TY - JOUR A1 - Behr, Daniel S. A1 - Peitsch, Wiebke K. A1 - Hametner, Christian A1 - Lasitschka, Felix A1 - Houben, Roland A1 - Schönhaar, Kathrin A1 - Michel, Julia A1 - Dollt, Claudia A1 - Goebeler, Matthias A1 - Marx, Alexander A1 - Goerdt, Sergij A1 - Schmieder, Astrid T1 - Prognostic value of immune cell infiltration, tertiary lymphoid structures and PD-L1 expression in Merkel cell carcinomas JF - International Journal of Clinical and Experimental Pathology N2 - Merkel cell carcinoma (MCC) is an aggressive, virus-associated, neuroendocrine tumor of the skin mainly affecting immunocompromised patients. Higher intratumoral infiltration with CD3 and CD8 positive T-cells is associated with a better prognosis, highlighting the relevance of the immune system for MCC development and progression. In this study 21 primary MCCs were stained with immune cell markers including CD3, CD4, CD8, CD68, CD20, and S100. Furthermore, tumor-infiltrating neutrophils, tertiary lymphoid structures and PD-L1 expression were analyzed and correlated with overall and recurrence free survival. All MCCs were Merkel Cell Polyomavirus positive. Overall and recurrence-free survival did not correlate with intra-and peritumoral CD3 and CD8 T-cell infiltration. In addition, no significant association regarding prognosis was found for tumor-associated neutrophils, tumor-associated macrophages or PD-L1 positivity in MCCs. Interestingly, the presence of tertiary lymphoid structures (TLS) in the tumor microenvironment significantly correlated with recurrence-free survival (P=0.025). In addition, TLS were significantly associated with a higher CD8/CD4 ratio in the tumor periphery (P=0.032), but not in the center of the tumor (P > 0.999). These results demonstrate for the first time that TLS, easily assessed in paraffin-embedded tissue in the tumor periphery of MCCs, may be a valuable prognostic factor indicating prolonged recurrence free survival. KW - CD8(+) KW - PD-L1 KW - tertiary lymphoid structures KW - immune cell infiltration KW - polymavirus KW - survival KW - lymphocytes KW - responses KW - lung cancer KW - B-cells KW - breast cancer KW - antitumor immunity KW - T-antigens KW - Merkel cell carcinoma Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117720 SN - 1936-2625 VL - 7 IS - 11 ER - TY - JOUR A1 - Ceteci, Fatih A1 - Ceteci, Semra A1 - Zanucco, Emanuele A1 - Thakur, Chitra A1 - Becker, Matthias A1 - El-Nikhely, Nefertiti A1 - Fink, Ludger A1 - Seeger, Werner A1 - Savai, Rajkumar A1 - Rapp, Ulf R. T1 - E-Cadherin Controls Bronchiolar Progenitor Cells and Onset of Preneoplastic Lesions in Mice JF - Neoplasia N2 - Although progenitor cells of the conducting airway have been spatially localized and some insights have been gained regarding their molecular phenotype, relatively little is known about the mechanisms regulating their maintenance, activation, and differentiation. This study investigates the potential roles of E-cadherin in mouse Clara cells, as these cells were shown to represent the progenitor/stem cells of the conducting airways and have been implicated as the cell of origin of human non-small cell lung cancer. Postnatal inactivation of E-cadherin affected Clara cell differentiation and compromised airway regeneration under injury conditions. In steady-state adult lung, overexpression of the dominant negative E-cadherin led to an expansion of the bronchiolar stem cells and decreased differentiation concomitant with canonical Wnt signaling activation. Expansion of the bronchiolar stem cell pool was associated with an incessant proliferation of neuroepithelial body-associated Clara cells that ultimately gave rise to bronchiolar hyperplasia. Despite progressive hyperplasia, only a minority of the mice developed pulmonary solid tumors, suggesting that the loss of E-cadherin function leads to tumor formation when additional mutations are sustained. The present study reveals that E-cadherin plays a critical role in the regulation of proliferation and homeostasis of the epithelial cells lining the conducting airways. KW - injury KW - lung cancer KW - stem cells KW - clara cell KW - gene expression KW - basal cell KW - in vivo KW - epithelium KW - airway KW - renewal Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-135407 VL - 14 IS - 12 ER - TY - JOUR A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Maier, Carina R. A1 - Fischer, Thomas A1 - Prieto-Garcia, Cristian A1 - Baluapuri, Apoorva A1 - Schwarz, Jessica A1 - Schmitz, Werner A1 - Garrido-Rodriguez, Martin A1 - Pahor, Nikolett A1 - Davies, Clare C. A1 - Bassermann, Florian A1 - Orian, Amir A1 - Wolf, Elmar A1 - Schulze, Almut A1 - Calzado, Marco A. A1 - Rosenfeldt, Mathias T. A1 - Diefenbacher, Markus E. T1 - Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease JF - Frontiers in Cell and Developmental Biology N2 - Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research. KW - non-small cell lung cancer KW - CRISPR-Cas9 KW - mouse model KW - lung cancer KW - MYC KW - JUN Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230949 SN - 2296-634X VL - 9 ER - TY - THES A1 - Iltzsche, Fabian T1 - The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer T1 - Die Rolle DREAM/MMB-vermittelter mitotischer Genexpression unterhalb von mutiertem K-Ras in Lungenkrebs N2 - The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far. In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters. Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis. This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer. N2 - Der evolutionär konservierte Myb-MuvB (MMB) Multiproteinkomplex hat eine wesentliche Rolle in der transkriptionellen Aktivierung mitotischer Gene. Zielgene des MMB sowie die MMB assoziierten Transkriptionsfaktoren B-Myb und FoxM1 sind hoch exprimiert in einer Bandbreite verschiedener Krebsarten. Die erhöhte Expression dieser Gene korreliert mit einem fortgeschrittenen Tumorstadium und einer geringen Prognose. Das weißt auf darauf hin, dass MMB an der Tumorentstehung beteiligt sein könnte indem es die Überexpression mitotischer Gene fördert. Obwohl MMB biochemisch eingehend untersucht wurde, ist die Erfordernis von MMB zur Tumorentstehung in vivo weitestgehend unbekannt und wurde bisher nicht direkt getestet. In dieser Studie hemmt der konditionale Knockout der MMB Kerneinheit Lin9 die Tumorbildung in vivo in einem Lungenkrebs-Mausmodell angetrieben durch onkogenes K-Ras und den Verlust von p53. Die unvollständige Rekombination welche in Tumoren beobachtet wurde deutet auf einen starken Selektionsdruck gegen den kompletten Verlust von Lin9 hin. Die Verminderung von Lin9 und der MMB- assoziierten Untereinheit B-Myb durch RNAi-Interferenz (RNAi) liefert Beweise dafür, dass MMB für die Expression mitotischer Gene in Lungenkrebszellen notwendig ist. Zudem wurde gezeigt, dass das Zellwachstum von Lungenkrebszellen stark von MMB abhängig ist. Weiterhin wurde der Zusammenhang zwischen MMB und dem p53-Tumorsuppressor in einer primären Lungenkrebszelllinie mit wiederherstellbarer p53-Funktion untersucht. Expressionsanalysen zeigen, dass mitotische Gene nach Re-expression von p53 runterreguliert werden. Außerdem induziert die Aktivierung von p53 die Bildung des repressiven DREAM-Komplexes und führt zu einer Anreicherung von DREAM an Promotoren mitotischer Gene. Im Gegenzug wird MMB an den Promotoren verdrängt. Basierend auf den Ergebnissen wird das folgende Model vorgeschlagen: In p53- negativen Zellen begünstigen mitogene Reize den Wechsel von DREAM zu MMB. Dadurch werden mitotische Gene überexprimiert und können so chromosomale Instabilität und Tumorentstehung fördern Diese Studie liefert Hinweise, dass MMB an der Hochregulation G2/M- Phasenspezifischer Gene in p53-negativen Zellen beteiligt ist und dass die Hemmung von MMB (oder seiner Zielgene) eine Strategie zur Behandlung von Lungenkrebs sein könnte. KW - Nicht-kleinzelliges Bronchialkarzinom (NSCLC) KW - Lungenkrebs KW - lung cancer KW - DREAM complex KW - MMB KW - K-Ras KW - mitotic gene expression KW - Mitose Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-154108 ER - TY - JOUR A1 - Kunz, Meik A1 - Göttlich, Claudia A1 - Walles, Thorsten A1 - Nietzer, Sarah A1 - Dandekar, Gudrun A1 - Dandekar, Thomas T1 - MicroRNA-21 versus microRNA-34: Lung cancer promoting and inhibitory microRNAs analysed in silico and in vitro and their clinical impact JF - Tumor Biology N2 - MicroRNAs are well-known strong RNA regulators modulating whole functional units in complex signaling networks. Regarding clinical application, they have potential as biomarkers for prognosis, diagnosis, and therapy. In this review, we focus on two microRNAs centrally involved in lung cancer progression. MicroRNA-21 promotes and microRNA-34 inhibits cancer progression. We elucidate here involved pathways and imbed these antagonistic microRNAs in a network of interactions, stressing their cancer microRNA biology, followed by experimental and bioinformatics analysis of such microRNAs and their targets. This background is then illuminated from a clinical perspective on microRNA-21 and microRNA-34 as general examples for the complex microRNA biology in lung cancer and its diagnostic value. Moreover, we discuss the immense potential that microRNAs such as microRNA-21 and microRNA-34 imply by their broad regulatory effects. These should be explored for novel therapeutic strategies in the clinic. KW - biomarker KW - microRNA–target interaction KW - microRNAs KW - lung cancer KW - therapeutic strategy KW - bioinformatics Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158399 VL - 39 IS - 7 ER - TY - JOUR A1 - Kunz, Meik A1 - Wolf, Beat A1 - Schulze, Harald A1 - Atlan, David A1 - Walles, Thorsten A1 - Walles, Heike A1 - Dandekar, Thomas T1 - Non-Coding RNAs in Lung Cancer: Contribution of Bioinformatics Analysis to the Development of Non-Invasive Diagnostic Tools JF - Genes N2 - Lung cancer is currently the leading cause of cancer related mortality due to late diagnosis and limited treatment intervention. Non-coding RNAs are not translated into proteins and have emerged as fundamental regulators of gene expression. Recent studies reported that microRNAs and long non-coding RNAs are involved in lung cancer development and progression. Moreover, they appear as new promising non-invasive biomarkers for early lung cancer diagnosis. Here, we highlight their potential as biomarker in lung cancer and present how bioinformatics can contribute to the development of non-invasive diagnostic tools. For this, we discuss several bioinformatics algorithms and software tools for a comprehensive understanding and functional characterization of microRNAs and long non-coding RNAs. KW - lung cancer KW - non-invasive biomarkers KW - miRNAs KW - lncRNAs KW - bioinformatics KW - early diagnosis KW - algorithm Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-147990 VL - 8 IS - 1 ER - TY - JOUR A1 - Kuon, Jonas A1 - Blasi, Miriam A1 - Unsöld, Laura A1 - Vogt, Jeannette A1 - Mehnert, Anja A1 - Alt-Epping, Bernd A1 - Oorschot, Birgitt van A1 - Sistermanns, Jochen A1 - Ahlborn, Miriam A1 - Ritterbusch, Ulrike A1 - Stevens, Susanne A1 - Kahl, Christoph A1 - Ruellan, Anne A1 - Matthias, Kathrin A1 - Kubin, Thomas A1 - Stahlhut, Kerstin A1 - Heider, Andrea A1 - Lordick, Florian A1 - Thomas, Michael T1 - Impact of molecular alterations on quality of life and prognostic understanding over time in patients with incurable lung cancer: a multicenter, longitudinal, prospective cohort study JF - Supportive Care in Cancer N2 - Purpose The purpose of this study is to investigate changes over time in quality of life (QoL) in incurable lung cancer patients and the impact of determinants like molecular alterations (MA). Methods In a prospective, longitudinal, multicentric study, we assessed QoL, symptom burden, psychological distress, unmet needs, and prognostic understanding of patients diagnosed with incurable lung cancer at the time of the diagnosis (T0) and after 3 (T1), 6 (T2) and 12 months (T3) using validated questionnaires like FACT-L, National Comprehensive Cancer Network (NCCN) Distress Thermometer (DT), PHQ-4, SCNS-SF-34, and SEIQoL. Results Two hundred seventeen patients were enrolled, 22 (10%) with reported MA. QoL scores improved over time, with a significant trend for DT, PHQ-4, and SCNS-SF-34. Significant determinants for stable or improving scores over time were survival > 6 months, performance status at the time of diagnosis, and presence of MA. Patients with MA showed better QoL scores (FACT-L at T1 104.4 vs 86.3; at T2 107.5 vs 90.0; at T3 100.9 vs 92.8) and lower psychological distress (NCCN DT at T1 3.3 vs 5; at T2 2.7 vs 4.5; at T3 3.7 vs 4.5; PHQ-4 at T1 2.3 vs 4.1; at T2 1.7 vs 3.6; at T3 2.2 vs 3.6), but also a worsening of the scores at 1 year and a higher percentage of inaccurate prognostic understanding (27 vs 17%) compared to patients without MA. Conclusion Patients with tumors harboring MA are at risk of QoL deterioration during the course of the disease. Physicians should adapt their communication strategies in order to maintain or improve QoL. KW - lung cancer KW - quality of life KW - molecular alterations KW - prognostic awareness Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-308211 SN - 0941-4355 SN - 1433-7339 VL - 30 IS - 4 ER - TY - THES A1 - Kühnemundt, Johanna T1 - Defined microphysiologic 3D tumour models with aspects from the tumour microenvironment for the evaluation of cellular immunotherapies T1 - Definierte mikrophysiologische 3D-Tumormodelle mit Aspekten aus der Tumormikroumgebung zur Evaluierung von zellulären Immuntherapien N2 - Adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells is highly effective in haematological malignancies. This success, however, has not been achieved in solid tumours so far. In contrast to hematologic malignancies, solid tumours include a hostile tumour microenvironment (TME), that poses additional challenges for curative effects and consistent therapeutic outcome. These challenges manifest in physical and immunological barriers that dampen efficacy of the CAR T cells. Preclinical testing of novel cellular immunotherapies is performed mainly in 2D cell culture and animal experiments. While 2D cell culture is an easy technique for efficacy analysis, animal studies reveal information about toxicity in vivo. However, 2D cell culture cannot fully reflect the complexity observed in vivo, because cells are cultured without anchorage to a matrix and only short-term periods are feasible. Animal studies provide a more complex tissue environment, but xenografts often lack human stroma and tumour inoculation occurs mostly ectopically. This emphasises the need for standardisable and scalable tumour models with incorporated TME-aspects, which enable preclinical testing with enhanced predictive value for the clinical outcome of immunotherapies. Therefore, microphysiologic 3D tumour models based on the biological SISmuc (Small Intestinal mucosa and Submucosa) matrix with preserved basement membrane were engaged and improved in this work to serve as a modular and versatile tumour model for efficacy testing of CAR T cells. In order to reflect a variety of cancer entities, TME-aspects, long-term stability and to enhance the read-out options they were further adapted to achieve scalable and standardisable defined microphysiologic 3D tumour models. In this work, novel culture modalities (semi-static, sandwich-culture) were characterised and established that led to an increased and organised tissue generation and long-term stability. Application of the SISmuc matrix was extended to sarcoma and melanoma models and serial bioluminescence intensity (BLI)-based in vivo imaging analysis was established in the microphysiologic 3D tumour models, which represents a time-efficient read-out method for quality evaluation of the models and treatment efficacy analysis, that is independent of the cell phenotype. Isolation of cancer-associated-fibroblasts (CAFs) from lung (tumour) tissue was demonstrated and CAF-implementation further led to stromal-enriched microphysiologic 3D tumour models with in vivo-comparable tissue-like architecture. Presence of CAFs was confirmed by CAF-associated markers (FAP, α-SMA, MMP-2/-9) and cytokines correlated with CAF phenotype, angiogenesis, invasion and immunomodulation. Additionally, an endothelial cell barrier was implemented for static and dynamic culture in a novel bioreactor set-up, which is of particular interest for the analysis of immune cell diapedesis. Studies in microphysiologic 3D Ewing’s sarcoma models indicated that sarcoma cells could be sensitised for GD2-targeting CAR T cells. After enhancing the scale of assessment of the microphysiologic 3D tumour models and improving them for CAR T cell testing, the tumour models were used to analyse their sensitivity towards differently designed receptor tyrosine kinase-like orphan receptor 1 (ROR1) CAR T cells and to study the effects of the incorporated TME-aspects on the CAR T cell treatment respectively. ROR1 has been described as a suitable target for several malignancies including triple negative breast cancer (TNBC), as well as lung cancer. Therefore, microphysiologic 3D TNBC and lung cancer models were established. Analysis of ROR1 CAR T cells that differed in costimulation, spacer length and targeting domain, revealed, that the microphysiologic 3D tumour models are highly sensitive and can distinguish optimal from sub-optimal CAR design. Here, higher affinity of the targeting domain induced stronger anti-tumour efficacy and anti-tumour function depended on spacer length, respectively. Long-term treatment for 14 days with ROR1 CAR T cells was demonstrated in dynamic microphysiologic 3D lung tumour models, which did not result in complete tumour cell removal, whereas direct injection of CAR T cells into TNBC and lung tumour models represented an alternative route of application in addition to administration via the medium flow, as it induced strong anti-tumour response. Influence of the incorporated TME-aspects on ROR1 CAR T cell therapy represented by CAF-incorporation and/or TGF-β supplementation was analysed. Presence of TGF-β revealed that the specific TGF-β receptor inhibitor SD-208 improves ROR1 CAR T cell function, because it effectively abrogated immunosuppressive effects of TGF-β in TNBC models. Implementation of CAFs should provide a physical and immunological barrier towards ROR1 CAR T cells, which, however, was not confirmed, as ROR1 CAR T cell function was retained in the presence of CAFs in stromal-enriched microphysiologic 3D lung tumour models. The absence of an effect of CAF enrichment on CAR T cell efficacy suggests a missing component for the development of an immunosuppressive TME, even though immunomodulatory cytokines were detected in co-culture models. Finally, improved gene-edited ROR1 CAR T cells lacking exhaustion-associated genes (PD-1, TGF-β-receptor or both) were challenged by the combination of CAF-enrichment and TGF-β in microphysiologic 3D TNBC models. Results indicated that the absence of PD-1 and TGF-β receptor leads to improved CAR T cells, that induce strong tumour cell lysis, and are protected against the hostile TME. Collectively, the microphysiologic 3D tumour models presented in this work reflect aspects of the hostile TME of solid tumours, engage BLI-based analysis and provide long-term tissue homeostasis. Therefore, they present a defined, scalable, reproducible, standardisable and exportable model for translational research with enhanced predictive value for efficacy testing and candidate selection of cellular immunotherapy, as exemplified by ROR1 CAR T cells. N2 - Die adoptive Immuntherapie mit chimären Antigenrezeptor (CAR) exprimierenden T-Zellen zeigt bei hämatologischen Krebsformen eine hohe Wirksamkeit. Bisher konnte dieser Erfolg für solide Tumore nicht erreicht werden. Im Gegensatz zu hämatologischen Krebsformen zeigen solide Tumore eine feindliche Tumormikroumgebung (TME), die zusätzliche Herausforderungen für die Erlangung kurativer Effekte und konsistenter Therapieergebnisse darstellen. Diese Herausforderungen äußern sich in physikalischen und immunologischen Barrieren, welche die Wirksamkeit der CAR-T-Zellen abschwächt. Zur präklinischen Testung neuartiger zellulärer Immuntherapien werden hauptsächlich 2D-Zellkulturen und Tierstudien durchgeführt. 2D-Zellkulturexperimente eignen sich vor allem für Wirksamkeitsanalysen, während Tierstudien Aufschluss über die Toxizität in-vivo geben können. Allerdings kann die 2D-Zellkultur die Komplexität der in-vivo Situation nicht vollständig widerspiegeln, da die Zellen ohne Verankerung an einer Matrix kultiviert werden und nur kurzfristige Zeiträume abgebildet werden können. Tierstudien bieten einen komplexeren Gewebekontext, wobei Xenografts aber oft das humane Stroma fehlt und die Tumorinokulation meist ektopisch erfolgt. Dies unterstreicht den Bedarf an standardisierbaren und skalierbaren Tumormodellen mit inkorporierten TME-Aspekten, die präklinische Testungen mit erhöhtem Vorhersagewert für den klinischen Erfolg von Immuntherapien ermöglichen. Daher wurden in dieser Arbeit mikrophysiologische 3D-Tumormodelle auf Basis der biologischen SISmuc (Small Intestinal mukosa und Submukosa)-Matrix mit erhaltener Basalmembran eingesetzt und verbessert, um als modulares und vielseitiges Tumormodell für die Wirksamkeitsprüfung von CAR T-Zellen zu dienen. Um eine Vielzahl von Krebsentitäten, TME-Aspekte und Langzeitstabilität abzubilden und um die Ausleseparamter zu verbessern, wurden die Tumormodelle weiter angepasst um skalierbare und standardisierbare definierte mikrophysiologische 3D Tumormodelle zu erhalten. In der vorliegenden Arbeit wurden neue Kulturmodalitäten (semistatische Kultur, Sandwich-Kultur) charakterisiert und etabliert, die zu einer vermehrten und erhöhten Gewebebildung sowie Langzeitstabilität der Modelle führen. Die Anwendung der SISmuc-Matrix wurde auf Sarkom- und Melanom-Modelle erweitert und in den mikrophysiologischen 3D-Tumormodellen wurde ein serielles Biolumineszenz-Intensitäts (BLI)-basiertes In-vivo-Analyse-Verfahren etabliert, welches eine zeiteffiziente Methode für die Qualitätsbewertung der Modelle sowie die Analyse der Therapiewirksamkeit darstellt, welche unabhängig vom Zell-Phänotyp ist. Die Isolation von Krebs-assoziierten Fibroblasten (CAFs) aus Lungen-(Tumor) Gewebe wurde demonstriert und die CAF-Implementierung führte des Weiteren zu stromal-angereicherten mikrophysiologischen 3D-Tumormodellen mit in-vivo vergleichbarer gewebeähnlicher Architektur. CAFs wurden mit Hilfe von CAF-assoziierten Markern (FAP, α-SMA, MMP-2/-9) und einer Zytokinanalyse in den Modellen identifiziert. Diese bestätigte ebenfalls Zytokine, welche mit Angiogenese, Invasion und Immunmodulation assoziiert sind. Zusätzlich wurde eine Endothelzellbarriere sowohl in statischer als auch in der dynamischen Kultur implementiert, wofür ein neuer Bioreaktoraufbau verwendet wurde, welcher insbesondere für die Analyse der Immunzelldiapedesis interessant ist. Studien in mikrophysiologischen 3D-Ewing-Sarkom-Modellen zeigten, dass diese für GD2-spezifische CAR-T-Zellen sensibilisiert werden können. Nach der Erweiterung des Untersuchungsumfangs der mikrophysiologischen 3D-Tumormodelle und deren Verbesserung für die CAR-T-Zell-Testung wurden die Tumormodelle verwendet, um ihre Sensitivität gegenüber unterschiedlich designten Rezeptor-Tyrosinkinase-like Orphan-Rezeptor 1 (ROR1) -spezifischen CAR-T-Zellen zu analysieren. Des Weiteren wurden die Auswirkungen der eingebauten TME-Aspekte auf die CAR-T-Therapie untersucht. ROR1 wurde als geeignetes Ziel für verschiedene maligne Erkrankungen beschrieben, darunter auch triple-negtive-breast-cancer (TNBC) und Lungenkrebs. Daher wurden mikrophysiologische 3D-TNBC- und Lungenkrebs-Modelle für die Testungen aufgebaut. Die Analyse von ROR1-CAR-T-Zellen, die sich in Kostimulation, Spacerlänge und der Ziel-Domäne unterschieden, zeigte, dass die mikrophysiologischen 3D-Tumormodelle eine hohe Sensitivität zur Unterscheidung von suboptimal und optimal designten CARs aufweisen. Dabei induzierte eine Ziel-Domäne mit höherer Affinität eine stärkere Anti-Tumor-Wirkung. Zusätzlich war die Anti-Tumor-Funktion abhängig von der Spacerlänge. In dynamischen mikrophysiologischen 3D-Lungentumormodellen wurde eine Langzeitbehandlung über 14 Tage mit ROR1-CAR-T-Zellen realisiert, die jedoch nicht zu einer vollständigen Entfernung der Tumorzellen führte. Die direkte Injektion von CAR-T-Zellen in TNBC- und Lungentumormodellen induzierte eine starke Anti-Tumorantwort und stellt somit neben der Zugabe über den Medienstrom einen alternativen Applikationsweg dar. Des Weiteren wurde der Einfluss der inkorporierten TME-Aspekte auf die ROR1 CAR T-Zelltherapie untersucht, welche sich durch CAF-Inkorporation und/oder TGF-β-Supplementierung darstellten. Die Zugabe von TGF-β zeigte, dass der spezifische TGF-β-Rezeptor-Inhibitor SD-208 die Funktion der ROR1 CAR T-Zellen verbesserte, da er die immunsuppressiven Effekte von TGF-β in TNBC-Modellen effektiv aufhob. Die Implementierung von CAFs sollte eine physikalische und immunologische Barriere gegenüber ROR1 CAR T-Zellen darstellen, was sich jedoch nicht bestätigte, da die Funktion der ROR1 CAR T-Zellen in Anwesenheit von CAFs in stromal-angereicherten mikrophysiologischen 3D-Lungentumormodellen erhalten blieb. Das Fehlen eines Effekts der CAF-Anreicherung auf die CAR T-Zell-Effektivität deutet auf eine fehlende Komponente für die Entwicklung eines immunsuppressiven TME hin, obwohl immunmodulatorische Zytokine in Co-Kultur-Modellen nachgewiesen wurden. Schließlich wurden verbesserte gen-editierte ROR1-CAR-T-Zellen, denen erschöpfungsassoziierte Gene (PD-1, TGF-β-Rezeptor oder beide) fehlten, durch die Kombination von CAF-Anreicherung und TGF-β in mikrophysiologischen 3D-TNBC-Modellen herausgefordert. Die Ergebnisse zeigten, dass ROR1 CAR T Zellen ohne PD-1 und TGF-β-Rezeptor überlegen sind, eine starke Tumorzell-Lyse induzieren und vor der feindlichen TME geschützt sind. Zusammenfassend spiegeln die in dieser Arbeit vorgestellten mikrophysiologischen 3D-Tumormodelle Aspekte der feindlichen TME solider Tumore wider, ermöglichen BLI-basierte Analysen und bieten eine langfristige Gewebehomöostase. Daher stellen sie ein definiertes, skalierbares, reproduzierbares, standardisierbares und exportierbares Modell für die translationale Forschung mit erhöhtem Vorhersagewert dar. Sie können für die Wirksamkeitsprüfung sowie Kandidatenauswahl von zellulären Immuntherapie verwendet werden, was vor allem am Beispiel der ROR1 CAR T-Zellen gezeigt wurde. KW - CAR T cell KW - immunotherapy KW - 3D tumour model KW - solid tumour KW - tumour microenvironment KW - TNBC KW - lung cancer KW - tumour stroma KW - microphysiologic 3D tumour model KW - Immuntherapie KW - Lungenkrebs KW - Stroma KW - Tumormikroumgebung Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-276674 ER - TY - JOUR A1 - Meder, Lydia A1 - König, Katharina A1 - Ozretić, Luka A1 - Schultheis, Anne M. A1 - Ueckeroth, Frank A1 - Ade, Carsten P. A1 - Albus, Kerstin A1 - Boehm, Diana A1 - Rommerscheidt-Fuss, Ursula A1 - Florin, Alexandra A1 - Buhl, Theresa A1 - Hartmann, Wolfgang A1 - Wolf, Jürgen A1 - Merkelbach-Bruse, Sabine A1 - Eilers, Martin A1 - Perner, Sven A1 - Heukamp, Lukas C. A1 - Buettner, Reinhard T1 - NOTCH, ASCL1, p53 and RB alterations define an alternative pathway driving neuroendocrine and small cell lung carcinomas JF - International Journal of Cancer N2 - Small cell lung cancers (SCLCs) and extrapulmonary small cell cancers (SCCs) are very aggressive tumors arising de novo as primary small cell cancer with characteristic genetic lesions in RB1 and TP53. Based on murine models, neuroendocrine stem cells of the terminal bronchioli have been postulated as the cellular origin of primary SCLC. However, both in lung and many other organs, combined small cell/non-small cell tumors and secondary transitions from non-small cell carcinomas upon cancer therapy to neuroendocrine and small cell tumors occur. We define features of "small cell-ness" based on neuroendocrine markers, characteristic RB1 and TP53 mutations and small cell morphology. Furthermore, here we identify a pathway driving the pathogenesis of secondary SCLC involving inactivating NOTCH mutations, activation of the NOTCH target ASCL1 and canonical WNT-signaling in the context of mutual bi-allelic RB1 and TP53 lesions. Additionaly, we explored ASCL1 dependent RB inactivation by phosphorylation, which is reversible by CDK5 inhibition. We experimentally verify the NOTCH-ASCL1-RB-p53 signaling axis in vitro and validate its activation by genetic alterations in vivo. We analyzed clinical tumor samples including SCLC, SCC and pulmonary large cell neuroendocrine carcinomas and adenocarcinomas using amplicon-based Next Generation Sequencing, immunohistochemistry and fluorescence in situ hybridization. In conclusion, we identified a novel pathway underlying rare secondary SCLC which may drive small cell carcinomas in organs other than lung, as well. KW - lung cancer KW - small cell lung cancer KW - achaete-scute homolog 1 KW - neurogenic locus notch homolog KW - retinoblastoma protein Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190853 VL - 138 IS - 4 ER - TY - THES A1 - Nguyen, Hoang Duong T1 - Vaccinia virus mediated expression of human erythropoietin in colonized human tumor xenografts results in faster tumor regression and increased red blood cell biogenesis in mice T1 - Expression von humanem Erythropietin in Vaccinia Virus-kolonisierten Tumorxenograftmodellen fördert die Tumorregression und die Biogenese roter Blutzellen N2 - Cancer-related anemia is prevalent in cancer patients. Anemia negatively affects normal mental and physical function capacity with common symptoms s like fatigue, headache, or depression. Human erythropoietin (hEPO), a glycoprotein hormone regulating red blood cell formation, is approved for the treatment of cancer-related anemia. It has shown benefits in correcting anemia, and subsequently improving health-related quality of life and/or enhancing radio-, and chemotherapy. Several recent clinical trials have suggested that recombinant hEPO (rhEPO) may promote tumor growth that raises the questions concerning the safety of using rhEPO for cancer treatment. However in others, such effects were not indicated. As of today, the direct functional effect of rhEPO in tumor models remains controversial and needs to be further analyzed. Based on the GLV-1h68 backbone, the hEPO-expressing recombinant VACV strains (EPO-VACVs) GLV-1h210, GLV-1h211, GLV-1h212 and GLV-1h213 were generated by replacing the lacZ expression cassette at the J2R locus with hEPO under the control of different vaccinia promoters p7.5, pSE, pSEL, pSL, respectively. Also, GLV-1h209 was generated, which is similar to GLV-1h210 but expresses a mutated non-functinal EPO (R103A). The EPO-VACV strains were characterized for their oncolytic efficacy in lung (A549) cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed hEPO in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated. As expected, EPO-VACVs enhanced red blood cell (RBC) formation in xenograft model. The number of RBCs and hemoglobin (Hb) levels were significantly increased in EPO-VACVs-treated mice compared to GLV-1h68-treated or untreated control mice. However, the mean size of RBC or Hb content per RBC remained normal. Furthermore, over-expression of hEPO did not significantly affect numbers of lymphocytes, monocytes, leucocytes or platelets in the peripheral blood stream. The expression of hEPO in colonized tumors of mice treated with EPO-VACVs was demonstrated by immunohistological staining. Interestingly, there were 9 - 10 hEPO isoforms detected either in tumors, cells, or supernatant, while 3-4 basic isoforms were missing in blood serum, where only six hEPO isoforms were found. Tumor-bearing mice after treatment with EPO-VACVs showed enhanced tumor regression compared to GLV-1h68. The virus titers in tumors in EPO-VACVs-treated mice were 3-4 fold higher compared to GLV-1h68-treated mice. Nevertheless, no significant difference in virus titers among EPO-VACVs was found. The blood vessels in tumors were significantly enlarged while the blood vessel density remained unchanged compared to the GLV-1h68 treated mice, indicating that hEPO did not affect endothelial cell proliferation in this model. Meanwhile, rhEPO (Epoetin alfa) alone or in combination with GLV-1h68 did not show any signs of enhanced tumor growth when compared to untreated controls and GLV-1h68 groups, while doses used were clinical relevant (500 U/kg). These findings suggested that hEPO did not promote angiogenesis or tumor growth in the A549 tumor xenograft model. Human EPO has been reported to function as an immune modulator. In this study, however, we did not find any involvement of hEPO in immune cytokine and chemokine expression or innate immune cell infiltration (leucocytes, B cells, macrophages and dendritic cells) into infected tumors. The degree of immune infiltration and cytokine expression was directly correlated to the number of virus particles. Increased virus replication, led to more recruited immune cells and secreted cytokines/chemokines. It was proposed that tumor regression was at least partially mediated through activation of innate immune mechanisms. In conclusion, the novel EPO-VACVs were shown to significantly increase the number of RBCs, Hb levels, and virus replication in tumors as well as to enhance tumor regression in the A549 tumor xenograft model. Moreover, locally expressed hEPO did not promote tumor angiogenesis, tumor growth, and immune infiltration but was shown to causing enlarged tumoral microvessels which facilitated virus spreading. It is conceivable that in a possible clinical application, anemic cancer patients could benefit from the EPO-VACVs, where they could serve as “wellness pills” to decrease anemic symptoms, while simultaneously destroying tumors. N2 - Blutarmut stellt eine häufige Begleiterscheinung in Krebspatienten dar. Anämie beeinträchtigt die normale mentale und körperliche Funktionsfähigkeit. Menschliches Erythropoetin (hEPO), welches die Bildung roter Blutzellen reguliert, ist klinisch zur Behandlung von Krebs-induzierter Blutarmut zugelassen. Wenn es zur Behandlung von Anämie benutzt wird, verbessert es den Gesundheitszustand sowie Bestrahlungs- und Chemotherapie. Verschiedene klinische zeigten, dass rekombinantes hEPO (rhEPO) das Tumorwachstum anregen kann, was die Frage nach Sicherheit der Anwendung von rhEPO aufbringt. In anderen Studien hingegen, gab es keine Anzeichen für eine Tumorwachstum anregenden Wirkung oder für ein Eingreifen in krebsspezifische Signalwege. Verschiedene hEPO exprimierende rekombinante VACV Stämme (EPO-VACV) wurden hergestellt, GLV-1h210, GLV-1h211 und GLV-1h213, in welchen die lacZ Expressionskassette im J2R Lokus durch das hEPO Gen unter der Kontrolle von verschiedenen Promotoren, p7.5, pSE und pSL, ersetzt wurde. Ebenfalls wurde GLV-1h209 hergestellt, welches ähnlich zu GLV-1h210 ist, jedoch ein mutiertes und nicht-funktionelles EPO Protein (R103A) exprimiert. Alle EPO-VACV Stämme wurden bezüglich ihrer onkolytischen Funktion in Zellkulturexperimenten sowie in in vivo Tumormodellen charakterisiert. Die Expression von zwei Markergene war in Zellkultur sowie in Tumorxenograften für alle EPO-VACV vergleichbar mit der des parentalen GLV-1h68 Virus. Unterschiede in hEPO Transkription und Translation der EPO-VACV war deutlich abhängig von der Promotorstärke und stieg an von p7.5, über pSE und pSL zu pSEL 12 h nach Infektion von Zellen. Darüberhinaus hatte die Insertion von hEPO in das virale Genom keinen Einfluss auf Replikation oder Zytotoxizität aller EPO-VACV in A549 oder NCI-H1299 Zelllinien, obwohl zu frühen Zeitpunkten (24-48 hpi) die Replikation der EPO-VACV etwas höher war, als die des GLV-1h68 Virus. Die A549 Zellen war zugänglicher für virale Infektion durch alle untersuchten Viren als die NCI-H1299 Zellen. Von besonderem Interesse ist, dass hypoxische Bedingungen (2% O2) die Replikation und damit Expression des Markergens gusA, sowie Zytotoxizität für alle untersuchten VACV unabhängig von hEPO Expression verlangsamte. Alle EPO-VACV erhöhen die Bildung von roten Blutzellen (RBC) in Mausmodellen. Anzahl und RBCs sowie Hämoglobin (Hb) Level waren signifikant erhöht im Vergleich zu unbehandelten oder GLV-1h68 behandelten Mäusen. Die Durchschnittsgröße einer RBC sowie der Hämoglobinanteil hingegen waren unverändert. Darüberhinaus hatte die Expression von hEPO keinen signifikanten Einfluss auf Lymphozyten, Monozyten, Leukozyten oder Blutplättchen im peripheren Blut. Die Expression von hEPO in EPO-VACV kolonisierten Tumoren wurde durch immunohistologische Färbungen bestätigt. Interessanterweise konnten 9-10 EPO Isoformen in Tumoren, Zellen oder Zellüberständen gefunden werden, während im Blutserum 3-4 basische Isoformen fehlten und nur 6 Isoformen auftraten. Tumortragende Mäuse, die mit EPO-VACV behandelt wurden, wiesen im Vergleich zu GLV-1h68 behandelten Mäusen eine erhöhte Tumorregression auf. Ausserdem waren virale Titer in EPO-VACV behandleten Tumoren 3-4 fach höher also in denen, die mit GLV-1h68 behandelt wurden. Kein signifikanter Unterschied hingegen wurde zwischen viralen Titern der verschiedenen EPO-VACV in Tumoren gefunden. Tumorale Blutgefäße waren im Vergleich zu GLV-1h68 behandelten Mäusen deutlich vergrößert, wohingegen die Dichte an Blutgefäßen unverändert war, was andeuted, dass keine Proliferation von Endothelzellen angeregt wurde. Rekombinant hergestelltes Epoetin alfa in klinisch relevanten Dosen allein oder in Kombination mit GLV-1h68 hatte keinen Einfluss auf Verbesserung der Tumorregression verglichen mit unbehandelten oder GLV-1h68 behandelten Mäusen. Diese Ergbnisse legen nahe, dass weder Angiogenese noch Tumorwachstum durch hEPO im A549 Tumormodell angeregt wurde. In dieser Studie hingegen wurde kein Einfluss von hEPO im Bezug auf Zytokin- oder Chemokinexpression sowie Immunzellinfiltration in Tumore nachgewiesen. Das Ausmass an Immunzellinfiltratrion und Zytokinexpression konnte direkt mit der Anzahl an viralen Partikeln korreliert werden. Es wurde angenommen, dass Tumorregression zumindest teiweise durch eine Aktivierung des angeborenen Immunsystems bedingt ist. Zusammenfassend kann gesagt werden, dass durch die neuartigen EPO-VACV die Bildung von RBC, die Level an Hb und die virale Replikation signifikant angeregt wurden sowie eine erhöhte Tumorregression im Xenograftmodell auftrat. Darüberhinaus leitete lokal exprimiertes hEPO keine Tumorangiogenese oder Tumorwachstum ein, aber führte zu einer Vergrößerung von Tumorblutgefäßen, was die virale Ausbreitung erleichtern könnte. Es ist vorstellbar, dass anämische Patienten von einer möglichen klinischen Anwendung der EPO-Viren profitieren würden. KW - Erythropoietin KW - Lungenkrebs KW - Anämie KW - onkolytische Virotherapie KW - erythropoietin KW - lung cancer KW - anemia KW - oncolytic therapy KW - Onkolyse Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85383 ER -