Yes / We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. / This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBERBBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Eukaryotic organisms that cannot be classified as animals, land plants, or fungi are termed protists. Despite the fact that protists represent the majority of eukaryotic diversity, these organisms have received relatively little attention from biological researchers beyond morphological characterization. Reasons that likely contributed to their neglect include their mostly microscopic nature, that only a few lineages are the causative agents of human disease, that laboratory cultivation can be challenging, and that species concepts for the majority of protists was vague in many lineages. Initial attempts to resolve relationships among eukaryotes produced the five kingdoms model. This model suggested protists were an evolutionary assemblage separate from the animals, plants, and fungi. Molecular systematics provided a more accurate view of relationships amongst eukaryotic taxa. Results from molecular phylogenetic studies demonstrated that protists were a polyphyletic group made of many assemblages, and that more complex lineages such as plants and animals were nested within these assemblages. This new evolutionary framework brought increased attention to protists. The application of molecular biology, especially genomic and transcriptomic sequencing to protists has allowed researchers to generate meaningful data on poorly understood lineages rapidly. Applying these techniques to understudied amoeboid protists, I demonstrated the presence of a complex life cycle in a well-studied group of opportunistic pathogens and their close relatives that was not previously known, as well as characterized new diversity within the group. I made methodological advances in the field of molecular systematics through development of a novel ortholog collection algorithm that I have included in a phylogenomic package capable of resolving ancient (>100 million years) divergences in the tree of eukaryotes. I used the algorithm to build the packages accompanying manually curated database of 240 homologs from 304 eukaryotic taxa. Using the newly developed software and manually curated gene set has yielded the most complete and highly resolved tree of eukaryotes to date. Finally, I used developmental transcriptomics to demonstrate the amoeba Copromyxa protea evolved a means of simple cooperative multicellularity independently from other more well studied multicellular lineages.

We report the design and assembly of three-dimensional (3D) multi-cellular liver models comprised of primary rat hepatocytes, liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs). LSECs and KCs in the liver model were separated from hepatocytes by a nanoscale, detachable, optically transparent chitosan and hyaluronic acid (HA) polyelectrolyte multilayer (PEM) film. The properties of the PEM were tuned to mimic the Space of Disse found in liver. The thickness of the detachable PEM was 650 to 1000 nm under hydrated conditions. The Young's modulus of the PEM was approximately 42 kPa, well within the range of modulus values reported for bulk liver. The 3D liver models comprised of all three cell types and a detachable PEM exhibited stable urea production and increased albumin secretion over a 12 day culture period. Additionally, the 3D liver model maintained the phenotype of both LSECs and KCs over the 12 day culture period, verified by CD32b and CD163 staining, respectively. Additionally, CYP1A1 enzyme activity increased significantly in the 3D liver models. The number of hepatocytes in the 3D liver model increased by approximately 60% on day 16 of culture compared to day 4 indicating. Furthermore, only the 3D hepatic model maintained cellular compositions virtually identical to those found in vivo. DNA microarray measurements were conducted on the hepatocyte fractions of the 3D liver mimic to obtain insights into hepatic processes. Gene sets up-regulated in the 3D liver model were related to proliferation, migration, and deposition of extracellular matrix, all functions observed in regenerating hepatocytes. Taken together, these results suggest that inter-cellular signaling between the different cell types in the 3D liver model led to increased hepatic functions. To the best of our knowledge, this is the first study where three of the major hepatic cell types have been incorporated into a model that closely mimics the structure of the sinusoid. These studies demonstrate that the multi-cellular liver models are physiologically relevant. Such models are very promising to conduct detailed investigations into hepatic inter-cellular signaling. / Ph. D.

The topic of this thesis has been built on previous work of our group, especially Rieger et al. 2008 and Čepl et al. 2010. We examined and described the regularity of morphogenesis of S. marcescens, morphotype F colonies (from "the fountain", because of a shape it resembles). Typical colony consists of elevated red navel, low non-pigmented ring and again elevated red rim. Structured profile of the colony together with changes in pigmentation of structures during development without need of artificial dying, provides considerable advantage in observation of the morphogenesis. Aims of this thesis were (i) to find other factors that affect the morphogenesis, (ii) to characterize interactions of S. marcescens colonies with other bacterial strains (S. rubidaea and E .coli) and finally (iii) to study the phenomenon of induced resistance to the antibiotics described in (Heal and Parsons, 2002; Lu 2004; Bernier et al. 2011) in our model organisms. (i) Mutual interactions of colonies on the plate indicates that morphogenesis is affected by autocrine signals, which diffuse into the medium and the atmosphere and affect the development of surrounding colonies. We have detected changes of pH of the medium during the development of colonies in their vicinity. At ...

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells.

Cellular redox potential is incredibly important for the control and regulation of a vast number of processes occurring in cells. Disruption of the fine redox balance within cells is has been associated with disease. Of particular interest to my research is the redox gradient that develops in cancer tumours, in which the internal regions are further from vascular blood supply and therefore become starved of oxygen and hypoxic. This makes treatment of these areas a lot more challenging, as radiotherapy approaches rely on the presence of oxygen and, with a poor vascular blood supply, drugs delivered through the blood stream will have poor access to these regions. Currently, there is limited knowledge regarding the quantitative nature of this redox gradient in cancerous tumours. To aid the development of drugs and therapies to overcome this problem, a system that enables quantitative mapping of redox potential through a tumour would be a vital tool. In this work redox sensitive molecules attached to gold nanoparticles (NPs) are delivered to cells and give signals using surface enhanced Raman scattering (SERS). Redox potential changes are monitored quantitatively by ratiometric changes in signal intensity of selected signals in the SER spectra acquired. Multicellular tumour spheroids (MTS) are used as a three dimensional (3D) in vitro tumour model, in which the 3D architecture and gradients observed in tumours in vivo develop. As redox potential is pH dependent and pH is another important physiological characteristic in its own right, a SERS pH sensor was developed and ultimately a system that multiplexes intracellular pH and redox measurement by SERS. Initially, simultaneous redox potential and pH measurements were performed in monolayer culture before extending this to MTS. Photothermal optical coherence tomography (OCT) was used to investigate overall 3D NP distribution in the MTS models. It was possible to control NP delivery to MTS to localise NPs to various regions. Redox potential and pH could then be measured using a fibre optic Raman probe, and spatial response to drug treatment monitored. Intracellular NP localisation was investigated using transmission electron microscopy (TEM), scanning electron microscopy (SEM), helium ion microscopy (HIM) and confocal fluorescence microscopy (CFM) and attempts were made to control NP delivery to particular intracellular compartments.

Doctor of Philosophy (PhD) / The toxicity of platinum anticancer drugs presents a major obstacle in the effective treatment of tumours. Much of the toxicity stems from a lack of specificity of the drugs for the sites at which they are able to exert maximum anticancer activity. An improved understanding of the behaviour of the drugs in the tumour environment may assist in the rational design of future platinum anticancer agents with enhanced specificity and reduced toxicity. In the work presented herein, the specificity of two classes of platinum anticancer agents was assessed (platinum(IV) cisplatin analogues and platinum(II) anthraquinone complexes). The interaction of the platinum(IV) agents with DNA, believed to be their main cellular target, was examined using XANES spectroscopy. This experiment was designed to assess the ability of the drugs to interact with DNA and thus exert their anticancer activity. It was shown that the platinum(IV) complexes were not reduced by DNA during 48 hr incubation. It was not possible to conclusively determine whether the interaction of the complexes with DNA was direct or platinum(II) catalysed, or whether interaction had occurred at all. The distribution of platinum(II) anthraquinone complexes and their corresponding anthraquinone ligands in tumour cells (A2780 ovarian and DLD-1 colon cancer cell lines) was investigated. The cytotoxicity of the compounds in DLD-1 cells was also assessed. It was found that the compounds were efficiently taken up into the cells and entered the lysosomal compartments almost exclusively. This suggested that the cytotoxicity of the drugs was caused by lysosomal disruption, or that the platinum complexes were degraded, leaving a platinum species to enter the cell nuclei and interact with DNA. Alternatively, the complexes may bind to proteins and transport into the nuclei of the cells, though with their fluorescence quenched by the protein. The penetration and distribution of platinum(IV) complexes was assessed in DLD-1 multicellular tumour spheroids (established models of solid tumours) using a number of synchrotron techniques, including micro-tomography, micro-SRIXE, and micro-XANES. The complexes were found to be capable of penetrating throughout the entire volume of the spheroids. Micro-XANES indicated that in central and peripheral spheroidal regions, bound platinum species were present largely as platinum(II).

<p>Positron Emission Tomography (PET) has gained an important roll in clinical for diagnosis, staging and prognosis of a range of cancer types. Utilization of PET for monitoring and evaluation of cancer treatment is an attractive but almost new concept. The proper choice of PET-tracer as a biomarker for treatment follow-up is crucial. The important characteristic for a suitable tracer is its ability to reflect the response to a treatment at an early stage, before any morphologically changes occurs. It would be an advantage to screen a battery of PET tracers in a preclinical model and introduce a few potential tracers in clinical trial. </p><p>The most conventional pre-clinical approach in PET-oncology utilizes xenografts in mice or rats and requires a large number of subjects. It would be a great advantage to introduce a less demanding but still reliable preclinical method for a more efficient planning of studies in animal model and then in human trials. </p><p>The Multicellular Tumour Spheroid (MTS) system represents an intermediary level between cells growing as monolayer and solid tumours in experimental animals or patients. It mimics the growth of naturally occurring human tumours before neovascularization and appears to be more informative than monolayer and more economical and more ethical than animal models.</p><p>The aim of this work was to establish, refine and evaluate the application of MTS model as a preclinical approach in PET oncology. The vision was to introduce a preclinical method to probe and select PET tracer for treatment monitoring of anticancer drugs, which can hopefully be applied for optimization in breast cancer treatment.</p><p>In this thesis, a number of basic experiments were performed to explore the character of 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (FDG) uptake in MTS. FDG as the most established PET tracer was an obvious initial option for the evaluation of the model. For further assess-ment, we studied effects on FDG uptake in MTS treated with five routinely used chemother-apy agents. For association of PET tracer uptake to size change of MTS, we developed a reliable and user-friendly method for size determination of MTS. The next step was to apply the MTS model to screen PET tracers for analysis of early response of chemotherapy in breast cancer. Finally the method was utilized for translational imaging exemplified with a new chemotherapy agent.</p><p>The results were encouraging and the MTS model was introduced and evaluated as a preclini-cal tool in PET oncology. The method was implicated to in vitro quickly assess a therapy profile of existing and newly developed anticancer drugs in order to investigate the effects of candidate drugs on tumour-growth, selection of appropriate PET tracer for treatment monitor-ing and finally understanding relation between growth inhibition and biomarkers as part of translational imaging activities.</p>

Positron Emission Tomography (PET) has gained an important roll in clinical for diagnosis, staging and prognosis of a range of cancer types. Utilization of PET for monitoring and evaluation of cancer treatment is an attractive but almost new concept. The proper choice of PET-tracer as a biomarker for treatment follow-up is crucial. The important characteristic for a suitable tracer is its ability to reflect the response to a treatment at an early stage, before any morphologically changes occurs. It would be an advantage to screen a battery of PET tracers in a preclinical model and introduce a few potential tracers in clinical trial. The most conventional pre-clinical approach in PET-oncology utilizes xenografts in mice or rats and requires a large number of subjects. It would be a great advantage to introduce a less demanding but still reliable preclinical method for a more efficient planning of studies in animal model and then in human trials. The Multicellular Tumour Spheroid (MTS) system represents an intermediary level between cells growing as monolayer and solid tumours in experimental animals or patients. It mimics the growth of naturally occurring human tumours before neovascularization and appears to be more informative than monolayer and more economical and more ethical than animal models. The aim of this work was to establish, refine and evaluate the application of MTS model as a preclinical approach in PET oncology. The vision was to introduce a preclinical method to probe and select PET tracer for treatment monitoring of anticancer drugs, which can hopefully be applied for optimization in breast cancer treatment. In this thesis, a number of basic experiments were performed to explore the character of 2-[fluorine-18]-fluoro-2-deoxy-d-glucose (FDG) uptake in MTS. FDG as the most established PET tracer was an obvious initial option for the evaluation of the model. For further assess-ment, we studied effects on FDG uptake in MTS treated with five routinely used chemother-apy agents. For association of PET tracer uptake to size change of MTS, we developed a reliable and user-friendly method for size determination of MTS. The next step was to apply the MTS model to screen PET tracers for analysis of early response of chemotherapy in breast cancer. Finally the method was utilized for translational imaging exemplified with a new chemotherapy agent. The results were encouraging and the MTS model was introduced and evaluated as a preclini-cal tool in PET oncology. The method was implicated to in vitro quickly assess a therapy profile of existing and newly developed anticancer drugs in order to investigate the effects of candidate drugs on tumour-growth, selection of appropriate PET tracer for treatment monitor-ing and finally understanding relation between growth inhibition and biomarkers as part of translational imaging activities.

Calcium signal can be found in many types of cell. It has been treated as a life and death signal in cell-level for triggering life at fertilization, controlling the development and differentiation of cells into specialized types, mediating the subsequent activity, and finally affecting the cell death. In tissues, intercellular calcium wave is thought to serve as a long-range signaling, affected by the cell architecture. The aim of this thesis is to provide insight into the intercellular calcium waves in multicellular complex structures subjected to mechano- or chemical-stimuli. In the mechano-stimulated study, we combine the development of theoretical and experimental study of the propagation of calcium signals in multicellular structures composed of human endothelial cells. This analysis provides evidence for an effect of architecture on the propagation of calcium signals and the effect of single and dual stimulation on the multicellular structures. A simple model was established based on the calcium release/intake reaction and diffusion through gap junction from stimulated cell to the downstream cells. The simulation result shows similar results as what is shown in experiments. In the chemical-stimulated model, we studied computationally the interdependence between intracellular calcium and inositol-1,4,5-trisphosphate (IP₃) pathway and cell-cell communication via gap junction. We investigate the influence of the microenvironment of cells on the frequency of intracellular calcium oscillation. The simulation result shows that the oscillation frequency of an isolated cell is lower than that of a cell embedded in a cell-chain. This phenomenon is attributed to retrograde diffusion of calcium and IP₃ originating from a widening range of cells in the chain undergoing oscillations. It further demonstrates the important influence of microenvironment on the bio-signaling propagation.