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Leong H.S.,Translational Prostate Cancer Research Group | Robertson A.E.,Translational Prostate Cancer Research Group | Stoletov K.,University of Alberta | Leith S.J.,Translational Prostate Cancer Research Group | And 12 more authors.
Cell Reports | Year: 2014

Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. We utilized a high-resolution time-lapse intravital imaging approach to visualize the dynamics of cancer cell extravasation invivo. During intravascular migration, cancer cells form protrusive structures identified as invadopodia by their enrichment of MT1-MMP, cortactin, Tks4, and importantly Tks5, which localizes exclusively toinvadopodia. Cancer cells extend invadopodia through the endothelium into the extravascular stroma prior to their extravasation at endothelial junctions. Genetic or pharmacological inhibition of invadopodia initiation (cortactin), maturation (Tks5), or function (Tks4) resulted in an abrogation of cancer cell extravasation and metastatic colony formation in an experimental mouse lung metastasis model. This provides direct evidence of a functional role for invadopodia during cancer cell extravasation and distant metastasis and reveals an opportunity for therapeutic intervention in this clinically important process. © 2014 The Authors.


Bendris N.,French National Center for Scientific Research | Bendris N.,Montpellier University | Bendris N.,Southwestern Medical Center | Cheung C.T.,French National Center for Scientific Research | And 8 more authors.
Cellular and Molecular Life Sciences | Year: 2014

Our previous work showed that Cyclin A2 deficiency promotes cell invasion in fibroblasts. Given that the majority of cancers emerge from epithelia, we explored novel functions for Cyclin A2 by depleting it in normal mammary epithelial cells. This caused an epithelial to mesenchymal transition (EMT) associated with loss of cell-to-cell contacts, decreased E-Cadherin expression and increased invasive properties characterized by a reciprocal regulation of RhoA and RhoC activities, where RhoA-decreased activity drove cell invasiveness and E-Cadherin delocalization, and RhoC-increased activity only supported cell motility. Phenotypes induced by Cyclin A2 deficiency were exacerbated upon oncogenic activated-Ras expression, which led to an increased expression of EMT-related transcriptional factors. Moreover, Cyclin A2-depleted cells exhibited stem cell-like properties and increased invasion in an in vivo avian embryo model. Our work supports a model where Cyclin A2 downregulation facilitates cancer cell EMT and metastatic dissemination. © Springer 2014.


Leong H.S.,St Pauls Hospital | Leong H.S.,Translational Prostate Cancer Research Group | Podor T.J.,St Pauls Hospital | Manocha B.,Translational Prostate Cancer Research Group | Lewis J.D.,Translational Prostate Cancer Research Group
Journal of Thrombosis and Haemostasis | Year: 2011

Background:Platelet microparticles (PMPs) are a promising prognostic marker for thrombotic disorders because of their release during platelet activation. The use of flow cytometry for the enumeration of PMPs in plasma has generated controversy due to their size, which is below the stated detection limits of conventional flow cytometry instruments. The potential impact of this is an underestimation of PMP counts. Objectives/Methods:To address this possibility, we used a combination of fluorescence-activated cell sorting (FACS) and atomic force microscopy (AFM) to determine the size distribution of PMPs present in plasma from acute myocardial infarction (AMI) patients and normal volunteers, and PMPs generated by expired platelet concentrates and washed platelets treated with agonists such as thrombin and calcium ionophore (A23187). Results:According to AFM image analysis, there was no statistically significant difference in height or volume distributions in PMPs from thrombin-activated, calcium ionophore-activated, expired platelet concentrates and plasma from healthy volunteers and AMI patients. Based on volume, expired platelets released the greatest proportion of exosomes (<1.0×10 -22L 3 in volume) in relation to the entire PMP population (29.7%) and the smallest proportion of exosomes was observed in AMI patient plasma (1.8%). Moreover, AFM imaging revealed that PMPs from expired platelets exhibited smooth surfaces compared with other PMP types but this was not statistically significant. Conclusions:We confirm that flow cytometry is capable of analyzing PMPs from plasma by using AFM to perform nanoscale measurements of individual PMP events isolated by FACS. This method also provided the first quantitative nanoscale images of PMP ultrastructure. © 2011 International Society on Thrombosis and Haemostasis.


Goulet B.,Translational Prostate Cancer Research Group | Chan G.,University of Montréal | Chambers A.F.,90 Commissioners Rd E | Chambers A.F.,University of Western Ontario | And 2 more authors.
Biochemistry and Cell Biology | Year: 2012

Maspin, a member of the serpin family of serine protease inhibitors, was originally identified as a tumor suppressor that is expressed in normal mammary epithelial cells but is reduced or absent in breast carcinomas. Early enthusiasm for maspin as a biomarker for disease progression has been tempered by clinical data that associates maspin with favourable outcomes in some studies and poor prognosis in others. Here, we review all of the published clinical studies for maspin in breast and ovarian cancers and propose that the apparent discordance between clinical reports is a consequence of differential cellular distribution of maspin. Indeed, it was thought that an extracellular pool of maspin possessed tumor suppressor activity, acting by inhibiting migration and increasing cell adhesion. Recent evidence from our group and others indicates, however, that the nuclear localization of maspin in cancer cells is necessary for its tumor suppressor activity. We provide additional data here to demonstrate that nuclear-localized maspin binds to chromatin and is required to effectively prevent cells from metastasizing. Our knowledge of other serpins that localize to the nucleus should help to inform future studies of nuclear maspin. Elucidation of the molecular mechanisms regulating the localization and activities of maspin should pave the way for the development of improved diagnostics and therapies for cancer. © 2011 Published by NRC Research Press.


Palmer T.D.,Vanderbilt University | Martinez C.H.,Translational Prostate Cancer Research Group | Vasquez C.,University of Alberta | Hebron K.E.,Vanderbilt University | And 11 more authors.
Cancer Research | Year: 2014

Normal physiology relies on the organization of transmembrane proteins by molecular scaffolds, such as tetraspanins. Oncogenesis frequently involves changes in their organization or expression. The tetraspanin CD151 is thought to contribute to cancer progression through direct interaction with the laminin-binding integrins α3β1 and α6β1. However, this interaction cannot explain the ability of CD151 to control migration in the absence of these integrins or on non-laminin substrates. We demonstrate that CD151 can regulate tumor cell migration without direct integrin binding and that integrin-free CD151 (CD151free) correlates clinically with tumor progression and metastasis. Clustering CD151free through its integrin-binding domain promotes accumulation in areas of cell-cell contact, leading to enhanced adhesion and inhibition of tumor cell motility in vitro and in vivo. CD151free clustering is a strong regulator of motility even in the absence of α3 expression but requires PKCα, suggesting that CD151 can control migration independent of its integrin associations. The histologic detection of CD151free in prostate cancer correlates with poor patient outcome. When CD151free is present, patients are more likely to recur after radical prostatectomy and progression to metastatic disease is accelerated. Multivariable analysis identifies CD151free as an independent predictor of survival. Moreover, the detection of CD151 free can stratify survivalamong patients with elevated prostate-specific antigen levels. Cumulatively, these studies demonstrate that a subpopulation of CD151 exists on the surface of tumor cells that can regulate migration independent of its integrin partner. The clinical correlation of CD151free with prostate cancer progression suggests that itmay contribute to the disease and predict cancer progression. © 2014 American Association for Cancer Research.


Leong H.S.,Translational Prostate Cancer Research Group | Steinmetz N.F.,University of California at San Diego | Steinmetz N.F.,Scripps Research Institute | Ablack A.,Translational Prostate Cancer Research Group | And 6 more authors.
Nature Protocols | Year: 2010

Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of CPMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application. © 2010 Nature America, Inc. All rights reserved.


Arpaia E.,Campbell University | Arpaia E.,University of Toronto | Blaser H.,Campbell University | Quintela-Fandino M.,Campbell University | And 17 more authors.
Oncogene | Year: 2012

Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130 Cas, which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130 Cas phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130 Cas/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades. © 2012 Macmillan Publishers Limited All rights reserved.


Steinmetz N.F.,Scripps Research Institute | Steinmetz N.F.,Case Western Reserve University | Ablack A.L.,Translational Prostate Cancer Research Group | Hickey J.L.,University of Western Ontario | And 5 more authors.
Small | Year: 2011

Multivalent nanoparticles have several key advantages in terms of solubility, binding avidity, and uptake, making them particularly well suited to molecular imaging applications. Herein is reported the stepwise synthesis and characterization of NIR viral nanoparticles targeted to gastrin-releasing peptide receptors that are over-expressed in human prostate cancers. The pan-bombesin analogue, [β-Ala11, Phe13, Nle14]bombesin-(7-14), is conjugated to cowpea mosaic virus particles functionalized with an NIR dye (Alexa Fluor 647) and polyethylene glycol (PEG) using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. Targeting and uptake in human PC-3 prostate cells is demonstrated in vitro. Tumor homing is observed using human prostate tumor xenografts on the chicken chorioallantoic membrane model using intravital imaging. Further development of this viral nanoparticle platform may open the door to potential clinical noninvasive molecular imaging strategies. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Steinmetz N.F.,Scripps Research Institute | Steinmetz N.F.,Case Western Reserve University | Cho C.-F.,Translational Prostate Cancer Research Group | Cho C.-F.,University of Western Ontario | And 4 more authors.
Nanomedicine | Year: 2011

Aims: Vimentin, a type III intermediate filament, is upregulated during epithelial-mesenchymal transition and tumor progression. Vimentin is surface-expressed on cells involved in inflammation; the function remains unknown. We investigated the expression of surface vimentin on cancer cells and evaluated targeting nanoparticles to tumors exploiting vimentin. Materials & methods: Cowpea mosaic virus nanoparticles that interact with surface vimentin were used as probes. Tumor homing was tested using the chick chorioallantoic membrane model with human tumor xenografts. Results & discussion: Surface vimentin levels varied during cell cycle and among the cell lines tested. Surface vimentin expression correlated with cowpea mosaic virus uptake, underscoring the utility of cowpea mosaic virus to detect invasive cancer cells. Targeting to tumor xenografts was observed; homing was based on the enhanced permeability and retention effect. Our data provide novel insights into the role of surface vimentin in cancer and targeting nanoparticles in vivo. © 2011 Future Medicine Ltd.


Leong H.S.,Translational Prostate Cancer Research Group | Lizardo M.M.,University of Western Ontario | Ablack A.,Translational Prostate Cancer Research Group | McPherson V.A.,Translational Prostate Cancer Research Group | And 4 more authors.
PLoS ONE | Year: 2012

The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters. © 2012 Leong et al.

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