Real-time Study of E-Cadherin and Membrane Dynamics in Living Animals: Implications for Disease Modeling and Drug Development Alan Serrels, 1 Paul Timpson, 2 Marta Canel, 1 Juliane P. Schwarz, 2 Neil O. Carragher, 3 Margaret C. Frame, 1 Valerie G. Brunton, 1 and Kurt I. Anderson 2 1 Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom; 2 Beatson Institute for Cancer Research, Glasgow, United Kingdom; and 3 Advanced Science and Technology Lab, AstraZeneca, Charnwood, United Kingdom 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 dasa- tinib, 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;69(7):2714–9] Introduction Animal models have rapidly become essential tools in cancer research; from the determination of basic biological mechanisms to the study of complex human diseases. The analysis of molecular dynamics in an intact host environment, however, remains a major challenge. Fluorescence microscopy has been used to probe molecular dynamics of key proteins within two-dimensional cell cultures (1, 2), but in vivo conditions are more restrictive (3–5), and it is unclear to what extent these in vitro techniques can be applied in vivo . It is therefore important and necessary to develop techniques for the quantification of molecular dynamics in vivo . Cell-cell interactions mediated by E-cadherin are central to maintaining normal tissue epithelial architecture and have been studied in great detail in vitro (6). The disruption and deregulation of E-cadherin–mediated cell-cell adhesions in cancer is a critical initiation step in the epithelial to mesenchymal transition associated with an invasive phenotype (7). Alterations in E-cadherin dynamics could therefore serve as an early molecular biomarker of metastasis. In order to study E-cadherin and membrane dynamics in their physiologic context, we have applied two complimentary techniques, photobleaching and photoactiva- tion of fluorescence, to visualize protein dynamics in cell-cell junctions and the plasma membrane in tumor xenografts (8–10). Photobleaching is frequently used for the characterization of molecular dynamics within cultured cells. In the simplest approach, the fluorescence within a small region of the sample is bleached using a laser and the recovery of fluorescence into the bleached region is measured over time (11). Photoactivation is a related technique, in which a nonfluorescent (caged) precursor becomes fluorescent upon activation (10). In both cases, two basic variables are derived, ( a ) the half-time of recovery, an indication of the rate at which probes move in or out of the analysis region, and (b) the immobile fraction, an indication of how much of the probe remains trapped and unable to move out of the analyzed region. Using this approach, we have successfully quantified E-cadherin dynamics in a three-dimensional host setting and provided a molecular readout of the early mobilization events in tumor cells. Our results highlight the necessity and advantages of live animal imaging in the study of tumor development and metastasis, and show that photobleaching and photoactivation can be used in vivo for the quantification of drug action in the treatment of cancer. Materials and Methods Plasmids. GFP-E-cadherin and PAGFP were kind gifts from Jennifer Stow (Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland, Australia) and Jennifer Lippincott-Schwartz (Cell Biology and Metabolism Branch, NIH, Bethesda, MD), respectively. PAGFPFarn2Palm was made by substituting GFP in the pEGFP-N1 vector with PAGFP-Farn2Palm (for cloning, see Supplementary Materials). Cell lines. A431 cells were obtained from (American Type Culture Collection), and maintained as described. The cell-derived matrix was generated as described previously (12). Cells (2 Â 10 5 ) were plated onto the cell-derived matrix and cells within colonies or migrating cells were targeted for photobleaching and analyzed as described in the Supplemen- tary Materials. Photoactivation, photobleaching, and analysis in vitro and in vivo. A detailed description of these techniques and analysis, as well as drug treatment in vitro and in vivo , can also be found online in the Supplementary Materials. Results and Discussion Photobleaching of GFP-E-cadherin dynamics in vitro corre- lates with cell migration. Cell-cell adhesion turnover is thought Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). A. Serrels and P. Timpson contributed equally to this work. Requests for reprints: Kurt I. Anderson, The Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, United Kingdom. Phone: 44-141-330- 2864; Fax: 44-141-942-6521; E-mail: k.anderson@beatson.gla.ac.uk. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-4308 Cancer Res 2009; 69: (7). April 1, 2009 2714 www.aacrjournals.org Priority Report Research. on February 7, 2022. © 2009 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst March 24, 2009; DOI: 10.1158/0008-5472.CAN-08-4308