ORCID Profile
0000-0001-5541-9747
Current Organisation
The University of Edinburgh
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Publisher: American Association for Cancer Research (AACR)
Date: 02-2011
DOI: 10.1158/0008-5472.CAN-10-2267
Abstract: The ability to observe changes in molecular behavior during cancer cell invasion in vivo remains a major challenge to our understanding of the metastatic process. Here, we demonstrate for the first time, an analysis of RhoA activity at a subcellular level using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer) imaging in a live animal model of pancreatic cancer. In invasive mouse pancreatic ductal adenocarcinoma (PDAC) cells driven by mutant p53 (p53R172H), we observed a discrete fraction of high RhoA activity at both the leading edge and rear of cells in vivo which was absent in two-dimensional in vitro cultures. Notably, this pool of active RhoA was absent in noninvasive p53fl knockout PDAC cells, correlating with their poor invasive potential in vivo. We used dasatanib, a clinically approved anti-invasive agent that is active in this model, to illustrate the functional importance of spatially regulated RhoA. Dasatanib inhibited the activity of RhoA at the poles of p53R172H cells in vivo and this effect was independent of basal RhoA activity within the cell body. Taken together, quantitative in vivo fluorescence lifetime imaging illustrated that RhoA is not only necessary for invasion, but also that subcellular spatial regulation of RhoA activity, as opposed to its global activity, is likely to govern invasion efficiency in vivo. Our findings reveal the utility of FLIM-FRET in analyzing dynamic biomarkers during drug treatment in living animals, and they also show how discrete intracellular molecular pools might be differentially manipulated by future anti-invasive therapies. Cancer Res 71(3) 747–57. ©2011 AACR.
Publisher: American Association for Cancer Research (AACR)
Date: 04-2009
DOI: 10.1158/0008-5472.CAN-08-4308
Abstract: The ability of tumor cells to invade and metastasize requires deregulation of interactions with adjacent cells and the extracellular matrix. A major challenge of cancer biology is to observe the dynamics of the proteins involved in this process in their functional and physiologic context. Here, for the first time, we have used photobleaching and photoactivation to compare the mobility of cell adhesion and plasma membrane probes in vitro and in tumors grown in mice (in vivo). We find differences between in vitro and in vivo recovery dynamics of two key molecules, the tumor suppressor E-cadherin and the membrane-targeting sequence of H-Ras. Our data show that E-cadherin dynamics are significantly faster in vivo compared with cultured cells, that the ratio of E-cadherin stabilized in cell-cell junctions is significantly higher in vivo, and that E-cadherin mobility correlates with cell migration. Moreover, quantitative imaging has allowed us to assess the effects of therapeutic intervention on E-cadherin dynamics using dasatinib, a clinically approved Src inhibitor, and show clear differences in the efficacy of drug treatment in vivo. Our results show for the first time the utility of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals. Furthermore, this work highlights critical differences in molecular dynamics in vitro and in vivo, which have important implications for the use of cultured disease models as surrogates for living tissue. [Cancer Res 2009 (7):2714–9]
Publisher: Springer Science and Business Media LLC
Date: 17-04-2014
DOI: 10.1038/NRC3724
Abstract: Integrating biological imaging into early stages of the drug discovery process can provide invaluable readouts of drug activity within complex disease settings, such as cancer. Iterating this approach from initial lead compound identification in vitro to proof-of-principle in vivo analysis represents a key challenge in the drug discovery field. By embracing more complex and informative models in drug discovery, imaging can improve the fidelity and statistical robustness of preclinical cancer studies. In this Review, we highlight how combining advanced imaging with three-dimensional systems and intravital mouse models can provide more informative and disease-relevant platforms for cancer drug discovery.
Publisher: Elsevier BV
Date: 2016
Publisher: MDPI AG
Date: 04-04-2011
Publisher: Wiley
Date: 28-06-2013
DOI: 10.1111/FEBS.12348
Abstract: The integration of signal transduction pathways plays a fundamental role in governing disease initiation, progression and outcome. It is therefore necessary to understand disease at the signalling level to enable effective treatment and to intervene in its progression. The recent extension of in vitro subcellular image-based analysis to live in vivo modelling of disease is providing a more complete picture of real-time, dynamic signalling processes or drug responses in live tissue. Intravital imaging offers alternative strategies for studying disease and embraces the biological complexities that govern disease progression. In the present review, we highlight how three-dimensional or live intravital imaging has uncovered novel insights into biological mechanisms or modes of drug action. Furthermore, we offer a prospective view of how imaging applications may be integrated further with the aim of understanding disease in a more physiological and functional manner within the framework of the drug discovery process.
Publisher: American Association for Cancer Research (AACR)
Date: 31-07-2013
DOI: 10.1158/0008-5472.CAN-12-4545
Abstract: Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy–fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation. Cancer Res 73(15) 4674–86. ©2013 AACR.
Publisher: Informa UK Limited
Date: 07-2011
Publisher: Wiley
Date: 18-12-2021
Abstract: A more comprehensive understanding of how cells respond to drug intervention, the likely immediate signalling responses and how resistance may develop within different microenvironments will help inform treatment regimes. The nonreceptor tyrosine kinase SRC regulates many cellular signalling processes, and pharmacological inhibition has long been a target of cancer drug discovery projects. Here, we describe the in vitro and in vivo characterisation of the small‐molecule SRC inhibitor AZD0424. We show that AZD0424 potently inhibits the phosphorylation of tyrosine‐419 of SRC (IC50 ~ 100 n m ) in many cancer cell lines however, inhibition of cell viability, via a G1 cell cycle arrest, was observed only in a subset of cancer cell lines in the low (on target) micromolar range. We profiled the changes in intracellular pathway signalling in cancer cells following exposure to AZD0424 and other targeted therapies using reverse‐phase protein array (RPPA) analysis. We demonstrate that SRC is activated in response to treatment of KRAS‐mutant colorectal cell lines with MEK inhibitors (trametinib or AZD6244) and that AZD0424 abrogates this. Cell lines treated with trametinib or AZD6244 in combination with AZD0424 had reduced EGFR, FAK and SRC compensatory activation, and cell viability was synergistically inhibited. In vivo , trametinib treatment of mice‐bearing HCT116 tumours increased phosphorylation of SRC on Tyr419, and, when combined with AZD0424, inhibition of tumour growth was greater than with trametinib alone. We also demonstrate that drug‐induced resistance to trametinib is not re‐sensitised by AZD0424 treatment in vitro , likely as a result of multiple compensatory signalling mechanisms however, inhibition of SRC remains an effective way to block invasion of trametinib‐resistant tumour cells. These data imply that SRC inhibition may offer a useful addition to MEK inhibitor combination strategies.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Neil Carragher.