Abstract 1500: Development of a Quantitative Immunofluorescence imaging assay to assess β-catenin translocation and multiplex biomarkers for Epithelial-Mesenchymal Transition (EMT) in tumor tissues.
Authors: Navas Tony , Kinders Robert J. , Mutreja Karun , Lawrence Scott M. , Butcher Donna , Hollingshead Melinda , Parchment Ralph E. , Tomaszewski Joseph E. , Doroshow James H. 
Published: August 2014

Abstract
Background: Wnt/ß-catenin signaling and cadherin-mediated cell adhesion are two processes that promote cell proliferation and epithelial-mesenchymal transition (EMT) of carcinomas. Therapeutic inhibitors of Wnt-induced tumor activation prevent stabilization and nuclear translocation of β-catenin and putatively lead to inhibition of tumor metastasis by upregulation of E-Cadherin and downregulation of N-Cadherin and Vimentin.
Methods: A multiplex quantitative immunofluorescence (qIFA) assay was developed to assess changes in levels of EMT biomarkers (E-Cadherin, N-Cadherin, Vimentin/Pancytokeratin) as well as monitor intracellular localization of β-catenin in tumor tissues. A panel of highly specific antibodies against EMT targets, directly-conjugated to various fluorophores, were optimized for multiplex staining of FFPE tissues using colorectal (CRC) and triple-negative breast (TNBC) cancer cell lines and xenograft tissues as tumor controls for target expression. Rat kidney was used as positive control for endogenous biomarker expression in tissues. Image acquisition was performed with Confocal and Epifluorescence microscopy. Image segmentation and quantitative multiplex analysis were performed using NIS Elements and Definiens Tissue Studio software to obtain total mean integrated signal per biomarker within a tissue region of interest.
Results: Fitness for purpose evaluation of the biomarkers was performed in vitro with several cell lines and compounds to demonstrate nuclear translocation of β-catenin as well as EMT-mediated changes in E- to N-Cadherin ratio. A431 squamous epithelial cell lines treated with Lysophosphatidic acid (LPA) induced Adherens Junction (AJ) dissociation of membrane E-Cadherin and β-catenin while co-treatment with specific inhibitors to glycogen synthase kinase-3β (GSK-3β) induced a 5-fold increase in the nuclear to cytoplasmic mean intensity ratio of β-catenin after 24h. TGFβ-treatment of the NMuMG mouse epithelial cell line induced a significant decrease in E- to N-Cadherin membrane intensity ratio after 48 and 72h in vitro. Studies with human TNBC or APC-mutated CRC mouse xenograft models using HDAC or Src/Abl kinase inhibitors to compare pre- and post-treatment effects on EMT biomarker staining will be presented.
Conclusion: We have developed a quantitative imaging assay that integrates multiplex changes in EMT biomarkers to assay pharmacodynamic activities of potential targeted agents currently in development at the National Cancer Institute (NCI). This assay could also be applied for retrospective analysis of clinical samples to document drug-induced changes in tumor phenotypes. Funded by NCI Contract No. HHSN261200800001E.
Citation Format: Tony Navas, Robert J. Kinders, Karun Mutreja, Scott M. Lawrence, Donna Butcher, Melinda Hollingshead, Ralph E. Parchment, Joseph E. Tomaszewski, James H. Doroshow. Development of a Quantitative Immunofluorescence imaging assay to assess β-catenin translocation and multiplex biomarkers for Epithelial-Mesenchymal Transition (EMT) in tumor tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1500. doi:10.1158/1538-7445.AM2013-1500