Nanobiology of Cancer

Modelling Biomolecular Signalling Pathways in Colorectal Cancer Cells by Using Multidimensional Correlative Imaging Techniques
In this project we endeavour to further elucidate the complex and dynamic machinery of membrane transport and signalling mechanisms that have been shown to be a key event in colorectal tumour cell survival and further metastatic outgrowth. In this project we will apply cutting-edge 3-D and 4-D microscopic methods in combination with correlative immunocytochemistry- and molecular biology techniques to address the aims of the study.
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X-Ray Micro-Computed Imaging of Bioreactor Liver Tissue: A Model for Colorectal Metastasis
In previous studies we successfully imaged liver tissue and its associated vasculature via soft X-ray micro-computed imaging. In this research project we aim to image, reconstruct and model colorectal cancer growth in a liver organoid bioreactor via X-ray micro-computed tomography. This approach will be an unique set-up in the study and screening of potential anti-cancer drugs.
Investigators: , Dr Matsuura Tomokazu
Collaborators: Carina Fernandes, Dr Keisuke Nagatsuma, Dr Masaya Saito

3-D Microscopic Analysis of Colorectal Cancer
This fundamental research project addresses problems of collecting currently unknown architectural tissue and (sub)cellular information by reconstructing and modelling colorectal liver metastasis pathways using novel correlative tomographic imaging methods. Identifying the mechanisms regu-lating the nanobiology of colorectal liver metastasis, as well as gaining a better understanding of the interaction between the metastatic tumour cell and the different types of liver cells, including the liver vasculature, will provide a foundation for new therapeutic approaches. The correlative data and models achieved will be a breakthrough in the study of liver tumours.
Investigators: , Dr Matsuura Tomokazu
Collaborators: Carina Fernandes, Dr Keisuke Nagatsuma

Apoptosis-inducing Anti-Actin Drugs: A Potential Approach for Suppressing the Onset of Hepatic Metastasis
Mounting literature evidence reveals that alterations of actin polymerization plays a pivotal role in regulating the metastatic behaviour of a malignant cell. In line, we found in a preliminary study by using actin-binding agents a relationship between actin-mediated fine structural changes and an in-crease in the number of apoptotic colorectal cancer cells. The identification of anti-actin drugs which exerts severe cytotoxic cellular effects by inducing apoptosis in colon carcinoma cancer cells are relatively unexplored and form the basis of this research project. This should in the long-term con-tribute to the development of new chemotherapeutic strategies.
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Development of Correlative Fluorescence- and Scanning, Transmission Electron Microscopy Imaging Methods for Biomolecular Investigation of Colorectal Metastasis
In this project we aim to develop novel imaging analysis techniques to detect and image the specific localisation of small biomolecules within subcellular structures of the same cancer cell(s) using ad-vanced immunogold techniques such as nano and decagold technology in conjunction with correlative confocal, X-ray and electron microscopy.
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Collaborators: Prof. John Robinson, Dr Willie Geerts

Transendothelial Transport Mechanisms of the Hepatic Sinusoid: From Cell to Molecule
The liver sinusoidal endothelium plays a central and active role in regulating the exchange of mac-romolecules, solutes, fluid and (cancer) cells between the blood and the surrounding tissues. The high permeability of the liver sinusoidal endothelium to these substances are reflected in the presence of special transporting systems represented by non-diaphragmed fenestrae, coated pits, caveolae, vesicle vacuolar organelles and receptor-mediated scavenger mechanisms. This ongoing research project endeavours to elucidate further the complex and dynamic machinery of liver transendothelial transport mechanisms by applying high-resolution 3-D and 4-D correlative microscopic methods
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Collaborators: Dr Willie Geerts, Prof. Peter Frederik

Mechanisms of Chemokinesis and Chemotaxis
We explored the heterogeneity of a lung cancer cell line and succeeded in isolating a supopulation of highly chemokinetic and invasive cells. Microarray analyses showed differential gene profiles between the chemokinetic subpopulation and the original cell line. Real-time PCR confirmed that a gene, reduced in-random motile (ROM), was down-regulated in chemokinetic cells as well as in a panel of lung cancer cells compared to control or normal cells. Disruption of ROM function by mutagenesis resulted in increased invasion and chemokinesis with no effect on chemotaxis. Ongoing studies serve to characterise the function of ROM in the regulation of speed, polarity, motility and metastasis in cancer cells. The proteins along the EGFR/Cdc42/N-WASP/Arp2/3 axis will also be evaluated for function in navigation or directionality of cancer cell chemotaxis along an EGF gradient.
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PhD Student: (thesis title: In-vitro Models of Cancer Cell Migration)

Mechanisms of Cancer Cell Invasion
This project incorporates the investigation of cancer cell invasion through collagen matrix by using high-resolution, live-cell imaging to study the morpho-dynamic changes involved. By varying the microenvironment of the cells, new data are obtained that demonstrate a requirement for metallo-proteinase (MMP) secretion for the invasion of amoeboid-like cancer cells. Furthermore, mesenchy-mal-like cancer cells migrated at greater levels compared to the amoeboid cells depending on the matrix environment. The findings also show that the amoeboid cancer cells were invasive while re-maining viable for up to 7 days but ceased to proliferate in that time. Confocal microscopy of FITC-labelled collagen demonstrated the presence of higher-intensity matrix areas at the leading front of invading cells suggesting some degree of matrix remodelling by the cells. Finger-like projections were also found to occur in the direction of invasion. The use of a cocktail of metalloproteinase (MMP) and protease inhibitors showed a reduction in cell invasion levels suggesting a positive role for MMPs in amoeboid cell invasion. The data contrast existing invasion models for these cells, which retain their ability to migrate through certain matrix preparations in the presence of protease inhibitors. Current work serves to determine the molecular regulation of novel migratory behaviours in amoeboid-like cancer cells.
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[1] Rik Thompson, Head, VBCRC Invasion and Metastasis Unit, St. Vincent's Institute and University of Melbourne Department of Surgery; Program Manager, Matrix Biology, Australian Tissue Engi-neering Centre, Bernard O'Brien Institute for Microsurgery Clinical Sciences Building, 29 Regent St., Fitzroy, 3065, Melbourne, Australia.
[2] Pascal Vallotton PhD, Leader, Biotech Imaging, CSIRO Mathematical & Information Sciences, Locked Bag 17, North Ryde, NSW, 1670, Australia.
PhD Student: (thesis title: Mechanisms of Cancer Cell Invasion).

Intravital Imaging of Tumour Cell Motility along Tumor and Microfabricated Collagen Fibres
Multiphoton imaging of live tumors is a powerful method of studying the motility and invasion of car-cinoma cells in vivo. Knockdown of ROM and N-WASP in fluorescent, GFP-expressing cancer cells will be accomplished by stable siRNA transfections. The function of these genes on the speed and direc-tionality of motility along collagen fibres will be studied. Micropatterning of linear collagen tracks will also be performed to simulate the linear collagen fibres observed in vivo. The premise for this study is that cancer cells may rely on contact guidance of fibres to not only walk linearly but also develop high speeds. In this model, cells adhered on collagen tracks of the appropriate thickness will become more elongated through contact guidance mechanisms, assuming the shape of the linear substrate. In the presence of chemotactic factors, elongated cells can become polarised with distinctive front and tail morphologies. When a certain degree of polarity is achieved and maintained, cells switch from low to high speeds and show more persistence (directionality) in their motility. To study this, we are using soft lithographic methods to generate microchannels of different widths that will be filled with collagen I and evaluate cancer cell migration parameters along these collagen tracks.
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[1] Prof. John Condeelis, Co-Chair of Anatomy & Structural Biology, Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, NY, USA.
[2] Dr Gary Rosengarten, Senior Lecturer, Thermofluids and Microfluidics School of Mechanical Engineering, University of New South Wales, Sydney, NSW, Australia.

Assay Development for the Simulation of Sharp Gradients for Investigating Rapid and Long-term Cancer Cell Processes
Rapid progress in the elucidation of candidate genes and proteins that play a role in disease processes such as cancer has been possible with advances and in the wide-use of genomics and proteomics in the last ten years. It will become critical to be able to transfer the vast knowledge from mass analytical techniques to visual techniques that enable spatial-temporal discernment of molecular events. This is significant, particularly for the study of pathways that lead to dynamic processes such as cell migration or early events associated with differentiation such as Ca2+ signalling. This work involves the use of techniques that create sharp growth factor gradients suitable for local activation of cognate cell surface receptors. The methods involve retardation of the direct flow and utilization of indirect flow to create steep gradients at the surface where cells are grown. We show, for example, that PC12 cells were stimulated to release Ca2+ release within short 1 min time-frames and MTLn3 breast carcinoma cells were induced to chemotax towards the gradient at a speed of 0.5 ┬Ám/s.
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[1] Prof. Ben Eggleton, CUDOS ARC Centre of Excellence for Ultrahigh-bandwidth Devices for Optical Systems, Physics School of Physics, The University of Sydney.
[2] Dr Peter Domachuk, Research Fellow at the Department of Biomedical Engineering, Tufts University, Medford, MA, USA.