320 research outputs found

    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/

    Natural or anthropogenic variability? A long-term pattern of the zooplankton communities in an ever-changing transitional ecosystem

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    The Venice Lagoon is an important site belonging to the Italian Long-Term Ecological Research Network (LTER). Alongside with the increasing trend of water temperature and the relevant morphological changes, in recent years, the resident zooplankton populations have also continued to cope with the colonization by alien species, particularly the strong competitor Mnemiopsis leidyi. In this work, we compared the dynamics of the lagoon zooplankton over a period of 20 years. The physical and biological signals are analyzed and compared to evaluate the hypothesis that a slow shift in the environmental balance of the site, such as temperature increase, sea level rise (hereafter called “marinization”), and competition between species, is contributing to trigger a drift in the internal equilibrium of the resident core zooplankton. Though the copepod community does not seem to have changed its state, some important modifications of structure and assembly mechanisms have already been observed. The extension of the marine influence within the lagoon has compressed the spatial gradients of the habitat and created a greater segregation of the niches available to some typically estuarine taxa and broadened and strengthened the interactions between marine species

    Relationship between synoptic circulations and the spatial distributions of rainfall in Zimbabwe

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    This study examines how the atmospheric circulation patterns in Africa south of the equator govern the spatial distribution of precipitation in Zimbabwe. The moisture circulation patterns are designated by an ample set of eight classified circulation types (CTs). Here it is shown that all wet CTs over Zimbabwe features enhanced cyclonic/convective activity in the southwest Indian Ocean. Therefore, enhanced moisture availability in the southwest Indian Ocean is necessary for rainfall formation in parts of Zimbabwe. The wettest CT in Zimbabwe is characterized by a ridging South Atlantic Ocean high-pressure, south of South Africa, driving an abundance of southeast moisture fluxes, from the southwest Indian Ocean into Zimbabwe. Due to the proximity of Zimbabwe to the Agulhas and Mozambique warm current, the activity of the ridging South Atlantic Ocean anticyclone is a dominant synoptic feature that favors above-average rainfall in Zimbabwe. Also, coupled with a weaker state of the Mascarene high, it is shown that a ridging South Atlantic Ocean high-pressure, south of South Africa, can be favorable for the southwest movement of tropical cyclones into the eastern coastal landmasses resulting in above-average rainfall in Zimbabwe. The driest CT is characterized by the northward track of the Southern Hemisphere mid-latitude cyclones leading to enhanced westerly fluxes in the southwest Indian Ocean, limiting moist southeast winds into Zimbabwe

    Deriving an in vitro source of canine corneal stromal cells for future studies of corneal disease and therapeutic applications

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    The cornea is the transparent tissue located at the front of the eye, that transmits and refracts light onto the retina. Despite great advances in corneal stem cell biology in human and laboratory animal research, no information is available in dogs. Corneal pathology, as corneal crystalline dystrophy has a prevalence of up to 15% and has been described in eight different canine breeds. Cholesterol and phospholipids are deposited in the stroma, similar to Schnyder’s dystrophy (SCD) in humans. Chronic corneal fibrosis is one of the leading causes of visual impairment in veterinary ophthalmology. Similar to the situation in human ophthalmology, there is a shortage of corneal donor tissue. Therefore, the overall aim was first to investigate whether corneal stromal stem cells exist in the canine cornea., The second aim was to determine the potential of deriving an in vitro source of corneal stroma cells from corneal stromal stem cells, adipose derived mesenchymal stromal cells (adMSC) and canine induced pluripotent stem cells (ciPSC), to provide a resource for studies investigating the pathogenesis of inherited stromal dystrophies, and for the development of novel cell-based therapies for dogs. First, a canine corneal stromal cell (CSC) population was characterised that demonstrated mesenchymal stromal cell properties, they differentiated into keratocyte-like cells (KDCs) in vitro and appeared to be immune privileged. Second, canine adMSC were differentiated into KDCs, but expressed high levels of a myofibroblastic marker, similar to those found in fibrotic tissue. Third, a modified protocol was established whereby ciPSCs were induced into neural crest (stem) cell lineages and then into KDCs. This led to the successful expression of some keratocyte associated markers in absence of a myofibroblastic expression. Taken together, a novel cell population was characterised in the canine corneal stroma. The differentiation protocols of adMSC and ciPSC led to preliminary results and built a basic foundation for future studies

    The 7th Annual Conference on "Relooking at Development, Value for Money and Public Service Delivery"

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    The fire services are a salient contestation which are often overlooked by the Social Science scholars. The Department of Cooperative Governance has ultimately promulgated a White Paper on fire services in May 2020. This study therefore aims to review and examine the legislation with a purpose of traversing the intricacies entrenched within the fire landscape. The review and analysis of policies have the potential to analyse the realities and the misnomer which are a perfect avenue to create dialogue. The theoretical framework of manipulation and elitism are employed for attributing meaning towards the study perspectives for practicality and simplicity. The paper follows a systematic procedure of reviewing documents, and policies to elicit information as a methodology adopted for the study. Gaps identified in the White Paper are uncovered and fully discussed. The content was studied, contextualised, and synthesised intellectually to derive meaning on all the aspects. It is the contention of this paper to attribute meaning to policy improvement in the fire services with a consequential contribution to the world of science for sustainable development.University of South AfricaDevelopment Studie
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