18 research outputs found

    Charakterisierung von CD4+ T-Zellen nach Priming und Boosting durch auf primären uvealen Melanom-Zellen basierenden MHCII-Vakzinen

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    Uveal melanoma is the most common primary malignancy of the eye in adults. Despite significant improvements in treatment of the primary tumor, to date none of these therapies prevent metastatic disease or improve overall survival. We are exploring immunotherapeutic options for metastatic uveal melanoma using MHC II uveal melanoma cell-based vaccines that target the activation of tumor-reactive CD4+ T cells. Previously, we showed that these uveal melanoma cell-based vaccines activate CD4+ T cells within total peripheral blood lymphocytes (PBMC). Since PBMC include professional antigen presenting cells, we now demonstrate that Mel202/DR1/CD80 vaccine cells directly activate a diverse repertoire of purified, naïve CD4+ T cells. The activated CD4+ T cells proliferated, secreted high amounts of interferon gamma (IFNγ) and produced a heterogeneous profile of Th1, Th2 and Th17 cytokines. Analysis of the TCR-Vβ-repertoire showed that a polyclonal T cell response was induced, suggesting the capacity of vaccine-activated CD4+ T cells to target multiple tumor (neo)antigens. In addition, a subset of the responding CD4+ T cells expressed forkhead box protein P3 (FoxP3), indicating that although a regulatory component of the vaccine-activated CD4+ T cell response was induced, the anti-tumor vaccine response was not limited by these regulatory CD4+ T cells. Finally, Mel202/DR1/CD80 uveal melanoma vaccine cells expressed the intercellular adhesion molecule 1 (ICAM-1) that was pivotal for CD4+ T cell activation via lymphocyte function-associated antigen 1(LFA-1). In conclusion, MHC II uveal melanoma vaccines activate purified CD4+ T cells and may serve as a novel immunotherapy for uveal melanoma patients

    Molecular insights and emerging strategies for treatment of metastatic uveal melanoma

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    Uveal melanoma (UM) is the most common intraocular cancer. In recent decades, major advances have been achieved in the diagnosis and prognosis of UM allowing for tailored treatments. However, nearly 50% of patients still develop metastatic disease with survival rates of less than 1 year. There is currently no standard of adjuvant and metastatic treatment in UM, and available therapies are ineffective resulting from cutaneous melanoma protocols. Advances and novel treatment options including liver-directed therapies, immunotherapy, and targeted-therapy have been investigated in UM-dedicated clinical trials on single compounds or combinational therapies, with promising results. Therapies aimed at prolonging or targeting metastatic tumor dormancy provided encouraging results in other cancers, and need to be explored in UM. In this review, the latest progress in the diagnosis, prognosis, and treatment of UM in adjuvant and metastatic settings are discussed. In addition, novel insights into tumor genetics, biology and immunology, and the mechanisms underlying metastatic dormancy are discussed. As evident from the numerous studies discussed in this review, the increasing knowledge of this disease and the promising results from testing of novel individualized therapies could offer future perspectives for translating in clinical use

    Role of Fibroblast Growth Factors in the Crosstalk of Hepatic Stellate Cells and Uveal Melanoma Cells in the Liver Metastatic Niche

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    Hepatic metastasis is the critical factor determining tumor-associated mortality in different types of cancer. This is particularly true for uveal melanoma (UM), which almost exclusively metastasizes to the liver. Hepatic stellate cells (HSCs) are the precursors of tumor-associated fibroblasts and support the growth of metastases. However, the underlying mechanisms are widely unknown. Fibroblast growth factor (FGF) signaling is dysregulated in many types of cancer. The aim of this study was to analyze the pro-tumorigenic effects of HSCs on UM cells and the role of FGFs in this crosstalk. Conditioned medium (CM) from activated human HSCs significantly induced proliferation together with enhanced ERK and JNK activation in UM cells. An in silico database analysis revealed that there are almost no mutations of FGF receptors (FGFR) in UM. However, a high FGFR expression was found to be associated with poor survival for UM patients. In vitro, the pro-tumorigenic effects of HSC-CM on UM cells were abrogated by a pharmacological inhibitor (BGJ398) of FGFR1/2/3. The expression analysis revealed that the majority of paracrine FGFs are expressed by HSCs, but not by UM cells, including FGF9. Furthermore, the immunofluorescence analysis indicated HSCs as a cellular source of FGF9 in hepatic metastases of UM patients. Treatment with recombinant FGF9 significantly enhanced the proliferation of UM cells, and this effect was efficiently blocked by the FGFR1/2/3 inhibitor BGJ398. Our study indicates that FGF9 released by HSCs promotes the tumorigenicity of UM cells, and thus suggests FGF9 as a promising therapeutic target in hepatic metastasis

    Bioengineering strategies for cancer therapy and modelling

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    Tese de doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células EstaminaisCancer is a global pandemic with a high incidence among the world population and effective treatments are for the most part elusive. The tumor microenvironment is a highly complex and heterotypic mixture of cells that interact to regulate central control mechanisms, driving immunosuppression and promoting both survival and invasion of cancer cells into surrounding tissues. It has been this complexity that has made finding effective therapeutics such a demanding task and therefore cancer still remains a burden worldwide in health as well as in economic terms. While the progression in the field of cancer research has been clear over the years, there are still several challenges that need to be addressed. Herein, two different sides to this disease are explored: treatment and in vitro models. Adoptive T cell therapy has shown impressive results, however not without its limitations. The use of the T cell mitogen IL-2 within culture systems is known to lead to early exhaustion of T cell subsets while high density of co-stimulating molecules has been linked to undesired immune responses. As an alternative, a nanoparticle system based on the natural polymer gellan-gum was proposed, with tailorable surface functionalization with co-stimulatory molecules. High levels of T cell expansion were observed over the studied period, with secreted IL-2 levels overcoming those of commercial alternatives. With this system, increased expression of cytotoxic molecules Granzyme B and Perforin were also detected in vitro. On the other spectrum, 3D cancer models have sustained a great number of developments observed by an increase in similarity towards native tissues; however, a requirement for even more complex architectures capable of better mimicking cellular interactions is still present. Therefore, an assembloid-based approach was proposed to develop a 3D in vitro melanoma model to further study cellular interactions. These heterotypic tumor assembloids presented a complex architecture capable of sustaining endothelial cell function as well as a high expression of stemness-related markers. These models were subjected to functionality assays where they showed a capacity for “cooperative invasion” which was coincident with an observed increased production of MMP-2. To further unravel the role of stromal cells in the invasive potential of cancer cells a 3D chemotaxis chamber was developed to study cellular interactions observed in the tumor microenvironment, where stem cells and fibroblasts showed to have a crucial role. Ultimately, this thesis allowed to explore biomedical engineering approaches to further contribute to the knowledge in the field opening new doors to be explored in the future.O cancro é uma pandemia global com uma elevada incidência e cujo desenvolvimento de tratamentos eficazes continua a ser difícil. O microambiente tumoral é uma mistura altamente complexa e heterotípica de células que interagem para regular mecanismos centrais que provocam imunossupressão promovendo a sobrevivência e invasão de células tumorais para os tecidos circundantes. É esta complexidade que tem tornado desafiante encontrar terapias eficazes, tornando esta doença um fardo a nível global em termos de saúde e economia. Enquanto a progressão na área da investigação oncológica tem sido clara ao longo dos anos, existem ainda vários desafios que precisam de serem encarados para permitir futuros desenvolvimentos. Aqui, foram exploradas duas vertentes diferentes desta doença: o tratamento e os modelos in vitro. A terapia celular adotiva tem demonstrado resultados impressionantes, no entanto não sem as suas limitações. O uso do mitogénio IL-2 nestes sistemas de cultura é conhecido por levar rapidamente à exaustão das células T, enquanto o uso de moléculas co-estimulatórias em elevadas densidades está associado a respostas imunes não desejadas. Como alternativa, foi proposto um sistema de nanopartículas baseado no polímero natural goma gelana e funcionalizado com moléculas co estimulatórias. Foram observados elevados níveis de expansão de células T e quantidade de IL-2 secretada superior à de alternativas comerciais. Foi ainda verificado in vitro um aumento de expressão das moléculas citotóxicas Grazima B e Perforina. No outro espectro, têm sido desenvolvidos modelos tumorais 3D com uma cada vez maior similaridade para tecidos nativos; no entanto, a necessidade de arquiteturas ainda mais complexas capazes de melhor representar interações celulares persiste. Assim, foi proposta uma abordagem baseada em “assemblóides” para obter modelos 3D in vitro de melanoma para estudar interações celulares. Estes “assemblóides” tumorais heterotípicos apresentaram uma arquitetura complexa capaz de suportar a função de células endoteliais, bem como a elevada expressão de marcadores de pluripotência. Estes modelos foram sujeitos a ensaios de funcionalidade onde mostraram a capacidade de “invasão cooperativa” que foi coincidente com uma produção aumentada de MMP-2. Para tornar mais claro o papel das células estaminais no potencial invasivo de células tumorais, uma câmara 3D de quimiotaxia foi desenvolvida para estudar as interações celulares observadas no microambiente tumoral onde as células estaminais e fibroblastos mostraram ter um papel determinante. Em última análise, esta tese permitiu explorar abordagens da engenharia biomédica de forma a contribuir para o conhecimento da área e abrir novas portas a serem exploradas no futuro

    Advances and Novel Treatment Options in Metastatic Melanoma

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    The book presents several studies reporting advances on melanoma pathogenesis, diagnosis and therapy. It represents a milestone on the state of the art, updated at 2021, and also presents the current knowledge on the future developments in melanoma field

    Zielgerichtetes transkriptionelles Ansprechen von Dendritischen Zellen als Ansatz für eine therapeutische Impfung gegen HIV-1

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    According to the World Health Organisation in 2021, more than 38 million people were living with the Human Immunodeficiency Virus (HIV). While antiretroviral therapy (ART) was a breakthrough in preventing progression into the acquired immunodeficiency syndrome (AIDS), there is still no cure available and traditional vaccination attempts have shown limited effects. More recently, dendritic cell (DC)-based vaccination strategies have sparked interest in controlling HIV infection. Targeting DCs for vaccination is particularly promising as they are extraordinarily potent inducers of strong and broad T cell responses. Currently, most DC vaccination attempts focus on cumbersome and expensive in vitro transduction of cells. Vectors targeting DCs in vivo would facilitate up-scaling and vaccine availability. Achieving these benefits without compromising on safety or risk pathogen tolerance requires new tactics to ensure targeting specifically only mature T cell stimulating DCs (mDCs). To address this challenge, this project aimed to investigate transcriptional targeting utilising the status-specific activity of the CD83 promoter to limit expression of HIV-1 antigens to mature DCs. We strived to create vectors based on the adenovirus serotype 5 (HAdV-5) that encode for HIV gag, a HIV gene as an antigen source, and a constitutive active variant of the IκB kinase (caIKK), an immune-stimulatory molecule to boost the DC activity. Expression of both those genes was controlled by the CD83 promoter. Before, however, we had to tackle limited transduction efficiency of DCs with HAdV-5. Towards the latter, LentiBOOST®/Polybrene (LeB/PB) significantly improved the transduction rate of human DCs. Additionally, LeB/PB enabled adenoviral transduction of human monocytes and murine bone marrow-derived DCs. Pivotally, cell viability, morphology and function was maintained in all assessed cell types despite exposure to the transduction enhancer. Next, we used the CD83 promoter to co-express two proteins within the same vector. By cloning the promoter twice into the vector or alternatively introducing an internal ribosomal entry site (IRES) and using fluorescent genes as placeholders, we created two vectors with comparable protein expression levels that provided the basis for the generation of HAdV-5 vectors for potential future use as vaccines: HAdV-5-IRES-gag-caIKK and HAdV-5-2xP-gag-caIKK (containing the CD83 promoter twice). qPCR confirmed protein expression to be restricted to mature DCs. DCs transduced with both viral vectors stimulated autologous T cell activity independently of the expression of antigen-specific T cells, as was demonstrated by upregulation of CD69 and increased production of pro-inflammatory cytokines including IL-6, IL-8, IL-12p70, and in addition IL-10. These results provide the basis in order to use the CD83 promoter to develop DC-based gene therapy against HIV-1.Im Jahr 2021 lebten nach Angaben der Weltgesundheitsorganisation mehr als 38 Millionen Menschen mit dem Humanen Immundefizienz-Virus (HIV). Während antiretrovirale Therapie ein Durchbruch für die Prävention des Fortschreitens der Erkrankung hin zum Erworbenen Immunschwäche-Synrom (Acquired Immunodeficiency Syndrome, AIDS) war, ist eine Heilung noch immer nur in Einzelfällen möglich und herkömmliche Impfversuche zeigen bislang nur begrenzt Wirkung. Impfstrategien gegen HIV-1 auf der Grundlage dendritischer Zellen (DCs) sorgten in den letzten Jahren vermehrt für Aufmerksamkeit. DC-basierte Impfstrategien gelten als vielversprechend, da sie außergewöhnlich starke und vielfältige T-Zell-Reaktionen auslösen können. Die meisten DC-Impfversuche setzen derzeit auf eine umständliche und teure in-vitro-Transduktion von Zellen, obwohl in vivo-einsetzbare Vektoren die Anwendung und Verfügbarkeit des Impfstoffs vereinfachen könnten. Um diese Vorteile zu erreichen, ohne die Sicherheit zu beeinträchtigen oder die Toleranz gegenüber Krankheitserregern zu riskieren, sind neue Taktiken erforderlich, die sicherstellen, dass ausschließlich reife DCs Antigene präsentieren. Dementsprechend wurde in diesem Projekt das transkriptionelle Targeting untersucht, um die Expression von HIV-1-Antigenen mit Hilfe der stadiums-spezifischen Aktivität des CD83-Promotors auf reife DCs zu beschränken. Adenovirus-Serotyp-5 (HAdV-5)-basierende Vektoren wurden entwickelt, die HIV gag als Antigen-Quelle und eine konstitutiv aktive Variante der IκB-Kinase (caIKK) als immunstimulierendes Molekül zur Steigerung der DC-Aktivität kodieren. Die Expression dieser beiden Gene wird durch den CD83-Promotor gesteuert. Mit LentiBOOST®/Polybrene (LeB/PB) konnte die Transduktionsrate humaner DCs mit HAdV-5-Vektoren deutlich verbessert werden. Außerdem ermöglichte LeB/PB die adenovirale Transduktion von humanen Monozyten und murinen DCs, ohne Viabilität, Morphologie und Funktion der Zellen einzuschränken. Die gleichzeitige Expression zweier Proteine unter der Kontrolle des CD83 Promoters gelang mit Hilfe einer internen ribosomalen Eintrittsstelle (IRES) oder durch zweifaches Einsetzen des Promotors. Fluoreszierende Proteine als Platzhalter für HIV-1 gag und caIKK demonstrierten ähnliche Level der Proteinexpression für beide Vektoren. Darauf basierend wurden Vektoren entwickelt, die die Grundlage für die Erzeugung von HAdV-5-Vektoren für eine mögliche zukünftige Verwendung als Impfstoffe bilden: HAdV-5-IRES-gag-caIKK und HAdV-5-2xP-gag-caIKK (mit zweimaligem CD83-Promotor). Die Restriktion der Genexpression auf reife DCs wurde mittles qPCR bestätigt. DCs, die mit einem der beiden viralen Vektoren transduziert wurden, stimulierten die Aktivität autologer T-Zellen unabhängig von der Expression antigenspezifischer T-Zellen. Dies wurde durch die Hochregulierung von CD69 und die erhöhte Produktion von pro-inflammatorischen Zytokinen wie IL-6, IL-8, IL-12p70 und IL-10 nachgewiesen. Diese Ergebnisse stellen die Basis zur Nutzung des CD83-Promotors für die Entwicklung einer DC-basierten Gentherapie gegen HIV-1 dar

    Glioblastoma: multifaceted immunosuppression mediated through galectin family members

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    A thesis submitted to the University of Wolverhampton in partial fulfilment of the requirements for awarding the degree of Doctor of Philosophy.Glioblastoma (GBM) is an aggressive brain cancer with a near-uniformly lethal prognosis. Anti-tumoural immunity remains low due to a highly suppressive tumour microenvironment. This project set out to identify immunomodulatory proteins secreted by glioblastoma, describe the distribution of intratumoural immune cells, and characterise the effects of secreted immunomodulatory on adaptive immune effector cells. Mass spectrometry data identified galectin family members in the glioblastoma secretome. ELISA assays confirmed galectin presence in GBM-derived liquid samples; immunofluorescence confirmed their presence in tumour sections, as well as the presence of T-cells, B-cells and macrophages. Changes in cell surface marker expression and phagocytosis following exposure to recombinant proteins were assayed using flow cytometry and pHrodo-conjugated Escherichia coli respectively. B-cells and macrophages were predominantly found in tumour bulk (p=0.0053 and 0.0087) with T-cells located in the perivascular niche (p=0.0932). Additionally, 22% of tumours contained T-cell aggregations. Galectins 1, 3, 4 and 7 were also found in the majority of GBM assayed; immune checkpoints galectin-9 and PD-L1 were found in 100% and 50% of assayed tumours, respectively. The perivascular location of galectin-1 approached significance (p=0.0844), whereas other galectins showed a more equitable distribution. Consistent, though insignificant, downregulation of MHCII and phagocytosis was observed in pro-inflammatory adult macrophages exposed to GBM-relevant galectin-1 concentrations. These results suggested a T-cell-specific mechanism of suppression resulting in reduced infiltration, whereas B-cells and macrophages were able to effectively infiltrate GBM. Antigen presentation by macrophages may be inhibited by perivascular galectin-1, thus limiting T-cell retention; encounter with known immunosuppressive proteins such as galectins 1, 3, 9 and PD-L1 following effector extravasation may be a key mechanism through which GBM immunosuppression is mediated. These results posit the perivascular region as a uniquely immunosuppressive environment, and the presence of galectins as a potential key mediator of immunosuppression within glioblastoma.The Colin Oliphant Charitable Trus
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