@phdthesis{Peindl2024,
  author    = {Peindl, Matthias},
  title     = {Refinement of 3D lung cancer models for automation and patient stratification with mode-of-action studies},
  doi       = {10.25972/OPUS-31069},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-310693},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Lung cancer is the main cause of cancer-related deaths worldwide. Despite the availability of several targeted therapies and immunotherapies in the clinics, the prognosis for lung cancer remains poor. A major problem for the low benefit of these therapies is intrinsic and acquired resistance, asking for pre-clinical models for closer investigation of predictive biomarkers for refined personalized medicine and testing of possible combination therapies as well as novel therapeutic approaches to break resistances. One third of all lung adenocarcinoma harbor mutations in the KRAS gene, of which 39 \% are transitions from glycine to cysteine in codon 12 (KRASG12C). Being considered "undruggable" in previous decades, KRASG12C-inhibitors now paved the way into the standard-of-care for lung adenocarcinoma treatment in the clinics. Still, the overall response rates as well as overall survival of patients treated with KRASG12C-inhibitors are sobering. Therefore, 3D KRASG12C-biomarker in vitro models were developed based on a decellularized porcine jejunum (SISmuc) using commercial and PDX-derived cell lines and characterized in regards of epithelial-mesenchymal-transition (EMT), stemness, proliferation, invasion and c-MYC expression as well as the sensitivity towards KRASG12C-inhibiton. The phenotype of lung tumors harboring KRAS mutations together with a c-MYC overexpression described in the literature regarding invasion and proliferation for in vivo models was well represented in the SISmuc models. A higher resistance towards targeted therapies was validated in the 3D models compared to 2D cultures, while reduced viability after treatment with combination therapies were exclusively observed in the 3D models. In the test system neither EMT, stemness nor the c-MYC expression were directly predictive for drug sensitivity. Testing of a panel of combination therapies, a sensitizing effect of the aurora kinase A (AURKA) inhibitor alisertib for the KRASG12C-inhibitor ARS-1620 directly correlating with the level of c-MYC expression in the corresponding 3D models was observed. Thereby, the capability of SISmuc tumor models as an in vitro test system for patient stratification was demonstrated, holding the possibility to reduce animal experiments. Besides targeted therapies the treatment of NSCLC with oncolytic viruses (OVs) is a promising approach. However, a lack of in vitro models to test novel OVs limits the transfer from bench to bedside. In this study, 3D NSCLC models based on the SISmuc were evaluated for their capability to perform efficacy and risk assessment of oncolytic viruses (OVs) in a pre-clinical setting. Hereby, the infection of cocultures of tumor cells and fibroblasts on the SISmuc with provided viruses demonstrated that in contrast to a wildtype herpes simplex virus 1 (HSV-1) based OV, the attenuated version of the OV exhibited specificity for NSCLC cells with a more advanced and highly proliferative phenotype, while fibroblasts were no longer permissive for infection. This approach introduced SISmuc tumor models as novel test system for in vitro validation of OVs. Finally, a workflow for validating the efficacy of anti-cancer therapies in 3D tumor spheroids was established for the transfer to an automated platform based on a two-arm-robot system. In a proof-of-concept process, H358 spheroids were characterized and treated with the KRASG12C-inhibitor ARS-1620. A time- and dose-dependent reduction of the spheroid area after treatment was defined together with a live/dead-staining as easy-to-perform and cost-effective assays for automated drug testing that can be readily performed in situ in an automated system.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Landwehr2023,
  author    = {Landwehr, Laura-Sophie},
  title     = {Steroid Hormones and Cancer Immunity - learning from Adrenocortical Carcinoma},
  doi       = {10.25972/OPUS-25189},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251895},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Adrenocortical carcinoma (ACC) is a rare, but highly aggressive endocrine malignancy. Tumor-related hypercortisolism is present in 60 \% of patients and associated with worse outcome. While cancer immunotherapies have revolutionized the treatment of many cancer entities, the results of initial studies of different immune checkpoint inhibitors in ACC were heterogeneous. Up to now, five small clinical trials with a total of 121 patients have been published and demonstrated an objective response in only 17 patients. However, one of the studies, by Raj et al., reported a clinically meaningful disease control rate of 52 \% and a median overall survival of almost 25 months suggesting that a subgroup of ACC patients may benefit from immunotherapeutic approaches. Following the hypothesis that some ACCs are characterized by a glucocorticoid-induced T lymphocytes depletion, several studies were performed as part of the presented thesis. First, the immune cell infiltration in a large cohort of 146 ACC specimens was investigated. It was demonstrated for the first time, and against the common assumption, that ACCs were infiltrated not only by FoxP3+ regulatory T cells (49.3 \%), but also that a vast majority of tumor samples was infiltrated by CD4+ TH cells (74 \%) and CD8+ cytotoxic T cells (84.3 \%), albeit the immune cell number varied heterogeneously and was rather low (median: 7.7 CD3+ T cells / high power field, range: 0.1-376). Moreover, the presence of CD3+-, CD4+- and CD8+ ACC-infiltrating lymphocytes was associated with an improved recurrence-free (HR: 0.31 95 \% CI 0.11-0.82) and overall survival (HR: 0.47 96 \% CI 0.25-0.87). Particularly, patients with tumor-infiltrating CD4+ TH cells without glucocorticoid excess had a significantly longer overall survival compared to patients with T cell-depleted ACC and hypercortisolism (121 vs. 27 months, p = 0.004). Hence, the impact of glucocorticoids might to some extent be responsible for the modest immunogenicity in ACC as hypercortisolism was reversely correlated with the number of CD4+ TH cells. Accordingly, CD3+ T cells co-cultured with steroidogenic NCI-H295R ACC cells demonstrated in vitro an enhanced anti-tumoral cytotoxicity by secreting 747.96 ±225.53 pg/ml IFN-γ in a therapeutically hormone-depleted microenvironment (by incubation with metyrapone), versus only 276.02 ±117.46 pg/ml IFN-γ in a standard environment with glucocorticoid excess. Other potential biomarkers to predict response to immunotherapies are the immunomodulatory checkpoint molecules, programmed cell death 1 (PD-1) and its ligand PD-L1, since both are targets of antibodies used therapeutically in different cancer entities. In a subcohort of 129 ACCs, expressions of both molecules were heterogeneous (PD-1 17.4 \%, range 1-15; PD-L1 24.4 \%, range 1 - 90) and rather low. Interestingly, PD-1 expression significantly influenced ACC patients´ overall (HR: 0.21 95 \% CI 0.53-0.84) and progression- free survival (HR: 0.30 95 \% CI 0.13-0.72) independently of established factors, like ENSAT tumor stage, resection status, Ki67 proliferation index and glucocorticoid excess, while PD-L1 had no impact. In conclusion, this study provides several potential explanations for the heterogeneous results of the immune checkpoint therapy in advanced ACC. In addition, the establishment of PD-1 as prognostic marker can be easily applied in routine clinical care, because it is nowadays anyway part of a detailed histo-pathological work-up. Furthermore, these results provide the rationale and will pave the way towards a combination therapy using immune checkpoint inhibitors as well as glucocorticoid blockers. This will increase the likelihood of re-activating the immunological anti-tumor potential in ACC. However, this will have to be demonstrated by additional preclinical in vivo experiments and finally in clinical trials with patients.},
  subject      = {Steroidhormon},
  language  = {en}
}
@phdthesis{Marquardt2023,
  author    = {Marquardt, Andr{\´e}},
  title     = {Machine-Learning-Based Identification of Tumor Entities, Tumor Subgroups, and Therapy Options},
  doi       = {10.25972/OPUS-32954},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-329548},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Molecular genetic analyses, such as mutation analyses, are becoming increasingly important in the tumor field, especially in the context of therapy stratification. The identification of the underlying tumor entity is crucial, but can sometimes be difficult, for example in the case of metastases or the so-called Cancer of Unknown Primary (CUP) syndrome. In recent years, methylome and transcriptome utilizing machine learning (ML) approaches have been developed to enable fast and reliable tumor and tumor subtype identification. However, so far only methylome analysis have become widely used in routine diagnostics. The present work addresses the utility of publicly available RNA-sequencing data to determine the underlying tumor entity, possible subgroups, and potential therapy options. Identification of these by ML - in particular random forest (RF) models - was the first task. The results with test accuracies of up to 99\% provided new, previously unknown insights into the trained models and the corresponding entity prediction. Reducing the input data to the top 100 mRNA transcripts resulted in a minimal loss of prediction quality and could potentially enable application in clinical or real-world settings. By introducing the ratios of these top 100 genes to each other as a new database for RF models, a novel method was developed enabling the use of trained RF models on data from other sources. Further analysis of the transcriptomic differences of metastatic samples by visual clustering showed that there were no differences specific for the site of metastasis. Similarly, no distinct clusters were detectable when investigating primary tumors and metastases of cutaneous skin melanoma (SKCM). Subsequently, more than half of the validation datasets had a prediction accuracy of at least 80\%, with many datasets even achieving a prediction accuracy of - or close to - 100\%. To investigate the applicability of the used methods for subgroup identification, the TCGA-KIPAN dataset, consisting of the three major kidney cancer subgroups, was used. The results revealed a new, previously unknown subgroup consisting of all histopathological groups with clinically relevant characteristics, such as significantly different survival. Based on significant differences in gene expression, potential therapeutic options of the identified subgroup could be proposed. Concludingly, in exploring the potential applicability of RNA-sequencing data as a basis for therapy prediction, it was shown that this type of data is suitable to predict entities as well as subgroups with high accuracy. Clinical relevance was also demonstrated for a novel subgroup in renal cell carcinoma. The reduction of the number of genes required for entity prediction to 100 genes, enables panel sequencing and thus demonstrates potential applicability in a real-life setting.},
  subject      = {Maschinelles Lernen},
  language  = {en}
}
@phdthesis{Zetzl2021,
  author    = {Zetzl, Teresa Margarete},
  title     = {Cancer-related fatigue intervention},
  doi       = {10.25972/OPUS-25166},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251662},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The incidence of cancer cases is rising steadily, while improved early detection and new cancer-specific therapies are reducing the mortality rate. In addition to curing cancer or prolonging life, increasing the quality of life is thus an important goal of oncology, which is why the burdens of cancer and treatment are becoming more important. A common side effect of cancer and its therapy is cancer-related fatigue, a tiredness that manifests itself on physical, emotional and cognitive levels and is not in proportion to previous physical efforts. Since the etiology of fatigue has not yet been fully clarified, symptom-oriented therapy is preferable to cause-specific therapy. In addition to activity management, sleep hygiene, and cognitive behavioral therapy, mind-body interventions such as yoga are recommended for reducing fatigue. Previous studies with small sample sizes were able to examine the efficacy of yoga regarding fatigue predominantly in patients with breast cancer. Long-term effects of yoga have rarely been studied and there have been no attempts to increase long-term effects through interventions such as reminder e-mails. This dissertation takes a closer look at these mentioned aspects of the study sample and long-term effects. An 8-week randomized controlled yoga intervention was conducted, including patients with different cancer types reporting mild to severe fatigue. Following the 8-week yoga therapy, a randomized group of participants received weekly reminder e-mails for 6 months for regular yoga practice, whereas the control group did not receive reminder e-mails. The first paper is a protocol article, which addresses the design and planned implementation of the research project this dissertation is based upon. This serves to ensure better replicability and comparability with other yoga studies. Due to a very low consent rate of patients in the pilot phase, it was necessary to deviate from the protocol article in the actual implementation and the planned inclusion criterion of fatigue >5 was reduced to fatigue >1. The second paper examines the efficacy of the eight-week yoga intervention. Patients in the intervention group who participated in the yoga classes seven times or more showed a significantly greater reduction in general and physical fatigue than those who participated less often. The efficacy of yoga was related to the number of attended yoga sessions. Women with breast cancer who participated in yoga reported greater reductions in fatigue than women with other cancer types. There was also an improvement for depression and quality of life after eight weeks of yoga therapy compared to no yoga therapy. These results imply that yoga is helpful in reducing depression and cancer-related fatigue, especially in terms of physical aspects and improving quality of life. The third paper focuses on the efficacy of reminder e-mails in terms of fatigue and practice frequency. Patients who received reminder e-mails reported greater reductions in general and emotional fatigue, as well as significant increases in practice frequency, compared to patients who did not receive reminder e-mails. Compared to fatigue scores before yoga, significantly lower fatigue and depression scores and higher quality of life were reported after yoga therapy and at follow-up six months later. Weekly e-mail reminders after yoga therapy may have positive effects on general and emotional fatigue and help cancer patients with fatigue establish a regular yoga practice at home. However, higher practice frequency did not lead to higher improvement in physical fatigue as found in Paper 2. This may indicate other factors that influence the efficacy of yoga practice on physical fatigue, such as mindfulness or side effects of therapy. This research project provides insight into the efficacy of yoga therapy for oncology patients with fatigue. It is important that such interventions be offered early, while fatigue symptoms are not too severe. Regular guided yoga practice can reduce physical fatigue, but subsequent yoga practice at home does not further reduce physical fatigue. Reminder emails after completed yoga therapy could only reduce patients' emotional fatigue. It may be that physical fatigue was reduced as much as possible by the previous yoga therapy and that there was a floor effect, or it may be that reminder emails are not suitable as an intervention to reduce physical fatigue at all. Further research is needed to examine the mechanisms of the different interventions in more detail and to find appropriate interventions that reduce all levels of fatigue equally.},
  subject      = {Erm{\"u}dungssyndrom},
  language  = {en}
}
@phdthesis{Wachtler2020,
  author    = {Wachtler, Stefan},
  title     = {Synthese und Charakterisierung von funktionalisierten Nanodiamantmaterialien f{\"u}r biomedizinische Anwendungen},
  doi       = {10.25972/OPUS-21075},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210757},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In dieser Arbeit ist die Synthese von funktionalisiertem Nanodiamant mit bioaktiven Substanzen, welche vor allem als Wirkstofftransporter eingesetzt werden sollen, beschrieben. Dazu werden zum einen bereits bekannte Anbindungsm{\"o}glichkeiten an Nanodiamant, wie zum Beispiel die Klick-Reaktion, sowie die Ausbildung von Amidbr{\"u}cken verwendet. Zum anderen werden neuartige Funktionalisierungsm{\"o}glichkeiten wie Protein Ligation und Thioharnstoffbr{\"u}cken verwendet und somit das Repertoire an bekannten Anbindungsreaktion erweitert. Des weiteren wurde ein multifunktionales Nanodiamantsystem synthetisiert. Dieses ist in der Lage, zwei verschiedene Molek{\"u}le auf einem Partikel zu immobilisieren. Die verwendeten Methoden erm{\"o}glichen die Anbindung verschiedener Substanzen aus unterschiedlichen Molek{\"u}lgruppen an Nanodiamanten und sind somit universell einsetzbar.},
  subject      = {Synthesediamant},
  language  = {de}
}
@phdthesis{Ackermann2020,
  author    = {Ackermann, Sabine},
  title     = {Auswirkungen der multimodalen Therapie und der Einf{\"u}hrung der Vorsorgekoloskopie auf die {\"U}berlebensraten beim Kolonkarzinom},
  doi       = {10.25972/OPUS-20611},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206118},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Ziel dieser Arbeit war es, die Auswirkungen der {\"A}nderungen der Therapiestandards in der Behandlung des Kolonkarzinoms und die Auswirkungen der Einf{\"u}hrung der Vorsorgekoloskopie auf die {\"U}berlebensraten der Patienten mit Kolonkarzinom zu untersuchen. Die umfassende Analyse der therapieabh{\"a}ngigen {\"U}berlebensraten von 1016 Patienten mit Kolonkarzinom aus 20 Jahren zeigt eine Verbesserung der {\"U}berlebenswahrscheinlichkeit durch den Einsatz adjuvanter Therapie und multimodaler Therapieregime. Durch Neuerungen in der Therapie konnten die 5-Jahres-{\"U}berlebensraten seit Anfang der 90er Jahre nahezu verdoppelt werden. Als wichtigste Pr{\"a}diktoren f{\"u}r das Langzeit{\"u}berleben stellten sich das Alter der Patienten bei Erstdiagnose, das UICC Stadium und die Art der adjuvanten Therapie heraus. Der {\"U}berlebenszeit verl{\"a}ngernde Effekt war f{\"u}r den Einsatz der heutigen Standardtherapie mit 5-Flourouracil (5-FU) schon signifikant und zeigt sich f{\"u}r die Kombination mit neueren Medikamenten, insbesondere Oxaliplatin, noch deutlicher. Neue Operationstechniken, Fortschritte in der Metastasenchirurgie, ein optimiertes supportives Management und weitere Erkenntnisse onkologischer Prinzipien beeinflussten die erzielten Erfolge synergistisch. Das Gesamt{\"u}berleben der Patienten, die per Vorsorgekoloskopie detektiert werden ist besser als das der Patienten, die aufgrund klinischer Symptome diagnostiziert werden. Neben dem signifikanten {\"U}berlebensvorteil der Fr{\"u}herkennungs-Patienten, der sich durch die niedrigeren UICC Stadien in dieser Gruppe ergibt, finden sich auch Trends bez{\"u}glich eines besseren Outcomes dieser Patienten innerhalb der selben UICC Stadien. Die Patienten, deren Tumor im Rahmen des Screenings detektiert wurde, waren signifikant j{\"u}nger, wiesen signifikant weniger Begleiterkrakungen auf und zeigten signifikant niedrigere Tumorstadien. Eine adjuvante Therapie wurde in der Screening-Gruppe signifikant h{\"a}ufiger durchgef{\"u}hrt. Mehr als einer von f{\"u}nf tumorbedingten Todesf{\"a}llen der Patienten, die augrund von Symptomen diagnostiziert wurden, h{\"a}tte in dieser Studienpopulation verhindert werden k{\"o}nnen, wenn eine routinem{\"a}ßige Vorsorgekoloskopie durchgef{\"u}hrt worden w{\"a}re. Das Fazit lautet: die Vorsorgekoloskopie ist effektiv. Die Tumorgenese kann durch Entfernung von Vor{\"a}uferl{\"a}sionen durchbrochen werden, Tumoren k{\"o}nnen in fr{\"u}hen asymptomatischen Stadien detektiert werden. Screeningprogramme sollten erweitert werden, um die Inzidenz und die Mortalit{\"a}t von Darmkrebs weiter zu senken.},
  subject      = {Kolonkarzinom},
  language  = {de}
}
@phdthesis{Mestermann2020,
  author    = {Mestermann, Katrin},
  title     = {Pharmacological control of CAR T-cells by dasatinib},
  doi       = {10.25972/OPUS-18056},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180562},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Cellular therapies using chimeric antigen receptor (CAR) modified T cells to eradicate tumor cells have been a major breakthrough in the treatment of hematologic malignancies. However, there are no measures to control CAR T cell activity after infusion, which is mostly required in cases of CAR T cell overreaction, e.g. cytokine release syndrome, or in the case of T cell failure, e.g. caused by exhaustion. In our study, we identified the tyrosine kinase inhibitor (TKI) dasatinib (© Sprycel) as a suitable agent to steer CAR T cells in vitro and in vivo. We show that single treatment of CD4+ and CD8+ CAR T cells with dasatinib conferred either partial or complete inhibition, depending on the applied concentration. The blockade was immediate and encompassed spe-cific lysis, cytokine secretion and proliferation following antigen encounter. The mechanism relied on reduced phosphorylation of key kinases in the CAR signaling cascade, which led to abrogation of nuclear factor of activated T-cells (NFAT) signaling. Importantly, inhibition was fully reversible by dasatinib withdrawal. In vivo, dasatinib blocked CAR T cell function without impairing the engraftment of CAR T cells or their subsequent anti tumor function once dasatinib administration was discontinued. We therefore introduce dasatinib as a new tool to efficiently block CAR T cells in vitro and in vivo, with data suggesting that dasatinib can be used in a clinical setting to mitigate toxicity after adaptive transfer of CAR modified T cells and other forms of T cell based immunotherapy. Additionally we show that intermittent inhibition of CAR T cells by dasatinib im-proves the efficacy of CAR T cell therapy. By pausing T cells for short periods of time in vi-vo, upregulation of programmed death protein 1 (PD-1) and subsequent induction of exhaus-tion was prevented, which increased the expansion of T cells and the rate of tumor eradica-tion. Our data therefore suggest that dasatinib can additionally be used to overcome T cell exhaustion that is induced by massive tumor burden and upregulation of inhibitory receptors.},
  subject      = {Immuntherapie},
  language  = {en}
}
@phdthesis{Alkhargi2020,
  author    = {Alkhargi, Manuel},
  title     = {Cancer And Living Meaningfully: eine qualitative Studie zur Treatment Integrity der CALM-Therapie im Vergleich zu einer Kontrollbedingung},
  doi       = {10.25972/OPUS-19939},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199390},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Hintergrund: Circa ein Drittel der Patientinnen und Patienten mit fortgeschrittenen Krebserkrankungen ist von psychischen Komorbidit{\"a}ten betroffen und circa die H{\"a}lfte weist eine psychische Belastung im klinisch signifikanten Bereich auf. Zur psychotherapeutischen Behandlung dieser Patientengruppe stehen unterschiedliche psychotherapeutische Interventionen zur Verf{\"u}gung. Die CALM-Therapie, eine manualisierte Kurzintervention im Einzelsetting, ist eine dieser Interventionen. Hier bilden vier Module, welche auf den wichtigsten Anliegen und Belastungsfaktoren von Patientinnen und Patienten mit fortgeschrittenen Krebserkrankungen basieren, den inhaltlichen Rahmen. Ziel: Die Treatment Integrity beschreibt das Maß, inwieweit eine psychotherapeutische Intervention wie vorgesehen umgesetzt wurde. F{\"u}r eine fundierte Interpretation psychotherapeutischer Interventionseffekte sind Kenntnisse {\"u}ber die Treatment Integrity entscheidend. Die vorliegende Arbeit untersuchte Teilaspekte der Treatment Integrity durchgef{\"u}hrter CALM-Therapien im Vergleich zu durchgef{\"u}hrten konventionellen psychoonkologischen Therapien, um einen Beitrag zu einer fundierten Interpretation von Interventionseffekten der CALM-Therapie zu leisten. Methoden: Transkriptionen von zwei CALM-Therapien und zwei Therapien einer konventionellen psychoonkologischen Intervention wurden anhand einer qualitativen Inhaltsanalyse nach P. Mayring untersucht. Im Zentrum stand hierbei ein selbst entwickeltes Kategoriensystem zur Analyse des gesamten Textmaterials. Zus{\"a}tzlich wurden Auff{\"a}lligkeiten bez{\"u}glich Ansprachen von Themenbereichen der CALM-Module unsystematisch beobachtet. Ergebnisse: Die Inhalte der untersuchten CALM-Therapien bezogen sich durchschnittlich zu 99,54\% und die der konventionellen psychoonkologischen Therapien durchschnittlich zu 98,71\% auf die Themenbereiche der CALM-Module. Die ermittelten Werte f{\"u}r einzelne Therapiesitzungen lagen f{\"u}r CALM-Sitzungen zwischen 98,12\% und 100\% und f{\"u}r Sitzungen der konventionellen psychoonkologischen Therapie zwischen 96,20\% und 100\%. Unsystematisch beobachtete Auff{\"a}lligkeiten zeigten, dass die Themenbereiche der CALM-Module zum Teil sehr spezifisch durch die CALM-Therapeutinnen und -Therapeuten angesprochen und vernetzt wurden. Schlussfolgerung: Unter Ber{\"u}cksichtigung von methodischen Grenzen zeigte sich bez{\"u}glich des Anteils von Themenbereichen der CALM-Module innerhalb der beiden untersuchten Therapiegruppen kein maßgeblicher Unterschied. Zus{\"a}tzlich liefert die vorliegende Arbeit Hinweise f{\"u}r einen spezifischen therapeutischen Umgang mit den Themenbereichen der CALM-Module innerhalb der untersuchten CALM-Therapien. Um ermittelte Interventionseffekte der CALM-Therapie fundiert interpretieren zu k{\"o}nnen, sollten zuk{\"u}nftige Untersuchungen unterschiedliche Umgangsweisen von Therapeutinnen und Therapeuten der beiden Therapiegruppen mit den Themenbereichen der CALM-Module genauer in den Blick nehmen.},
  subject      = {Psychoonkologie},
  language  = {de}
}
@phdthesis{WeinstockgebPattschull2019,
  author    = {Weinstock [geb. Pattschull], Grit},
  title     = {Crosstalk between the MMB complex and YAP in transcriptional regulation of cell cycle genes},
  doi       = {10.25972/OPUS-17086},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170866},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The Myb-MuvB (MMB) multiprotein complex is a master regulator of cell cycle-dependent gene expression. Target genes of MMB are expressed at elevated levels in several different cancer types and are included in the chromosomal instability (CIN) signature of lung, brain, and breast tumors. This doctoral thesis showed that the complete loss of the MMB core subunit LIN9 leads to strong proliferation defects and nuclear abnormalities in primary lung adenocarcinoma cells. Transcriptome profiling and genome-wide DNA-binding analyses of MMB in lung adenocarcinoma cells revealed that MMB drives the expression of genes linked to cell cycle progression, mitosis, and chromosome segregation by direct binding to promoters of these genes. Unexpectedly, a previously unknown overlap between MMB-dependent genes and several signatures of YAP-regulated genes was identified. YAP is a transcriptional co-activator acting downstream of the Hippo signaling pathway, which is deregulated in many tumor types. Here, MMB and YAP were found to physically interact and co-regulate a set of mitotic and cytokinetic target genes, which are important in cancer. Furthermore, the activation of mitotic genes and the induction of entry into mitosis by YAP were strongly dependent on MMB. By ChIP-seq and 4C-seq, the genome-wide binding of MMB upon YAP overexpression was analyzed and long-range chromatin interaction sites of selected MMB target gene promoters were identified. Strikingly, YAP strongly promoted chromatin-association of B-MYB through binding to distal enhancer elements that interact with MMB-regulated promoters through chromatin looping. Together, the findings of this thesis provide a so far unknown molecular mechanism by which YAP and MMB cooperate to regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Monjezi2018,
  author    = {Monjezi, Razieh},
  title     = {Engineering of chimeric antigen receptor T cells with enhanced therapeutic index in cancer immunotherapy using non-viral gene transfer and genome editing},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152521},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The advances in genetic engineering have enabled us to confer T cells new desired functions or delete their specific undesired endogenous properties for improving their antitumor function. Due to their efficient gene delivery, viral vectors have been successfully used in T-cell engineering to provide gene transfer medicinal products for the treatment of human disease. One example is adoptive cell therapy with T cells that were genetically modified with gamma-retroviral and lentiviral (LV) delivery vectors to express a CD19-specific chimeric antigen receptor (CAR) for cancer treatment. This therapeutic approach has shown remarkable results against B-cell malignancies in pilot clinical trials. Consequently, there is a strong desire to make CAR T cell therapy scalable and globally available to patients. However, there are persistent concerns and limitations with the use of viral vectors for CAR T cell generation with regard to safety, cost and scale of vector production. In order to address these concerns, we aimed to improve non-viral gene transfer and genome editing tools as an effective, safe and broadly applicable alternative to viral delivery methods for T-cell engineering. In the first part of the study, we engineered CAR T cells through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles rather than conventional SB plasmids. This novel approach dramatically increased stable gene transfer rate and cell viability and resulted in higher yield of CAR+ T cells without the need of long ex vivo expansion to generate therapeutic doses of CAR+ T cells. Importantly, CD19-CAR T cells modified by MC-based SB transposition were equally effective as LV transduced CD19-CAR T cells in vitro and in a murine xenograft model (NSG/Raji-ffLuc), where a single administration of CD8+ and CD4+ CAR T cells led to complete eradication of lymphoma and memory formation of CAR T cells after lymphoma clearance. To characterize the biosafety profile of the CAR T cell products, we did the most comprehensive genomic insertion site analysis performed so far in T cells modified with SB. The data showed a close-to-random integration profile of the SB transposon with a higher number of insertions in genomic safe harbors compared to LV integrants. We developed a droplet digital PCR assay that enables rapid determination of CAR copy numbers for clinical applications. In the second part of the study, we ablated expression of PD-1, a checkpoint and negative regulator of T cell function to improve the therapeutic index of CAR T cells. This was accomplished using non-viral CRISPR/Cas9 via pre-assemble Cas9 protein and in vitro-transcribed sgRNA (Cas9 RNP). Finally, we combined our developed Cas9 RNP tool with CAR transposition from MC vectors into a single-step protocol and successfully generated PD-1 knockout CAR+ T cells. Based on the promising results achieved from antibody-mediated PD-1 blockade in the treatment of hematological and solid tumors, we are confident that PD-1 knockout CAR T cells enhance the potency of CAR T cell therapies for treatment of cancers without the side effects of antibody-based therapies. In conclusion, we provide a novel platform for virus-free genetic engineering of CAR T cells that can be broadly applied in T-cell cancer therapy. The high level of gene transfer rate and efficient genome editing, superior safety profile as well as ease-of-handling and production of non-viral MC vectors and Cas9 RNP position our developed non-viral strategies to become preferred approaches in advanced cellular and gene-therapy.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Wolter2015,
  author    = {Wolter, Patrick},
  title     = {Characterization of the mitotic localization and function of the novel DREAM target GAS2L3 and Mitotic kinesins are regulated by the DREAM complex, often up-regulated in cancer cells, and are potential targets for anti-cancer therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122531},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The recently discovered human DREAM complex (for DP, RB-like, E2F and MuvB complex) is a chromatin-associated pocket protein complex involved in cell cycle- dependent gene expression. DREAM consists of five core subunits and forms a complex either with the pocket protein p130 and the transcription factor E2F4 to repress gene expression or with the transcription factors B-MYB and FOXM1 to promote gene expression. Gas2l3 was recently identified by our group as a novel DREAM target gene. Subsequent characterization in human cell lines revealed that GAS2L3 is a microtubule and F-actin cross-linking protein, expressed in G2/M, plays a role in cytokinesis, and is important for chromosomal stability. The aim of the first part of the study was to analyze how expression of GAS2L3 is regulated by DREAM and to provide a better understanding of the function of GAS2L3 in mitosis and cytokinesis. ChIP assays revealed that the repressive and the activating form of DREAM bind to the GAS2L3 promoter. RNA interference (RNAi) mediated GAS2L3 depletion demonstrated the requirement of GAS2L3 for proper cleavage furrow ingression in cytokinesis. Immunofluorescence-based localization studies showed a localization of GAS2L3 at the mitotic spindle in mitosis and at the midbody in cytokinesis. Additional experiments demonstrated that the GAS2L3 GAR domain, a putative microtubule- binding domain, is responsible for GAS2L3 localization to the constriction zones in cytokinesis suggesting a function for GAS2L3 in the abscission process. DREAM is known to promote G2/M gene expression. DREAM target genes include several mitotic kinesins and mitotic microtubule-associated proteins (mitotic MAPs). However, it is not clear to what extent DREAM regulates mitotic kinesins and MAPs, so far. Furthermore, a comprehensive study of mitotic kinesin expression in cancer cell lines is still missing. Therefore, the second major aim of the thesis was to characterize the regulation of mitotic kinesins and MAPs by DREAM, to investigate the expression of mitotic kinesins in cancer cell line panels and to evaluate them as possible anti-cancer targets. ChIP assays together with RNAi mediated DREAM subunit depletion experiments demonstrated that DREAM is a master regulator of mitotic kinesins. Furthermore, expression analyses in a panel of breast and lung cancer cell lines revealed that mitotic kinesins are up-regulated in the majority of cancer cell lines in contrast to non-transformed controls. Finally, an inducible lentiviral-based shRNA system was developed to effectively deplete mitotic kinesins. Depletion of selected mitotic kinesins resulted in cytokinesis failures and strong anti-proliferative effects in several human cancer cell lines. Thus, this system will provide a robust tool for future investigation of mitotic kinesin function in cancer cells.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Kober2015,
  author    = {Kober, Christina},
  title     = {Characterization of Murine GL261 Glioma Models for Oncolytic Vaccinia Virus Therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118556},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Glioblastoma multiforme (GBM) is one of the most frequent and malignant forms of brain cancer in adults. The prognosis is poor with a median survival time of 12-15 months. There is a broad range of alternative treatment options studied in preclinical and clinical trials for GBM. One alternative treatment option is oncolytic virotherapy, defined as the use of replication-competent viruses that selectively infect and destroy cancer cells while leaving, non-transformed cells unharmed. Vaccinia virus (VACV) is one favorable candidate. Although oncolytic viruses can kill tumor cells grown in vitro with high efficiency, they often exhibit reduced replication capacity in vivo suggesting that physiological aspects of the tumor microenvironment decrease the virus' therapeutic potential. The percentage and composition of immune cells varies between cancer types and patients and is investigated as a biomarker in several studies. Making oncolytic virotherapy successful for GBM, it is necessary to understand the individual tumor biology, the interaction with the microenvironment and immune system. It was demonstrated that the attenuated VACV wild-type (wt) isolate LIVP 1.1.1 replicate and lyse the murine GL261 glioma cell line in vitro. In the following, the replication efficacy was characterized in a comparative approach in vivo. Immunocompetent C57BL/6 (wt) mice and immunodeficient mouse strains of different genetic background C57BL/6 athymic and Balb/c athymic mice were used. In addition, subcutaneous and intracranial locations were compared. The results revealed viral replication exclusively in Balb/c athymic mice with subcutaneous tumors but in none of the other models. In the following, the tumor microenvironment of the subcutaneous tumor models at the time of infection was performed. The study showed that implantation of the same tumor cells in different mouse strains resulted in a different tumor microenvironment with a distinct composition of immune cells. Highest differences were detected between immunodeficient and immunocompetent mice. The study showed major differences in the expression of MHCII with strongest expression in C57BL/6 wt and weakest in Balb/c athymic tumors. In the following, the influence of the phenotypic change associated with the upregulation of MHCII on GL261 tumor cells on viral replication was analyzed. Comparison of C57BL/6 wt and C57BL/6 IFN-γ knockout mice revealed endogenous IFN-γ levels to upregulate MHCII on GL261 tumor cells and to reduce viral replication in C57BL/6 wt mice. Analysis of single cell suspensions of tumor homogenates of C57BL/6 and Balb/c athymic mice showed that the IFN-γ-mediated anti-tumor effect was a reversible effect. Furthermore, reasons for inhibition of virus replication in orthotopic glioma models were elucidated. By immunohistochemical analysis it was shown that intratumoral amounts of Iba1+ microglia and GFAP+ astrocytes in Gl261 gliomas was independent from intratumoral VACV injection. Based on these findings virus infection in glioma, microglia and astrocytes was compared and analyzed in cell culture. In contrast to the GL261 glioma cells, replication was barely detectable in BV-2 microglia and IMA2.1 astrocytic cells. Co-culture experiments revealed that microglia compete for virus uptake in cell culture. It was further shown that BV-2 cells showed apoptotic characteristics after VACV infection while GL261 cells showed signs of necrotic cell death. Additionally, in BV-2 cells with M1-phenotype a further reduction of viral replication and inhibition of cell lysis was detected. Infection of IMA 2.1 cells was independent of the M1/M2-phenotype. Application of BV-2 microglia with M1-phenotype onto organotypic slice cultures with implanted GL261 tumors resulted in reduced infection of BV-2 cells with LIVP 1.1.1, whereas GL261 cells were significantly infected. Taken together, the analyzed GL261 tumors were imprinted by the immunologic and genetic background in which they grow. The experimental approach applied in this thesis can be used as suitable model which reflects the principles of personalized medicine In an additional project, based on gene expression data and bioinformatic analyses, the biological role and function of the anti-apoptotic factor AVEN was analyzed with regard to oncolytic VACV therapy. Besides a comparison of the replication efficacy of GLV-1h68 and VACV-mediated cell killing of four human tumor cell lines, it was shown that AVEN was expressed in all analyzed cells. Further, shown for HT-29 and 1936-MEL, the knockdown of AVEN by siRNA in cell culture resulted in an increase of apoptotic characteristics and a decrease of VACV infection. These findings provide essential insights for future virus development.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{MeirgebRother2015,
  author    = {Meir [geb. Rother], Juliane},
  title     = {Influence of oncolytic vaccinia viruses on metastases of human and murine tumors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118530},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Cancer is one of the leading causes of death. 90\% of all deaths are caused by the effects of metastases. It is of major importance to successfully treat the primary tumor and metastases. Tumors and metastases often differ in their properties and therefore, treatment is not always successful. In contrast, those therapeutic agents can even promote formation and growth of metastases. Hence, it is indispensable to find treatment options for metastatic disease. One promising candidate represents the oncolytic virus therapy with vaccinia viruses. The aim of this work was to analyze two cell lines regarding their metastatic abilities and to investigate whether oncolytic vaccinia viruses are useful therapy options. The cell lines used were the human cervical cancer cell line C33A implanted into immune-compromised mice and the murine melanoma cell line B16F10, implanted into immune-competent mice. The initial point of the investigations was the observation of enlarged lumbar und renal lymph nodes in C33A tumor-bearing mice 35 days post implantation of C33A cells subcutaneously into immune-compromised nude mice. Subsequently, the presence of human cells in enlarged lymph nodes was demonstrated by RT-PCR. To facilitate the monitoring of cancer cell spreading, the gene encoding for RFP was inserted into the genome of C33A cells. In cell culture experiments, it was possible to demonstrate that this insertion did not negatively affect the susceptibility of the cells to virus infection, replication and virus-mediated cell lysis. The analysis of the metastatic process in a xenografted mouse model revealed the continuous progression of lumbar (LN) and renal (RN) lymph node metastasis after C33A-RFP tumor cell implantation. The lymph node volume and the amount of RFP-positive LNs and RNs was increasing from week to week in accordance with the gain of the primary tumor volume. Moreover, the metastatic spread of cancer cells in lymph vessels between lumbar and renal lymph nodes was visualized. Additionally, the haematogenous way of cancer cell migration was demonstrated by RFP positive cancer cells in blood vessels. The haematogenous route of spreading was confirmed by detecting micrometastases in lungs of tumor bearing mice. The next step was to investigate whether the recombinant oncolytic vaccinia virus GLV-1h68 is a suitable candidate to cure the primary tumor and metastases. Therefore, GLV-1h68 was systemically injected into C33A-RFP tumor bearing mice 21 days after tumor cell implantation. It was demonstrated that the volume of the primary tumor was drastically reduced, and the volume and the amount of RFP positive lumbar and renal lymph nodes were significantly decreasing compared to the untreated control group. Subsequently, this process was analyzed further by investigating the colonization pattern in the C33A-RFP model. It was shown that first the primary tumor was colonized with highest detectable virus levels, followed by LN and RN lymph nodes. Histological analyses revealed the proliferative status of tumor cells in the tumor and lymph nodes, the amount of different immune cell populations and the vascular permeability in primary tumors and lymph nodes having an influence on the colonization pattern of the virus. Whereby, the vascular permeability seems to have a crucial impact on the preferential colonization of tumors compared to lymph node metastases in this tumor model. C33A turned out to be a useful model to study the formation and therapy of metastases. However, a metastatic model in which the influence of the immune system on tumors and especially on tumor therapy can be analyzed would be preferable. Therefore, the aim of the second part was to establish a syngeneic metastatic mouse model. Accordingly, the murine melanoma cell line B16F10 was analyzed in immunocompetent mice. First, the highly attenuated GLV 1h68 virus was compared to its parental strain LIVP 1.1.1 concerning infection, replication and cell lysis efficacy in cell culture. LIVP 1.1.1 was more efficient than GLV-1h68 and was subsequently used for following mouse studies. Comparative studies were performed, comparing two different implantation sites of the tumor cells, subcutaneously and footpad, and two different mouse strains, FoxN1 nude and C57BL/6 mice. Implantation into the footpad led to a higher metastatic burden in lymph nodes compared to the subcutaneous implantation site. Finally, the model of choice was the implantation of B16F10 into the footpad of immune-competent C57BL/6 mice. Furthermore, it was inevitable to deliver the virus as efficient as possible to the tumor and metastases. Comparison of two different injection routes, intravenously and intratumorally, revealed, that the optimal injection route was intratumorally. In summary, the murine B16F10 model is a promising model to study the effects of the immune system on vaccinia virus mediated therapy of primary tumors and metastases.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Junker2015,
  author    = {Junker, Markus},
  title     = {Development and characterization of monoclonal antibodies to GDF-15 for potential use in cancer therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132424},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Background GDF-15 is a divergent member of the TGF-superfamily, which was first described as macrophage inhibitory cytokine-1 (MIC-1), revealing an immune modulatory function. GDF-15 is a soluble protein which is, under physiological conditions, highly expressed in the placenta and found in elevated levels in blood sera of pregnant women. Apart from the placenta, GDF-15 is expressed in healthy tissue, albeit to a lower extent and overexpressed in many solid tumors. A variety of different functions are attributed to GDF-15 in healthy as well as diseased humans. On the one hand, GDF-15 is required for successful pregnancy and low GDF-15 serum levels during pregnancy correlate with fetal abortion. On the other hand, overexpression of GDF-15, which can be observed in several malignancies is correlated with a poor prognosis. Furthermore, tumor derived GDF-15 leads to cancer associated anorexia-cachexia syndrome in mice. The aim of my PhD thesis was to further investigate the role of GDF-15 as an immune modulatory factor in cancer, in particular, by inhibiting the target molecule in vitro and in vivo. Therefore, the main focus was placed on the generation and characterization of monoclonal GDF-15 specific blocking antibodies, which were tested in vitro and in vivo, which represents a substantial part of my work. Results Here, GDF-15 was shown to be highly expressed in human gynecological cancer and brain tumors. We could then demonstrate that GDF-15 modulates effector immune cells in vitro. GDF-15 mediated a slight downregulation of the activating NKG2D receptor on NK and CD8+ T cells, which is crucial for proper anti-tumoral immune responses. Furthermore, we could demonstrate that GDF-15 reduces the adhesion of CD4+ and CD8+ T cells on endothelial cells in vitro. A negatively affected trans-endothelial migration of leukocytes into inflamed tissue could explain the low T cell infiltration in GDF-15 expressing tumors, which were observed in vivo, where mice bearing (shRNA mediated) GDF-15 deficient glioma cells revealed enhanced immune cell infiltrates in the tumor microenvironment, compared with the GDF-15 expressing control group. Those animals further exhibited a decreased tumor growth and prolonged survival. GDF-15 is a soluble protein, secreted by more than 50 \% of solid tumors and associated with grade of malignancy. Therefore a neutralizing monoclonal antibody to GDF-15 was assumed to be an auspicious therapeutically anti-cancer tool. Such an antibody was thus generated in GDF-15 knock out mice against human GFD-15. Amongst many clones, the GDF-15 antibody clone B1-23 was found to be applicable in Western Blot as well as in ELISA techniques, detecting a three-dimensional epitope of the mature GDF-15 dimer with high affinity and specificity. To enable the humanization for a later administration in humans, the variable regions of antibody B1-23 were identified by a special PCR method using degenerate primers and cloned into a sequencing vector. The sequence obtained thereby enabled the generation of chimeric and humanized B1-23 variants. After further comprehensive characterization, the original mouse antibody B1-23 as well as the chimeric antibody (ChimB1-23) and the humanized B1-23 antibody (H1L5) were applied in a melanoma xenograft study in vivo. None of the antibodies could significantly inhibit tumor growth. .However of utmost importance, body weight loss mediated by tumor derived GDF-15 could be significantly prevented upon administration of all three GDF-15 specific antibodies, which confirmed the antagonizing functionality of the immunoglobulin. Conclusion GDF-15 is a promising cancer target, involved in tumor progression and cancer related cachexia. A monoclonal GDF-15 antibody was generated, which served on one hand as a tool for molecular biological applications (Western Blot, ELISA, etc.) and on the other hand was applied as an antagonizing antibody in vitro and in vivo. Even though tumor growth inhibition by GDF-15 depletion in T cell deficient athymic mice failed using B1-23, the same antibody and derivates thereof (chimeric and humanized) impressively prevented tumor associated cachexia in UACC-257 melanoma bearing nude mice. The missing anti-tumor effect in our own melanoma model in nude mice can only partially be explained by the missing secondary immunity, in particular cytotoxic T cells, in the athymic animals, since in a similar melanoma model, performed by an external company, a tumor reduction in immunocompromised animals was observed, when B1-23 was administered. These findings support the idea that T cells are substantial for an effective tumor immunity and are in line with the results of the syngeneic, T cell comprising, mouse glioma model, where silencing of tumor expressed GDF-15 led to an enhanced intratumoral T cell infiltration and a prolonged survival. Taken together our data allow for the conclusion that tumor associated cachexia can be combatted with the GDF-15 antibody B1-23. Further, B1-23 might elicit direct anti-tumor effects in immune competent models, which contain T cells, rather than in an athymic, T cell deficient nude mouse model.},
  subject      = {Growth-differentiation Factor 15},
  language  = {en}
}
@phdthesis{Huang2013,
  author    = {Huang, Ting},
  title     = {Vaccinia Virus-mediated Therapy of Solid Tumor Xenografts: Intra-tumoral Delivery of Therapeutic Antibodies},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-91327},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Over the past 30 years, much effort and financial support have been invested in the fight against cancer, yet cancer still represents the leading cause of death in the world. Conventional therapies for treatment of cancer are predominantly directed against tumor cells. Recently however, new treatments options have paid more attention to exploiting the advantage of targeting the tumor stroma instead. Vaccinia virus (VACV) has played an important role in human medicine since the 18th century as a vaccination against smallpox. In our laboratory, the recombinant, replication-competent vaccinia virus, GLV-1h68, was shown to enter, colonize and destroy cancer cells both in cell culture, and in vivo, in xenograft models (Zhang, Yu et al. 2007). In addition, combined therapy of GLV-1h68 and anti-VEGF immunotherapy significantly enhanced antitumor therapy in vivo (Frentzen, Yu et al. 2009). In this study, we constructed several new recombinant VACVs carrying genes encoding different antibodies against fibroblast activation protein (FAP) in stroma (GLV-1h282), nanobody against the extracellular domain of epidermal growth factor receptor (EGFR, GLV-1h442) or antibodies targeting both vascular endothelial growth factor (VEGF) and EGFR (GLV-1h444) or targeting both VEGF and FAP (GLV-1h446). The expression of the recombinant proteins was first verified using protein analytical methods, SDS-gel electrophoresis, Western blot analysis, immunoprecipitation (IP) assays and ELISA assays. The proteins were detected after infection of the cells with the different VACVs and the recombinant proteins purified by affinity adsorption. The purified antibodies were shown to specifically bind to their respective antigens. Secondly, the infection and replication capability of all the virus strains was analyzed in cell culture using several human tumor cell lines (A549, FaDu or DU145), revealing that all the new recombinant VACVs were able to infect cancer cells with comparable efficiency to the parental viruses from which they were derived. Thirdly, the antitumor efficacy of the new recombinant VACVs was evaluated in vivo using several human cancer xenograft models in mice. In A549 and DU145 xenografts, the new recombinant VACVs exhibited an enhanced therapeutic efficacy compared to GLV-1h68 with no change in toxicity in mice. In the FaDu xenograft, treatment with GLV-1h282 (anti-FAP) significantly slowed down the speed of tumor growth compared to GLV-1h68. Additionally, treatment with the recombinant VACVs expressed the various antibodies achieved comparable or superior therapeutic effects compared to treatment with a combination of GLV-1h68 and the commercial therapeutic antibodies, Avastin, Erbitux or both. Next, the virus distribution in tumors and organs of treated mice was evaluated. For most of the viruses, the virus titer in tumors was not signficantly diffferent than GLV-1h68. However, for animals treated with GLV-1h282, the virus titer in tumors was significantly higher than with GLV-1h68. This may be the reason for enhanced antitumor efficacy of GLV-1h282 in vivo. Lastly, the underlying mechanisms of therapeutic antibody-enhanced antitumor effects were investigated by immunohistochemistry. Blood vessels density and cell proliferation in tumors were suppressed after treatment with the antibody-encoded VACVs. The results indicated that the suppression of angiogenesis or cell proliferation in tumors may cause the observed therapeutic effect. In conclusion, the results of the studies presented here support the hypothesis that the treatment of solid tumors with a combination of oncolytic virotherapy and immunotherapy has an additive effect over each treatment alone. Moreover, expression of the immunotherapeutic antibody by the oncolytic VACV locally in the tumor enhances the antitumor effect over systemic treatment with the same antibody. Combined, these results indicate that therapy with oncolytic VACVs expressing-therapeutic antibodies may be a promising approach for the treatment of cancer.},
  subject      = {Vaccinia-Virus},
  language  = {en}
}
@phdthesis{FiebeckgebApfel2014,
  author    = {Fiebeck [geb. Apfel], Johanna Natalie},
  title     = {Etablierung eines fluoreszenzbasierten Zellassays zum Screening potentieller Krebstherapeutika des Wnt-Signalwegs},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-93101},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Der Wnt Signalweg spielt eine entscheidende Rolle in der Embryogenese durch Steuerung der Proliferation, Apoptose, Differenzierung und der Festlegung der K{\"o}rperachsen im fr{\"u}hen Embryo. Eine Fehlregulation des Signalwegs durch Mutationen in einem der Proteine und Gene dieser hochkomplexen Signalkaskade kann fatale Folgen haben, und ist ein erster Schritt auf dem Weg der Krebsentstehung. Dabei spielt das Protein β-Catenin eine Schl{\"u}sselrolle im kanonischen Zweig des Wnt Signalwegs. Durch Steuerung seiner Konzentration im Zytoplasma wird die Expression seiner direkten Zielgene reguliert, da β-Catenin im aktiven Signalweg als Co-Transkriptionsfaktor agiert. Durch Sichtbarmachung dieses Proteins durch fluoreszierende Reportergenkonstrukte kann der Aktivit{\"a}tsstatus des Wnt Signalwegs in der Zelle beobachtet werden. Das erm{\"o}glicht zum einen genaue Analysen des Signalwegs, wie zum Beispiel das Studium des Zusammenspiels mit anderen Signalwegen. Vor allem aber erlaubt es die gezielte Suche nach Wnt-Signalwegs-modulierenden Substanzen als potentielle Wirkstoffe in der Krebsmedikamentenentwicklung. In der vorliegenden Arbeit wurden mehrere Reportergenkonstrukte f{\"u}r die stabile Transfektion von Zelllinien entwickelt und hinsichtlich eines m{\"o}glichen Einsatzes sowohl in der Forschung, als auch in Wirkstoffscreenings validiert. Dies umfasst sowohl mehrere Reporter mit β-Catenin als Fusionsprotein, als auch Wnt-Promoter-regulierte eGFP-Reporter, die den Akitvit{\"a}tsstatus des Wnt-Signalwegs anzeigen. Mit Hilfe dieser Reporter konnten Untersuchungen zur Wirkung des Wnt-Signalwegs auf die Morphologie von transfizierten und nicht-transfizierten MDCK-Zellen durchgef{\"u}hrt werden. {\"U}berdies wurde ein promotorregulierter eGFP-Reporter konstruiert, mit welchem transfizierte Zellen mit aktiviertem Wnt-Signalweg aus einem Zellpool gefischt werden k{\"o}nnen. Diese Methode ist sowohl f{\"u}r den Einsatz in kultivierten Zelllinien, als auch in der Diagnostik nach der Transfektion prim{\"a}rer Zellen geeignet. Auf Grundlage der neuen Zelllinien wurde weiterhin ein neuer Screeningansatz f{\"u}r potentielle Wnt-Signalwegsinhibitoren entwickelt, der auf dem Ausbleichen der Fluoreszenz in einem Well einer Multiwell-Kulturplatte beruht.},
  subject      = {Wnt-Proteine},
  language  = {de}
}
@phdthesis{Hess2013,
  author    = {Heß, Michael},
  title     = {Vaccinia virus-encoded bacterial beta-glucuronidase as a diagnostic biomarker for oncolytic virotherapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86789},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Oncolytic virotherapy represents a promising approach to revolutionize cancer therapy. Several preclinical and clinical trials display the safety of oncolytic viruses as wells as their efficiency against solid tumors. The development of complementary diagnosis and monitoring concepts as well as the optimization of anti-tumor activity are key points of current virotherapy research. Within the framework of this thesis, the diagnostic and therapeutic prospects of beta-glucuronidase expressed by the oncolytic vaccinia virus strain GLV-1h68 were evaluated. In this regard, a beta-glucuronidase-based, therapy-accompanying biomarker test was established which is currently under clinical validation. By using fluorescent substrates, the activity of virally expressed beta-glucuronidase could be detected and quantified. Thereby conclusions about the replication kinetics of oncolytic viruses in animal models and virus-induced cancer cell lysis could be drawn. These findings finally led to the elaboration and establishment of a versatile biomarker assay which allows statements regarding the replication of oncolytic viruses in mice based on serum samples. Besides the analysis of retrospective conditions, this test is able to serve as therapy-accompanying monitoring tool for virotherapy approaches with beta-glucuronidase-expressing viruses. The newly developed assay also served as complement to routinely used plaque assays as well as reference for virally expressed anti-angiogenic antibodies in additional preclinical studies. Further validation of this biomarker test is currently taking place in the context of clinical trials with GL-ONC1 (clinical grade GLV-1h68) and has already shown promising preliminary results. It was furthermore demonstrated that fluorogenic substrates in combination with beta-glucuronidase expressed by oncolytic viruses facilitated the optical detection of solid tumors in preclinical models. In addition to diagnostic purposes, virus-encoded enzymes could also be combined with prodrugs resulting in an improved therapeutic outcome of oncolytic virotherapy. In further studies, the visualization of virus-induced immune reactions as well as the establishment of innovative concepts to improve the therapeutic outcome of oncolytic virotherapy could be accomplished. In conclusion, the results of this thesis provide crucial findings about the influence of virally expressed beta-glucuronidase on various diagnostic concepts in the context of oncolytic virotherapy. In addition, innovative monitoring and therapeutic strategies could be established. Our preclinical findings have important clinical influence, particularly by the development of a therapy-associated biomarker assay which is currently used in different clinical trials.},
  subject      = {Vaccinia-Virus},
  language  = {en}
}
@inproceedings{SchartlSchartlAnders1981,
  author    = {Schartl, A. and Schartl, Manfred and Anders, F.},
  title     = {Phenotypic conversion of malignant melanoma to benign melanoma and vice versa in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86662},
  year      = {1981},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{SchartlSchartl1990,
  author    = {Schartl, Angelika and Schartl, Manfred},
  title     = {Genes and cancer: Molecular biology of the melanoma oncogene of Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72670},
  year      = {1990},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{RiehlSchartlAnders1985,
  author    = {Riehl, R{\"u}diger and Schartl, Manfred and Anders, Fritz},
  title     = {An ultrastructural study of melanoma in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70978},
  year      = {1985},
  abstract  = {Melanotic melanoma (MM) of Xiphophorus (Teleostei: Poeciliidae) was studied by conventional preparations and freeze-etch preparations for electron microscopy. MM of Xiphophorus exhibits tightly packed pigment cells with prominent dendritic processes and interdigitations of their plasma membranes. The most impressive feature of MM cells is the occurrence of Iarge lobulated nuclei with numerous nuclear pores and some nuclear pockets. Abundant spheroidal or ellipsoidal melanosomes (diameter 200-650 nm) and vesicular structures are distributed throughout the cellular dendrites, whereas the perinucJear cytoplasm is free of melanosomes. A further characteristic feature of melanoma cells in fish is the occurrence of melanosome complexes (i.e., "compound melanosomes"). These melanosome complexes consist of a few to numerous melanosomes, which are enveloped by a separate rnembrane. Pinocytotic vesicles couJd be demonstrated with distinct differences in frequency and distribution patterns, indicating differences in the metabolic activities of the cells in the same melanoma. Intercellular junctions are lacking in the MM cells. The conventional TEM technique showed clear advantages in the demonstration of intemal architecture of organelles, whereas FE bad considerable potential in respect to the visualization of membrane surface specializations.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}