8,229 research outputs found

    T-cell receptor determinants of response to chemoradiation in locally-advanced HPV16-driven malignancies

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    BackgroundThe effect of chemoradiation on the anti-cancer immune response is being increasingly acknowledged; however, its clinical implications in treatment responses are yet to be fully understood. Human papillomavirus (HPV)-driven malignancies express viral oncogenic proteins which may serve as tumor-specific antigens and represent ideal candidates for monitoring the peripheral T-cell receptor (TCR) changes secondary to chemoradiotherapy (CRT).MethodsWe performed intra-tumoral and pre- and post-treatment peripheral TCR sequencing in a cohort of patients with locally-advanced HPV16-positive cancers treated with CRT. An in silico computational pipeline was used to cluster TCR repertoire based on epitope-specificity and to predict affinity between these clusters and HPV16-derived epitopes.ResultsIntra-tumoral repertoire diversity, intra-tumoral and post-treatment peripheral CDR3β similarity clustering were predictive of response. In responders, CRT triggered an increase peripheral TCR clonality and clonal relatedness. Post-treatment expansion of baseline peripheral dominant TCRs was associated with response. Responders showed more baseline clustered structures of TCRs maintained post-treatment and displayed significantly more maintained clustered structures. When applying clustering by TCR-specificity methods, responders displayed a higher proportion of intra-tumoral TCRs predicted to recognise HPV16 peptides.ConclusionsBaseline TCR characteristics and changes in the peripheral T-cell clones triggered by CRT are associated with treatment outcome. Maintenance and boosting of pre-existing clonotypes are key elements of an effective anti-cancer immune response driven by CRT, supporting a paradigm in which the immune system plays a central role in the success of CRT in current standard-of-care protocols

    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Converging organoids and extracellular matrix::New insights into liver cancer biology

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    Primary liver cancer, consisting primarily of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a heterogeneous malignancy with a dismal prognosis, resulting in the third leading cause of cancer mortality worldwide [1, 2]. It is characterized by unique histological features, late-stage diagnosis, a highly variable mutational landscape, and high levels of heterogeneity in biology and etiology [3-5]. Treatment options are limited, with surgical intervention the main curative option, although not available for the majority of patients which are diagnosed in an advanced stage. Major contributing factors to the complexity and limited treatment options are the interactions between primary tumor cells, non-neoplastic stromal and immune cells, and the extracellular matrix (ECM). ECM dysregulation plays a prominent role in multiple facets of liver cancer, including initiation and progression [6, 7]. HCC often develops in already damaged environments containing large areas of inflammation and fibrosis, while CCA is commonly characterized by significant desmoplasia, extensive formation of connective tissue surrounding the tumor [8, 9]. Thus, to gain a better understanding of liver cancer biology, sophisticated in vitro tumor models need to incorporate comprehensively the various aspects that together dictate liver cancer progression. Therefore, the aim of this thesis is to create in vitro liver cancer models through organoid technology approaches, allowing for novel insights into liver cancer biology and, in turn, providing potential avenues for therapeutic testing. To model primary epithelial liver cancer cells, organoid technology is employed in part I. To study and characterize the role of ECM in liver cancer, decellularization of tumor tissue, adjacent liver tissue, and distant metastatic organs (i.e. lung and lymph node) is described, characterized, and combined with organoid technology to create improved tissue engineered models for liver cancer in part II of this thesis. Chapter 1 provides a brief introduction into the concepts of liver cancer, cellular heterogeneity, decellularization and organoid technology. It also explains the rationale behind the work presented in this thesis. In-depth analysis of organoid technology and contrasting it to different in vitro cell culture systems employed for liver cancer modeling is done in chapter 2. Reliable establishment of liver cancer organoids is crucial for advancing translational applications of organoids, such as personalized medicine. Therefore, as described in chapter 3, a multi-center analysis was performed on establishment of liver cancer organoids. This revealed a global establishment efficiency rate of 28.2% (19.3% for hepatocellular carcinoma organoids (HCCO) and 36% for cholangiocarcinoma organoids (CCAO)). Additionally, potential solutions and future perspectives for increasing establishment are provided. Liver cancer organoids consist of solely primary epithelial tumor cells. To engineer an in vitro tumor model with the possibility of immunotherapy testing, CCAO were combined with immune cells in chapter 4. Co-culture of CCAO with peripheral blood mononuclear cells and/or allogenic T cells revealed an effective anti-tumor immune response, with distinct interpatient heterogeneity. These cytotoxic effects were mediated by cell-cell contact and release of soluble factors, albeit indirect killing through soluble factors was only observed in one organoid line. Thus, this model provided a first step towards developing immunotherapy for CCA on an individual patient level. Personalized medicine success is dependent on an organoids ability to recapitulate patient tissue faithfully. Therefore, in chapter 5 a novel organoid system was created in which branching morphogenesis was induced in cholangiocyte and CCA organoids. Branching cholangiocyte organoids self-organized into tubular structures, with high similarity to primary cholangiocytes, based on single-cell sequencing and functionality. Similarly, branching CCAO obtain a different morphology in vitro more similar to primary tumors. Moreover, these branching CCAO have a higher correlation to the transcriptomic profile of patient-paired tumor tissue and an increased drug resistance to gemcitabine and cisplatin, the standard chemotherapy regimen for CCA patients in the clinic. As discussed, CCAO represent the epithelial compartment of CCA. Proliferation, invasion, and metastasis of epithelial tumor cells is highly influenced by the interaction with their cellular and extracellular environment. The remodeling of various properties of the extracellular matrix (ECM), including stiffness, composition, alignment, and integrity, influences tumor progression. In chapter 6 the alterations of the ECM in solid tumors and the translational impact of our increased understanding of these alterations is discussed. The success of ECM-related cancer therapy development requires an intimate understanding of the malignancy-induced changes to the ECM. This principle was applied to liver cancer in chapter 7, whereby through a integrative molecular and mechanical approach the dysregulation of liver cancer ECM was characterized. An optimized agitation-based decellularization protocol was established for primary liver cancer (HCC and CCA) and paired adjacent tissue (HCC-ADJ and CCA-ADJ). Novel malignancy-related ECM protein signatures were found, which were previously overlooked in liver cancer transcriptomic data. Additionally, the mechanical characteristics were probed, which revealed divergent macro- and micro-scale mechanical properties and a higher alignment of collagen in CCA. This study provided a better understanding of ECM alterations during liver cancer as well as a potential scaffold for culture of organoids. This was applied to CCA in chapter 8 by combining decellularized CCA tumor ECM and tumor-free liver ECM with CCAO to study cell-matrix interactions. Culture of CCAO in tumor ECM resulted in a transcriptome closely resembling in vivo patient tumor tissue, and was accompanied by an increase in chemo resistance. In tumor-free liver ECM, devoid of desmoplasia, CCAO initiated a desmoplastic reaction through increased collagen production. If desmoplasia was already present, distinct ECM proteins were produced by the organoids. These were tumor-related proteins associated with poor patient survival. To extend this method of studying cell-matrix interactions to a metastatic setting, lung and lymph node tissue was decellularized and recellularized with CCAO in chapter 9, as these are common locations of metastasis in CCA. Decellularization resulted in removal of cells while preserving ECM structure and protein composition, linked to tissue-specific functioning hallmarks. Recellularization revealed that lung and lymph node ECM induced different gene expression profiles in the organoids, related to cancer stem cell phenotype, cell-ECM integrin binding, and epithelial-to-mesenchymal transition. Furthermore, the metabolic activity of CCAO in lung and lymph node was significantly influenced by the metastatic location, the original characteristics of the patient tumor, and the donor of the target organ. The previously described in vitro tumor models utilized decellularized scaffolds with native structure. Decellularized ECM can also be used for creation of tissue-specific hydrogels through digestion and gelation procedures. These hydrogels were created from both porcine and human livers in chapter 10. The liver ECM-based hydrogels were used to initiate and culture healthy cholangiocyte organoids, which maintained cholangiocyte marker expression, thus providing an alternative for initiation of organoids in BME. Building upon this, in chapter 11 human liver ECM-based extracts were used in combination with a one-step microfluidic encapsulation method to produce size standardized CCAO. The established system can facilitate the reduction of size variability conventionally seen in organoid culture by providing uniform scaffolding. Encapsulated CCAO retained their stem cell phenotype and were amendable to drug screening, showing the feasibility of scalable production of CCAO for throughput drug screening approaches. Lastly, Chapter 12 provides a global discussion and future outlook on tumor tissue engineering strategies for liver cancer, using organoid technology and decellularization. Combining multiple aspects of liver cancer, both cellular and extracellular, with tissue engineering strategies provides advanced tumor models that can delineate fundamental mechanistic insights as well as provide a platform for drug screening approaches.<br/

    Investigating the molecular drivers of CNS disease in a murine model of infant leukaemia

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    Infant leukaemia is a rare, aggressive entity with poor outcomes. Infant leukaemia is defined by the age of the patient, less than one year, and is commonly associated with rearrangements of the MLL gene. There are many unique features that distinguishMLLrearranged infant leukaemia from other paediatric leukaemias, including a high rate of central nervous system (CNS) involvement. CNS involvement is typically a leukaemic infiltrate of themeninges, a niche that is very different fromthe primary site of disease, the bone marrow (BM), in terms of nutrient abundance and cellular composition. While our understanding of how leukaemia cells survive and propagate in the CNS has progressed, there are many unanswered questions about infant leukaemia-specific features and the contribution of the niche. This thesis explores the cellular dynamics of leukaemia in the CNS niche, the differential regulation of immune cell interactions and growth factor pathways through transplantation assays, bulk RNA sequencing and functional experiments. In previous studies, two microRNAs (miR-128a and miR-130b) were identified as being upregulated in MLL-AF4+ infant patient samples. Overexpression of these microRNAs individually in mouse fetal liver haematopoietic stem and progenitor cells resulted in microRNA-dependent lineage-specific acute leukaemias in the context of Mll- AF4. Terminal leukaemia development in these immunocompetent models of infant leukaemia was associated with CNS involvement in a leptomeningeal distribution representative of human disease. These mouse models form the foundation of the investigation of CNS niche-specific features in infant leukaemia in this thesis. The functional properties of leukaemia propagating cells (LPC) in both niches, in either model, were explored. Data are presented that show different LPCs are very similar, are able to give rise to one another and are all represented in the CNS niche. Further transplantation assays show a lasting functional impact on LPCs and their ability to repopulate leukaemia following exposure to the CNS niche in both model systems. The thesis goes on to strengthen these findings by describing the transcriptomic differiv ences between these LPCs. The RNA sequencing data generated are also used to validate the miR-128a overexpression Mll-AF4, or pro-B infant acute lymphoblastic leukaemia, model. The next section of the thesis outlines transcriptomic differences between LPCs from the CNS and BM niches in both models. The discussion focuses on comparisons to existing datasets and on two novel differentially regulated processes; interaction with immune cells and growth factor signalling. Subsequent sections present functional followup experiments exploring these two novel themes. The data shown describe nichespecific differences in macrophage, T cell and NK cell dynamics and, of particular interest, imply suppression of anti-leukaemia macrophage and T cell responses in the CNS niche. In the final section, increased activation of the PI3K pathway in CNS-derived compared with BM-derived leukaemia cells is explored through downregulation of the inhibitory regulator PTEN.MiR-93, which targets PTENand CDKN1A, a downstream target of the PI3K pathway, is proposed as a global regulator of this cell-intrinsic niche-specific pathway activation. miR-93 is shown to be upregulated in CNS-derived leukaemia cells in the miR-128a overexpressionMll-AF4 model and three independent infant leukaemia patient-derived xenograft models. To conclude this section, suppression of miR-93 activity is shown to impair leukaemia cell engraftment to the CNS niche. Overall, this thesis will put forward new insights into cell-intrinsic and cell-extrinsic mechanisms of leukaemia cell survival and propagation within the CNS niche in infant leukaemia murine models

    Quantum-Classical hybrid systems and their quasifree transformations

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    The focus of this work is the description of a framework for quantum-classical hybrid systems. The main emphasis lies on continuous variable systems described by canonical commutation relations and, more precisely, the quasifree case. Here, we are going to solve two main tasks: The first is to rigorously define spaces of states and observables, which are naturally connected within the general structure. Secondly, we want to describe quasifree channels for which both the Schrödinger picture and the Heisenberg picture are well defined. We start with a general introduction to operator algebras and algebraic quantum theory. Thereby, we highlight some of the mathematical details that are often taken for granted while working with purely quantum systems. Consequently, we discuss several possibilities and their advantages respectively disadvantages in describing classical systems analogously to the quantum formalism. The key takeaway is that there is no candidate for a classical state space or observable algebra that can be put easily alongside a quantum system to form a hybrid and simultaneously fulfills all of our requirements for such a partially quantum and partially classical system. Although these straightforward hybrid systems are not sufficient enough to represent a general approach, we use one of the candidates to prove an intermediate result, which showcases the advantages of a consequent hybrid ansatz: We provide a hybrid generalization of classical diffusion generators where the exchange of information between the classical and the quantum side is controlled by the induced noise on the quantum system. Then, we present solutions for our initial tasks. We start with a CCR-algebra where some variables may commute with all others and hence generate a classical subsystem. After clarifying the necessary representations, our hybrid states are given by continuous characteristic functions, and the according state space is equal to the state space of a non-unital C*-algebra. While this C*-algebra is not a suitable candidate for an observable algebra itself, we describe several possible subsets in its bidual which can serve this purpose. They can be more easily characterized and will also allow for a straightforward definition of a proper Heisenberg picture. The subsets are given by operator-valued functions on the classical phase space with varying degrees of regularity, such as universal measurability or strong*-continuity. We describe quasifree channels and their properties, including a state-channel correspondence, a factorization theorem, and some basic physical operations. All this works solely on the assumption of a quasifree system, but we also show that the more famous subclass of Gaussian systems fits well within this formulation and behaves as expected

    The development of bioinformatics workflows to explore single-cell multi-omics data from T and B lymphocytes

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    The adaptive immune response is responsible for recognising, containing and eliminating viral infection, and protecting from further reinfection. This antigen-specific response is driven by T and B cells, which recognise antigenic epitopes via highly specific heterodimeric surface receptors, termed T-cell receptors (TCRs) and B cell receptors (BCRs). The theoretical diversity of the receptor repertoire that can be generated via homologous recombination of V, D and J genes is large enough (>1015 unique sequences) that virtually any antigen can be recognised. However, only a subset of these are generated within the human body, and how they succeed in specifically recognising any pathogen(s) and distinguishing these from self-proteins remains largely unresolved. The recent advances in applying single-cell genomics technologies to simultaneously measure the clonality, surface phenotype and transcriptomic signature of pathogen- specific immune cells have significantly improved understanding of these questions. Single-cell multi-omics permits the accurate identification of clonally expanded populations, their differentiation trajectories, the level of immune receptor repertoire diversity involved in the response and the phenotypic and molecular heterogeneity. This thesis aims to develop a bioinformatic workflow utilising single-cell multi-omics data to explore, quantify and predict the clonal and transcriptomic signatures of the human T-cell response during and following viral infection. In the first aim, a web application, VDJView, was developed to facilitate the simultaneous analysis and visualisation of clonal, transcriptomic and clinical metadata of T and B cell multi-omics data. The application permits non-bioinformaticians to perform quality control and common analyses of single-cell genomics data integrated with other metadata, thus permitting the identification of biologically and clinically relevant parameters. The second aim pertains to analysing the functional, molecular and immune receptor profiles of CD8+ T cells in the acute phase of primary hepatitis C virus (HCV) infection. This analysis identified a novel population of progenitors of exhausted T cells, and lineage tracing revealed distinct trajectories with multiple fates and evolutionary plasticity. Furthermore, it was observed that high-magnitude IFN-γ CD8+ T-cell response is associated with the increased probability of viral escape and chronic infection. Finally, in the third aim, a novel analysis is presented based on the topological characteristics of a network generated on pathogen-specific, paired-chain, CD8+ TCRs. This analysis revealed how some cross-reactivity between TCRs can be explained via the sequence similarity between TCRs and that this property is not uniformly distributed across all pathogen-specific TCR repertoires. Strong correlations between the topological properties of the network and the biological properties of the TCR sequences were identified and highlighted. The suite of workflows and methods presented in this thesis are designed to be adaptable to various T and B cell multi-omic datasets. The associated analyses contribute to understanding the role of T and B cells in the adaptive immune response to viral-infection and cancer

    Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea

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    ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy action’s effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (−10% per year) trend after adoption and enforcement of these laws (β2 = 0.18, p-value = 0.07; β3 = −0.10, p-value = 0.02). SHS exposure at home (β2 = 0.10, p-value = 0.09; β3 = −0.03, p-value = 0.14) and the primary cigarette smoking rate (β2 = 0.03, p-value = 0.10; β3 = 0.008, p-value = 0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK

    Investigation of host factors in influenza a virus infection

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    Influenza A virus (IAV) causes a contagious respiratory disease which can be fatal in at risk populations. Seasonal epidemics of IAV infection are responsible for as many as 650,000 human deaths annually. Antiviral drugs currently available for the treatment of IAV infection target viral polypeptides. However, as an RNA virus, IAV is able to mutate and rapidly develop resistance to these drugs. For this reason, new approaches which focus on targeting the host are being explored. IAV interacts with many host proteins which are necessary for efficient completion of the viral lifecycle; these host factors potentially make attractive drug targets for the treatment of IAV, as development of resistance to host-targeted treatments is predicted to be slower. Host factors required for IAV replication have been identified through a number of routes including targeted research based on previous knowledge of the virus and the host, and large scale screens. The work presented in this thesis aimed to investigate three cellular proteins that had been previously proposed as pro-viral host factors for IAV. Using RNA interference (RNAi) and chemical inhibitors, I sought to dissect the role of these proteins in the viral lifecycle and to assess their suitability as drug targets in the treatment of IAV infection. The arginine demethylase JMJD6 had been suggested to have a proviral role in IAV replication. In the process of refining previously developed RNAi procedures for depleting JMJD6, I found that a published siRNA used to study JMJD6 induced an interferon (IFN) response in cells. Here, I showed that IAV was able to replicate normally in cells in which JMJD6 was depleted by alternative RNAi methods that did not induce an IFN response and in JMJD6⁻/⁻ cells, strongly suggesting that the previous finding was likely an artefact of the approach used to deplete JMJD6. Thus JMJD6 was determined not to be a host factor of IAV or a suitable target for the treatment of IAV infection. Next, a host factor that had been identified in a genome-wide CRISPR screen was investigated. The cap 2’-O-methyltransferase, CMTR1, had been proposed to be required for preventing detection of viral mRNA via the IFN signalling pathway. In my hands, knockdown of CMTR1 using RNAi did not lead to enhanced IFN response to infection, nor did it inhibit the replication of IAV in cell culture. This did not support CMTR1 alone as an effective target for IAV treatment. However, preliminary results investigating CMTR1 knockdown in combination with the viral cap-dependent endonuclease inhibitor Baloxavir, suggested that future work should examine CMTR1 as a target to enhance other antiviral treatment. Finally, the N6-methyladenosine (m⁶A) reader protein IGF2BP1 was examined. IAV RNAs, both genomic and messenger, are known to contain the m⁶A modification, which is required for optimal replication efficiency. IGF2BP1, which binds to m⁶A modified mRNA to enhance stability, has been linked to IAV replication in host interaction screens. However, using RNAi and chemical inhibition, I did not find that IGF2BP1 was required for IAV replication or spread. Furthermore, overexpression of IGF2BP1 showed no effect on IAV replication. Together these findings rule out IGF2BP1 as an IAV host factor and drug target. Overall, the work presented in this study did not support the three host proteins investigated as being suitable drug targets for the treatment of IAV, although CMTR1 could still be investigated as a target for combination therapy. The ruling out of JMJD6 and IGF2BP1 can help to focus future work investigating host targets for IAV treatment

    Software Design Change Artifacts Generation through Software Architectural Change Detection and Categorisation

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    Software is solely designed, implemented, tested, and inspected by expert people, unlike other engineering projects where they are mostly implemented by workers (non-experts) after designing by engineers. Researchers and practitioners have linked software bugs, security holes, problematic integration of changes, complex-to-understand codebase, unwarranted mental pressure, and so on in software development and maintenance to inconsistent and complex design and a lack of ways to easily understand what is going on and what to plan in a software system. The unavailability of proper information and insights needed by the development teams to make good decisions makes these challenges worse. Therefore, software design documents and other insightful information extraction are essential to reduce the above mentioned anomalies. Moreover, architectural design artifacts extraction is required to create the developer’s profile to be available to the market for many crucial scenarios. To that end, architectural change detection, categorization, and change description generation are crucial because they are the primary artifacts to trace other software artifacts. However, it is not feasible for humans to analyze all the changes for a single release for detecting change and impact because it is time-consuming, laborious, costly, and inconsistent. In this thesis, we conduct six studies considering the mentioned challenges to automate the architectural change information extraction and document generation that could potentially assist the development and maintenance teams. In particular, (1) we detect architectural changes using lightweight techniques leveraging textual and codebase properties, (2) categorize them considering intelligent perspectives, and (3) generate design change documents by exploiting precise contexts of components’ relations and change purposes which were previously unexplored. Our experiment using 4000+ architectural change samples and 200+ design change documents suggests that our proposed approaches are promising in accuracy and scalability to deploy frequently. Our proposed change detection approach can detect up to 100% of the architectural change instances (and is very scalable). On the other hand, our proposed change classifier’s F1 score is 70%, which is promising given the challenges. Finally, our proposed system can produce descriptive design change artifacts with 75% significance. Since most of our studies are foundational, our approaches and prepared datasets can be used as baselines for advancing research in design change information extraction and documentation
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