The Normal or Cancerous Cell Mechanical Properties Alteration After Epithelial-Mesenchymal Transition

  • 吳 宗憲

Student thesis: Doctoral Thesis


The epithelial-mesenchymal transition (EMT) is known to involve several physiological and pathological phenomena and endows cells with invasive and migratory properties However to identify the alteration of mechanical property of cell undergoing EMT needs further elucidation Moreover the effects of substrate stiffness and topography on the migration of cells before or after transforming growth factor-β1 (TGF-β1)-induced EMT are unknown The cell mechanical properties (CMs) can be used to identify not only cancer EMT but also the cancer malignant transforming process Previous in situ study revealed that EMT taken place and affected cancer cells in seconds during intravasation and extravasation However there is a scarcity of defined tumor CMs with which to predict which noninvasive tumors will progress to malignancy and which cancers will metastasize Therefore there were two purposes in this study Firstly this study utilized a microplate measurement system (MMS) approach based on the deflection of a flexible micro-cantilever was built to measure cell stiffness (in Pa) and adhesion force (in nN) of a single cell during EMT with nN resolution Herein the migration alteration of EMT cell model were measured on the 2D patterns consisted of 1 μm or 5 μm line-widths and groove or cone patterns on either soft (1 96±0 48 MPa) or stiff (3 70±0 74 MPa) polydimethylsiloxane (PDMS) substrates Secondly in bridging across physical and physiological disciplines this study reconciled single-cell measurements in whether recurrent or not tumors and reveal CMs can be used to predict cancer malignant transformation The results demonstrated that after EMT mesenchymal cells became stiffer due to thicker and more abundant F-actin and displayed stronger vinculin accumulation after long-term cell-substrate adhesion The MMS could distinguish differences in compressive stiffness (500 0 and 616 6 Pa) tensile stiffness (469 3 and 517 5 Pa) and adhesion force (153 2 and 173 9 nN) between cells before and after EMT However without proper development of the F-actin or myosin II structure following cytochalasin D (cyto-D) or ML-7 inhibition the mechanical differences were diminished The increased expression of ?-smooth muscle actin (?-SMA) with vinculin in focal adhesion (FA) sites led to an acceleration of EMT cell motility On the 2 MPa substrate the most influenced substrate was the 1 μm cone-patterned substrate where the EMT cells motility decelerated by 0 13 μm/min (36% slower than the cells on groove pattern) However on the 5 μm groove-patterned substrate where the EMT cells demonstrated the most rapid motility relative to the control cells with a increment of 0 18 μm/min (100%) Using MMS combined immunostaining the discrete results demonstrated the intrinsic heterogeneous composition of Rec tumor: including soft lewis lung cancer cell (LLC) stiff EMT LLC and endothelial cells Also using atomic force microscopy (AFM) force mapping the identified stiffer and more complex composition in Rec tumors were observed By flowcytometry differentiation the tumor-retrieval cells were mainly identified as LLC In vitro experiment showed LLC acquired stiffer properties after EMT as well Additionally the rich angiogenesis in Rec tumors offer the stiffer stroma and cytokines for tumor malignant transformation and aggressive invasion ability A comprehensive effort to correlate the CMs of tumor-retrieval cells to specific tumor-prognosis markers were conducted in this study Trivially the decreased body weight gain ratio (BWG) and increased tumor weight (TW) correlated with the stiffer and adhesive CMs of tumors as well as the recurrence and metastasis possibility After EMT the cells with increased stiffness and cell-substrate adhesion force were benefited by faster migratiion and higher invasiveness Thus this technology has the potential to benefit researches on cancer diagnosis drug development and cell-substrate interactions In terms of clinical application the local tumor excision followed by MMS analysis offer the predictability of tumor recurrence and metastasis
Date of Award2014 Apr 25
Original languageEnglish
SupervisorMing-Long Yeh (Supervisor)

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