In vitro vasculogenesis in 3D

Angiogenesis and vasculogenesis are essential neovascularisation processes. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 identified as key components. The PhD project “In vitro vasculogenesis in 3D” tested the effect of parameters such as support cells, matrix composition and physiological hypoxia on the morphology and aggregation of ECs in 3D collagen hydrogels. Different aggregation patterns were identified depending on the culture conditions tested, and these were found to reflect the different developmental pathways that ECs take to form different sized tubular structures. ECs formed contiguous sheets in collagen only hydrogels, analogous to the ‘wrapping’ pathway in development. In contrast, in co-cultures in 3D collagen-laminin cultures, end-to-end networks formed, mimicking cord hollowing and cell hollowing. A relationship between matrix composition, growth factors and VEGF receptor levels in 3D collagen hydrogels was shown for the first time in this study. Results showed a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in end-to-end network aggregation in HBMSC-HUVEC co-cultures. The effect of physiological hypoxia on EC aggregation was also tested by lowering the oxygen tension to 5% O2 using a controlled culture environment. Angiogenic growth factors were quantified using ELISA and their levels were correlated to EC morphological progression within 3D collagen hydrogels. Overall, the findings here showed how different parameters affected EC morphology and aggregation in 3D in vitro collagen hydrogels. The study provides an understanding of how these individual parameters influence EC morphology and show the mechanisms of how this is achieved in 3D in vitro

Therapeutic enhancement of a cytotoxic agent using Photochemical internalisation in 3D compressed collagen constructs of ovarian cancer

Photochemical internalisation (PCI) is a method for enhancing delivery of drugs to their intracellular target sites of action. In this study we investigated the efficacy of PCI using a porphyrin photosensitiser and a cytotoxic agent on spheroid and non-spheroid compressed collagen 3D constructs of ovarian cancer versus conventional 2D culture. The therapeutic responses of two human carcinoma cell lines (SKOV3 and HEY) were compared using a range of assays including optical imaging. The treatment was shown to be effective in non-spheroid constructs of both cell lines causing a significant and synergistic reduction in cell viability measured at 48 or 96 hours post-illumination. In the larger spheroid constructs, PCI was still effective but required higher saporin and photosensitiser doses. Moreover, in contrast to the 2D and non-spheroid experiments, where comparable efficacy was found for the two cell lines, HEY spheroid constructs were found to be more susceptible to PCI and a lower dose of saporin could be used. PCI treatment was observed to induce death principally by apoptosis in the 3D constructs compared to the mostly necrotic cell death caused by PDT. At low oxygen levels (1%) both PDT and PCI were significantly less effective in the constructs

The anti-angiogenic tyrosine kinase inhibitor Pazopanib kills cancer cells and disrupts endothelial networks in biomimetic 3D renal tumouroids

Pazopanib is a tyrosine kinase inhibitor used to treat renal cell carcinoma. Few in vitro studies investigate its effects towards cancer cells or endothelial cells in the presence of cancer. We tested the effect of Pazopanib on renal cell carcinoma cells (CAKI-2,786-O) in two-dimensional and three-dimensional tumouroids made of dense extracellular matrix, treated in normoxia and hypoxia. Finally, we engineered complex tumouroids with a stromal compartment containing fibroblasts and endothelial cells. Simple CAKI-2 tumouroids were more resistant to Pazopanib than 786-O tumouroids. Under hypoxia, while the more ‘resistant’ CAKI-2 tumouroids showed no decrease in viability, 786-O tumouroids required higher Pazopanib concentrations to induce cell death. In complex tumouroids, Pazopanib exposure led to a reduction in the overall cell viability (p < 0.0001), disruption of endothelial networks and direct killing of renal cell carcinoma cells. We report a biomimetic multicellular tumouroid for drug testing, suitable for agents whose primary target is not confined to cancer cells

Tissue-Engineering the Fibrous Pancreatic Tumour Stroma Capsule in 3D Tumouroids to Demonstrate Paclitaxel Response

Pancreatic cancer is a unique cancer in that up to 90% of its tumour mass is composed of a hypovascular and fibrotic stroma. This makes it extremely difficult for chemotherapies to be delivered into the core of the cancer mass. We tissue-engineered a biomimetic 3D pancreatic cancer ("tumouroid") model comprised of a central artificial cancer mass (ACM), containing MIA Paca-2 cells, surrounded by a fibrotic stromal compartment. This stromal compartment had a higher concentration of collagen type I, fibronectin, laminin, and hyaluronic acid (HA) than the ACM. The incorporation of HA was validated with alcian blue staining. Response to paclitaxel was determined in 2D MIA Paca-2 cell cultures, the ACMs alone, and in simple and complex tumouroids, in order to demonstrate drug sensitivity within pancreatic tumouroids of increasing complexity. The results showed that MIA Paca-2 cells grew into the complex stroma and invaded as cell clusters with a maximum distance of 363.7 µm by day 21. In terms of drug response, the IC50 for paclitaxel for MIA Paca-2 cells increased from 0.819 nM in 2D to 3.02 nM in ACMs and to 5.87 nM and 3.803 nM in simple and complex tumouroids respectively, indicating that drug penetration may be significantly reduced in the latter. The results demonstrate the need for biomimetic models during initial drug testing and evaluation

Basement membrane protein orchestrates network formation by endothelial cells in 3D: Laminin the conductor

Engineering vascular networks within 3D tissue models is paramount for the survival of cells in large 3D constructs. This is critical for both in vitro and in vivo survival. Our work has focused on network formation by endothelial cells in co-cultures with Human bone marrow stromal cells (HBMSCs) in 3D. We have examined the effect of matrix composition on endothelial cell morphology and our results emphasize the importance of basement membrane proteins for inducing network formation by endothelial cells

Photogrammetric re-discovery of the hidden long-term landscapes of western Thessaly, central Greece

This paper introduces a novel workflow for the reconstruction of nowadays disappeared cultural landscapes based on the extraction of morphological information from historic aerial photographs. This methodology has been applied for the first time for the detection, classification and characterisation of upstanding, flattened and buried archaeological sites and various off-site ancient landscape features in the plain of Karditsa, western Thessaly. Although Thessaly has been the focus of prehistoric, and especially Neolithic, research in Greece, since the beginning of the 20th century, western Thessaly has not received as much archaeological attention and its archaeological record remains rather scanty. Moreover, an extensive land reclamation project implemented in the western Thessalian plain during the early 1970s resulted in the flattening of habitation tells and funerary sites of all periods. Thus, recognition of archaeological sites and relict landscape features becomes extremely difficult, whereas standard landscape analysis and application of mainstream Remote Sensing (RS) techniques based on multispectral satellite images are problematic. Digital photogrammetric reconstruction techniques and the subsequent GIS-based treatment of the results allowed overcoming these challenging limitations: the combined use of pre-1970s aerial photographs with later imagery provided a powerful means to reconstruct the landscape before the land reclamation process, using a workflow designed to highlight photogrammetry-derived topographic differences and multi-temporal imagery analysis. Hundreds of previously unknown mounded archaeological sites, as well as other ancient landscape traits such as roads, city grids and field systems were detected. More importantly, invaluable insights into the type and character of these archaeological features were gained, which would have been impossible to obtain by conventional RS techniques

Acceptability and feasibility study of patient-specific ‘tumouroids’ as personalised treatment screening tools: protocol for prospective tissue and data collection of participants with confirmed or suspected renal cell carcinoma

Introduction: ‘Personalised medicine’ aims to tailor interventions to the individual, and has become one of the fastest growing areas of cancer research. One of these approaches is to harvest cancer cells from patients and grow them in the laboratory, which can then be subjected to treatments and the response assessed. We have developed a 3D tumour model with a complex protein matrix that mimics the tumour stroma, cell to cell and cell-matrix interactions seen in vivo, called a tumouroid. In this study, we test the acceptability and feasibility of using this model to establish patient-derived tumouroids. Methods and analysis This is a first in-human study using prospective tissue and data collection of adult participants with confirmed or suspected renal cell carcinoma. The goals of the study are to assess patient acceptability to the use of patient-derived tumour models for future treatment decisions, and to assess the feasibility of generating patient-specific renal cancer tumouroids that can be challenged with drugs. These goals will be realised through the collection of tumour samples (expected n=10), participant-completed questionnaires (expected n=10), and in-depth semi-structured interviews with patients (expected n=5). Collected multiregional tumour samples will be dissociated to isolate primary cells which are then expanded in vitro and incorporated into tumouroids. Drug challenge will ensue and the response will be categorised into “responder”, “weak responder”, and “non-responder”. Statistical analysis will be descriptive. Ethics and dissemination: The study has ethical approval (REC reference 17/LO/1744). Findings will be made available to patients, clinicians, funders, and the National Health Service (NHS) through presentations at national and international meetings, peer-reviewed publications, social media and patient support groups. Trial Registration: Registered on ClinicalTrials.gov (NCT03300102)

Discovering Conformational Sub-States Relevant to Protein Function

Background: Internal motions enable proteins to explore a range of conformations, even in the vicinity of native state. The role of conformational fluctuations in the designated function of a protein is widely debated. Emerging evidence suggests that sub-groups within the range of conformations (or sub-states) contain properties that may be functionally relevant. However, low populations in these sub-states and the transient nature of conformational transitions between these substates present significant challenges for their identification and characterization. Methods and Findings: To overcome these challenges we have developed a new computational technique, quasianharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions to identify sub-states in the conformational landscape. Further, the focus on anharmonicity allows identification of conformational fluctuations that enable transitions between sub-states. QAA applied to equilibrium simulations of human ubiquitin and T4 lysozyme reveals functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA characterizes conformational sub-states associated with cis/trans peptidyl-prolyl isomerization catalyzed by the enzyme cyclophilin A. In these three proteins, QAA allows identification of conformational sub-states, with critical structural and dynamical features relevant to protein function. Conclusions: Overall, QAA provides a novel framework to intuitively understand the biophysical basis of conformational diversity and its relevance to protein function. © 2011 Ramanathan et al

Pushing the high count rate limits of scintillation detectors for challenging neutron-capture experiments

One of the critical aspects for the accurate determination of neutron capture cross sections when combining time-of-flight and total energy detector techniques is the characterization and control of systematic uncertainties associated to the measuring devices. In this work we explore the most conspicuous effects associated to harsh count rate conditions: dead-time and pile-up effects. Both effects, when not properly treated, can lead to large systematic uncertainties and bias in the determination of neutron cross sections. In the majority of neutron capture measurements carried out at the CERN n\_TOF facility, the detectors of choice are the C$_{6}$D$_{6}$ liquid-based either in form of large-volume cells or recently commissioned sTED detector array, consisting of much smaller-volume modules. To account for the aforementioned effects, we introduce a Monte Carlo model for these detectors mimicking harsh count rate conditions similar to those happening at the CERN n\_TOF 20~m fligth path vertical measuring station. The model parameters are extracted by comparison with the experimental data taken at the same facility during 2022 experimental campaign. We propose a novel methodology to consider both, dead-time and pile-up effects simultaneously for these fast detectors and check the applicability to experimental data from $^{197}$Au($n$,$\gamma$), including the saturated 4.9~eV resonance which is an important component of normalization for neutron cross section measurements