CANCER CELL MIGRATION IN 3D COLLAGEN GEL MATRIX SYSTEM SUN WEI (B.Eng Tsinghua Univ, Beijing) (MSc, NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements My sincere thanks must first go to my supervisor, Professor Lim Chwee Teck He is not only my academic advisor, but also a role model for us – a young generation of engineers who are determined to pursue a career out of the conventional engineering domain, by solving long standing questions in life sciences and medical sciences with engineering approaches or by adding to the human’s understanding of lives and diseases from new perspectives Although he never advocated with words, his achievements greatly inspired me when I made up my mind to switch my career, otherwise I will probably be designing an energy system for a building right at this moment I believe he as a good example will attract more and more young people with engineering background to contribute to improving the health of mankind He first directed me to this topic – studying cancer cell migration in 3D – in Feb 2007 Through guiding me to read relevant literatures, introducing me to the group leading the field, Professor Lim opened the door to a great new world for me and brought me the compass He is very supportive while I went through the period of protocol optimization, through generously funding the reagents, consumables, microscope and accessories and also investing in a very expensive software package for image analysis I had never ever worried about purchasing anything for the experiments throughout the years, all thanks to his great support As the research progressed, he went through the difficulties, unforeseeable adversaries together with me, keeping me encouraged all the time To solve technical problems, we had many discussions within the group and also with scientists of similar interests, who offered constructive advice To my surprise, he is not only making his best effort, but also engaging his friends to make their best effort to help my research When I was confused about choosing the direction, he helped me to set realistic goals His insight and detailed guidance ensured that the research is always on the right track and the project is making progresses i All in all, without Professor Lim as my supervisor, I will not be able to take this endeavour to explore cancer metastasis, not able to find the right paths, to proceed and complete the project, never mention presenting this thesis to all I am also deeply indebted to my co-supervisor Professor Raj Rajagopalan Among the committee who interviewed me for admission into NGS, it is his support on me that made everything possible Ever since joining his research group in the second year of my study, he treated me same as the graduate students that he guides as main supervisor On the project requiring collaboration between Nicholas and me, he invested tremendous time and energy to guide us His foresight of frontier science, his wisdom, enthusiasm and intelligence guided my research like a lighthouse Whenever I had difficulties in anything during the study, I would like to turn to him for advice because I know he is kind and ready to help me He always outlined the tasks and dissected them into detailed steps which were in turn explained in clarity with his experience As a result, I often came into his office confused, with fear over the challenges, and left his office with confidence, courage, and sometimes a big smile – thanks to his humour He really cares about the development of his graduate students and gives us career guide from time to time I benefitted from Prof Raj’s supervision a great deal and really feel that he is a great academic father to me All in all, I feel lucky to have good supervisors in the years working towards a PhD Besides, I am very grateful to all the past and current lab officers: Ms Tan Phay Shing, Eunice, Mr Hairul Nizam Bin Ramli and Ms Charlene Wang (Prof Seeram Ramakrishna’s Laboratory) for their good maintenance to the laboratory and their timely help throughout the course of my PhD study Since I became a member of NanoBiomechanics Lab four years ago, I often feel lucky to have a wonderful lab environment In this friendly and conducive lab, I am able to conduct experiments smoothly, to have my bench work time more enjoyable, my everyday life filled with fun My gratitude goes to everyone of my lab friends, for being kind colleagues to work ii with, for offering me help whenever requested, sharing your knowledge and experience to me without reservation, encouraging me in many ways and sharing countless cheerful occasions with me I appreciate being a student of NUS Graduate School for Integrative Sciences & Engineering (NGS), the privileged graduate school in Singapore, for a few reasons NGS offers me not only generous funding, but also a great opportunity to build up the background and carry out interdisciplinary research, because we are empowered by the innovative curriculum setting and the encouraging environment in NGS The community of NGS did take care of each of us postgraduate students with attention I also benefitted from support of the NGS office, for which I can never forget the kind assistance and guidance from Ms Ivy Wee, Ms Irene Chuan and Ms Neo Cheng Bee and many others, during the course of my stay in NGS Last, but most importantly, I would also like to thank my family for their love and understanding throughout My husband, parents and parents-in-law all supported my study whole-heartedly Their encouragement and caring kept me accompanied all the time despite that I was thousands of miles away Without my family standing behind, it would not be possible for me to start and complete the PhD study iii Table of Contents Acknowledgements i Table of Contents iv Summary vii List of Tables viii List of Figures ix List of Symbols xvi Chapter 1: Introduction 1.1 The significance of cancer metastasis research 1.1.1 The urgency of cancer research 1.1.2 Cancer metastasis research 1.2 Quantitative in vitro 3D cell migration assays 1.2.1 Values of in vitro drug assays 1.2.2 Advantages of 3D cell-based drug assay 1.2.3 Fundamentals of cell migration 1.2.4 Advantages of 3D cell invasive migration assays 1.2.5 Summary 11 1.3 Tissue mechanical factors in 3D cell migration assays 12 1.4 Objective 13 1.5 Scope and structure of the thesis 13 Chapter 2: Literature Review 15 2.1 In vitro 2D and 3D cancer metastasis studies 15 2.1.1 Tracking 2D cell migration 16 2.1.2 Micro-pore-based cell migration/ invasion assays 21 2.1.3 3D cell migration models 24 2.1.4 Discussion on cell migration assays 31 2.2 General anti-cancer drug assays in 3D 33 2.2.1 Introduction 33 2.2.2 Background: why 2D cell culture is insufficient for anti-cancer drug testing 34 iv 2.2.3 Techniques in 3D anti-cancer drug assays 36 2.2.4 Specific research questions 39 2.2.5 Comparing 3D substrates with different structures / properties 45 2.2.6 Summary 46 2.3 3D anti-migratory drug assays 47 2.4 Summary 49 Chapter 3: Materials and Methods 50 3.1 Malignant breast cancer cells 50 3.2 3D collagen model for cell invasion 51 3.3 Structure and mechanical property of collagen gel 53 3.3.1 Mechanical characterization 53 3.3.2 Scanning electron microscopy 55 3.4 Microscopy methods for cell migration study 56 3.4.1 Imaging and tracking cells 56 3.4.2 Cell track data analysis 59 3.4.3 Statistical methods 61 3.5 Summary of the chapter 63 Chapter 4: Mechanical Modulation of the Collagen Gel Matrix 64 4.1 Modulation of bulk elasticity of the gel 64 4.2 Non-linear elasticity of collagen 66 4.3 Micro-architecture of collagen matrices 69 4.3.1 Dependence of collagen fibril thickness on polymerization pH 70 4.3.2 Pore-size of the collagen network 73 4.4 Discussion 76 Chapter 5: 3D Cell Migration and Drug Effects 79 5.1 Innate cell migration 79 5.2 MMP inhibition by GM6001 89 5.3 ROCK-inhibition 96 5.4 Actin destabilization 104 5.5 Microtubules destabilization 111 v 5.6 Discussions on drug effects 118 Chapter 6: Conclusions and Future Work 122 6.1 Conclusions 122 6.2 Further modulation of the ECM environment 125 References 127 Appendix A The Time Dependence of Cell Motility 138 Appendix B Results of Statistical Tests 140 vi Summary Cancer has long been one of the leading causes of death in the industrial world The main reason for its high mortality is due to inefficient early detection which results in the spread of cancer cells to other distant sites in the body, via a process known as metastasis Metastasis causes the dissemination of cancer cells and accounts for 90% of cancer-induced deaths, thus requiring better therapeutic treatments The thesis examines a functional assay to study cancer cell invasive migration inside a three-dimensional (3D) collagen-I hydrogel, focusing on its micro-architecture and mechanics First part of the project is the tuning of 3D collagen hydrogel to achieve varying fibre network structure and gel mechanical properties Embedded in such collagen gels, breast cancer cells were tracked using live confocal imaging followed by quantitative 3D cell track analysis The second part evaluated drug effects on cell migration using the model Pharmacological inhibitions of cell-activated collagen matrix degradation and cytoskeletal reorganization reduced cell movement speed and directionality Interestingly, the drug effects depended on the matrix micro-structure and elasticity The study found that tissue mechanics is important in anti-migratory cancer drug assays, and incorporated ECM factors in interpreting results of drug assays vii List of Tables Table 2.1 In vitro cell migration assays 16 Table 2.2 Categorizing 3D cell migration assay 25 Table 4.1 The list of parameter values in the exponential models that G’ data were fitted with 68 Table 5.1 Case index of migration assays 79 Table 5.2 Effects of GM6001 on cell movement speed and track straightness 91 Table 5.3 Effects of Y27632 on cell movement speed and track straightness 99 Table 5.4 Effects of CytoD on cell movement speed and track straightness 106 Table 5.5 Effects of nocodazole on cell movement speed and track straightness 113 Table 5.6 Recommended collagen-I gel mechanical property for drug assay designs 120 Table B.1 Comparing cell speed in the five control conditions; p-values obtained from K-S tests 141 Table B.2 Comparing cell straightness in the five control conditions; p-values obtained from K-S tests 141 viii References Cidadao AJ 1989 Interactions between fibronectin, glycosaminoglycans and native collagen fibrils: an EM study in artificial three-dimensional extracellular matrices Eur J Cell Biol 48(2):303-12 Cleek RL, Rege AA, Denner LA, Eskin SG, Mikos AG 1997 Inhibition of smooth muscle cell growth in vitro by an antisense oligodeoxynucleotide released from poly(DL-lactic-co-glycolic acid) microparticles J Biomed Mater Res 35(4):525-30 Condeelis J, Segall JE 2003 Intravital imaging of cell movement in tumours Nat Rev Cancer 3(12):921-30 Connolly L, Maxwell P 2002 Image analysis of Transwell assays in the assessment of invasion by malignant cell lines Br J Biomed Sci 59(1):11-4 Croix BS, Rak JW, Kapitain S, Sheehan C, Graham CH, Kerbel RS 1996 Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells J Natl Cancer Inst 88(18):1285-96 Debeir O, Megalizzi V, Warzee N, Kiss R, Decaestecker C 2008 Videomicroscopic extraction of specific information on cell proliferation and migration in vitro Exp Cell Res 314(16):2985-98 Debnath J, Walker SJ, Brugge JS 2003 Akt activation disrupts mammary acinar architecture and enhances proliferation in an mTOR-dependent manner J Cell Biol 163(2):315-26 Decaestecker C, Debeir O, Van Ham P, Kiss R 2007 Can anti-migratory drugs be screened in vitro? A review of 2D and 3D assays for the quantitative analysis of cell migration Med Res Rev 27(2):149-76 Di Lullo GA, Sweeney SM, Korkko J, Ala-Kokko L, San Antonio JD 2002 Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen J Biol Chem 277(6):4223-31 Dikovsky D, Bianco-Peled H, Seliktar D 2008 Defining the role of matrix compliance and proteolysis in three-dimensional cell spreading and remodeling Biophys J 94(7):2914-25 Eccles SA, Box C, Court W 2005 Cell migration/invasion assays and their application in cancer drug discovery Biotechnol Annu Rev 11:391-421 Engler AJ, Sen S, Sweeney HL, Discher DE 2006 Matrix elasticity directs stem cell lineage specification Cell 126(4):677-89 Even-Ram S, Yamada KM 2005 Cell migration in 3D matrix Curr Opin Cell Biol 17(5):524-32 Fischbach C, Chen R, Matsumoto T, Schmelzle T, Brugge JS, Polverini PJ, Mooney DJ 2007 Engineering tumours with 3D scaffolds Nat Methods 4(10):855-60 Fjellbirkeland L, Laerum OD, Eide GE, Bjerkvig R 1998 Invasiveness by lacZ transfected non-small-cell lung cancer cells into human bronchial tissues in vitro Lung Cancer 21(1):7-19 Folkman J 1971 Tumour angiogenesis: therapeutic implications N Engl J Med 285(21):1182-6 Fotos JS, Patel VP, Karin NJ, Temburni MK, Koh JT, Galileo DS 2006 Automated time-lapse microscopy and high-resolution tracking of cell migration Cytotechnology 51(1):7-19 Friedl P, Brocker EB 2000 The biology of cell locomotion within three-dimensional extracellular matrix Cell Mol Life Sci 57(1):41-64 Friedl P, Wolf K 2003 Tumour-cell invasion and migration: diversity and escape mechanisms Nat Rev Cancer 3(5):362-74 128 References Friedl P, Wolf K 2010 Plasticity of cell migration: a multiscale tuning model J Cell Biol 188(1):11-9 Friedl P, Zanker KS, Brocker EB 1998 Cell migration strategies in 3-D extracellular matrix: differences in morphology, cell matrix interactions, and integrin function Microsc Res Tech 43(5):369-78 Friedlander DR, Zagzag D, Shiff B, Cohen H, Allen JC, Kelly PJ, Grumet M 1996 Migration of brain tumour cells on extracellular matrix proteins in vitro correlates with tumour type and grade and involves alphaV and beta1 integrins Cancer Res 56(8):1939-47 Fung YC 1993 Biomechanics: mechanical properties of living tissues New York, NY: Springer Geiger B, Spatz JP, Bershadsky AD 2009 Environmental sensing through focal adhesions Nat Rev Mol Cell Biol 10(1):21-33 Green KL, Southgate TD, Mulryan K, Fairbairn LJ, Stern PL, Gaston K 2006 Diffusible VP22-E2 protein kills bystander cells and offers a route for cervical cancer gene therapy Hum Gene Ther 17(2):147-57 Griffith LG, Swartz MA 2006 Capturing complex 3D tissue physiology in vitro Nat Rev Mol Cell Biol 7(3):211-24 Grinnell F 1990 The activated keratinocyte: up regulation of cell adhesion and migration during wound healing J Trauma 30(12 Suppl):S144-9 Grinnell F, Rocha LB, Iucu C, Rhee S, Jiang H 2006 Nested collagen matrices: a new model to study migration of human fibroblast populations in three dimensions Exp Cell Res 312(1):86-94 Gunzer M, Friedl P, Niggemann B, Brocker EB, Kampgen E, Zanker KS 2000 Migration of dendritic cells within 3-D collagen lattices is dependent on tissue origin, state of maturation, and matrix structure and is maintained by proinflammatory cytokines J Leukoc Biol 67(5):622-9 Guo WH, Frey MT, Burnham NA, Wang YL 2006 Substrate rigidity regulates the formation and maintenance of tissues Biophys J 90(6):2213-20 Hanahan D, Weinberg RA 2000 The hallmarks of cancer Cell 100(1):57-70 Harley BA, Freyman TM, Wong MQ, Gibson LJ 2007 A new technique for calculating individual dermal fibroblast contractile forces generated within collagen-GAG scaffolds Biophys J 93(8):2911-22 Harley BA, Hastings AZ, Yannas IV, Sannino A 2006 Fabricating tubular scaffolds with a radial pore size gradient by a spinning technique Biomaterials 27(6):866-74 Harley BA, Kim HD, Zaman MH, Yannas IV, Lauffenburger DA, Gibson LJ 2008 Microarchitecture of three-dimensional scaffolds influences cell migration behavior via junction interactions Biophys J 95(8):4013-24 Harris MP, Kim E, Weidow B, Wikswo JP, Quaranta V 2008 Migration of isogenic cell lines quantified by dynamic multivariate analysis of single-cell motility Cell Adh Migr 2(2):127-36 Hayot C, Debeir O, Van Ham P, Van Damme M, Kiss R, Decaestecker C 2006 Characterization of the activities of actin-affecting drugs on tumour cell migration Toxicol Appl Pharmacol 211(1):30-40 Hicks KO, Fleming Y, Siim BG, Koch CJ, Wilson WR 1998 Extravascular diffusion of tirapazamine: effect of metabolic consumption assessed using the multicellular layer model Int J Radiat Oncol Biol Phys 42(3):641-9 129 References Hoffman RM 1991 Three-dimensional gel-supported native-state histoculture for evaluation of tumour-specific pharmacological activity: principles, practice and possibilities Journal of Cell Pharmacology 2:189-201 Hooper S, Marshall JF, Sahai E 2006 Tumour cell migration in three dimensions Methods Enzymol 406:625-43 Horodyski J, Powell RJ 1996 Effect of aprotinin on smooth muscle cell proliferation, migration, and extracellular matrix synthesis J Surg Res 66(2):115-8 Huang Q, Shen HM, Ong CN 2005 Emodin inhibits tumour cell migration through suppression of the phosphatidylinositol 3-kinase-Cdc42/Rac1 pathway Cell Mol Life Sci 62(10):1167-75 Ingber DE 2008 Can cancer be reversed by engineering the tumour microenvironment? Semin Cancer Biol 18(5):356-64 Irimia D, Toner M 2009 Spontaneous migration of cancer cells under conditions of mechanical confinement Integr Biol (Camb) 1(8-9):506-12 Jiang C, Ganther H, Lu J 2000 Monomethyl selenium specific inhibition of MMP-2 and VEGF expression: implications for angiogenic switch regulation Mol Carcinog 29(4):236-50 Jiang X, Bruzewicz DA, Wong AP, Piel M, Whitesides GM 2005 Directing cell migration with asymmetric micropatterns Proc Natl Acad Sci U S A 102(4):975-8 Jones PA, De Clerck YA 1982 Extracellular matrix destruction by invasive tumour cells Cancer Metastasis Rev 1(4):289-317 Jung SP, Siegrist B, Wade MR, Anthony CT, Woltering EA 2001 Inhibition of human angiogenesis with heparin and hydrocortisone Angiogenesis 4(3):175-86 Kang H, Wen Q, Janmey PA, Tang JX, Conti E, MacKintosh FC 2009 Nonlinear elasticity of stiff filament networks: strain stiffening, negative normal stress, and filament alignment in fibrin gels J Phys Chem B 113(12):3799-805 Karp G, editor 2008 Cell and molecular biology: concepts and experiments 5th ed ed: John Wiley & Sons Keese CR, Wegener J, Walker SR, Giaever I 2004 Electrical wound-healing assay for cells in vitro Proc Natl Acad Sci U S A 101(6):1554-9 Kerbel RS, St Croix B, Florenes VA, Rak J 1996 Induction and reversal of cell adhesion-dependent multicellular drug resistance in solid breast tumours Hum Cell 9(4):257-64 Kim YJ, Bae HI, Kwon OK, Choi MS 2009 Three-dimensional gastric cancer cell culture using nanofiber scaffold for chemosensitivity test Int J Biol Macromol 45(1):65-71 Knapp DM, Barocas VH, Moon AG, Yoo K, Petzold LR, Tranquillo RT 1997 Rheology of reconstituted type I collagen gel in confined compression Journal of Rheology 41(5):971-993 Kobayashi H, Man S, Graham CH, Kapitain SJ, Teicher BA, Kerbel RS 1993 Acquired multicellular-mediated resistance to alkylating agents in cancer Proc Natl Acad Sci U S A 90(8):3294-8 Koblinski JE, Kaplan-Singer BR, VanOsdol SJ, Wu M, Engbring JA, Wang S, Goldsmith CM, Piper JT, Vostal JG, Harms JF and others 2005 Endogenous osteonectin/SPARC/BM-40 expression inhibits MDA-MB-231 breast cancer cell metastasis Cancer Res 65(16):7370-7 Kohno T, Matsutani M, Hoshino T, Takakura K 1985 [Effects of anticancer drugs on multicellular spheroid of 9L rat brain tumour] No To Shinkei 37(10):991-7 130 References Kornyei Z, Czirok A, Vicsek T, Madarasz E 2000 Proliferative and migratory responses of astrocytes to in vitro injury J Neurosci Res 61(4):421-9 Kuh HJ, Jang SH, Wientjes MG, Weaver JR, Au JL 1999 Determinants of paclitaxel penetration and accumulation in human solid tumour J Pharmacol Exp Ther 290(2):871-80 Kumar S, Weaver VM 2009 Mechanics, malignancy, and metastasis: the force journey of a tumour cell Cancer Metastasis Rev 28(1-2):113-27 Lafleur MA, Handsley MM, Knauper V, Murphy G, Edwards DR 2002 Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs) J Cell Sci 115(Pt 17):3427-38 Lamy S, Beaulieu E, Labbe D, Bedard V, Moghrabi A, Barrette S, Gingras D, Beliveau R 2008 Delphinidin, a dietary anthocyanidin, inhibits platelet-derived growth factor ligand/receptor (PDGF/PDGFR) signaling Carcinogenesis 29(5):1033-41 Lamy S, Blanchette M, Michaud-Levesque J, Lafleur R, Durocher Y, Moghrabi A, Barrette S, Gingras D, Beliveau R 2006 Delphinidin, a dietary anthocyanidin, inhibits vascular endothelial growth factor receptor-2 phosphorylation Carcinogenesis 27(5):989-96 Lauffenburger DA, Horwitz AF 1996 Cell migration: a physically integrated molecular process Cell 84(3):359-69 Lee GY, Lim CT 2007 Biomechanics approaches to studying human diseases Trends Biotechnol 25(3):111-8 Lee J, Cuddihy MJ, Kotov NA 2008a Three-dimensional cell culture matrices: state of the art Tissue Eng Part B Rev 14(1):61-86 Lee TH, Avraham HK, Jiang S, Avraham S 2003 Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability J Biol Chem 278(7):5277-84 Lee WJ, Chen WK, Wang CJ, Lin WL, Tseng TH 2008b Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta integrin function in MDA-MB-231 breast cancer cells Toxicol Appl Pharmacol 226(2):178-91 Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K, Giaccia A, Weninger W and others 2009 Matrix Crosslinking Forces Tumour Progression by Enhancing Integrin Signaling Cell Liao G, Nagasaki T, Gundersen GG 1995 Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: implications for the role of dynamic microtubules in cell locomotion J Cell Sci 108 ( Pt 11):3473-83 Liu JX, Wang XG, Fu J, Luo RC 2006 [Bevacizumab (Avastin) inhibits lung cancer-induced pulmonary microvascular angiogenesis] Nan Fang Yi Ke Da Xue Xue Bao 26(7):1027-9, 1043 Lo CM, Wang HB, Dembo M, Wang YL 2000 Cell movement is guided by the rigidity of the substrate Biophys J 79(1):144-52 Lutolf MP 2009 Integration column: artificial ECM: expanding the cell biology toolbox in 3D Integr Biol (Camb) 1(3):235-41 Malkin AY 1994 Rheology Fundamentals Ontario, Canada: ChemTec Publishing Malliri A, Symons M, Hennigan RF, Hurlstone AF, Lamb RF, Wheeler T, Ozanne BW 1998 The transcription factor AP-1 is required for EGF-induced 131 References activation of rho-like GTPases, cytoskeletal rearrangements, motility, and in vitro invasion of A431 cells J Cell Biol 143(4):1087-99 Mareel MM, Storme GA, De Bruyne GK, Van Cauwenberge RM 1982 Vinblastine, Vincristine and Vindesine: anti-invasive effect on MO4 mouse fibrosarcoma cells in vitro Eur J Cancer Clin Oncol 18(2):199-210 Mazzoleni G, Di Lorenzo D, Steimberg N 2009 Modelling tissues in 3D: the next future of pharmaco-toxicology and food research? Genes Nutr 4(1):13-22 Menezes GC, Miron-Mendoza M, Ho CH, Jiang H, Grinnell F 2008 Oncogenic Ras-transformed human fibroblasts exhibit differential changes in contraction and migration in 3D collagen matrices Exp Cell Res 314(16):3081-91 Meshel AS, Wei Q, Adelstein RS, Sheetz MP 2005 Basic mechanism of three-dimensional collagen fibre transport by fibroblasts Nat Cell Biol 7(2):157-64 Mesri M, Morales-Ruiz M, Ackermann EJ, Bennett CF, Pober JS, Sessa WC, Altieri DC 2001 Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting Am J Pathol 158(5):1757-65 Mi K, Wang G, Liu Z, Feng Z, Huang B, Zhao X 2009 Influence of a self-assembling peptide, RADA16, compared with collagen I and Matrigel on the malignant phenotype of human breast-cancer cells in 3D cultures and in vivo Macromol Biosci 9(5):437-43 Michalickova K, Susic M, Willing MC, Wenstrup RJ, Cole WG 1998 Mutations of the alpha2(V) chain of type V collagen impair matrix assembly and produce ehlers-danlos syndrome type I Hum Mol Genet 7(2):249-55 Millipore 2008 GM6001 MMP Inhibitor product data, http://www.millipore.com/catalogue/item/CC1010 Miron-Mendoza M, Seemann J, Grinnell F 2008 Collagen fibril flow and tissue translocation coupled to fibroblast migration in 3D collagen matrices Mol Biol Cell 19(5):2051-8 Mitrofanova E, Hagan C, Qi J, Seregina T, Link C, Jr 2003 Sodium iodide symporter/radioactive iodine system has more efficient antitumour effect in three-dimensional spheroids Anticancer Res 23(3B):2397-404 Mosadegh B, Huang C, Park JW, Shin HS, Chung BG, Hwang SK, Lee KH, Kim HJ, Brody J, Jeon NL 2007 Generation of stable complex gradients across two-dimensional surfaces and three-dimensional gels Langmuir 23(22):10910-2 Muir CP, Adams MA, Graham CH 2006 Nitric oxide attenuates resistance to doxorubicin in three-dimensional aggregates of human breast carcinoma cells Breast Cancer Res Treat 96(2):169-76 Nakagawa H, Yoshioka K, Miyahara E, Fukushima Y, Tamura M, Itoh K 2005 Intrathecal administration of Y-27632, a specific rho-associated kinase inhibitor, for rat neoplastic meningitis Mol Cancer Res 3(8):425-33 Nelson CM, Bissell MJ 2005 Modeling dynamic reciprocity: engineering three-dimensional culture models of breast architecture, function, and neoplastic transformation Semin Cancer Biol 15(5):342-52 Newman SA, Frenz DA, Tomasek JJ, Rabuzzi DD 1985 Matrix-driven translocation of cells and nonliving particles Science 228(4701):885-9 Niggemann B, Drell TLt, Joseph J, Weidt C, Lang K, Zaenker KS, Entschladen F 2004 Tumour cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix Exp Cell Res 298(1):178-87 132 References Nikolai G, Niggemann B, Werner M, Zanker KS, Friedl P 1998 Direct and rapid induction of migration in human CD4+ T lymphocytes within three-dimensional collagen matrices mediated by signalling via CD3 and/or CD2 Immunology 95(1):62-8 Ozerdem B, Tozeren A 1995 Physical response of collagen gels to tensile strain J Biomech Eng 117(4):397-401 Padron JM, van der Wilt CL, Smid K, Smitskamp-Wilms E, Backus HH, Pizao PE, Giaccone G, Peters GJ 2000 The multilayered postconfluent cell culture as a model for drug screening Crit Rev Oncol Hematol 36(2-3):141-57 Palm D, Lang K, Brandt B, Zaenker KS, Entschladen F 2005 In vitro and in vivo imaging of cell migration: two interdepending methods to unravel metastasis formation Semin Cancer Biol 15(5):396-404 Park HJ, Kong D, Iruela-Arispe L, Begley U, Tang D, Galper JB 2002 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of RhoA Circ Res 91(2):143-50 Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, Reinhart-King CA, Margulies SS, Dembo M, Boettiger D and others 2005 Tensional homeostasis and the malignant phenotype Cancer Cell 8(3):241-54 Paweletz CP, Charboneau L, Liotta LA 2001 Overview of metastasis assays Curr Protoc Cell Biol Chapter 19:Unit 19 Pedersen JA, Swartz MA 2005 Mechanobiology in the third dimension Ann Biomed Eng 33(11):1469-90 Poincloux R, Lizarraga F, Chavrier P 2009 Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia J Cell Sci 122(Pt 17):3015-24 Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White JG, Keely PJ 2006 Collagen reorganization at the tumour-stromal interface facilitates local invasion BMC Med 4(1):38 Provenzano PP, Inman DR, Eliceiri KW, Keely PJ 2009 Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage Oncogene 28(49):4326-43 Provenzano PP, Inman DR, Eliceiri KW, Trier SM, Keely PJ 2008 Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization Biophys J 95(11):5374-84 Punathil T, Katiyar SK 2009 Inhibition of non-small cell lung cancer cell migration by grape seed proanthocyanidins is mediated through the inhibition of nitric oxide, guanylate cyclase, and ERK1/2 Mol Carcinog 48(3):232-42 Raeber GP, Lutolf MP, Hubbell JA 2005 Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration Biophys J 89(2):1374-88 Raeber GP, Lutolf MP, Hubbell JA 2007 Mechanisms of 3-D migration and matrix remodeling of fibroblasts within artificial ECMs Acta Biomater 3(5):615-29 Rao MA 1999 Rheology of Fluid and Semisolid Foods: Principles and Applications Gaithersburg, MD: Aspen Publishers, Inc Rehfeldt F, Engler AJ, Eckhardt A, Ahmed F, Discher DE 2007 Cell responses to the mechanochemical microenvironment implications for regenerative medicine and drug delivery Adv Drug Deliv Rev 59(13):1329-39 Roeder BA, Kokini K, Sturgis JE, Robinson JP, Voytik-Harbin SL 2002 Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure J Biomech Eng 124(2):214-22 133 References Rowe RG, Li XY, Hu Y, Saunders TL, Virtanen I, Garcia de Herreros A, Becker KF, Ingvarsen S, Engelholm LH, Bommer GT and others 2009 Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs J Cell Biol 184(3):399-408 Roy P, Petroll WM, Chuong CJ, Cavanagh HD, Jester JV 1999 Effect of cell migration on the maintenance of tension on a collagen matrix Ann Biomed Eng 27(6):721-30 Ruoslahti E, Pierschbacher MD 1987 New perspectives in cell adhesion: RGD and integrins Science 238(4826):491-7 Ryan EA, Mockros LF, Weisel JW, Lorand L 1999 Structural origins of fibrin clot rheology Biophys J 77(5):2813-26 Sabeh F, Shimizu-Hirota R, Weiss SJ 2009 Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited J Cell Biol 185(1):11-9 Sahai E 2007 Illuminating the metastatic process Nat Rev Cancer 7(10):737-49 Sahai E, Marshall CJ 2003 Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis Nat Cell Biol 5(8):711-9 Saltzman WM, Radomsky ML, Whaley KJ, Cone RA 1994 Antibody diffusion in human cervical mucus Biophys J 66(2 Pt 1):508-15 Scanlon EF, Murthy S 1991 The process of metastasis CA Cancer J Clin 41(5):301-5 Schroyens W, Tchao R 1985 Migration inhibition of an epithelial cell line by s-Con A and the effect on its invasiveness Virchows Arch B Cell Pathol Incl Mol Pathol 49(2):175-81 Shamloo A, Ma N, Poo MM, Sohn LL, Heilshorn SC 2008 Endothelial cell polarization and chemotaxis in a microfluidic device Lab Chip 8(8):1292-9 Shekhar MP, Werdell J, Tait L 2000 Interaction with endothelial cells is a prerequisite for branching ductal-alveolar morphogenesis and hyperplasia of preneoplastic human breast epithelial cells: regulation by estrogen Cancer Res 60(2):439-49 Sigma-Aldrich 2009 Product data http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=85 9958|ALDRICH&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC Sodek KL, Brown TJ, Ringuette MJ 2008 Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration BMC Cancer 8:223 Sonnenschein C, Soto AM 2008 Theories of carcinogenesis: an emerging perspective Semin Cancer Biol 18(5):372-7 Sporn MB 1996 The war on cancer Lancet 347(9012):1377-81 Stevenson DP, Collins WP, Farzaneh F, Hata K, Miyazaki K 1998a Thymidine phosphorylase activity and prodrug effects in a three-dimensional model of angiogenesis: implications for the treatment of ovarian cancer Am J Pathol 153(5):1573-8 Stevenson DP, Milligan SR, Collins WP 1998b Effects of platelet-derived endothelial cell growth factor/thymidine phosphorylase, substrate, and products in a three-dimensional model of angiogenesis Am J Pathol 152(6):1641-6 Stossel TP 1994 The E Donnall Thomas Lecture, 1993 The machinery of blood cell movements Blood 84(2):367-79 134 References Sutherland RM 1988 Cell and environment interactions in tumour microregions: the multicell spheroid model Science 240(4849):177-84 Takashima A, Grinnell F 1985 Fibronectin-mediated keratinocyte migration and initiation of fibronectin receptor function in vitro J Invest Dermatol 85(4):304-8 Teranishi F, Takahashi N, Gao N, Akamo Y, Takeyama H, Manabe T, Okamoto T 2009 Phosphoinositide 3-kinase inhibitor (wortmannin) inhibits pancreatic cancer cell motility and migration induced by hyaluronan in vitro and peritoneal metastasis in vivo Cancer Sci 100(4):770-7 Terzis AJ, Fiskerstrand T, Refsum H, Ueland PM, Arnold H, Bjerkvig R 1993 Proliferation, migration and invasion of human glioma cells exposed to antifolate drugs Int J Cancer 54(1):112-8 Thiery JP 2002 Epithelial-mesenchymal transitions in tumour progression Nat Rev Cancer 2(6):442-54 Thiery JP, Sleeman JP 2006 Complex networks orchestrate epithelial-mesenchymal transitions Nat Rev Mol Cell Biol 7(2):131-42 Turley EA, Erickson CA, Tucker RP 1985 The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices Dev Biol 109(2):347-69 Vial E, Sahai E, Marshall CJ 2003 ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumour cell motility Cancer Cell 4(1):67-79 Wang XG, Fu J, Zheng H, Li GX, Luo RC 2006 [Effects of pirarubicin chemotherapy combined with hyperthermia on gastric cancer in vitro] Nan Fang Yi Ke Da Xue Xue Bao 26(10):1487-90 Watanabe M, Yano W, Kondo S, Hattori Y, Yamada N, Yanai R, Nishida T 2003 Up-regulation of urokinase-type plasminogen activator in corneal epithelial cells induced by wounding Invest Ophthalmol Vis Sci 44(8):3332-8 Winer JP, Oake S, Janmey PA 2009 Non-linear elasticity of extracellular matrices enables contractile cells to communicate local position and orientation PLoS One 4(7):e6382 Wolf K, Alexander S, Schacht V, Coussens LM, von Andrian UH, van Rheenen J, Deryugina E, Friedl P 2009a Collagen-based cell migration models in vitro and in vivo Semin Cell Dev Biol 20(8):931-41 Wolf K, Alexander S, Schacht V, Coussens LM, von Andrian UH, van Rheenen J, Deryugina E, Friedl P 2009b Collagen-based cell migration models in vitro and in vivo Semin Cell Dev Biol Wolf K, Friedl P 2009 Mapping proteolytic cancer cell-extracellular matrix interfaces Clin Exp Metastasis 26(4):289-98 Worthylake RA, Lemoine S, Watson JM, Burridge K 2001 RhoA is required for monocyte tail retraction during transendothelial migration J Cell Biol 154(1):147-60 Yamada KM, Pankov R, Cukierman E 2003 Dimensions and dynamics in integrin function Braz J Med Biol Res 36(8):959-66 Yamakawa S, Furuyama Y, Oku N 2000 Development of a Simple Cell Invasion Assay System Biol Pharm Bull 23(10):1264-6 Yamamura N, Sudo R, Ikeda M, Tanishita K 2007 Effects of the mechanical properties of collagen gel on the in vitro formation of microvessel networks by endothelial cells Tissue Eng 13(7):1443-53 Yamauchi M, Shiiba M 2008 Lysine hydroxylation and cross-linking of collagen Methods Mol Biol 446:95-108 135 References Yao LQ, Feng YJ, Ding JX, Xu CJ, Jin HY, Yin LH 2005 [Primary study of vasculogenic mimicry induced by hypoxia in epithelial ovarian carcinoma] Zhonghua Fu Chan Ke Za Zhi 40(10):662-5 Zagzag D, Shiff B, Jallo GI, Greco MA, Blanco C, Cohen H, Hukin J, Allen JC, Friedlander DR 2002 Tenascin-C promotes microvascular cell migration and phosphorylation of focal adhesion kinase Cancer Res 62(9):2660-8 Zajchowski DA, Bartholdi MF, Gong Y, Webster L, Liu HL, Munishkin A, Beauheim C, Harvey S, Ethier SP, Johnson PH 2001 Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells Cancer Res 61(13):5168-78 Zaman MH, Matsudaira P, Lauffenburger DA 2007 Understanding effects of matrix protease and matrix organization on directional persistence and translational speed in three-dimensional cell migration Ann Biomed Eng 35(1):91-100 Zaman MH, Trapani LM, Sieminski AL, Mackellar D, Gong H, Kamm RD, Wells A, Lauffenburger DA, Matsudaira P 2006 Migration of tumour cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis Proc Natl Acad Sci U S A 103(29):10889-94 Zicha D, Dunn GA, Brown AF 1991 A new direct-viewing chemotaxis chamber J Cell Sci 99 ( Pt 4):769-75 136 Appendix 137 Appendix A The Time Dependence of Cell Motility We have examined the cell speed at different time points, and found that 1) cell speed of each track did not vary considerably over hours of live confocal fluorescence microscopy (Figure A.1); 2) cells maintained active migration from Day to Day 16, but decreased after that (Figure A.2), suggesting that the current assay is able to maintain reproducibility for at least two weeks On such basis, we averaged cell speed over each track to reflect the motility of an individual cell, and performed further analysis using these cell speed averaged by track Considering the assay stability is proved for the period from Day 10 to Day 16, all cell tracking and drug tests were performed between Day 10 and Day 14 cell speed at each 10 interval (μm/hr) 30 25 23.5 20 20.0 15 10 18.8 × 10 mins 13 19 25 31 37 43 cell speed at each 10 interval (μm/hr) 30 25 20 15 10 23.5 20.0 18.8 × 10 mins 13 19 25 31 37 43 Figure A.1 Cell speed calculated at each 10 interval, over hours of monitoring via confocal fluorescence microscopy The three lines represent three typical tracks in one test Cell speed did not show any trend of variation over time The three tracks maintained their relative positions across the entire period, and their difference can be reflected by the average speed, as labelled on the right of the chart 138 Cell speed (μm/hr) 12 10 Day-10 Day-16 Day-24 Figure A.2 Averaged cell speed on different days into the assay All data were collected from one experiment using pH7-2.5 mg/ml collagen gel Each sample contained more than 450 data, and the variances were < 0.02 μm/hr, which are not presented in the chart 139 Appendix B Results of Statistical Tests The cell speed and track straightness data not follow normal distribution Paired t-test or ANOVA is not applicable to compare the data in our study Mann-Whitney U test is an alternative to t-test when the data are not normally distributed, which is a test of population medians and the shape of the distribution functions, assuming that the data are from the same type of distributions However, this assumption was not valid here, since among these sets of cell speed data we found inconsistency in the types of distribution As a result, Mann-Whitney U test was not selected Instead, two-sample Kolmogorov-Smirnov (K-S) test was conducted using MATLAB (The two data vectors x1 and x2 The null hypothesis is that x1 and x2 are from the same MathWorks, Inc., Natick, MA, USA) K-S test compares the distributions of the values in the continuous distribution The alternative hypothesis is that they are from different continuous distributions The test statistic is: 𝑚𝑎𝑥(|𝐹1(𝑥) − 𝐹2(𝑥)|) of x1 values less than or equal to x and F2(x) is the proportion of x2 values less than or equal where F1 is the cumulative distribution function (CDF) of vector x1, F1(x) is the proportion to x So, the test statistic is the maximum difference between the two CDF curves The test does not specify what that common distribution is, thus it sets no limit to the form of data distribution To conclude from the K-S test, most of the cell speed data were from different distributions, through paired comparisons; and most drug-treated cell migration speed distributions differed from the corresponding control counterpart 140 Table B.1 Comparing cell speed in the five control conditions; p-values obtained from K-S tests p-value pH9-2.5 mg/ml pH7-4.0 mg/ml pH7-2.5 mg/ml pH7-1.5 mg/ml pH6-2.5 mg/ml pH9-50 0* 0 pH7-80 0 0 pH7-50 0.49 pH7-30 *When p450 The same applies to all tables in Appendix B Table B.2 Comparing cell straightness in the five control conditions; p-values obtained from K-S tests p-value pH9-50 pH7-80 pH7-50 pH7-30 pH9-2.5 mg/ml pH7-4.0 mg/ml pH7-2.5 mg/ml pH7-1.5 mg/ml pH6-2.5 mg/ml 0 0 0 0 0.81 141 Figure B.1 Summary of K-S test of cell speed data Column height = means the data from the paired drug treated– control conditions were from different distributions, suggesting the drug effect is statistically significant Column height = means the pair of data were not drawn from different distributions All tests were performed at a significance level = 0.05 Figure B.2 Summary of K-S test of cell track straightness data Column height = means the data from the paired drug treated–control conditions were from different distributions, suggesting the drug effect is statistically significant Column height = means the pair of data were not drawn from different distributions All tests were performed at a significance level = 0.05 142 ... studying migration- inducing ingredients in ECM To study the pathways involved in hyaluronan-induced pancreatic cancer cell motility, inhibitors were tested for effects on reducing cell migration. .. of cell distribution in a nested collagen gel after 12 days, showing cells spreading out from the inner -gel (the dashed line enclosed area) to the surrounding, originally cell- free outer gel. .. must be well defined and there is a need for modulating matrix mechanics in these assays 2.1 In vitro 2D and 3D cancer metastasis studies Research into cell migration involved in cancer metastasis