@article{SzalayWeibelHofmannetal.2013, author = {Szalay, Aladar A and Weibel, Stephanie and Hofmann, Elisabeth and Basse-Luesebrink, Thomas Christian and Donat, Ulrike and Seubert, Carolin and Adelfinger, Marion and Gnamlin, Prisca and Kober, Christina and Frentzen, Alexa and Gentschev, Ivaylo and Jakob, Peter Michael}, title = {Treatment of malignant effusion by oncolytic virotherapy in an experimental subcutaneous xenograft model of lung cancer}, series = {Journal of Translational Medicine}, journal = {Journal of Translational Medicine}, doi = {doi:10.1186/1479-5876-11-106}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96016}, year = {2013}, abstract = {Background Malignant pleural effusion (MPE) is associated with advanced stages of lung cancer and is mainly dependent on invasion of the pleura and expression of vascular endothelial growth factor (VEGF) by cancer cells. As MPE indicates an incurable disease with limited palliative treatment options and poor outcome, there is an urgent need for new and efficient treatment options. Methods In this study, we used subcutaneously generated PC14PE6 lung adenocarcinoma xenografts in athymic mice that developed subcutaneous malignant effusions (ME) which mimic pleural effusions of the orthotopic model. Using this approach monitoring of therapeutic intervention was facilitated by direct observation of subcutaneous ME formation without the need of sacrificing mice or special imaging equipment as in case of MPE. Further, we tested oncolytic virotherapy using Vaccinia virus as a novel treatment modality against ME in this subcutaneous PC14PE6 xenograft model of advanced lung adenocarcinoma. Results We demonstrated significant therapeutic efficacy of Vaccinia virus treatment of both advanced lung adenocarcinoma and tumor-associated ME. We attribute the efficacy to the virus-mediated reduction of tumor cell-derived VEGF levels in tumors, decreased invasion of tumor cells into the peritumoral tissue, and to viral infection of the blood vessel-invading tumor cells. Moreover, we showed that the use of oncolytic Vaccinia virus encoding for a single-chain antibody (scAb) against VEGF (GLAF-1) significantly enhanced mono-therapy of oncolytic treatment. Conclusions Here, we demonstrate for the first time that oncolytic virotherapy using tumor-specific Vaccinia virus represents a novel and promising treatment modality for therapy of ME associated with advanced lung cancer.}, subject = {Lungenkrebs}, language = {en} } @article{DraganovSantidrianMinevetal.2019, author = {Draganov, Dobrin D. and Santidrian, Antonio F. and Minev, Ivelina and Duong, Nguyen and Kilinc, Mehmet Okyay and Petrov, Ivan and Vyalkova, Anna and Lander, Elliot and Berman, Mark and Minev, Boris and Szalay, Aladar A.}, title = {Delivery of oncolytic vaccinia virus by matched allogeneic stem cells overcomes critical innate and adaptive immune barriers}, series = {Journal of Translational Medicine}, volume = {17}, journal = {Journal of Translational Medicine}, issn = {100}, doi = {10.1186/s12967-019-1829-z}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226312}, year = {2019}, abstract = {Background Previous studies have identified IFNγ as an important early barrier to oncolytic viruses including vaccinia. The existing innate and adaptive immune barriers restricting oncolytic virotherapy, however, can be overcome using autologous or allogeneic mesenchymal stem cells as carrier cells with unique immunosuppressive properties. Methods To test the ability of mesenchymal stem cells to overcome innate and adaptive immune barriers and to successfully deliver oncolytic vaccinia virus to tumor cells, we performed flow cytometry and virus plaque assay analysis of ex vivo co-cultures of stem cells infected with vaccinia virus in the presence of peripheral blood mononuclear cells from healthy donors. Comparative analysis was performed to establish statistically significant correlations and to evaluate the effect of stem cells on the activity of key immune cell populations. Results Here, we demonstrate that adipose-derived stem cells (ADSCs) have the potential to eradicate resistant tumor cells through a combination of potent virus amplification and sensitization of the tumor cells to virus infection. Moreover, the ADSCs demonstrate ability to function as a virus-amplifying Trojan horse in the presence of both autologous and allogeneic human PBMCs, which can be linked to the intrinsic immunosuppressive properties of stem cells and their unique potential to overcome innate and adaptive immune barriers. The clinical application of ready-to-use ex vivo expanded allogeneic stem cell lines, however, appears significantly restricted by patient-specific allogeneic differences associated with the induction of potent anti-stem cell cytotoxic and IFNγ responses. These allogeneic responses originate from both innate (NK)- and adaptive (T)- immune cells and might compromise therapeutic efficacy through direct elimination of the stem cells or the induction of an anti-viral state, which can block the potential of the Trojan horse to amplify and deliver vaccinia virus to the tumor. Conclusions Overall, our findings and data indicate the feasibility to establish simple and informative assays that capture critically important patient-specific differences in the immune responses to the virus and stem cells, which allows for proper patient-stem cell matching and enables the effective use of off-the-shelf allogeneic cell-based delivery platforms, thus providing a more practical and commercially viable alternative to the autologous stem cell approach.}, language = {en} } @article{MinevLanderFelleretal.2019, author = {Minev, Boris R. and Lander, Elliot and Feller, John F. and Berman, Mark and Greenwood, Bernadette M. and Minev, Ivelina and Santidrian, Antonio F. and Nguyen, Duong and Draganov, Dobrin and Killinc, Mehmet O. and Vyalkova, Anna and Kesari, Santosh and McClay, Edward and Carabulea, Gabriel and Marincola, Francesco M. and Butterfield, Lisa H. and Szalay, Aladar A.}, title = {First-in-human study of TK-positive oncolytic vaccinia virus delivered by adipose stromal vascular fraction cells}, series = {Journal of Translational Medicine}, volume = {17}, journal = {Journal of Translational Medicine}, doi = {10.1186/s12967-019-2011-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224105}, year = {2019}, abstract = {Background ACAM2000, a thymidine kinase (TK)-positive strain of vaccinia virus, is the current smallpox vaccine in the US. Preclinical testing demonstrated potent oncolytic activity of ACAM2000 against several tumor types. This Phase I clinical trial of ACAM2000 delivered by autologous adipose stromal vascular fraction (SVF) cells was conducted to determine the safety and feasibility of such a treatment in patients with advanced solid tumors or acute myeloid leukemia (AML). Methods Twenty-four patients with solid tumors and two patients with AML participated in this open-label, non-randomized dose-escalation trial. All patients were treated with SVF derived from autologous fat and incubated for 15 min to 1 h with ACAM2000 before application. Six patients received systemic intravenous application only, one patient received intra-tumoral application only, 15 patients received combination intravenous with intra-tumoral deployment, 3 patients received intravenous and intra-peritoneal injection and 1 patient received intravenous, intra-tumoral and intra-peritoneal injections. Safety at each dose level of ACAM2000 (1.4 × 106 plaque-forming units (PFU) to 1.8 × 107 PFU) was evaluated. Blood samples for PK assessments, flow cytometry and cytokine analysis were collected at baseline and 1 min, 1 h, 1 day, 1 week, 1 month, 3 months and 6 months following treatment. Results No serious toxicities (> grade 2) were reported. Seven patients reported an adverse event (AE) in this study: self-limiting skin rashes, lasting 7 to 18 days—an expected adverse reaction to ACAM2000. No AEs leading to study discontinuation were reported. Viral DNA was detected in all patients' blood samples immediately following treatment. Interestingly, in 8 patients viral DNA disappeared 1 day and re-appeared 1 week post treatment, suggesting active viral replication at tumor sites, and correlating with longer survival of these patients. No major increase in cytokine levels or correlation between cytokine levels and skin rashes was noted. We were able to assess some initial efficacy signals, especially when the ACAM2000/SVF treatment was combined with checkpoint inhibition. Conclusions Treatment with ACAM2000/SVF in patients with advanced solid tumors or AML is safe and well tolerated, and several patients had signals of an anticancer effect. These promising initial clinical results merit further investigation of therapeutic utility. Trial registration Retrospectively registered (ISRCTN\#10201650) on October 22, 2018.}, language = {en} } @article{TsonevaMinevFrentzenetal.2017, author = {Tsoneva, Desislava and Minev, Boris and Frentzen, Alexa and Zhang, Qian and Wege, Anja K. and Szalay, Aladar A.}, title = {Humanized Mice with Subcutaneous Human Solid Tumors for Immune Response Analysis of Vaccinia Virus-Mediated Oncolysis}, series = {Molecular Therapy Oncolytics}, volume = {5}, journal = {Molecular Therapy Oncolytics}, doi = {10.1016/j.omto.2017.03.001}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170786}, pages = {41-61}, year = {2017}, abstract = {Oncolytic vaccinia virus (VACV) therapy is an alternative cancer treatment modality that mediates targeted tumor destruction through a tumor-selective replication and an induction of anti-tumor immunity. We developed a humanized tumor mouse model with subcutaneous human tumors to analyze the interactions of VACV with the developing tumors and human immune system. A successful systemic reconstitution with human immune cells including functional T cells as well as development of tumors infiltrated with human T and natural killer (NK) cells was observed. We also demonstrated successful in vivo colonization of such tumors with systemically administered VACVs. Further, a new recombinant GLV-1h376 VACV encoding for a secreted human CTLA4-blocking single-chain antibody (CTLA4 scAb) was tested. Surprisingly, although proving CTLA4 scAb's in vitro binding ability and functionality in cell culture, beside the significant increase of CD56\(^{bright}\) NK cell subset, GLV-1h376 was not able to increase cytotoxic T or overall NK cell levels at the tumor site. Importantly, the virus-encoded β-glucuronidase as a measure of viral titer and CTLA4 scAb amount was demonstrated. Therefore, studies in our "patient-like" humanized tumor mouse model allow the exploration of newly designed therapy strategies considering the complex relationships between the developing tumor, the oncolytic virus, and the human immune system.}, language = {en} } @article{HaddadSocciChenetal.2016, author = {Haddad, Dana and Socci, Nicholas and Chen, Chun-Hao and Chen, Nanhai G and Zhang, Qian and Carpenter, Susanne G and Mittra, Arjun and Szalay, Aladar A and Fong, Yuman}, title = {Molecular network, pathway, and functional analysis of-time dependent gene changes associated with pancreatic cancer susceptibility to oncolytic vaccinia virotherapy}, series = {Molecular Therapy — Oncolytics}, volume = {3}, journal = {Molecular Therapy — Oncolytics}, doi = {10.1038/mto.2016.8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165855}, pages = {16008}, year = {2016}, abstract = {Background: Pancreatic cancer is a fatal disease associated with resistance to conventional therapies. This study aimed to determine changes in gene expression patterns associated with infection and susceptibility of pancreatic cancer cells to an oncolyticvaccinia virus, GLV-1h153, carrying the human sodium iodide symporter for deep tissue imaging of virotherapy. Methods: Replication and susceptibility of pancreatic adenocarcinoma PANC-1 cells to GLV-1h153 was confirmed with replication and cytotoxicity assays. PANC-1 cells were then infected with GLV-1h153 and near-synchronous infection confirmed via flow cytometry of viral-induced green fluorescent protein (GFP) expression. Six and 24 hours after infection, three samples of each time point were harvested, and gene expression patterns assessed using HG-U133A cDNA microarray chips as compared to uninfected control. Differentially expressed genes were identified using Bioconductor LIMMA statistical analysis package. A fold change of 2.0 or above was used as a cutoff, with a P value of 0.01. The gene list was then analyzed using Ingenuity Pathways Analysis software. Results: Differential gene analysis revealed a total of 12,412 up- and 11,065 downregulated genes at 6 and 24 hours postinfection with GLV-1h153 as compared to control. At 6 hours postinfection. A total of 139 genes were either up or downregulated >twofold (false discovery rate < 0.05), of which 124 were mapped by Ingenuity Pathway Analysis (IPA). By 24 hours postinfection, a total of 5,698 genes were identified and 5,563 mapped by IPA. Microarray revealed gene expression changes, with gene networks demonstrating downregulation of processes such as cell death, cell cycle, and DNA repair, and upregulation of infection mechanisms (P < 0.01). Six hours after infection, gene changes involved pathways such as HMGB-1, interleukin (IL)-2, IL-6, IL-8, janus kinase/signal tranducer and activator of transcription (JAK/STAT), interferon, and ERK 5 signaling (P < 0.01). By 24 hours, prominent pathways included P53- and Myc-induced apoptotic processes, pancreatic adenocarcinoma signaling, and phosphoinositide 3-kinase/v-akt murine thymoma vial oncogene homolog 1 (PI3/AKT) pathways. Conclusions: Our study reveals the ability to assess time-dependent changes in gene expression patterns in pancreatic cancer cells associated with infection and susceptibility to vaccinia viruses. This suggests that molecular assays may be useful to develop safer and more efficacious oncolyticvirotherapies and support the idea that these treatments may target pathways implicated in pancreatic cancer resistance to conventional therapies.}, language = {en} } @article{CecilGentschevAdelfingeretal.2019, author = {Cecil, Alexander and Gentschev, Ivaylo and Adelfinger, Marion and Dandekar, Thomas and Szalay, Aladar A.}, title = {Vaccinia virus injected human tumors: oncolytic virus efficiency predicted by antigen profiling analysis fitted boolean models}, series = {Bioengineered}, volume = {10}, journal = {Bioengineered}, number = {1}, doi = {10.1080/21655979.2019.1622220}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200507}, pages = {190-196}, year = {2019}, abstract = {Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a promising approach for cancer therapy. Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a therapeutic potential in treating human prostate and hepatocellular carcinomas in xenografted mice. In this study, we describe the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus-injected human tumors. Antigen profiling data of vaccinia virus GLV-1h68-injected human xenografted mice were obtained, analyzed and used to calculate differences in the tumor growth signaling network by tumor type and gender. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, the T-killer cell mediated cell death, Interferon and Interleukin signaling networks. The in silico findings conform very well with in vivo findings of tumor growth. Similar to a previously published analysis of vaccinia virus-injected canine tumors, we were able to confirm the suitability of our boolean modeling for prediction of human tumor growth after virus infection in the current study as well. In summary, these findings indicate that our boolean models could be a useful tool for testing of the efficacy of VACV-mediated cancer therapy already before its use in human patients.}, language = {en} } @article{PetrovGentschevVyalkovaetal.2020, author = {Petrov, Ivan and Gentschev, Ivaylo and Vyalkova, Anna and Elashry, Mohamed I. and Klymiuk, Michele C. and Arnhold, Stefan and Szalay, Aladar A.}, title = {Canine Adipose-Derived Mesenchymal Stem Cells (cAdMSCs) as a "Trojan Horse" in Vaccinia Virus Mediated Oncolytic Therapy against Canine Soft Tissue Sarcomas}, series = {Viruses}, volume = {12}, journal = {Viruses}, number = {7}, doi = {10.3390/v12070750}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-236007}, year = {2020}, abstract = {Several oncolytic viruses (OVs) including various human and canine adenoviruses, canine distemper virus, herpes-simplex virus, reovirus, and members of the poxvirus family, such as vaccinia virus and myxoma virus, have been successfully tested for canine cancer therapy in preclinical and clinical settings. The success of the cancer virotherapy is dependent on the ability of oncolytic viruses to overcome the attacks of the host immune system, to preferentially infect and lyse cancer cells, and to initiate tumor-specific immunity. To date, several different strategies have been developed to overcome the antiviral host defense barriers. In our study, we used canine adipose-derived mesenchymal stem cells (cAdMSCs) as a "Trojan horse" for the delivery of oncolytic vaccinia virus Copenhagen strain to achieve maximum oncolysis against canine soft tissue sarcoma (CSTS) tumors. A single systemic administration of vaccinia virus-loaded cAdMSCs was found to be safe and led to the significant reduction and substantial inhibition of tumor growth in a CSTS xenograft mouse model. This is the first example that vaccinia virus-loaded cAdMSCs could serve as a therapeutic agent against CSTS tumors.}, language = {en} } @article{YeKeicherGentschevetal.2021, author = {Ye, Mingyu and Keicher, Markus and Gentschev, Ivaylo and Szalay, Aladar A.}, title = {Efficient selection of recombinant fluorescent vaccinia virus strains and rapid virus titer determination by using a multi-well plate imaging system}, series = {Biomedicines}, volume = {9}, journal = {Biomedicines}, number = {8}, issn = {2227-9059}, doi = {10.3390/biomedicines9081032}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-245104}, year = {2021}, abstract = {Engineered vaccinia virus (VACV) strains are used extensively as vectors for the development of novel cancer vaccines and cancer therapeutics. In this study, we describe for the first time a high-throughput approach for both fluorescent rVACV generation and rapid viral titer measurement with the multi-well plate imaging system, IncuCyte\(^®\)S3. The isolation of a single, well-defined plaque is critical for the generation of novel recombinant vaccinia virus (rVACV) strains. Unfortunately, current methods of rVACV engineering via plaque isolation are time-consuming and laborious. Here, we present a modified fluorescent viral plaque screening and selection strategy that allows one to generally obtain novel fluorescent rVACV strains in six days, with a minimum of just four days. The standard plaque assay requires chemicals for fixing and staining cells. Manual plaque counting based on visual inspection of the cell culture plates is time-consuming. Here, we developed a fluorescence-based plaque assay for quantifying the vaccinia virus that does not require a cell staining step. This approach is less toxic to researchers and is reproducible; it is thus an improvement over the traditional assay. Lastly, plaque counting by virtue of a fluorescence-based image is very convenient, as it can be performed directly on the computer.}, language = {en} } @article{YeWilhelmGentschevetal.2021, author = {Ye, Mingyu and Wilhelm, Martina and Gentschev, Ivaylo and Szalay, Alad{\´a}r}, title = {A modified limiting dilution method for monoclonal stable cell line selection using a real-time fluorescence imaging system: A practical workflow and advanced applications}, series = {Methods and Protocols}, volume = {4}, journal = {Methods and Protocols}, number = {1}, doi = {10.3390/mps4010016}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228896}, year = {2021}, abstract = {Stable cell lines are widely used in laboratory research and pharmaceutical industry. They are mainly applied in recombinant protein and antibody productions, gene function studies, drug screens, toxicity assessments, and for cancer therapy investigation. There are two types of cell lines, polyclonal and monoclonal origin, that differ regarding their homogeneity and heterogeneity. Generating a high-quality stable cell line, which can grow continuously and carry a stable genetic modification without alteration is very important for most studies, because polyclonal cell lines of multicellular origin can be highly variable and unstable and lead to inconclusive experimental results. The most commonly used technologies of single cell originate monoclonal stable cell isolation in laboratory are fluorescence-activated cell sorting (FACS) sorting and limiting dilution cloning. Here, we describe a modified limiting dilution method of monoclonal stable cell line selection using the real-time fluorescence imaging system IncuCyte\(^®\)S3.}, language = {en} } @article{SalihogluSrivastavaLiangetal.2023, author = {Salihoglu, Rana and Srivastava, Mugdha and Liang, Chunguang and Schilling, Klaus and Szalay, Aladar and Bencurova, Elena and Dandekar, Thomas}, title = {PRO-Simat: Protein network simulation and design tool}, series = {Computational and Structural Biotechnology Journal}, volume = {21}, journal = {Computational and Structural Biotechnology Journal}, issn = {2001-0370}, doi = {10.1016/j.csbj.2023.04.023}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350034}, pages = {2767-2779}, year = {2023}, abstract = {PRO-Simat is a simulation tool for analysing protein interaction networks, their dynamic change and pathway engineering. It provides GO enrichment, KEGG pathway analyses, and network visualisation from an integrated database of more than 8 million protein-protein interactions across 32 model organisms and the human proteome. We integrated dynamical network simulation using the Jimena framework, which quickly and efficiently simulates Boolean genetic regulatory networks. It enables simulation outputs with in-depth analysis of the type, strength, duration and pathway of the protein interactions on the website. Furthermore, the user can efficiently edit and analyse the effect of network modifications and engineering experiments. In case studies, applications of PRO-Simat are demonstrated: (i) understanding mutually exclusive differentiation pathways in Bacillus subtilis, (ii) making Vaccinia virus oncolytic by switching on its viral replication mainly in cancer cells and triggering cancer cell apoptosis and (iii) optogenetic control of nucleotide processing protein networks to operate DNA storage. Multilevel communication between components is critical for efficient network switching, as demonstrated by a general census on prokaryotic and eukaryotic networks and comparing design with synthetic networks using PRO-Simat. The tool is available at https://prosimat.heinzelab.de/ as a web-based query server.}, language = {en} }