Neue in vitro und in vivo Modelle für Kaposi-Sarkome zur Untersuchung der Viruserhaltung und zur Validierung von antiviralen Substanzen

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi’s sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of antiviral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study conditionally immortalized human endothelial HuARLT cells were validated as a system to study KSHV-induced pathogenesis and testing of novel antiviral drugs. Systematic analysis of cellular marker expression as well as cellular functions in 2D and 3D cell culture conditions showed that HuARLT cells closely mimic KSHV infection of primary cells. A humanized mouse model for KSHV based on the cell line develop lesions which histologically resemble KS. Importantly, invasive properties and tumor formation were completely reverted by purging cells from KSHV confirming for the first time that tumor formation is primarily dependent on viral infection, rather than being a consequence of irreversible transformation of the infected cells. The cell line was used to investigate the molecular mechanisms of KSHV maintenance in endothelial cells. This study proved that lack of cell proliferation is not sufficient to maintain the episomal virus, as was believed before. Rather, it demonstrated that 3D culture conditions (in vitro or in vivo) are required for efficient viral maintenance. Transcriptome analysis and pharmacological studies showed that increased activity of the PI3K/mTOR pathway governs viral maintenance in 3D cell culture. A subset of 26 compounds from a natural compound library was screened for reducing the episomal viral copy number and the tumorigenic properties of KSHV-infected cells in 2D and 3D in vitro cell culture systems. Selected compounds were subsequently tested for the ability to reduce the tumor size in xenotransplanted mice. Based on the assay three drug candidates showed significant tumor reduction. Thus, this study shows that the combined use of antiviral and antitumor assays based on the same cell system is indicative for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi’s sarcoma.Das Kaposi-Sarkom assoziierte Herpesvirus (KSHV) ist ein humanspezifischer Krankheitserreger, der zur Bildung von Kaposi-Sarkomen (KS) führt. Bis heute gibt es keine Impfstoffe und keine gezielte Therapie gegen KS. Die Identifizierung von antiviralen Substanzen wird dadurch erschwert, dass es keine skalierbaren Zellkultursysteme gibt, die die Eigenschaften der Virus-transformierten Patientenzellen widerspiegeln. Weiterhin verhindert der enge Wirtstropismus die Entwicklung von in vivo Modellsystemen. In der vorliegenden Studie wurden konditional immortalisierte humane HuARLT-Endothelzellen zur Untersuchung der durch KSHV ausgelösten Pathogenese sowie für die Testung neuer antiviraler Substanzen validiert. Die systematische Analyse der KSHV-infizierten HuARLT-Zellen unter 2D- und 3D- Kultur¬bedingungen zeigte, dass sie die Eigenschaften und Funktionen von infizierten primären Zellen sehr gut reflektieren. Ein durch Xenotransplantation der infizierten Zellen generiertes humanisiertes Mausmodell zeigte die Ausbildung von Läsionen, deren histologische Charakterisierung große Übereinstimmung mit KS aufwies. Die Zelllinie wurde eingesetzt, um die molekularen Mechanismen des Erhalts des Virus in Endothelzellen zu untersuchen. Dies zeigte, dass die Unterbindung der Zellproliferation nicht ausreicht, um den in vitro beobachteten Verlust des episomalen Virusgenoms zu verhindern, wodurch eine langjährige Hypothese widerlegt ist. Vielmehr konnte gezeigt werden, dass 3D-Kulturbedingungen (in vitro, aber auch in vivo) die Erhaltung des Virus unterstützen. Transkriptomanalysen und begleitende Inhibitionsstudien deckten auf, dass der PI3K/mTor-Signalweg essentiell für den Erhalt des Virus in 3D Zellkultur ist. Aus einer Wirkstoffbank wurden 26 Moleküle in vitro in 2D und 3D Zellkultursystemen bezüglich ihrer Fähigkeit untersucht, die Zahl der episomalen Virusgenome sowie die tumorigenen Eigenschaften der KSHV-infizierten Zellen zu reduzieren. Ausgewählte Wirkstoffe wurden dann bezüglich ihrer Fähigkeit getestet, die virusinduzierten Läsionen im Mausmodell zu verringern. Drei neue Wirkstoffkandidaten schränkten das Tumorwachstum im Mausmodell signifikant ein. Zusammenfassend zeigt diese Studie, dass der kombinierte Einsatz der in dieser Arbeit entwickelten Nachweissysteme es erlaubt, die Viruspathogenese zu untersuchen und auch antivirale und antitumorigene Wirkstoffe zu identifizieren, die die Tumorbildung in vivo mit hoher Effizienz reduzieren. Somit bieten sich diese Testsysteme für die Wirkstoffsuche gegen KS an

Targeting Kaposi's Sarcoma-Associated Herpesvirus ORF21 Tyrosine Kinase and Viral Lytic Reactivation by Tyrosine Kinase Inhibitors Approved for Clinical Use

Kaposi's Sarcoma-associated herpesvirus (KSHV) is the cause of three human malignancies, Kaposi's Sarcoma, Primary Effusion Lymphoma and the plasma cell variant of Multicentric Castleman's Disease. Previous research has shown that several cellular tyrosine kinases play crucial roles during several steps in the virus replication cycle. Two KSHV proteins also have protein kinase function: open reading frame (ORF) 36 encodes a serin-threonine kinase, while ORF21 encodes a thymidine kinase (TK), which has recently been found to be an efficient tyrosine kinase. In this study, we explore the role of the ORF21 tyrosine kinase function in KSHV lytic replication. By generating a recombinant KSHV mutant with an enzymatically inactive ORF21 protein we show that the tyrosine kinase function of ORF21/TK is not required for the progression of the lytic replication in tissue culture, but that it is essential for the phosphorylation and activation to toxic moieties of the antiviral drugs zidovudine and brivudine. In addition, we identify several tyrosine kinase inhibitors, already in clinical use against human malignancies, which potently inhibit not only ORF21 TK kinase function, but also viral lytic reactivation and the development of KSHV-infected endothelial tumors in mice. As they target both cellular tyrosine kinases and a viral kinase, some of these compounds might find a use in the treatment of KSHV-associated malignancies.Importance: Our findings address the role of KSHV ORF21 as a tyrosine kinase during lytic replication and the activation of prodrugs in KSHV-infected cells. We also show the potential of selected clinically approved tyrosine kinase inhibitors to inhibit KSHV TK, KSHV lytic replication, infectious virions release and the development of an endothelial tumor. Since they target both cellular tyrosine kinases supporting productive viral replication and a viral kinase, these drugs, which are already approved for clinical use, may be suitable for repurposing for the treatment of KSHV-related tumors in AIDS patients or transplant recipients

3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance

Macrophage entrapped silica coated superparamagnetic iron oxide particles for controlled drug release in a 3D cancer model.

Targeted delivery of drugs is a major challenge in treatment of diverse diseases. Systemically administered drugs demand high doses and are accompanied by poor selectivity and side effects on non-target cells. Here, we introduce a new principle for targeted drug delivery. It is based on macrophages as transporters for nanoparticle-coupled drugs as well as controlled release of drugs by hyperthermia mediated disruption of the cargo cells and simultaneous deliberation of nanoparticle-linked drugs. Hyperthermia is induced by an alternating electromagnetic field (AMF) that induces heat from silica-coated superparamagnetic iron oxide nanoparticles (SPIONs). We show proof-of-principle of controlled release by the simultaneous disruption of the cargo cells and the controlled, AMF induced release of a toxin, which was covalently linked to silica-coated SPIONs via a thermo-sensitive linker. Cells that had not been loaded with SPIONs remain unaffected. Moreover, in a 3D co-culture model we demonstrate specific killing of associated tumour cells when employing a ratio as low as 1:40 (SPION-loaded macrophage: tumour cells). Overall, our results demonstrate that AMF induced drug release from macrophage-entrapped nanoparticles is tightly controlled and may be an attractive novel strategy for targeted drug release

An endothelial cell line infected by Kaposi's sarcoma-associated herpes virus (KSHV) allows the investigation of Kaposi's sarcoma and the validation of novel viral inhibitors in vitro and in vivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of anti-viral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus-transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study, we exploited a conditionally immortalized human endothelial cell line for establishment of in vitro 2D and 3D KSHV latency models and the generation of KS-like xenograft tumors in mice. Importantly, the invasive properties and tumor formation could be completely reverted by purging KSHV from the cells, confirming that tumor formation is dependent on the continued presence of KSHV, rather than being a consequence of irreversible transformation of the infected cells. Upon testing a library of 260 natural metabolites, we selected the compounds that induced viral loss or reduced the invasiveness of infected cells in 2D and 3D endothelial cell culture systems. The efficacy of selected compounds against KSHV-induced tumor formation was verified in the xenograft model. Together, this study shows that the combined use of anti-viral and anti-tumor assays based on the same cell line is predictive for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi's sarcoma. KEY MESSAGES: Novel 2D, 3D, and xenograft mouse models mimic the consequences of KSHV infection. KSHV-induced tumorigenesis can be reverted upon purging the cells from the virus. A 3D invasiveness assay is predictive for tumor reduction in vivo. Chondramid B, epothilone B, and pretubulysin D diminish KS-like lesions in vivo