufficient proinflammatory signals to induce acute inflammation, the presence of viral replication in the oncolytic xenograft model provides the "tissue-specific trigger" that activates the immune response and in concordance with the hypothesis, the ICR is activated when chronic inflammation is switched into an acute one. Thus, in conditions in which a switch from a chronic to an acute inflammatory process can be induced by other factors like the immune-stimulation induced by the presence of a virus in the target tissue, adaptive immune responses may not be necessary and immune-mediated rejection can occur without the assistance of T or B cells. However, in the regression study using neu expressing MMC in absence of a stimulus such as a virus and infected cancer cells thereafter, adaptive immunity is needed to provoke the switch into an acute inflammation and initiate tissue rejection. Taken together, this work is supportive of the hypothesis that the mechanisms prompting TSD differ among immune pathologies but the effect phase converges and central molecules can be detected over and over every time TSD occurs. It could be shown that in presence of a trigger such as infection with VACV and functional danger signaling pathways of the infected tumor cells, innate immunity is sufficient to orchestrate rejection of manifested tumors. N2 - Ziel dieser Arbeit war, die Beteiligung des Wirts-eigenen Immunsystems bei der Tumoregression zu analysieren. Mittels eines Wildtyp-Regressionsmodells, wurde der Anteil des adaptiven Immunsystems studiert (Research-Artikel 1). Mit Hilfe von Organismus-spezifischen Mikroarrays und Genexpressionsanalysen konnte in einem Nacktmausmodell gezeigt werden, dass erfolgreiche, durch onkolytische VACV-vermittelte Tumortherapie auch ohne Beteiligung des adaptiven Immunsystems möglich ist (Research Artikel 2). In einer dritten Studie wurden 75 humane Tumorzelllinien auf ihren intrinsischen Entzündungsstatus hin getestet und bezüglich eines Zusammenhanges von diesem mit der Replikationsfähigkeit von VACV und Adenovirus 5 (Ad5) analysiert (Manuskript für den Research-Artikel 3). Obwohl Xenografts allein kein ausreichendes „Gefahrsignal“ geben und durch das Fehlen einer pro-inflammatorischen Stimulierung keine akute Entzündung verursachen können, ist die Infektion mit onkolytischem VACV ausreichend, um den Gewebe-spezifischen „Trigger“ darzustellen. In diesem Fall wird die Immunantwort aktiviert und nach der Hypothese des „Immunologic Constant of Rejection“ (ICR) geschieht dies, wenn eine chronische in eine akute Inflammation verändert wird. In dem beschriebenen onkolytischen Regressionsmodell ist die Präsenz des Virus ausreichend, um das Immunsystem zu aktivieren, d.h. die chronische Entzündung im Tumor in eine akute umzuwandeln. Dabei ist die adaptive Immunität mit T- und B-Zell-Aktivierung nicht notwendig für die Rückbildung des Tumors. In Abwesenheit eines solchen Stimulus, wie in der ersten Studie mit neu-exprimierenden MMCs, wird die Spezifität der adaptiven Immunantwort benötigt, um die akute Inflammation anzustoßen und die Tumorregression voranzutreiben. Zusammengefasst unterstützt diese Arbeit die Hypothese, dass die Mechanismen, die zu „tissue specific destruction“ (TSD) führen, in verschiedenen immunologischen Erkrankungen zwar divergieren, der Effektor-Mechanismus aber stets der Gleiche ist. Es zeigte sich, dass in Anwesenheit eines „triggers“, wie z.B. der VACV-Infektion und intakten „danger signaling pathways“ der Tumorzellen, die angeborene Immunität allein ausreicht, um die Tumorrückbildung zu vermitteln. KW - Tumorimmunologie KW - Tumor KW - Vaccinia-Virus KW - Interferon KW - Interferon Regulator Faktor 1 KW - Tumorregression KW - HT-29 KW - GI-101A KW - tissue-specific destruction Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-45338 ER - TY - JOUR A1 - Ascierto, Maria Libera A1 - Worschech, Andrea A1 - Yu, Zhiya A1 - Adams, Sharon A1 - Reinboth, Jennifer A1 - Chen, Nanhai G A1 - Pos, Zoltan A1 - Roychoudhuri, Rahul A1 - Di Pasquale, Giovanni A1 - Bedognetti, Davide A1 - Uccellini, Lorenzo A1 - Rossano, Fabio A1 - Ascierto, Paolo A A1 - Stroncek, David F A1 - Restifo, Nicholas P A1 - Wang, Ena A1 - Szalay, Aladar A A1 - Marincola, Francesco M T1 - Permissivity of the NCI-60 cancer cell lines to oncolytic Vaccinia Virus GLV-1h68 JF - BMC Cancer N2 - Background: Oncolytic viral therapy represents an alternative therapeutic strategy for the treatment of cancer. We previously described GLV-1h68, a modified Vaccinia Virus with exclusive tropism for tumor cells, and we observed a cell line-specific relationship between the ability of GLV-1h68 to replicate in vitro and its ability to colonize and eliminate tumor in vivo. Methods: In the current study we surveyed the in vitro permissivity to GLV-1h68 replication of the NCI-60 panel of cell lines. Selected cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain. In order to identify correlates of permissity to viral infection, we measured transcriptional profiles of the cell lines prior infection. Results: We observed highly heterogeneous permissivity to VACV infection amongst the cell lines. The heterogeneity of permissivity was independent of tissue with the exception of B cell derivation. Cell lines were also tested for permissivity to another Vaccinia Virus and a vesicular stomatitis virus (VSV) strain and a significant correlation was found suggesting a common permissive phenotype. While no clear transcriptional pattern could be identified as predictor of permissivity to infection, some associations were observed suggesting multifactorial basis permissivity to viral infection. Conclusions: Our findings have implications for the design of oncolytic therapies for cancer and offer insights into the nature of permissivity of tumor cells to viral infection. KW - gene-therapy KW - adenovirus KW - receptor KW - identification KW - infection KW - CD9 KW - panel Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-141503 VL - 11 IS - 451 ER - TY - JOUR A1 - Weibel, Stephanie A1 - Raab, Viktoria A1 - Yu, Yong A. A1 - Worschech, Andrea A1 - Wang, Ena A1 - Marincola, Francesco M. A1 - Szalay, Aladar A. T1 - Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection N2 - Background: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). Methods: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. Results: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. Conclusions: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome. KW - Virusinfektion KW - Krebs Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68691 ER - TY - JOUR A1 - Gowda, Madhu A1 - Godder, Kamar A1 - Kmieciak, Maciej A1 - Worschech, Andrea A1 - Ascierto, Maria-Libera A1 - Wang, Ena A1 - Francesco M., Marincola A1 - Manjili, Masoud H. T1 - Distinct signatures of the immune responses in low risk versus high risk neuroblastoma JF - Journal of Translational Medicine N2 - Background: Over 90% of low risk (LR) neuroblastoma patients survive whereas less than 30% of high risk (HR) patients are long term survivors. Age (children younger than 18 months old) is associated with LR disease. Considering that adaptive immune system is well developed in older children, and that T cells were shown to be involved in tumor escape and progression of cancers, we sought to determine whether HR patients may tend to show a signature of adaptive immune responses compared to LR patients who tend to have diminished T-cell responses but an intact innate immune response. Methods: We performed microarray analysis of RNA extracted from the tumor specimens of HR and LR patients. Flow cytometry was performed to determine the cellular constituents in the blood while multiplex cytokine array was used to detect the cytokine profile in patients' sera. A HR tumor cell line, SK-N-SH, was also used for detecting the response to IL-1 beta, a cytokines which is involved in the innate immune responses. Results: Distinct patterns of gene expression were detected in HR and LR patients indicating an active T-cell response and a diminished adaptive immune response, respectively. A diminished adaptive immune response in LR patients was evident by higher levels of IL-10 in the sera. In addition, HR patients had lower levels of circulating myeloid derived suppressor cells (MDSC) compared with a control LR patient. LR patients showed slightly higher levels of cytokines of the innate immune responses. Treatment of the HR tumor line with IL-1b induced expression of cytokines of the innate immune responses. Conclusions: This data suggests that adaptive immune responses may play an important role in the progression of HR disease whereas innate immune responses may be active in LR patients. KW - Neural precursor cells KW - Retinoic acid KW - Ifn-gamma KW - Progenitor cells KW - Breast-cancer KW - T-lymphocytes KW - IN-VIVO KW - Differentiation KW - Pathway KW - Activation KW - Neuroblastoma KW - innate immunity KW - adaptive immunity KW - prognostic biomarkers Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135147 VL - 9 IS - 170 ER - TY - JOUR A1 - De Giorgi, Valeria A1 - Buonaguro, Luigi A1 - Worschech, Andrea A1 - Tornesello, Maria Lina A1 - Izzo, Francesco A1 - Marincola, Francesco M. A1 - Wang, Ena A1 - Buonaguro, Franco M. T1 - Molecular Signatures Associated with HCV-Induced Hepatocellular Carcinoma and Liver Metastasis JF - PLoS ONE N2 - Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. In Italy, particularly Southern Italy, chronic hepatitis C virus (HCV) infection represents the main cause of HCC. Using high-density oligoarrays, we identified consistent differences in gene-expression between HCC and normal liver tissue. Expression patterns in HCC were also readily distinguishable from those associated with liver metastases. To characterize molecular events relevant to hepatocarcinogenesis and identify biomarkers for early HCC detection, gene expression profiling of 71 liver biopsies from HCV-related primary HCC and corresponding HCV-positive non-HCC hepatic tissue, as well as gastrointestinal liver metastases paired with the apparently normal peri-tumoral liver tissue, were compared to 6 liver biopsies from healthy individuals. Characteristic gene signatures were identified when normal tissue was compared with HCV-related primary HCC, corresponding HCV-positive non-HCC as well as gastrointestinal liver metastases. Pathway analysis classified the cellular and biological functions of the genes differentially expressed as related to regulation of gene expression and post-translational modification in HCV-related primary HCC; cellular Growth and Proliferation, and Cell-To-Cell Signaling and Interaction in HCV-related non HCC samples; Cellular Growth and Proliferation and Cell Cycle in metastasis. Also characteristic gene signatures were identified of HCV-HCC progression for early HCC diagnosis. Conclusions: A diagnostic molecular signature complementing conventional pathologic assessment was identified. KW - identification KW - hepatitis C virus KW - United States KW - gene expression KW - class I KW - endoplasmic reticulum KW - motile phenotype KW - bladder cancer KW - up-regulation KW - target Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131155 VL - 8 IS - 2 ER -