Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification

Reflection interference contrast microscopy (RICM) allows the visualization of the cell’s adhesion topology on substrates. Here it is applied as a new label-free method to measure adhesion forces between tumor cells and their substrate without any external manipulation, i.e., the application of force or adjustments in the substrate elasticity. Malignant cancer transformation is closely associated with the down-regulation of adhesion proteins and the consequent reduction of adhesion forces. By analyzing the size and distribution of adhesion patches from a benign and a malignant human pancreatic tumor cell line, we established a model for calculating the adhesion strength based on RICM images. Further, we could show that the cell’s spread area does not necessarily scale with adhesion strength. Despite the larger projected cell area of the malignant cell line, adhesion strength was clearly reduced. This underscores the importance of adhesion patch analysis. The calculated force values were verified by microfluidic detachment assays. Static and dynamic RICM measurements produce numerous adhesion-related parameters from which characteristic cell signatures can be derived. Such a cellular fingerprint can refine the process of categorizing cell lines according to their grade of differentiation.

[1]  Andrés J. García,et al.  Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly. , 2005, Molecular biology of the cell.

[2]  T. Friede,et al.  Adhesion Maturation of Neutrophils on Nanoscopically Presented Platelet Glycoprotein Ibα , 2013, ACS nano.

[3]  Joachim P. Spatz,et al.  Marker-Free Phenotyping of Tumor Cells by Fractal Analysis of Reflection Interference Contrast Microscopy Images , 2013, Nano letters.

[4]  G. J. Fleer,et al.  Stationary dynamics approach to analytical approximations for polymer coexistence curves. , 2004, Physical review. E, Statistical, nonlinear, and soft matter physics.

[5]  D. Gingell,et al.  Interference reflection microscopy. A quantitative theory for image interpretation and its application to cell-substratum separation measurement. , 1979, Biophysical journal.

[6]  L. Addadi,et al.  Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates , 2001, Nature Cell Biology.

[7]  H. Jin,et al.  Integrins: roles in cancer development and as treatment targets , 2004, British Journal of Cancer.

[8]  Thilo Stehle,et al.  Crystal Structure of the Extracellular Segment of Integrin αVβ3 , 2001, Science.

[9]  K. Beningo,et al.  An in vitro correlation of mechanical forces and metastatic capacity , 2011, Physical biology.

[10]  M. Cates,et al.  Imaging optical thicknesses and Separation distances of phospholipid vesicles at solid surfaces , 2011 .

[11]  Clare M. Waterman,et al.  Adhesive F-actin Waves: A Novel Integrin-Mediated Adhesion Complex Coupled to Ventral Actin Polymerization , 2011, PloS one.

[12]  Arnoud Sonnenberg,et al.  Integrins in regulation of tissue development and function , 2003, The Journal of pathology.

[13]  W. T. Chen,et al.  Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts , 1982, The Journal of cell biology.

[14]  P. Davies,et al.  Endothelial cell adhesion in real time. Measurements in vitro by tandem scanning confocal image analysis. , 1993, The Journal of clinical investigation.

[15]  L. Liotta,et al.  General mechanisms of metastasis , 1997, Cancer.

[16]  K. Jacobson,et al.  The composition and dynamics of cell-substratum adhesions in locomoting fish keratocytes. , 1997, Journal of cell science.

[17]  E. Sackmann,et al.  Adhesion-induced domain formation by interplay of long-range repulsion and short-range attraction force: a model membrane study. , 1997, Biophysical journal.

[18]  H. Kessler,et al.  Hydrogel Micropillars with Integrin Selective Peptidomimetic Functionalized Nanopatterned Tops: A New Tool for the Measurement of Cell Traction Forces Transmitted through αvβ3- or α5β1-Integrins , 2013, Advanced materials.

[19]  E. Sackmann,et al.  Microinterferometric Study of the Structure, Interfacial Potential, and Viscoelastic Properties of Polyelectrolyte Multilayer Films on a Planar Substrate , 2004 .

[20]  A. Sonnenberg,et al.  Erratum: Integrins in regulation of tissue development and function. J Pathol; 200: 471–480 , 2003 .

[21]  P Bongrand,et al.  Cell fitting to adhesive surfaces: A prerequisite to firm attachment and subsequent events. , 2002, European cells & materials.

[22]  Jörg Schilling,et al.  Absolute interfacial distance measurements by dual-wavelength reflection interference contrast microscopy. , 2004, Physical review. E, Statistical, nonlinear, and soft matter physics.

[23]  V. Barr,et al.  Interference Reflection Microscopy , 2009, Current protocols in cell biology.

[24]  David R Critchley,et al.  Biochemical and structural properties of the integrin-associated cytoskeletal protein talin. , 2009, Annual review of biophysics.

[25]  D. G. T. Strange,et al.  Extracellular-matrix tethering regulates stem-cell fate. , 2012, Nature materials.

[26]  Jens Friedrichs,et al.  Revealing early steps of alpha2beta1 integrin-mediated adhesion to collagen type I by using single-cell force spectroscopy. , 2007, Molecular biology of the cell.

[27]  Jens Friedrichs,et al.  Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy , 2007 .

[28]  V. Rotter,et al.  Epithelial to Mesenchymal Transition of a Primary Prostate Cell Line with Switches of Cell Adhesion Modules but without Malignant Transformation , 2008, PloS one.

[29]  Laurent Limozin,et al.  Quantitative reflection interference contrast microscopy (RICM) in soft matter and cell adhesion. , 2009, Chemphyschem : a European journal of chemical physics and physical chemistry.

[30]  Wenjun Guo,et al.  The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells , 2008, Cell.

[31]  Dennis E Discher,et al.  Topographical pattern dynamics in passive adhesion of cell membranes. , 2004, Biophysical journal.

[32]  Kristyn S Masters,et al.  Measurement of single-cell adhesion strength using a microfluidic assay , 2010, Biomedical microdevices.

[33]  A. De Arcangelis,et al.  Integrin and ECM functions: roles in vertebrate development. , 2000, Trends in genetics : TIG.

[34]  H. Elsässer,et al.  Establishment and characterisation of two cell lines with different grade of differentiation derived from one primary human pancreatic adenocarcinoma , 1992, Virchows Archiv. B, Cell pathology including molecular pathology.

[35]  Maryam Tabrizian,et al.  Sensing surfaces : Challenges in studying the cell adhesion process and the cell adhesion forces on biomaterials , 2008 .

[36]  Axel Rosenhahn,et al.  Quantification of the adhesion strength of fibroblast cells on ethylene glycol terminated self-assembled monolayers by a microfluidic shear force assay. , 2010, Physical chemistry chemical physics : PCCP.

[37]  J. Spatz,et al.  Synthesis of Quasi‐Hexagonal Ordered Arrays of Metallic Nanoparticles with Tuneable Particle Size , 2008 .

[38]  G. Whitesides,et al.  Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane). , 1998, Analytical chemistry.

[39]  A. S. G. Curtis,et al.  THE MECHANISM OF ADHESION OF CELLS TO GLASS , 1964, The Journal of cell biology.

[40]  Michael W. Davidson,et al.  Nanoscale architecture of integrin-based cell adhesions , 2010, Nature.

[41]  N. Balaban,et al.  Calculation of forces at focal adhesions from elastic substrate data: the effect of localized force and the need for regularization. , 2002, Biophysical journal.

[42]  Y. Fung,et al.  Vascular Endothelium‐Leukocyte Interaction: STICKING SHEAR FORCE IN VENULES , 1975, Circulation research.

[43]  Kevin T. Turner,et al.  Methods to Measure the Strength of Cell Adhesion to Substrates , 2010 .

[44]  D. Lauffenburger,et al.  Receptor-mediated adhesion phenomena. Model studies with the Radical-Flow Detachment Assay. , 1990, Biophysical journal.

[45]  Benjamin Geiger,et al.  Focal Contacts as Mechanosensors Externally Applied Local Mechanical Force Induces Growth of Focal Contacts by an Mdia1-Dependent and Rock-Independent Mechanism , 2001 .

[46]  P. Fromherz,et al.  No correlation of focal contacts and close adhesion by comparing GFP-vinculin and fluorescence interference of DiI , 2001, European Biophysics Journal.

[47]  E. Evans.  Detailed mechanics of membrane-membrane adhesion and separation. I. Continuum of molecular cross-bridges. , 1985, Biophysical journal.

[48]  Michael L. Dustin,et al.  The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling , 2000, Nature Immunology.

[49]  Jean-Jacques Meister,et al.  Force transmission in migrating cells , 2010, The Journal of cell biology.

[50]  M. Abercrombie,et al.  Adhesions of fibroblasts to substratum during contact inhibition observed by interference reflection microscopy. , 1975, Experimental cell research.

[51]  Z. Kam,et al.  Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing , 2011, Nature Cell Biology.

[52]  M. Lerch,et al.  Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model , 2009, BMC Cancer.