933 research outputs found
Converging organoids and extracellular matrix::New insights into liver cancer biology
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/
Raman Spectroscopy Techniques for the Detection and Management of Breast Cancer
Breast cancer has recently become the most common cancer worldwide, and with increased incidence, there is increased pressure on health services to diagnose and treat many more patients. Mortality and survival rates for this particular disease are better than other cancer types, and part of this is due to the facilitation of early diagnosis provided by screening programmes, including the National Health Service breast screening programme in the UK. Despite the benefits of the programme, some patients undergo negative experiences in the form of false negative mammograms, overdiagnosis and subsequent overtreatment, and even a small number of cancers are induced by the use of ionising radiation. In addition to this, false positive mammograms cause a large number of unnecessary biopsies, which means significant costs, both financially and in terms of clinicians' time, and discourages patients from attending further screening. Improvement in areas of the treatment pathway is also needed. Surgery is usually the first line of treatment for early breast cancer, with breast conserving surgery being the preferred option compared to mastectomy. This type of operation achieves the same outcome as mastectomy - removal of the tumour - while allowing the patient to retain the majority of their normal breast tissue for improved aesthetic and psychological results. Yet, re-excision operations are often required when clear margins are not achieved, i.e. not all of the tumour is removed. This again has implications on cost and time, and increases the risk to the patient through additional surgery.
Currently lacking in both the screening and surgical contexts is the ability to discern specific chemicals present in the breast tissue being assessed/removed. Specifically relevant to mammography is the presence of calcifications, the chemistry of which holds information indicative of pathology that cannot be accessed through x-rays. In addition, the chemical composition of breast tumour tissue has been shown to be different to normal tissue in a variety of ways, with one particular difference being a significant increase in water content. Raman spectroscopy is a rapid, non-ionising, non-destructive technique based on light scattering. It has been proven to discern between chemical types of calcification and subtleties within their spectra that indicate the malignancy status of the surrounding tissue, and differentiate between cancerous and normal breast tissue based on the relative water contents.
Furthermore, this thesis presents work aimed at exploring deep Raman techniques to probe breast calcifications at depth within tissue, and using a high wavenumber Raman probe to discriminate tumour from normal tissue predominantly via changes in tissue water content. The ability of transmission Raman spectroscopy to detect different masses and distributions of calcified powder inclusions within tissue phantoms was tested, as well as elucidating a signal profile of a similar inclusion through a tissue phantom of clinically relevant thickness. The technique was then applied to the measurement of clinically active samples of bulk breast tissue from informed and consented patients to try to measure calcifications. Ex vivo specimens were also measured with a high wavenumber Raman probe, which found significant differences between tumour and normal tissue, largely due to water content, resulting in a classification model that achieved 77.1% sensitivity and 90.8% specificity. While calcifications were harder to detect in the ex vivo specimens, promising results were still achieved, potentially indicating a much more widespread influence of calcification in breast tissue, and to obtain useful signal from bulk human tissue is encouraging in itself. Consequently, this work demonstrates the potential value of both deep Raman techniques and high wavenumber Raman for future breast screening and tumour margin assessment methods
Analytical validation of innovative magneto-inertial outcomes: a controlled environment study.
peer reviewe
Artificial intelligence in histopathology image analysis for cancer precision medicine
In recent years, there have been rapid advancements in the field of computational
pathology. This has been enabled through the adoption of digital pathology
workflows that generate digital images of histopathological slides, the publication
of large data sets of these images and improvements in computing infrastructure.
Objectives in computational pathology can be subdivided into two categories,
first the automation of routine workflows that would otherwise be performed by
pathologists and second the addition of novel capabilities. This thesis focuses on
the development, application, and evaluation of methods in this second category,
specifically the prediction of gene expression from pathology images and the
registration of pathology images among each other.
In Study I, we developed a computationally efficient cluster-based technique to
perform transcriptome-wide predictions of gene expression in prostate cancer
from H&E-stained whole-slide-images (WSIs). The suggested method
outperforms several baseline methods and is non-inferior to single-gene CNN
predictions, while reducing the computational cost with a factor of approximately
300. We included 15,586 transcripts that encode proteins in the analysis and
predicted their expression with different modelling approaches from the WSIs. In
a cross-validation, 6,618 of these predictions were significantly associated with
the RNA-seq expression estimates with FDR-adjusted p-values <0.001. Upon
validation of these 6,618 expression predictions in a held-out test set, the
association could be confirmed for 5,419 (81.9%). Furthermore, we demonstrated
that it is feasible to predict the prognostic cell-cycle progression score with a
Spearman correlation to the RNA-seq score of 0.527 [0.357, 0.665].
The objective of Study II is the investigation of attention layers in the context of
multiple-instance-learning for regression tasks, exemplified by a simulation study
and gene expression prediction. We find that for gene expression prediction, the
compared methods are not distinguishable regarding their performance, which
indicates that attention mechanisms may not be superior to weakly supervised
learning in this context.
Study III describes the results of the ACROBAT 2022 WSI registration challenge,
which we organised in conjunction with the MICCAI 2022 conference. Participating
teams were ranked on the median 90th percentile of distances between
registered and annotated target landmarks. Median 90th percentiles for eight
teams that were eligible for ranking in the test set consisting of 303 WSI pairs
ranged from 60.1 µm to 15,938.0 µm. The best performing method therefore has a
score slightly below the median 90th percentile of distances between first and
second annotator of 67.0 µm.
Study IV describes the data set that we published to facilitate the ACROBAT
challenge. The data set is available publicly through the Swedish National Data
Service SND and consists of 4,212 WSIs from 1,153 breast cancer patients.
Study V is an example of the application of WSI registration for computational
pathology. In this study, we investigate the possibility to register invasive cancer
annotations from H&E to KI67 WSIs and then subsequently train cancer detection
models. To this end, we compare the performance of models optimised with
registered annotations to the performance of models that were optimised with
annotations generated for the KI67 WSIs. The data set consists of 272 female
breast cancer cases, including an internal test set of 54 cases. We find that in this
test set, the performance of both models is not distinguishable regarding
performance, while there are small differences in model calibration
30th European Congress on Obesity (ECO 2023)
This is the abstract book of 30th European Congress on Obesity (ECO 2023
Microscopic study of RNA-lipoplex-mediated cell targeting using 2D and 3D models
Advanced mRNA therapeutics are not only emerging as vaccines, but are also being investigated for cancer therapy. The purpose of RNA lipoplexes (lipid nanoparticle-based RNA therapeutics) in tumor immunotherapy is to encode tumor-associated antigens and deliver this information to antigen-presenting cells (APCs). Besides the systemic application of RNA-LPX with the intention of targeting APCs in organs of the lymphatic system such as the spleen or lymph nodes, local applications in the direct tumor environment are also being studied.
The presented work describes the cell targeting of an RNA-lipoplex based therapeutic in different modalities. The focus is placed on in vitro and in vivo studies that reflect direct as well as systemic application. For data acquisition, microscopic analyses were used with a special focus on 3D microscopy. The studies revealed varying colocalizations with different cell types in the different study models.
In the described in vitro melanoma model, different cell type specific RNA reporter expression could be observed in classical 2D cell culture as well as in 3D mono and cocultures. The 2D cell cultures indicated an increased expression in the coculture compared to monocultures. In 3D monocultures, increased eGFP (RNA reporter) signal was detected in the SK-MEL 28 cells. In the 3D coculture system, a distribution within the spheroid could be depicted, where in addition to the CK 14 (basal keratinocytes) positive cells localised in the rim area, also eGFP was expressed in the spheroid nucleus.
Due to its central role in the secondary lymphatic system, the spleen was analysed as a target organ for systemic application, both in conventional 2D sections and in 3D whole mounts. Based on the results of the 2D sections, we found that both CD11c positive and CD169 positive cells show strong colocalizations with the RNA reporter signal. However, among APCs an increased colocalizations with CD169 positive cells could be detected.
For a comparison between classical 2D analyses and state-of-the-art 3D analyses, different techniques were tested and a method was adapted to the special requirements of the target organ (spleen) and the detection of eGFP (RNA reporter). These studies have provided important insights into the strength of the tested tissue clearing techniques. Furthermore, colocalization between eGFP and CD169-positive cells in 3D provided a first insight into quantitative analyses like those from 2D tissue sections. The described method can be implemented using standard tools and thereby opens the way for future 3D studies of lipid nanoparticle-based RNA therapeutics
Disease progression and genetic risk factors in the primary tauopathies
The primary tauopathies are a group of progressive neurodegenerative diseases within the frontotemporal lobar degeneration spectrum (FTLD) characterised by the accumulation of misfolded, hyperphosphorylated microtubule-associated tau protein (MAPT) within neurons and glial cells. They can be classified according to the underlying ratio of three-repeat (3R) to four-repeat (4R) tau and include Pick’s disease (PiD), which is the only 3R tauopathy, and the 4R tauopathies the most common of which are progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). There are no disease modifying therapies currently available, with research complicated by the wide variability in clinical presentations for each underlying pathology, with presentations often overlapping, as well as the frequent occurrence of atypical presentations that may mimic other non-FTLD pathologies. Although progress has been made in understanding the genetic contribution to disease risk in the more common 4R tauopathies (PSP and CBD), very little is known about the genetics of the 3R tauopathy PiD.
There are two broad aims to this thesis; firstly, to use data-driven generative models of disease progression to try and more accurately stage and subtype patients presenting with PSP and corticobasal syndrome (CBS, the most common presentation of CBD), and secondly to identify genetic drivers of disease risk and progression in PiD. Given the rarity of these disorders, as part of this PhD I had to assemble two large cohorts through international collaboration, the 4R tau imaging cohort and the Pick’s disease International Consortium (PIC), to build large enough sample sizes to enable the required analyses.
In Chapter 3 I use a probabilistic event-based modelling (EBM) approach applied to structural MRI data to determine the sequence of brain atrophy changes in clinically diagnosed PSP - Richardson syndrome (PSP-RS). The sequence of atrophy predicted by the model broadly mirrors the sequential spread of tau pathology in PSP post-mortem staging studies, and has potential utility to stratify PSP patients on entry into clinical trials based on disease stage, as well as track disease progression.
To better characterise the spatiotemporal heterogeneity of the 4R tauopathies, I go on to use Subtype and Stage Inference (SuStaIn), an unsupervised machine algorithm, to identify population subgroups with distinct patterns of atrophy in PSP (Chapter 4) and CBS (Chapter 5). The SuStaIn model provides data-driven evidence for the existence of two spatiotemporal subtypes of atrophy in clinically diagnosed PSP, giving insights into the relationship between pathology and clinical syndrome. In CBS I identify two distinct imaging subtypes that are differentially associated with underlying pathology, and potentially a third subtype that if confirmed in a larger dataset may allow the differentiation of CBD from both PSP and AD pathology using a baseline MRI scan.
In Chapter 6 I investigate the association between the MAPT H1/H2 haplotype and PiD, showing for the first time that the H2 haplotype, known to be strongly protective against developing PSP or CBD, is associated with an increased risk of PiD. This is an important finding and has implications for the future development of MAPT isoform-specific therapeutic strategies for the primary tauopathies.
In Chapter 7 I perform the first genome wide association study (GWAS) in PiD, identifying five genomic loci that are nominally associated with risk of disease. The top two loci implicate perturbed GABAergic signalling (KCTD8) and dysregulation of the ubiquitin proteosome system (TRIM22) in the pathogenesis of PiD.
In the final chapter (Chapter 8) I investigate the genetic determinants of survival in PiD, by carrying out a Cox proportional hazards genome wide survival study (GWSS). I identify a genome-wide significant association with survival on chromosome 3, within the NLGN1 gene. which encodes a synaptic scaffolding protein located at the neuronal pre-synaptic membrane. Loss of synaptic integrity with resulting dysregulation of synaptic transmission leading to increased pathological tau accumulation is a plausible mechanism though which NLGN1 dysfunction could impact on survival in PiD
Development of polarization-resolved optical scanning microscopy imaging techniques to study biomolecular organizations
Light, as electromagnetic radiation, conveys energy through space and time via fluctuations in electric and magnetic fields. This thesis explores the interaction of light and biological structures through polarization-resolved imaging techniques. Light microscopy, and polarization analysis enable the examination of biological entities. Biological function often centers on chromatin, the genetic material composed of DNA wrapped around histone proteins within cell nuclei. This structure's chiral nature gives rise to interactions with polarized light. This research encompasses three main aspects. Firstly, an existing multimodal Circular Intensity Differential Scattering (CIDS) and fluorescence microscopy are upgraded into an open configuration to be integrated with other modalities. Secondly, a novel cell classification method employing CIDS and a phasor representation is introduced. Thirdly, polarization analysis of fluorescence emission is employed for pathological investigations.
Accordingly, the thesis is organized into three chapters. Chapter 1 lays the theoretical foundation for light propagation and polarization, outlining the Jones and Stokes-Mueller formalisms. The interaction between light and optical elements, transmission, and reflection processes are discussed. Polarized light's ability to reveal image contrast in polarizing microscopes, linear and nonlinear polarization-resolved microscopy, and Mueller matrix microscopy as a comprehensive technique for studying biological structures are detailed. Chapter 2 focuses on CIDS, a label-free light scattering method, including a single point angular spectroscopy mode and scanning microscopy imaging. A significant upgrade of the setup is achieved, incorporating automation, calibration, and statistical analysis routines. An intuitive phasor approach is proposed, enabling image segmentation, cell discrimination, and enhanced interpretation of polarimetric contrast. As a result, image processing programs have been developed to provide automated measurements using polarization-resolved laser scanning microscopy imaging integrated with confocal fluorescence microscopy of cells and chromatin inside cell nuclei, including the use of new types of samples such as progeria cells. Chapter 3 applies a polarization-resolved two-photon excitation fluorescence (2PEF) microscopy to study multicellular cancerous cells. A homemade 2PEF microscope is developed for colon cancer cell analysis. The integration of polarization and fluorescence techniques leads to a comprehensive understanding of the molecular orientation within samples, particularly useful for cancer diagnosis.
Overall, this thesis presents an exploration of polarization-resolved imaging techniques for studying biological structures, encompassing theory, experimental enhancements, innovative methodologies, and practical applications
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