River Rapids Conference Proceedings of the Society for Experimental Mechanics Series Mechanics of Biological Systems and Materials, Volume 6 Chad S. Korach Srinivasan Arjun Tekalur Pablo Zavattieri Proceedings of the 2016 Annual Conference on Experimental and Applied Mechanics River Publishers
Conference Proceedings of the Society for Experimental Mechanics Series Series Editor Kristin B. Zimmerman, Ph.D. Society for Experimental Mechanics, Inc. Bethel, CT, USA
River Publishers Chad S. Korach • Srinivasan Arjun Tekalur • Pablo Zavattieri Editors Mechanics of Biological Systems and Materials, Volume 6 Proceedings of the 2016 Annual Conference on Experimental and Applied Mechanics
Published, sold and distributed by: River Publishers Broagervej 10 9260 Gistrup Denmark www.riverpublishers.com ISBN 978-87-7004-940-5 (eBook) Conference Proceedings of the Society for Experimental Mechanics An imprint of River Publishers © The Society for Experimental Mechanics, Inc. 2017 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, or reproduction in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Preface Mechanics of Biological Systems and Materials represents one of ten volumes of technical papers presented at the SEM 2016 Annual Conference and Exposition on Experimental and Applied Mechanics organized by the Society for Experimental Mechanics and held in Orlando, FL, June 6–9, 2016. The complete proceedings also includes volumes onDynamic Behavior of Materials; Challenges in Mechanics of Time-Dependent Materials; Advancement of Optical Methods in Experimental Mechanics; Experimental and Applied Mechanics; Micro-and Nanomechanics; Mechanics of Composite and Multifunctional Materials; Fracture, Fatigue, Failure and Damage Evolution; Residual Stress, Thermomechanics and Infrared Imaging, Hybrid Techniques and Inverse Problems; and Joining Technologies for Composites and Dissimilar Materials. Each collection presents early findings from experimental and computational investigations on an important area within experimental mechanics, the mechanics of biological systems and materials being one of these areas. This volume summarizes the exchange of ideas and information among scientists and engineers involved in the research and analysis of how mechanical loads interact with the structure, properties, and function of living organisms and their tissues. The scope includes experimental, imaging, numerical, and mathematical techniques and tools spanning various lengths and time scales. Establishing this symposium at the Annual Meeting of the Society for Experimental Mechanics provides a venue where state-of-the-art experimental methods can be leveraged in the study of biomechanics. A major goal of the symposium was for participants to collaborate in the asking of fundamental questions and the development of new techniques to address bio-inspired problems in society, human health, and the natural world. The organizers would like to thank all the speakers and staff at SEM for enabling a successful program. Stony Brook, NY, USA Chad S. Korach East Lansing, MI, USA Srinivasan Arjun Tekalur West Lafayette, IN, USA Pablo Zavattieri v
Contents 1 Mechanic Adaptability of Metastatic Cells in Colon Cancer................................. 1 V. Palmieri, D. Lucchetti, M. Papi, F. Calapa`, G. Ciasca, A. Sgambato, and M. De Spirito 2 Nano-Mechanical Response of Red Blood Cells ........................................... 11 Massimiliano Papi, Gabriele Ciasca, Valentina Palmieri, Giuseppe Maulucci, Cristina Rossi, Eleonora Minelli, and Marco De Spirito 3 Scale Dependence of the Mechanical Properties of Interfaces in Crustaceans Thin Films ........... 17 Devendra Verma, Tao Qu, and Vikas Tomar 4 Dynamic Analysis of Human Knee.................................................... 25 S. Yoshida, U. Tiwari, A. Saladino, M. Nguyen, D. Hollander, B. Boudreaux, and B. Hadley 5 Viscohyperelastic Calibration in Mechanical Characterization of Soft Matter................... 33 E. Ficarella, L. Lamberti, M. Papi, M. De Spirito, and C. Pappalettere 6 Contact Zone Evaluation of Dental Implants Using Digital Photoelasticity...................... 39 M.P. Hariprasad and K. Ramesh 7 Evolution of the Skin Microstructural Organization During a Mechanical Assay................. 45 B. Lynch, S. Bancelin, C. Bonod-Bidaud, F. Ruggiero, M.-C. Schanne-Klein, and J.-M. Allain 8 A Numerical Study of a Biaxial Sollicitation to Set-Up the Displacement Field Measurement of Ex Vivo Mouse Skin.............................................. 53 J.-S. Affagard, F. Wijanto, R. Rubio Amador, C. Bonod-Bidaud, F. Ruggiero, and J.-M. Allain 9 Dynamic Polarization Microscopy for In Situ Measurements of Collagen Fiber Realignment During Impact ......................................................... 61 Xianyu Wu, Hsiao-Ying Shadow Huang, Mark Pankow, and Kara Peters 10 Self-Shifting Neutral Axis and Negative Poisson’s Ratio in Hierarchical Structured Natural Composites: Bamboo........................................................ 67 Shaowen Xu, Aniruddha Mitra, Stephen Migues, Jacob Mayfield, Michael Shinall, Bessenbacher Derek, Davis Linley, and Spratlin Russell 11 High-Speed Holography for In-Vivo Measurement of Acoustically Induced Motions of Mammalian Tympanic Membrane.................................................. 75 Payam Razavi, Jeffrey Tao Cheng, Cosme Furlong, and John J. Rosowski 12 Rheology of Soft and Rigid Micro Particles in Curved Microfluidic Channels ................... 83 Jia Liu, Yuhao Qiang, Michael Mian, Weihe Xu, and E. Du 13 Microfluidic Approaches for Biomechanics of Red Blood Cells ............................... 89 E. Du 14 Custom Indentation System for Mechanical Characterization of Soft Matter.................... 95 Chelsey Simmons, Andres Rubiano, Daniel Stewart, and Brandey Andersen vii
15 Experimental Evaluation of Blast Loadings on the Ear and Head with and Without Hearing Protection Devices .......................................................... 101 Tim J. Walilko, Ryan D. Lowe, Ted F. Argo, G. Doug Meegan, Nathaniel T. Greene, and Daniel J. Tollin 16 A Mechano-Hydraulic Model of Intracranial Pressure Dynamics ............................. 111 D. Evans, C. Drapaca, and J.P. Cusumano 17 Regional Variations in the Mechanical Strains of the Human Optic Nerve Head................. 119 Dan E. Midgett, Mary E. Pease, Harry A. Quigley, Mohak Patel, Christian Franck, and Thao D. Nguyen 18 Experimental Electromechanics of Red Blood Cells Using Dielectrophoresis-Based Microfluidics ..................................................................... 129 Yuhao Qiang, Jia Liu, Michael Mian, and E. Du 19 Microbuckling of Fibrous Matrices Enables Long Range Cell Mechanosensing.................. 135 Brian Burkel, Ayelet Lesman, Phoebus Rosakis, David A. Tirrell, Guruswami Ravichandran, and Jacob Notbohm 20 The Growth and Mechanical Properties of Abalone Nacre Mesolayer......................... 143 Anqi Zhang, Yan Chen, MariAnne Sullivan, and Barton C. Prorok 21 Evaluation of Precise Optimal Cyclic Strain for Tenogenic Differentiation of MSCs .............. 149 Yasuyuki Morita, Toshihiro Sato, Sachi Watanabe, and Yang Ju 22 Effect of Fiber Architecture on the Cell Functions of Electrospun Fiber Membranes .............. 157 F. Sultana, M. Vaughan, and M. Khandaker 23 Controlling hESC-CM Cell Morphology on Patterned Substrates Over a Range of Stiffness . . . . . . . . 161 Brett N. Napiwocki, Max R. Salick, Randolph S. Ashton, and Wendy C. Crone 24 Cytoskeletal Perturbing Drugs and Their Effect on Cell Elasticity............................ 169 Martha E. Grady, Russell J. Composto, and David M. Eckmann viii Contents
Chapter 1 Mechanic Adaptability of Metastatic Cells in Colon Cancer V. Palmieri, D. Lucchetti, M. Papi, F. Calapa`, G. Ciasca, A. Sgambato, and M. De Spirito Abstract Tumour microenvironment contributes importantly to the phenotype development of cancer cells, which receive and adapt to chemical and physical signals. It is hypothesized that remodelling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signalling and permit the acquisition of metastatic properties such as anchorage-independent growth and enhanced cell migration. Among different cancer cell lines, the SW480 and SW620 colon cells, derived from the same patient at different stages of tumour progression, represent a unique model to study the changes in mechanical properties during metastases. The SW480 derive from a primary colon tumour, while the SW620 originate from a population of malignant cells appeared in a lymph node metastases months after the initial surgery. In this work we measured by AFM, combined with Electron and Confocal Microscopy, the mechanic adaptability of cancer cell which reached in vivo the lymph node environment. We demonstrate how to resist to apoptotic stimuli in the lymphatic circulation, where the flow shear stress reduce cell survival, the metastatic cell increase their non-specific adhesion while retaining a flexible and soft cytoskeleton, related to uncontrolled growth properties. These features allow accomplishing the spreading in the circulation aim of metastatic cells. Keywords Cancer mechanics • Colon • AFM • SEM 1.1 Introduction Cancer mechanics is the study of forces involved in the intricate interplay between cells and extracellular environment. The emerging idea in this research field is that forces acting on cells can control biochemical signals responsible for cell proliferation, migration and apoptosis [1]. Cells sense and respond to the mechanical properties of the environment, such as the stiffness of the extracellular matrix (ECM) or the compression exerted by neighbouring tissues, by balancing external forces with changes in cytoskeleton organization and shape remodelling. This response leads to the activation of signalling pathways of cells spreading, growth, motility and death [2]. The understanding of the forces that link environment and tumour cells can help to understand the metastatic process, leading cause of mortality among cancer patients. During this process, cells detach from neighbours of the primary tumor, remodel the external matrix and migrate towards the vasculature and lymphatics. These processes require dynamic modulation of cell shape and cytoskeleton together with changes in gene expression [2]. For example metastatic cells can detach by altering surface proteins expression and reducing adhesion or by altering cytoskeleton polymerization in order to acquire a higher plasticity to squeeze easily through ECM [1]. Specific cellular and extra-cellular mechanical properties have recently been exploited as possible biomarkers of disease [3]. In particular, in cancer progression, mechanical properties would allow identifying physical attributes of cells that are more likely to metastasize [3, 4]. In this study we compared the mechanical and morphological features of colon cancer cell populations isolated from a primary tumor and from its lymph-node metastases. We used the SW480 and SW620 colon cancer cell lines that offer the unique advantage of having been isolated from the same patient at different stages of cancer progression. In particular SW480 cell line originates from a Dukes’ stage B colon carcinoma while the SW620 cell line has been derived from a lymph V. Palmieri (*) • M. Papi • G. Ciasca • M. De Spirito Institute of Physics, Universita` Cattolica del Sacro Cuore, L.go Francesco Vito 1, 00168 Rome, Italy e-mail: vplabcemi@gmail.com D. Lucchetti (*) • F. Calapa` • A. Sgambato Institute of Pathology, Universita` Cattolica del Sacro Cuore, L.go Francesco Vito 1, 00168 Rome, Italy e-mail: dnlucchetti@gmail.com #The Society for Experimental Mechanics, Inc. 2017 C.S. Korach et al. (eds.), Mechanics of Biological Systems and Materials, Volume 6, Conference Proceedings of the Society for Experimental Mechanics Series, DOI 10.1007/978-3-319-41351-8_1 1
node metastasis developed in the same patient few months later the surgical removal of the tumor [5]. For their limited genetic variability, the SW480 and SW620 lines represent a unique model to analyse biophysical changes occurring during metastatic progression in vivo [5]. The mechanical and morphological properties of both cell lines have been studied by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Confocal Microscopy and a set of morphological parameters concerning cell shape, membrane roughness and protrusions appearance has been analysed. We elucidated the cytoskeleton organization associated with metastasis and provided a further understanding of the mechanical aspects implied in malignant transformation of cells that can represent possible targets for the prediction, treatment and even prevention of cancer. 1.2 Results and Discussion It has been previously reported that SW480 and SW620 cells have different appearance in culture: most SW480 cells have a spreading, epithelial-type morphology (E-type), while a small fraction displays a rounded morphology (R-type). In contrast, SW620 cells are known to display an ovoid morphology and form small aggregates [6]. We observed clear morphological differences in SEM micrographs (Fig. 1.1) with a prevailing rounded shape for SW620 cells (Fig. 1.1b) and a mixed elongated and rounded morphology in the SW480 cultures (Fig. 1.1a). E-type and R-type cells in the SW480 line were analysed separately as previously described [7]: cells with an Aspect Ratio higher than 1.5 were considered E-type cells (Fig. 1.1c). Fig. 1.1 SEM Morphological characterization of SW480 (E-type or R type) and SW620 cells. Cells have different morphologies: SW480 cells comprise an elongated (E) and a rounded (R) (a) population both showing a large lamellipodia area and fine protrusions emanating from it. On the contrary, SW620 cells have a rounded morphology with a reduced number of protrusions and a small lamellipodia area (b). (c) The SW480 line can be divided in two distinct populations using a threshold based on Aspect Ratio of the cells. Indeed elongated cells (E-type) have a mean AR around two while rounded cells (R-type) of one. The SW620 cells have all an aspect ratio always less than 1.5 so they were not divided in sub-populations. Both E-type and R-type cells display a larger lamellipodia area (d) and a higher density of filopodia (e) compared to SW620 cells. Results are statistically significant (p <0.001 one-way ANOVA and Tukey’s multiple comparison tests) 2 V. Palmieri et al.
In SW620 cells the AR never exceeded 1.5 (Fig. 1.1c). This criterion has been used also for measurements performed with AFM and Confocal Microscopy. The membrane protrusions of SW480 and SW620 cells appeared markedly different in SEM micrographs. In Fig. 1.1a, a large area around the cell body of SW480 cells is visible with thin filaments protruding from it. These structures are respectively the lamellipodia and filopodia, highly dynamic thin cell wall extensions of actin filaments. On the other hand, SW620 cells displayed a less extended lamellipodia area (Fig. 1.1b). Differences observed in membrane protrusions were quantified by means of two parameters: density of filopodia along the cell perimeter (ρF) and Lamellipodia Area (AL). The SW480 cells displayed a higher ρF and a larger AL in respect to SW620 cells (Fig. 1.1d, e). It is important to note also a sharp difference in cell surface roughness in SEM micrographs. Indeed SW620 display a much smoother membrane compared to both populations of SW480 line. Cell mechanics was analysed by means of Atomic Force Microscopy (AFM), recording strain-stress characteristics (force deformation curves) on cell surface and Hertz Model to calculate cell elasticity. From the obtained curves, cell stiffness (Young Modulus) and non-specific cell adhesion were derived (Fig. 1.2). Young Moduli of SW480 E-type or R-type and SW620 cells are shown in Fig. 1.2a–c respectively. SW480 E-type cells displayed the highest values of Young modulus, with a wide distribution peaked at 1060 Pa. Both R-type SW480 and SW620 cells exhibited a narrower distribution peaked at 500 Pa, hence displaying a “softer” cytoskeleton. The low values of Young moduli measured (below 1 kPa) for all cell lines considered in this study, indicate a high deformability and compliance, typical of malignant phenotypes [8]. The adhesion distributions of SW480 E-type, SW480 R-type and SW620 cells are shown in Fig. 1.2d–f, respectively. The metastatic line SW620 displayed the highest adhesion with a distribution peaked at 95 pN. The two populations of SW480 cell line showed comparable adhesion values with averages around 50 pN, in accordance to the similar surface roughness observed in SEM micrographs which is known to be strongly related to cell adhesion properties at the nanoscale [9]. Fig. 1.2 Young Moduli of SW480 cells (E-type in (a) and R-type in (b)) compared to SW620 cells (c). Cells with the highest Young modulus were SW480 E-type, which exhibited a larger distribution peaked at 1200 Pa. Both R-type SW480 and SW620 cells exhibited a narrower distribution peaked at 500 Pa, hence displaying a “softer” cytoskeleton. Non-specific cell adhesion of SW620 (f) was significatively higher than in E-type (d) and R-type (e) SW480 cells 1 Mechanic Adaptability of Metastatic Cells in Colon Cancer 3
The high values of adhesion, the smooth surface topography and the decreased number of protrusions observed in the SW620 cells suggested an altered actin organization, which has been then analysed by confocal imaging (Fig. 1.3). In Fig. 1.3 representative confocal micrographs of SW480 and SW620 cells are shown with actin in green and cell nuclei in blue. In order to establish cytoskeleton organization differences among different cell lines, two parameters were considered: actin fibers Coherency and density of junctions of actin network (ρJ). Coherency is calculated from the structure tensor of each pixel in the image and is bounded between 0 (isotropic areas) and 1 (highly oriented structures) [10]. In Fig. 1.4a–c three representative confocal images of skeletonized actins filaments Fig. 1.3 Representative confocal images of Phalloidin labeled actin in SW480 and SW620 cells. Both elongated and rounded cells are visible in the microscope field in SW480 sample (left), while only rounded cells are visible in SW620 micrograph (right). Cells nuclei are labelled in blue withDAPI Fig. 1.4 Representative confocal images of skeletonized actin in SW480 E-type (a), SW480 R-type (b) and SW620 cells (c). Skeleton branches are labelled in orange, junctions in purple and end points in blue. On the right, skeletons are superimposed on the original confocal images of SW480 E-type (d), SW480 R-type (e) and SW620 (f) cells. Scale Bar is 22μmin (d, e) and10μmin (f). In theinsets detail of cytoskeleton of each cell are shown 4 V. Palmieri et al.
acquired on SW480 E-type (a), SW480 R-type (b) and SW620 cells (c) are shown. The same images were superimposed to the corresponding original confocal images in Fig. 1.4d–e. Analysis of skeletonized images allowed to highlight actin branches (orange), junctions (purple) and isolated points (blue) (inset in Fig. 1.4c). Analysis of Coherency was also calculated on the original confocal images (Fig. 1.4d–f) according to the procedure described in the Experimental section. Image analysis revealed that, as expected from the lower stiffness values, the rounded cells have a decreased Coherency (Table 1.1). Indeed we measured a mean value of Coherency of 0.4 for E-type SW480 cells and around 0.2 for R-type SW480 cells and SW620 cells. On the other hand, the increased adhesion together with the reduced number of junctions are markers of an actin network destructuration in SW620 cells (Table 1.1). To control their migratory and invasive capabilities, cancer cells are able to reorganize both their membrane protrusions and cytoskeleton [11, 12]. Filopodia and lamellipodia protrusions drive cell migration by attaching to the substrate and generating forces to pull the cell body forward, while cytoskeleton organization influences cell shape and mechanics as well as cell response to external forces [13]. Protrusions and, more importantly, cytoskeleton subversion in cancer cells can lead to changes in cell growth, stiffness, movement and invasiveness [14]. In order to analyse cytoskeleton of metastatic and non-metastatic cells, different high resolution techniques were used in this study. Filopodia and lamellipodia protrusions and cell surface morphology were analyzed by Scanning Electron Microscopy. Mechanical cell parameters (elasticity and cell adhesion) were obtained recording Force-Distance Curves on cell surface by Atomic Force Microscopy and actin cytoskeleton organization was quantified by measuring fibers anisotropy (Coherency) and networking (Junctions density) by image analysis of Confocal Microscopy measurements. The specialized functions of SW480 populations, i.e. proliferate at the primary site (SW480 R-type) or metastasize (SW480 E-type), are reflected in cell shape and mechanical properties. We observed that SW480 R-type display a rounded morphology with a decreased stiffness and actin anisotropy (Coherency) compared to E-type cells. The decrease of cell stiffness, that can be a consequence of a destructuration of actin bundles (reduced Coherency), can represent a mechanistic pathway that drives uncontrolled growth and evasion of apoptosis [1]. Indeed, the actin cytoskeleton is a major determinant of cell polarity and cell-to-cell junctions, essential for normal tissue homeostasis. When cell polarity is lost, tissue integrity is compromised, resulting in overgrowth, aberrant invasive behaviour and promotion of tumours [14]. The higher cell deformability and actin isotropy in rounded cells are also markers of their ability to grow in culture without anchorage [6, 15]. Anchorage-independent growth is a condition where cell proliferation does not depend on culture substrate or cell-to-cell contact and has been observed for SW480 R-type and SW620 cells but not for SW480 E-type cells [6]. It has been recently demonstrated that the HCT-8 colon cancer cells become rounded and “soft” when, losing the mechano-sensitivity to the surrounding environment, gain this ability to grow “independently” [16]. Similarly, SW480 R-type cells have lost sensitiveness to the environment and grow uncontrollably while SW480 E-type cells, with a higher stiffness, are still capable of sensing neighbouring tissues and invade them. Cells derived from lymph node metastasis (SW620 cells) have a rounded morphology similarly to SW480 R-type cells and share with them the ability to grow without anchorage which is reflected in the mean cell elasticity and a reduced fibers Coherency which are comparable to the values obtained for SW480 R-type cells. Conversely, cells surface appeared markedly altered in the metastatic SW620 cells, which displayed a smoother surface and a decreased filopodia density and lamellipodia area in comparison to SW480 primary tumor cells. SW620 cells are known to have a limited migration capacity compared to both populations of SW480 cells line [15]. This feature was reflected in a decreased number of protrusions on their surface. The smoothness of cells surface can be a consequence of the reduction of filopodia structures and/or a marker of an impairment of organization of cortical layer of cytoskeleton underlying the membrane of SW620 cells [17]. SW620 cells are representative of cancer cells that have detached from primary tumor and have invaded lymph nodes, therefore they must have acquired specific properties to survive in the lymphatic stream. Indeed, once in the vasculature, Table 1.1 Analysis of actin organization from confocal images ρJ (μm 2) Coherency SW480-E 1.59 0.52 0.4 0.14 SW480-R 1.82 1.07 0.19 0.13 SW620 0.82 0.54 0.17 0.11 Average density of cytoskeleton junctions, normalized to cell area, obtained after skeletonization of actin network on confocal images ρJ. The number of junctions is highly reduced only in the SW620 cells. Coherency of cytoskeleton obtained with OrientationJ plugin. Coherency is bounded between 0 and 1, with 1 indicating highly oriented structures and 0 indicating isotropic areas. The coherency is markedly reduced in SW480-R and SW620 cells 1 Mechanic Adaptability of Metastatic Cells in Colon Cancer 5
cancer cells are exposed to fluid shear forces of the stream. The majority of circulating cancer cells are rapidly and lethally damaged in the microvasculature. To survive, circulating cancer cells must adhere to the vessel walls and eventually penetrate it [18]. We observed an increase in cell adhesion of metastatic SW620 cells which can be consequence of an altered arrangement of the cortical actin network underlying membrane, as demonstrated by Mescola et al. [19]. Indeed, actin network of SW620 cells, imaged with Confocal microscopy, displayed a reduction in the number of junctions compared to SW480 cells. This actin network “weakening” linearly correlates with the AFM values of adhesion. Therefore, we suggest that modification of the actin organization observed in SW620 cells is necessary to become more “sticky” to vasculature walls and be more stable in the lymphatic and blood vessels. 1.3 Experimental 1.3.1 Cell Cultures The SW480 and SW620 human colon carcinoma-derived cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and were maintained in RPMI supplemented with 10 % fetal bovine serum, penicillinstreptomycin (100 U/ml), and 2 mML-glutamine at 37 C, in a humid 5 % CO2 atmosphere, as previously reported [20]. All image analysis measurements were performed on 60 cells per sample on three independent samples. 1.3.2 Scanning Electron Microscopy (SEM) For SEM imaging, cells on coverslips were washed three times with 0.1 M Sodium Cacodylate buffer (pH 7.4) and then incubated with 0.1 M Sodium Cacodylate buffer and 2.5 % glutaraldehyde for 4 h. Then cells were dehydrated serially in 30, 60, 80 and 100 % ethanol. Finally samples were fixed for 30 min with 2 % osmium tetra-oxide (OsO4). The SEM procedures were completed by drying of the samples, and sputtering of 8 nm gold layer as reported previously [21]. Micrographs were acquired with a Zeiss Supra 25 microscope (Germany) with a secondary electron detector. In order to quantify cell morphology, cell axes were measured by means of ImageJ software. From these values, the ellipse fitting each single cell was drawn and perimeter (P) and area (A) were calculated as follows P¼2π ffiffiffiffiffiffiffiffiffiffiffiffiffiffifi a2 þb2 2 s ð1:1Þ A¼πab ð1:2Þ where a and b are the major and minor semi axis of the cell. The number of filopodia per cell (NF) was extracted from images and normalized by the cell perimeter to obtain the density of filopodia (ρF) along the cell membrane: ρF ¼ NF P ð 1:3Þ In order to quantify the lamellipodia area, cell major (aB) and minor (bB) axis, were measured without taking into account the lamellipodia extensions. From these parameters, the cell “body” area was calculated and subtracted from the total Area (A) to recover the Lamellipodia Area (AL): AL ¼A πaBbB ð1:4Þ 6 V. Palmieri et al.
In the SW480 cell line, cells belonging to the R-type or E-type were distinguished on the basis of the Aspect Ratio (AR) defined as: AR¼ aB bB ð 1:5Þ Cells with AR higher than 1.5 were considered E-type cells. 1.3.3 Atomic Force Microscopy (AFM) Cells were kept in the cell culture medium at a constant temperature (37 C) throughout data acquisition. Cantilevers with a silica conical tip characterized by an end radius of ~10 nm and a half conical angle of 20 have been used (CSC16MikroMasch). All these cantilevers, with a nominal spring constant of k ¼~0.02 N/m, were accurately calibrated by using thermal method. Force-distance (F-D) curves were obtained using a fixed force set point and keeping a constant speed of 4.0μm/s [22]. The total vertical displacement was set to 12μm. F–D curves were analysed using the data processing supplied with the JPK Nanowizard AFM system by applying baseline subtraction, conversion to tip-sample separation, identification and fitting of each jump in the retraction curve to determine the quantitative parameters described in the text. To recover the local cell Young Modulus (E) all the reaction forces F(δ) obtained from the approaching curves were fitted to the Hertz curve: F δð Þ¼ 2E tan αð Þ π 1 v2 ð Þ δ2 ð1:6Þ whereδis the indentation depth, ν ¼0.5 is the cells Poisson’s ratio andα ¼20 the half opening angle of the AFM tip apex. Local cell adhesion has been extracted directly from the absolute value of the force minimum in the retract curve [19, 23, 24]. 1.3.4 Confocal Microscopy For immunofluorescence cells were stained with FITC-labeled Phalloidin (Life Technologies) and DAPI (fluoromount G with DAPI, Electron Microscopy Sciences) as reported previously [24, 25]. Immunofluorescence images were obtained with a multichannel white light source with DAPI or FITC filter settings on a CARV II spinning-disk microscope (Crisel Instruments, Rome, Italy) by using a 60 oil immersion objective (NA 1.4). Z-stacks were acquired for each sample. Background values (defined as intensities below 7 % of the maximum intensity) were set to zero and coloured black as previously reported [26]. Image processing and analysis was performed with ImageJ software [27]. After having measured the Aspect Ratio of each cell in order to distinguish between elongated and rounded cells (AR threshold ¼1.5), actin organization was analysed with different ImageJ plugins. Firstly, the OrientationJ plugin has been used to obtain the Coherency parameter as described previously [10]. The analysis of Coherency was performed on maximum intensity projections of the acquired Z stacks. OrientationJ evaluates the local orientation of every pixel of an image by calculating the structure tensor for each pixel. The Coherency (C) parameter is the ratio between the difference and the sum of the structure tensor eigenvalues and is bounded between 0 (isotropic areas) and 1 (highly oriented structures) [10]. To analyse the number of knots of actin network the Skeletonize tool of ImageJ has been used in order to segment 2D Z-projections. For each cell the number of junctions was calculated and normalized to cell area to obtain the density of Junctions (ρJ). 1.3.5 Statistical Analysis Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison test. A value of p <0.001 was considered statistically significant. 1 Mechanic Adaptability of Metastatic Cells in Colon Cancer 7
1.4 Conclusions A better understanding of cells mechanics can help to predict growth and spreading ability of cancer cells. In order to investigate structural differences in cellular architecture between non-metastatic and metastatic cancer cells, we analysed the colon cancer SW480 and SW620 cell lines, which offer the unique advantage of representing different stages of disease progression of the same cancer. In fact, SW480 and SW620 cell lines were derived, respectively, from the primary tumour and a lymph-node metastasis from the same patient. Two distinct sub-populations have been described within the SW480 cell line, the E-type and R-type cells, whose name reflects their Elongated and Rounded morphology, respectively [7, 28]. Though being both derived from a primary tumour, these populations display opposite invasive properties when inoculated in nude mice: while the E-type cells have the ability to metastasize but form spontaneously regressive primary tumours, R-type cells form large primary tumors without invasion or nodal metastases [28]. On the other hand, the SW620 cell line comprises a single population of rounded metastatic cells [5]. In this article we demonstrated that cell mechanics can be related to actin organization and showed that a decrease in cell elasticity is accompanied by a high isotropy of actin fibers (SW480 R-type and SW620 cells) while a decreased number of actin network junctions is related to an increase in cell adhesion (SW620 cells) and to a smooth cell surface. We also demonstrated that mechanical stiffness and cell adhesion are modulated by cancer cells during the metastatic process. We hypothesize that regulation of cell mechanics can allow cancer cells to acquire specialized functions essential for their ability to grow, invade surrounding tissues and metastasize. In particular soft rounded cells, insensitive to the environment, are responsible for the increase of the tumour volume thanks to their uncontrolled growth without anchorage On the other hand, elongated and highly motile cells are more prone to invade neighbouring tissues due to their high plasticity coupled to sensitiveness to the ECM. In order to cope with shear forces of the lymphatic and hematic flow, cells organize a more “flexible” cortical actin network to increase their adhesion to vessels: then once a metastatic cell reaches a lymph node, again it modulates its mechanical properties to survive in a new environment [18]. Acknowledgements This research has been supported by Universita` Cattolica del Sacro Cuore of Rome. 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Chapter 2 Nano-Mechanical Response of Red Blood Cells Massimiliano Papi, Gabriele Ciasca, Valentina Palmieri, Giuseppe Maulucci, Cristina Rossi, Eleonora Minelli, and Marco De Spirito Abstract In their physiological function, red blood cells (RBCs) need to undergo large deformations in order to pass through capillaries and small vessels. In several pathological conditions, including diabetes mellitus and Alzheimer’s disease, this extreme deformability appears to be deeply impaired and an increase in the RBCs stiffness is usually detected. Given the key role played by the mechanical properties of RBCs, we investigated their viscous-elastic response by AFM nano-mapping. High-resolution maps demonstrate that healthy erythrocytes are stiffer in their canter and softer at the periphery. The RBC stiffness profile shows a cylindrical symmetry that appears to be strongly correlated with their typical biconcave shape. Our measurements show that the Young’s modulus is strongly depending on the indentation rate, demonstrating that viscous forces have a key role in determining their mechanical response. The importance of viscous forces is further stressed by the comparison between healthy and pathological erythrocyte. Our data show that pathological RBCs are not simply stiffer than healthy ones. Conversely they display a different dependence on the indentation rate that leads to an apparent increase in stiffness. Taken together our results show that both the local stiffness distribution and the viscoelastic response provide important information on RBC biomechanics. Keywords Atomic force microscopy • Stiffness • Dissipation • Biomechanics • Red blood cells 2.1 Introduction Red blood cells (RBC) have a typical biconcave shape with dimensions of approximately 8 μm in diameter and 2 μm in thickness and contain an interior viscous liquid enclosed by a viscoelastic membrane [1–7]. Such membrane consists of a nearly incompressible lipid bilayer attached to a spectrin protein network, held together by short actin filaments [7]. The peculiar RBC membrane structure ensures the integrity of the cell in narrow capillaries whose cross-section is smaller than the size of the cells. Under physiological conditions, indeed, RBCs must undergo repeated and severe deformations when travelling through small capillaries with diameter not more than 3–5 μm. Moreover, when passing through the spleen RBCs are required to traverse extremely narrow slits with sizes less than 1 μm [8]. There is a growing evidence that this extreme deformability is significantly altered in pathological conditions, such as diabetes mellitus, essential hypertension, arteriosclerosis and coronary artery disease [5, 9, 10]. On the other hand, an altered red blood cell deformability may contribute to the onset and the development of many pathologies. For example, membranopathies and hemoglobinopathies are known to alter the RBC deformability, affecting the blood flow in large and small vessels [11–13]. On the one hand, RBC modifications occur at the whole cell level, on the other hand they are closely related to changes in the molecular composition and organization of the cell that, in their turn, occur at the nanoscale. This has made it necessary to develop quantitative tools able to probe RBC changes at nanometer and piconewton scales. In this regard, Atomic Force Microscopy (AFM) is an extremely powerful technique as it permits to probe cells, tissues and molecule at the nanoscale in nearly physiological conditions [6, 14–25]. One of the key characteristics to look at RBC biomechanical properties by AFM is the Young’s modulus (E) that provides information on cell stiffness. The nanoscale mapping of E has been proven to be effective in distinguishing between normal M. Papi (*) • G. Ciasca • V. Palmieri • G. Maulucci • E. Minelli • M. De Spirito Institute of Physics, Catholic University of Sacred Heart, Largo F. Vito 1, 00168 Rome, Italy e-mail: massimiliano.papi@unicatt.it C. Rossi Institute of Biochemistry and Clinical Biochemistry, Catholic University of Sacred Heart, Largo F. Vito 1, 00168 Rome, Italy #The Society for Experimental Mechanics, Inc. 2017 C.S. Korach et al. (eds.), Mechanics of Biological Systems and Materials, Volume 6, Conference Proceedings of the Society for Experimental Mechanics Series, DOI 10.1007/978-3-319-41351-8_2 11
and pathological erythrocytes in several disease conditions [6–9, 17–19], opening the way to the development of novel diagnostic tools. Many biomechanics AFM studies rely on the basic assumption that RBCs behaves like an elastic body: dissipative forces are neglected and Young’s modulus is treated as unaffected by probe dynamics. As recently demonstrated in [6], this assumption cannot be considered strictly valid and dissipative forces play a key role in the biomechanical response of red blood cells at the nanoscale level. This result is in close agreement with the peculiar structure of the RBC membrane that can be modelled as a network of viscoelastic springs mediating elastic and viscous response. One of the major properties to evaluate quantitatively the contribution of dissipative forces is to measure the percentage of energy dissipated during the AFM indentation process or Hysteresis (H). Hysteresis is rarely considered in AFM experiments on the biomechanical behaviour of red blood cells. Nevertheless, the nanoscale mapping of H can provide valuable information on the RCB modifications occurring at the molecular level at the onset of many pathologies. In this work we describe a novel scanning probe-based nanoscale mapping methodology that generates Hysteresis maps of cells and tissues, which have been subjected to AFM indentation. To test this method we used red blood cells extracted from an healthy donors and patients with iron overload and hyperferritinemia, showing that H may provide highly valuable information with potential application in the clinical practice. 2.2 Material and Methods The blood was obtained from healthy donor volunteers and patients with iron overload and hyperferritinemia, anticoagulated with heparin and centrifuged to separate blood form serum. Erythrocytes were than dissolved in physiological solution and deposited on a poly-L-lysine coated petri dish. After 1 h incubation, the poly-L-lysine coated petri dish was gently washed in physiological solution to remove unattached red blood cells. Subjects affected by iron overload and hyperferritinemia were selected because increased serum ferritin concentration is associated with inflammation processes, which lead to a number of changes in the biophysical properties of red blood cells, including aggregation, sedimentation and deformability [2, 26]. Measurements were performed at 37 C in liquid environment using a JPK Nanowizard II atomic force microscope (JPK instruments, berlin Germany) coupled to an optical microscope (Axio observer, Carl Zeiss, Milan, Italy). MikroMasch silicon cantilevers with a spring constant of approximately 0.05 N/m and a tip radius of about 10 nm were used (CSC38, MikroMasch). The cantilever spring constant was computed for each measurement by thermal calibration. Force curves were acquired by using an indentation force of 0.5 nN. To evaluate quantitatively the contribution of dissipative forces, we estimated the energy dissipated during the deformation process (or Hysteresis H). H was computed as the difference between the area (AE) under the extension force curveFE(δ) and that (AR) under the retract curve (FR(δ)) normalized by AE: H¼ Z δ 0 FE δð Þdδ Z δ 0 FR δð Þdδ Z δ 0 FE δð Þdδ ¼ AE AR AE ð 2:1Þ The nanoscale mapping of H was obtained by a homemade Labview software that allows us for the contemporaneous determination of hysteresis and work of adhesion. 2.3 Results In Fig. 2.1a, b two representative approach-retract cycles acquired on a healthy (Fig. 2.1a) and a pathological RBC are shown (Fig. 2.1b). The coloured area between the approach and the retract curve provides a graphical representation of H. One can observe that the pathological red blood cell displays a larger H value than the healthy one. However, the typical biconcave shape of red blood cell hints at a spatial inhomogeneous biomechanical response, suggesting that a single point measure could not be representative of the overall behaviour of the cells. 12 M. Papi et al.
As far as RBC stiffness is concerned, this hypothesis was recently confirmed in Ref. [6]. The high resolution nanoscale mapping of E values acquired in physiological solution unveiled that healthy erythrocytes are stiffer in their centre and softer at the cell periphery [6]. Therefore we probed the local response of RBCs by acquiring force-distance curves at different positions over the cell surfaces and evaluating the perceptual of energy dissipated during the indentation process, trough the estimation of H (Eq. (2.1)). In Fig. 2.2a, b we reported two hysteresis maps acquired on an healthy (a) and pathological red blood cell (b). H values range between 0 and 0.8 for both cells, indicating that a perceptual energy ranging from 0 to 80 % is dissipated during the indentation process. As far as the healthy RBC is concerned, the nanoscale mapping of H shows a cylindrical symmetry. The cell centre behaves approximately as a pure elastic body, showing H values ranging from 0 to 0.1. An increase in H values can be observed at the cell periphery, where H can be as high as 0.8. The cylindrical distribution of H values appears to be strongly correlated with the typical biconcave shape of healthy red blood cells. Moreover such distribution well correlates with the Young’s modulus distribution detected in healthy red blood cells [6]. A significantly different result is obtained in the pathological case. In this case, the nanoscale map appears to be homogeneously brighter than that of the healthy RBCs indicating that a larger amount of energy is dissipated during the indentation process. Moreover, the cylindrical distribution of H values on healthy RBCs is not observed. Conversely, an almost uniform distribution can be detected. Fig. 2.1 Two representative approach-retract cycles acquired on healthy (a) and pathological (b) RBCs. The green coloured area of the cycle represent the hysteresis Fig. 2.2 High-resolution hysteresis maps of healthy (a) and pathological (b) RBCs. Both maps are represented with the same colour scale. These maps display the presence of a cylindrical distribution of H for the healthy RBC, correlated with the biconcave shape of the cell, and the lack of this spatial symmetry for the pathological one. Indeed normal RBC is characterized by value of H in the range 0–0.1 in the centre, and an increase of H in the periphery, while the pathological erythrocyte shows higher value of H, uniformly distributed 2 Nano-Mechanical Response of Red Blood Cells 13
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