Katja Simon-Keller

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Targeting the fetal acetylcholine receptor in rhabdomyosarcoma.

Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Recent efforts to enhance overall survival of patients with clinically advanced RMS have failed and there is a demand for conceptually novel treatments. Immune therapeutic options targeting the fetal nicotinic acetylcholine receptor (fnAChR), which is broadly expressed on RMS, are novel approaches to overcome the therapeutic resistance of RMS. Expression of the fnAChR is restricted to developing fetal muscles, some apparently dispensable ocular muscle fibers and thymic myoid cells. Therefore, after-birth fnAChR is a tumor-associated and almost tumor-specific antigen on RMS cells. Areas covered: This review gives an overview on nAChR function and expression pattern in RMS tumor cells, and deals with the immunological significance of fnAChR-expressing cells, including the risk of anti-nAChR autoimmunity as a potential side effect of fnAChR-directed immunotherapies. The article also addresses the advantages and disadvantages of vaccination strategies, immunotoxins and chimeric T cells targeting the fnAChR. Expert opinion: Finally, we suggest technical and biological strategies to improve the available immunotherapeutic tools including increasing the in vivo expression of the target fnAChR on RMS cells.

Gene expression profiling reveals aryl hydrocarbon receptor as a possible target for photobiomodulation when using blue light

Photobiomodulation (PBM) with blue light induces a biphasic dose response curve in proliferation of immortalized human keratinocytes (HaCaT), with a maximum anti-proliferative effect reached with 30min (41.4 J/cm2). The aim of this study was to test the photobiomodulatory effect of 41.4 J/cm2 blue light irradiation on ROS production, apoptosis and gene expression at different time points after irradiation of HaCaT cells in vitro and assess its safety. ROS concentration was increased 30 min after irradiation. However, already 1 h after irradiation, cells were able to reduce ROS and balance the concentration to a normal level. The sudden increase in ROS did not damage the cells, which was demonstrated with FACS analysis where HaCaT cells did not show any sign of apoptosis after blue light irradiation. Furthermore, a time course could be seen in gene expression analysis after blue light, with an early response of stimulated genes already 1 h after blue light irradiation, leading to the discovery of the aryl hydrocarbon receptor as possible target for blue light irradiation.

Stabilin-1 is expressed in human breast cancer and supports tumor growth in mammary adenocarcinoma mouse model

Stabilin-1 is a multifunctional scavenger receptor expressed on alternatively-activated macrophages. Stabilin-1 mediates phagocytosis of “unwanted-self” components, intracellular sorting, and endocytic clearance of extracellular ligands including SPARC that modulates breast cancer growth. The expression of stabilin-1 was found on tumor-associated macrophages (TAM) in mouse and human cancers including melanoma, lymphoma, glioblastoma, and pancreatic insulinoma. Despite its tumor-promoting role in mouse models of melanoma and lymphoma the expression and functional role of stabilin-1 in breast cancer was unknown. Here, we demonstrate that stabilin-1 is expressed on TAM in human breast cancer, and its expression is most pronounced on stage I disease. Using stabilin-1 knockout (ko) mice we show that stabilin-1 facilitates growth of mouse TS/A mammary adenocarcinoma. Endocytosis assay on stabilin-1 ko TAM demonstrated impaired clearance of stabilin-1 ligands including SPARC that was capable of inducing cell death in TS/A cells. Affymetrix microarray analysis on purified TAM and reporter assays in stabilin-1 expressing cell lines demonstrated no influence of stabilin-1 expression on intracellular signalling. Our results suggest stabilin-1 mediated silent clearance of extracellular tumor growth-inhibiting factors (e.g. SPARC) as a mechanism of stabilin-1 induced tumor growth. Silent clearance function of stabilin-1 makes it an attractive candidate for delivery of immunomodulatory anti-cancer therapeutic drugs to TAM.

Vascular architecture as a diagnostic marker for differentiation of World Health Organization thymoma subtypes and thymic carcinoma.

Thymomas and thymic squamous cell carcinomas (TSQCCs) are rare thymic epithelial tumours. Data on angiogenesis and vascular phenotype in these tumours are limited, and no study has taken histological World Health Organization (WHO) subtypes into account. The aim of this study was to compare vascularization, pericytes coverage and expression of angiogenic growth factors in different WHO‐defined subtypes of thymoma

[Adoptive T-cell therapy of rhabdomyosarcoma].

ZusammenfassungZielsetzungUm das Überleben bei fortgeschrittenen Rhabdomyosarkomen (RMS) zu verbessern, versuchen wir, Bedingungen für den adoptiven Transfer chimärer T-Zellen zu verbessern, die eine Spezifität für den fetalen Acetylcholinrezeptor, ein RMS-spezifisches Oberflächenmolekül, haben.MethodenZur Optimierung der Rezeptorzytotoxizität wurde dem ursprünglich vorhandenen chimären Rezeptor, bestehend aus einem humanen Anti-fAChR-Antikörper, einer Fc-Hinge-Region und einer humanen CD3ζ-Kette, eine CD28-Domäne zugefügt. Periphere Blutlymphozyten wurden mittels retroviraler Transduktion modifiziert und die Expression des chimären Rezeptors mittels Durchflusszytometrie verifiziert. Das zytotoxische Potenzial der modifizierten T-Zellen gegenüber RMS-Zellen wurde über MTT-Zytotoxizitätstests bestimmt. Die Expression kostimulatorischer Moleküle und antiapoptotischer Faktoren wurde durchflusszytometrisch bzw. mithilfe quantitativer PCR untersucht.ErgebnisseDie geringen Expressionslevel kostimulatorischer Moleküle auf RMS-Zellen führten zu der Überlegung, einen chimären Antigenrezeptor (CAR) mit einer CD28-CD3ζ-Signaldomäne zu generieren. Die erfolgreiche Modifikation der T-Zellen mit der „zweiten Generation“ des AChR-spezifischen CAR zeigte trotz guter Expressionsraten geringe Lyseraten gegenüber RMS, im Vergleich zu CD20-positiven Lymphom- oder CEA-exprimierenden Adenokarzinomzelllinien mit CD20- bzw. CEA-spezifischem CAR.SchlussfolgerungDie verminderten Lyseraten der RMS-Zellen durch einen fAChR-spezifischen CAR lassen eine Resistenz der RMS-Zellen gegenüber der T-Zell-Antwort vermuten. Die Inhibierung antiapoptotischer Stoffwechselwege könnte die Sensitivität dieser Zellen gegenüber konventioneller und auch gegenüber T-Zell-basierten Therapien verbessern.AbstractAimsTo improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen.MethodsA “second generation” chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis.ResultsCo-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the “second generation” AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR.ConclusionsData suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics.

Adoptive T-Zell-Therapie des Rhabdomyosarkoms

ZusammenfassungZielsetzungUm das Überleben bei fortgeschrittenen Rhabdomyosarkomen (RMS) zu verbessern, versuchen wir, Bedingungen für den adoptiven Transfer chimärer T-Zellen zu verbessern, die eine Spezifität für den fetalen Acetylcholinrezeptor, ein RMS-spezifisches Oberflächenmolekül, haben.MethodenZur Optimierung der Rezeptorzytotoxizität wurde dem ursprünglich vorhandenen chimären Rezeptor, bestehend aus einem humanen Anti-fAChR-Antikörper, einer Fc-Hinge-Region und einer humanen CD3ζ-Kette, eine CD28-Domäne zugefügt. Periphere Blutlymphozyten wurden mittels retroviraler Transduktion modifiziert und die Expression des chimären Rezeptors mittels Durchflusszytometrie verifiziert. Das zytotoxische Potenzial der modifizierten T-Zellen gegenüber RMS-Zellen wurde über MTT-Zytotoxizitätstests bestimmt. Die Expression kostimulatorischer Moleküle und antiapoptotischer Faktoren wurde durchflusszytometrisch bzw. mithilfe quantitativer PCR untersucht.ErgebnisseDie geringen Expressionslevel kostimulatorischer Moleküle auf RMS-Zellen führten zu der Überlegung, einen chimären Antigenrezeptor (CAR) mit einer CD28-CD3ζ-Signaldomäne zu generieren. Die erfolgreiche Modifikation der T-Zellen mit der „zweiten Generation“ des AChR-spezifischen CAR zeigte trotz guter Expressionsraten geringe Lyseraten gegenüber RMS, im Vergleich zu CD20-positiven Lymphom- oder CEA-exprimierenden Adenokarzinomzelllinien mit CD20- bzw. CEA-spezifischem CAR.SchlussfolgerungDie verminderten Lyseraten der RMS-Zellen durch einen fAChR-spezifischen CAR lassen eine Resistenz der RMS-Zellen gegenüber der T-Zell-Antwort vermuten. Die Inhibierung antiapoptotischer Stoffwechselwege könnte die Sensitivität dieser Zellen gegenüber konventioneller und auch gegenüber T-Zell-basierten Therapien verbessern.AbstractAimsTo improve survival of patients with advanced rhabdomyosarcomas (RMS), we aimed to adoptively transfer T-cells with redirected specificity for the fetal acetylcholine receptor (AChR), an RMS-specific cell surface antigen.MethodsA “second generation” chimeric antigen receptor (CAR) with a combined CD28-CD3ζ signaling domain was derived from our previously described chimeric antigen receptor composed of an extracellular human anti-fAChR antibody fragment, an Fc hinge region, and the intracellular T-cell receptor zeta chain. Lymphocytes from the peripheral blood were modified by retroviral transduction and monitored by FACS analysis. Cytotoxicity of modified T-cells towards RMS cells was recorded by MTT-based viability tests; expression of co-stimulatory molecules and anti-apoptotic genes was studied by FACS and qRT-PCR analysis.ResultsCo-stimulatory molecules were expressed in low levels on RMS cells giving the rationale to generate a CD28-CD3ζ signalling CAR (chimeric antigen receptor) for redirecting T-cells. T-cells were successfully engineered with the “second generation” AChR-specific chimeric antigen receptor. Despite of high CAR expression engineered T-cells showed low killing efficiency towards RMS compared to redirected killing of CD20+ lymphoma or CEA-expressing adenocarcinoma cell lines when redirected by CD20- and/or CEA-specific CAR.ConclusionsData suggest that RMS cells exhibit resistance to a T-cell attack redirected by a fAChR-specific CAR. Inhibition of anti-apoptotic pathways in those cells may improve sensitivity to conventional as well as T-cell-based therapeutics.

LEF1 reduces tumor progression and induces myodifferentiation in a subset of rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and show characteristics of skeletal muscle differentiation. The two major RMS subtypes in children are alveolar (ARMS) and embryonal RMS (ERMS). We demonstrate that approximately 50% of ARMS and ERMS overexpress the LEF1/TCF transcription factor LEF1 when compared to normal skeletal muscle and that LEF1 can restrain aggressiveness especially of ARMS cells. LEF1 knockdown experiments in cell lines reveal that depending on the cellular context, LEF1 can induce pro-apoptotic signals. LEF1 can also suppress proliferation, migration and invasiveness of RMS cells both in vitro and in vivo. Furthermore, LEF1 can induce myodifferentiation of the tumor cells. This may involve regulation of other LEF1/TCF factors i.e. TCF1, whereas β-catenin activity plays a subordinate role. Together these data suggest that LEF1 rather has tumor suppressive functions and attenuates aggressiveness in a subset of RMS.

Myf5 and Myogenin in the development of thymic myoid cells — Implications for a murine in vivo model of myasthenia gravis

Myasthenia gravis (MG) is caused by autoantibodies against the neuromuscular junction of striated muscle. Most MG patients have autoreactive T- and B-cells directed to the acetylcholine receptor (AChR). To achieve immunologic tolerance, developing thymocytes are normally eliminated after recognition of self-antigen-derived peptides. Presentation of muscle-specific antigens is likely achieved through two pathways: on medullary thymic epithelial cells and on medullary dendritic cells cross-presenting peptides derived from a unique population of thymic myoid cells (TMC). Decades ago, it has been hypothesized that TMC play a key role in the induction of immunological tolerance towards skeletal muscle antigens. However, an experimental model to address this postulate has not been available. To generate such a model, we tested the hypothesis that the development of TMC depends on myogenic regulatory factors. To this end, we utilized Myf5-deficient mice, which lack the first wave of muscle cells but form normal skeletal muscles later during development, and Myogenin-deficient mice, which fail to form differentiated myofibers. We demonstrate for the first time that Myf5- and Myogenin-deficient mice showed a partial or complete, respectively, loss of TMC in an otherwise regularly structured thymus. To overcome early postnatal lethality of muscle-deficient, Myogenin-knockout mice we transplanted Myogenin-deficient fetal thymuses into Foxn1(nu/nu) mice that lack their own thymus anlage. We found that the transplants are functional but lack TMC. In combination with established immunization strategies (utilizing AChR or Titin), this model should enable us in the future testing the hypothesis that TMC play an indispensable role in the development of central tolerance towards striated muscle antigens.

Characterization of an anti-fetal AChR monoclonal antibody isolated from a myasthenia gravis patient

We report here the sequence and functional characterization of a recombinantly expressed autoantibody (mAb 131) previously isolated from a myasthenia gravis patient by immortalization of thymic B cells using Epstein-Barr virus and TLR9 activation. The antibody is characterized by a high degree of somatic mutations as well as a 6 amino acid insertion within the VHCDR2. The recombinant mAb 131 is specific for the γ-subunit of the fetal AChR to which it bound with sub-nanomolar apparent affinity, and detected the presence of fetal AChR on a number of rhabdomyosarcoma cell lines. Mab 131 blocked one of the two α-bungarotoxin binding sites on the fetal AChR, and partially blocked the binding of an antibody (mAb 637) to the α-subunit of the AChR, suggesting that both antibodies bind at or near one ACh binding site at the α/γ subunit interface. However, mAb 131 did not reduce fetal AChR ion channel currents in electrophysiological experiments. These results indicate that mAb 131, although generated from an MG patient, is unlikely to be pathogenic and may make it a potentially useful reagent for studies of myasthenia gravis, rhabdomyosarcoma and arthrogryposis multiplex congenita which can be caused by fetal-specific AChR-blocking autoantibodies.