Advertisement

Two-Dimensional vs. Three-Dimensional In Vitro Tumor Migration and Invasion Assays

  • Miriam Zimmermann
  • Carol Box
  • Suzanne A. Eccles
Part of the Methods in Molecular Biology book series (MIMB, volume 986)

Abstract

Motility and invasion are key hallmarks that distinguish benign from malignant tumors, enabling cells to cross tissue boundaries, disseminate in blood and lymph and establish metastases at distant sites. Similar properties are also utilized by activated endothelial cells during tumor-induced angiogenesis. It is now appreciated that these processes might provide a rich source of novel molecular targets with the potential for inhibitors to restrain both metastasis and neoangiogenesis. Such therapeutic strategies require assays that can rapidly and quantitatively measure cell movement and the ability to traverse physiological barriers. The need for high-throughput, however, must be balanced by assay designs that accommodate, as far as possible, the complexity of the in vivo tumor microenvironment. This chapter aims to give an overview of some commonly used migration and invasion assays to aid in the selection of a balanced portfolio of techniques for the rapid and accurate evaluation of novel therapeutic agents.

Key words

Migration Haptotaxis Chemotaxis Invasion Motility Matrix protein 3-dimensional cultures Spheroid 

Abbreviations

2-D

Two-dimensional

3-D

Three-dimensional

BBB

Blood–brain barrier

BME

Basement membrane extract

CAM

Chorioallantoic membrane

CAM-DR

Cell adhesion-mediated drug resistance

EC

Endothelial cell

ECM

Extracellular matrix

EGF

Epidermal growth factor

EHS

Engelbreth–Holm–Swarm

EMT

Epithelial-to-mesenchymal transition

FAK

Focal adhesion kinase

FCS

Fetal calf serum

GBM

Glioblastoma

GFP

Green fluorescent protein

HCC

Hepatocellular carcinoma

HGF

Hepatocyte growth factor

HIF-1

Hypoxia-inducible factor-1

HT(S)

High-throughput (screening)

HUVEC

Human umbilical vein endothelial cell

MMP

Matrix metalloproteinase

MMPi

Matrix metalloproteinase inhibitors

PD

Pharmacodynamic

PET

Polyethylene terephthalate

RFP

Red fluorescent protein

s.c.

Subcutaneously

SCC

Squamous cell carcinoma

TIMP

Tissue inhibitor of metalloproteinases

VEGF

Vascular endothelial growth factor

Notes

Acknowledgments

The authors are funded by Cancer Research UK grant number C309/A8274 (S.E.) and by the Oracle Cancer Trust (M.Z. and C.B.). We acknowledge NHS funding to the NIHR Biomedical Research Centre. We thank Maria Vinci (funded by the National Centre for the Replacement, Refinement and Reduction of Animals in Research; G1000121 ID no. 94513) and other members of the Tumour Biology and Metastasis group for critical reading of the manuscript and constructive advice.

References

  1. 1.
    Francia G, Cruz-Munoz W, Man S et al (2011) Mouse models of advanced spontaneous metastasis for experimental therapeutics. Nat Rev Cancer 11:135–141PubMedCrossRefGoogle Scholar
  2. 2.
    Eccles SA, Welch DR (2007) Metastasis: recent discoveries and novel treatment strategies. Lancet 369:1742–1757PubMedCrossRefGoogle Scholar
  3. 3.
    Coleman R (2011) The use of bisphosphonates in cancer treatment. Ann N Y Acad Sci 1218:3–14PubMedCrossRefGoogle Scholar
  4. 4.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674PubMedCrossRefGoogle Scholar
  5. 5.
    Palmer TD, Ashby WJ, Lewis JD et al (2011) Targeting tumor cell motility to prevent metastasis. Adv Drug Deliv Rev 63:568–581PubMedCrossRefGoogle Scholar
  6. 6.
    Eccles SA (2004) Parallels in invasion and angiogenesis provide pivotal points for therapeutic intervention. Int J Dev Biol 48:583–598PubMedCrossRefGoogle Scholar
  7. 7.
    Eccles SA, Court W, Patterson L et al (2009) In vitro assays for endothelial cell functions related to angiogenesis: proliferation, motility, tubular differentiation, and proteolysis. Methods Mol Biol 467:159–181PubMedCrossRefGoogle Scholar
  8. 8.
    Brader S, Eccles SA (2004) Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. Tumori 90:2–8PubMedGoogle Scholar
  9. 9.
    Eccles SA, Massey A, Raynaud FI et al (2008) NVP-AUY922: a novel heat shock protein 90 inhibitor active against xenograft tumor growth, angiogenesis, and metastasis. Cancer Res 68:2850–2860PubMedCrossRefGoogle Scholar
  10. 10.
    Friedl P, Gilmour D (2009) Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Biol 10:445–457PubMedCrossRefGoogle Scholar
  11. 11.
    Sahai E, Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5:711–719PubMedCrossRefGoogle Scholar
  12. 12.
    Strongin AY (2010) Proteolytic and non-proteolytic roles of membrane type-1 matrix metalloproteinase in malignancy. Biochim Biophys Acta 1803:133–141PubMedCrossRefGoogle Scholar
  13. 13.
    Ebos JM, Kerbel RS (2011) Antiangiogenic therapy: impact on invasion, disease progression, and metastasis. Nat Rev Clin Oncol 8:210–221PubMedCrossRefGoogle Scholar
  14. 14.
    Collins CS, Hong J, Sapinoso L et al (2006) A small interfering RNA screen for modulators of tumor cell motility identifies MAP4K4 as a promigratory kinase. Proc Natl Acad Sci USA 103:3775–3780PubMedCrossRefGoogle Scholar
  15. 15.
    Roussos ET, Condeelis JS, Patsialou A (2011) Chemotaxis in cancer. Nat Rev Cancer 11:573–587PubMedCrossRefGoogle Scholar
  16. 16.
    Sawyer C, Sturge J, Bennett DC et al (2003) Regulation of breast cancer cell chemotaxis by the phosphoinositide 3-kinase p110delta. Cancer Res 63:1667–1675PubMedGoogle Scholar
  17. 17.
    Breckenridge MT, Egelhoff TT, Baskaran H (2010) A microfluidic imaging chamber for the direct observation of chemotactic transmigration. Biomed Microdevices 12:543–553PubMedCrossRefGoogle Scholar
  18. 18.
    Albini A, Noonan DM (2010) The ‘chemoinvasion’ assay, 25 years and still going strong: the use of reconstituted basement membranes to study cell invasion and angiogenesis. Curr Opin Cell Biol 22:677–689PubMedCrossRefGoogle Scholar
  19. 19.
    Eccles SA, Box C, Court W (2005) Cell migration/invasion assays and their application in cancer drug discovery. Biotechnol Annu Rev 11:391–421PubMedCrossRefGoogle Scholar
  20. 20.
    Maliakal JC (2002) Quantitative high throughput endothelial cell migration and invasion assay system. Methods Enzymol 352:175–182PubMedCrossRefGoogle Scholar
  21. 21.
    Marshall J (2011) Transwell((R)) invasion assays. Methods Mol Biol 769:97–110PubMedCrossRefGoogle Scholar
  22. 22.
    van Roosmalen W, Le Devedec SE, Zovko S et al (2011) Functional screening with a live cell imaging-based random cell migration assay. Methods Mol Biol 769:435–448PubMedCrossRefGoogle Scholar
  23. 23.
    Ng MR, Brugge JS (2009) A stiff blow from the stroma: collagen crosslinking drives tumor progression. Cancer Cell 16:455–457PubMedCrossRefGoogle Scholar
  24. 24.
    Kusama T, Mukai M, Tatsuta M et al (2006) Inhibition of transendothelial migration and invasion of human breast cancer cells by preventing geranylgeranylation of Rho. Int J Oncol 29:217–223PubMedGoogle Scholar
  25. 25.
    Burleson KM, Hansen LK, Skubitz AP (2004) Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers. Clin Exp Metastasis 21:685–697PubMedCrossRefGoogle Scholar
  26. 26.
    Jung S, Kim HW, Lee JH et al (2002) Brain tumor invasion model system using organotypic brain-slice culture as an alternative to in vivo model. J Cancer Res Clin Oncol 128:469–476PubMedCrossRefGoogle Scholar
  27. 27.
    Quintavalle M, Elia L, Price JH et al (2011) A cell-based high-content screening assay reveals activators and inhibitors of cancer cell invasion. Sci Signal 4:ra49PubMedCrossRefGoogle Scholar
  28. 28.
    Nystrom ML, Thomas GJ, Stone M et al (2005) Development of a quantitative method to analyse tumour cell invasion in organotypic culture. J Pathol 205:468–475PubMedCrossRefGoogle Scholar
  29. 29.
    Brekhman V, Neufeld G (2009) A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion. BMC Cancer 9:415PubMedCrossRefGoogle Scholar
  30. 30.
    Harma V, Virtanen J, Makela R et al (2010) A comprehensive panel of three-dimensional models for studies of prostate cancer growth, invasion and drug responses. PLoS One 5:e10431PubMedCrossRefGoogle Scholar
  31. 31.
    Duong HS, Le AD, Zhang Q et al (2005) A novel 3-dimensional culture system as an in vitro model for studying oral cancer cell invasion. Int J Exp Pathol 86:365–374PubMedCrossRefGoogle Scholar
  32. 32.
    David L, Dulong V, Le Cerf D et al (2004) Reticulated hyaluronan hydrogels: a model for examining cancer cell invasion in 3D. Matrix Biol 23:183–193PubMedCrossRefGoogle Scholar
  33. 33.
    Truong HH, de Sonneville J, Ghotra VP et al (2012) Automated microinjection of cell-polymer suspensions in 3D ECM scaffolds for high-throughput quantitative cancer invasion screens. Biomaterials 33:181–188PubMedCrossRefGoogle Scholar
  34. 34.
    Echeverria V, Meyvantsson I, Skoien A et al (2010) An automated high-content assay for tumor cell migration through 3-dimensional matrices. J Biomol Screen 15:1144–1151PubMedCrossRefGoogle Scholar
  35. 35.
    Mareel MM, Van Roy FM, Messiaen LM et al (1987) Qualitative and quantitative analysis of tumour invasion in vivo and in vitro. J Cell Sci Suppl 8:141–163PubMedGoogle Scholar
  36. 36.
    Bracke ME, Boterberg T, Mareel MM (2001) Chick heart invasion assay. Methods Mol Med 58:91–102PubMedGoogle Scholar
  37. 37.
    Woodward JK, Nichols CE, Rennie IG et al (2002) An in vitro assay to assess uveal melanoma invasion across endothelial and basement membrane barriers. Invest Ophthalmol Vis Sci 43:1708–1714PubMedGoogle Scholar
  38. 38.
    Kataoka T, Umeda M, Shigeta T et al (2010) A new in vitro model of cancer invasion using AlloDerm, a human cadaveric dermal equivalent: a preliminary report. Kobe J Med Sci 55:E106–E115PubMedGoogle Scholar
  39. 39.
    Andjelkovic AV, Zochowski MR, Morgan F et al (2001) Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction. In Vitro Cell Dev Biol Anim 37:111–120PubMedCrossRefGoogle Scholar
  40. 40.
    Pilkington GJ, Bjerkvig R, De Ridder L et al (1997) In vitro and in vivo models for the study of brain tumour invasion. Anticancer Res 17:4107–4109PubMedGoogle Scholar
  41. 41.
    Yates C, Shepard CR, Papworth G et al (2007) Novel three-dimensional organotypic liver bioreactor to directly visualize early events in metastatic progression. Adv Cancer Res 97:225–246PubMedCrossRefGoogle Scholar
  42. 42.
    Hsiao AY, Torisawa YS, Tung YC et al (2009) Microfluidic system for formation of PC-3 prostate cancer co-culture spheroids. Biomaterials 30:3020–3027PubMedCrossRefGoogle Scholar
  43. 43.
    Unger RE, Halstenberg S, Sartoris A et al (2011) Human endothelial and osteoblast co-cultures on 3D biomaterials. Methods Mol Biol 695:229–241PubMedCrossRefGoogle Scholar
  44. 44.
    Sieh S, Lubik AA, Clements JA et al (2010) Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model. Organogenesis 6:181–188PubMedCrossRefGoogle Scholar
  45. 45.
    Mastro AM, Vogler EA (2009) A three-dimensional osteogenic tissue model for the study of metastatic tumor cell interactions with bone. Cancer Res 69:4097–4100PubMedCrossRefGoogle Scholar
  46. 46.
    Niggemann B, Drell TL IV, Joseph J et al (2004) Tumor cell locomotion: differential dynamics of spontaneous and induced migration in a 3D collagen matrix. Exp Cell Res 298:178–187PubMedCrossRefGoogle Scholar
  47. 47.
    Joyce JA, Pollard JW (2009) Microenvironmental regulation of metastasis. Nat Rev Cancer 9:239–252PubMedCrossRefGoogle Scholar
  48. 48.
    Nyga A, Cheema U, Loizidou M (2011) 3D tumour models: novel in vitro approaches to cancer studies. J Cell Commun Signal 5:239–248PubMedCrossRefGoogle Scholar
  49. 49.
    Shirinifard A, Gens JS, Zaitlen BL et al (2009) 3D multi-cell simulation of tumor growth and angiogenesis. PLoS One 4:e7190PubMedCrossRefGoogle Scholar
  50. 50.
    Smalley KS, Lioni M, Herlyn M (2006) Life isn’t flat: taking cancer biology to the next dimension. In Vitro Cell Dev Biol Anim 42:242–247PubMedCrossRefGoogle Scholar
  51. 51.
    Green SK, Francia G, Isidoro C et al (2004) Antiadhesive antibodies targeting E-cadherin sensitize multicellular tumor spheroids to chemotherapy in vitro. Mol Cancer Ther 3:149–159PubMedGoogle Scholar
  52. 52.
    Birgersdotter A, Sandberg R, Ernberg I (2005) Gene expression perturbation in vitro—a growing case for three-dimensional (3D) culture systems. Semin Cancer Biol 15:405–412PubMedCrossRefGoogle Scholar
  53. 53.
    Cukierman E, Pankov R, Stevens DR et al (2001) Taking cell-matrix adhesions to the third dimension. Science 294:1708–1712PubMedCrossRefGoogle Scholar
  54. 54.
    Kunz-Schughart LA, Freyer JP, Hofstaedter F et al (2004) The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J Biomol Screen 9:273–285PubMedCrossRefGoogle Scholar
  55. 55.
    Takagi A, Watanabe M, Ishii Y et al (2007) Three-dimensional cellular spheroid formation provides human prostate tumor cells with tissue-like features. Anticancer Res 27:45–53PubMedGoogle Scholar
  56. 56.
    Hirschhaeuser F, Menne H, Dittfeld C et al (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148:3–15PubMedCrossRefGoogle Scholar
  57. 57.
    Scott RW, Hooper S, Crighton D et al (2010) LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells. J Cell Biol 191:169–185PubMedCrossRefGoogle Scholar
  58. 58.
    Hardelauf H, Frimat JP, Stewart JD et al (2011) Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits. Lab Chip 11:419–428PubMedCrossRefGoogle Scholar
  59. 59.
    Friedrich J, Seidel C, Ebner R et al (2009) Spheroid-based drug screen: considerations and practical approach. Nat Protoc 4:309–324PubMedCrossRefGoogle Scholar
  60. 60.
    Tung YC, Hsiao AY, Allen SG et al (2011) High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 136:473–478PubMedCrossRefGoogle Scholar
  61. 61.
    Vinci M, Gowan S, Boxall F et al (2012) Advances in establishment and analysis of 3D tumour spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10:29. doi: 10.1186/1741-7007-10-29 PubMedCrossRefGoogle Scholar
  62. 62.
    Lai Y, Asthana A, Kisaalita WS (2011) Biomarkers for simplifying HTS 3D cell culture platforms for drug discovery: the case for cytokines. Drug Discov Today 16:293–297PubMedCrossRefGoogle Scholar
  63. 63.
    Ghosh S, Spagnoli GC, Martin I et al (2005) Three-dimensional culture of melanoma cells profoundly affects gene expression profile: a high density oligonucleotide array study. J Cell Physiol 204:522–531PubMedCrossRefGoogle Scholar
  64. 64.
    Fischbach C, Kong HJ, Hsiong SX et al (2009) Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement. Proc Natl Acad Sci U S A 106:399–404PubMedCrossRefGoogle Scholar
  65. 65.
    Vaira V, Fedele G, Pyne S et al (2010) Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors. Proc Natl Acad Sci USA 107:8352–8356PubMedCrossRefGoogle Scholar
  66. 66.
    Keith B, Simon MC (2007) Hypoxia-inducible factors, stem cells, and cancer. Cell 129:465–472PubMedCrossRefGoogle Scholar
  67. 67.
    Rodriguez-Jimenez FJ, Moreno-Manzano V, Lucas-Dominguez R et al (2008) Hypoxia causes downregulation of mismatch repair system and genomic instability in stem cells. Stem Cells 26:2052–2062PubMedCrossRefGoogle Scholar
  68. 68.
    Lin Q, Yun Z (2010) Impact of the hypoxic tumor microenvironment on the regulation of cancer stem cell characteristics. Cancer Biol Ther 9:949–956PubMedCrossRefGoogle Scholar
  69. 69.
    Benita Y, Kikuchi H, Smith AD et al (2009) An integrative genomics approach identifies hypoxia inducible factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res 37:4587–4602PubMedCrossRefGoogle Scholar
  70. 70.
    Lu X, Kang Y (2010) Hypoxia and hypoxia-inducible factors: master regulators of metastasis. Clin Cancer Res 16:5928–5935PubMedCrossRefGoogle Scholar
  71. 71.
    DeClerck K, Elble RC (2010) The role of hypoxia and acidosis in promoting metastasis and resistance to chemotherapy. Front Biosci 15:213–225PubMedCrossRefGoogle Scholar
  72. 72.
    Rohwer N, Cramer T (2011) Hypoxia-mediated drug resistance: novel insights on the functional interaction of HIFs and cell death pathways. Drug Resist Updat 14:191–201PubMedCrossRefGoogle Scholar
  73. 73.
    Li Z, Rich JN (2010) Hypoxia and hypoxia inducible factors in cancer stem cell maintenance. Curr Top Microbiol Immunol 345:21–30PubMedCrossRefGoogle Scholar
  74. 74.
    Yu H, Zhang CM, Wu YS (2010) Research progress in cancer stem cells and their drug resistance. Chin J Cancer 29:261–264PubMedCrossRefGoogle Scholar
  75. 75.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E et al (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115PubMedCrossRefGoogle Scholar
  76. 76.
    Bartholoma P, Impidjati, Reininger-Mack A et al (2005) A more aggressive breast cancer spheroid model coupled to an electronic capillary sensor system for a high-content screening of cytotoxic agents in cancer therapy: 3-dimensional in vitro tumor spheroids as a screening model. J Biomol Screen 10:705–714PubMedCrossRefGoogle Scholar
  77. 77.
    Zhang X, Wang W, Yu W et al (2005) Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing. Biotechnol Prog 21:1289–1296PubMedCrossRefGoogle Scholar
  78. 78.
    Weiswald LB, Richon S, Validire P et al (2009) Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness. Br J Cancer 101:473–482PubMedCrossRefGoogle Scholar
  79. 79.
    Koo BK, Stange DE, Sato T et al (2012) Controlled gene expression in primary Lgr5 organoid cultures. Nat Methods 9:81–83CrossRefGoogle Scholar
  80. 80.
    Kim JB (2005) Three-dimensional tissue culture models in cancer biology. Semin Cancer Biol 15:365–377PubMedCrossRefGoogle Scholar
  81. 81.
    Pampaloni F, Reynaud EG, Stelzer EH (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839–845PubMedCrossRefGoogle Scholar
  82. 82.
    Mazzoleni G, Di Lorenzo D, Steimberg N (2009) Modelling tissues in 3D: the next future of pharmaco-toxicology and food research? Genes Nutr 4:13–22PubMedCrossRefGoogle Scholar
  83. 83.
    Haycock JW (2011) 3D cell culture: a review of current approaches and techniques. Methods Mol Biol 695:1–15PubMedCrossRefGoogle Scholar
  84. 84.
    Prestwich GD, Liu Y, Yu B et al (2007) 3-D culture in synthetic extracellular matrices: new tissue models for drug toxicology and cancer drug discovery. Adv Enzyme Regul 47:196–207PubMedCrossRefGoogle Scholar
  85. 85.
    Yamada KM, Cukierman E (2007) Modeling tissue morphogenesis and cancer in 3D. Cell 130:601–610PubMedCrossRefGoogle Scholar
  86. 86.
    Wartenberg M, Donmez F, Ling FC et al (2001) Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells. FASEB J 15:995–1005PubMedCrossRefGoogle Scholar
  87. 87.
    Unsworth BR, Lelkes PI (1998) Growing tissues in microgravity. Nat Med 4:901–907PubMedCrossRefGoogle Scholar
  88. 88.
    Del Duca D, Werbowetski T, Del Maestro RF (2004) Spheroid preparation from hanging drops: characterization of a model of brain tumor invasion. J Neurooncol 67:295–303PubMedCrossRefGoogle Scholar
  89. 89.
    Eicher C, Dewerth A, Kirchner B et al (2011) Development of a drug resistance model for hepatoblastoma. Int J Oncol 38:447–454PubMedGoogle Scholar
  90. 90.
    Zhang Q, Nguyen AL, Shi S et al (2011) Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis. Stem Cells Dev 21(6):937–947PubMedCrossRefGoogle Scholar
  91. 91.
    Ivascu A, Kubbies M (2006) Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. J Biomol Screen 11:922–932PubMedCrossRefGoogle Scholar
  92. 92.
    Lee GY, Kenny PA, Lee EH et al (2007) Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4:359–365PubMedCrossRefGoogle Scholar
  93. 93.
    McMillin DW, Delmore J, Weisberg E et al (2010) Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity. Nat Med 16:483–489PubMedCrossRefGoogle Scholar
  94. 94.
    Holliday DL, Brouilette KT, Markert A et al (2009) Novel multicellular organotypic models of normal and malignant breast: tools for dissecting the role of the microenvironment in breast cancer progression. Breast Cancer Res 11:R3PubMedCrossRefGoogle Scholar
  95. 95.
    Rhee HW, Zhau HE, Pathak S et al (2001) Permanent phenotypic and genotypic changes of prostate cancer cells cultured in a three-dimensional rotating-wall vessel. In Vitro Cell Dev Biol Anim 37:127–140PubMedCrossRefGoogle Scholar
  96. 96.
    Walter-Yohrling J, Pratt BM, Ledbetter S et al (2003) Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: a novel in vitro tumor model. Cancer Chemother Pharmacol 52:263–269PubMedCrossRefGoogle Scholar
  97. 97.
    Li Q, Chen C, Kapadia A et al (2011) 3D models of epithelial-mesenchymal transition in breast cancer metastasis: high-throughput screening assay development, validation, and pilot screen. J Biomol Screen 16:141–154PubMedCrossRefGoogle Scholar
  98. 98.
    Fischbach C, Chen R, Matsumoto T et al (2007) Engineering tumors with 3D scaffolds. Nat Methods 4:855–860PubMedCrossRefGoogle Scholar
  99. 99.
    Agudelo-Garcia PA, De Jesus JK, Williams SP et al (2011) Glioma cell migration on three-dimensional nanofiber scaffolds is regulated by substrate topography and abolished by inhibition of STAT3 signaling. Neoplasia 13:831–840PubMedGoogle Scholar
  100. 100.
    de Ridder L, Cornelissen M, de Ridder D (2000) Autologous spheroid culture: a screening tool for human brain tumour invasion. Crit Rev Oncol Hematol 36:107–122PubMedCrossRefGoogle Scholar
  101. 101.
    Biggs T, Foreman J, Sundstrom L et al (2011) Antitumor compound testing in glioblastoma organotypic brain cultures. J Biomol Screen 16:805–817PubMedCrossRefGoogle Scholar
  102. 102.
    Bruyere F, Melen-Lamalle L, Blacher S et al (2008) Modeling lymphangiogenesis in a three-dimensional culture system. Nat Methods 5:431–437PubMedCrossRefGoogle Scholar
  103. 103.
    O-Charoenrat P, Rhys-Evans P, Modjtahedi H et al (2000) Overexpression of epidermal growth factor receptor in human head and neck squamous carcinoma cell lines correlates with matrix metalloproteinase-9 expression and in vitro invasion. Int J Cancer 86:307–317PubMedCrossRefGoogle Scholar
  104. 104.
    Kleinman HK, Jacob K (2001) Invasion assays. Curr Protoc Cell Biol  Chapter 12 :Unit 12 12
  105. 105.
    Decaestecker C, Debeir O, Van Ham P et al (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 27:149–176PubMedCrossRefGoogle Scholar
  106. 106.
    Pickl M, Ries CH (2009) Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab. Oncogene 28:461–468PubMedCrossRefGoogle Scholar
  107. 107.
    Weaver VM, Petersen OW, Wang F et al (1997) Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol 137:231–245PubMedCrossRefGoogle Scholar
  108. 108.
    Leslie K, Gao SP, Berishaj M et al (2010) Differential interleukin-6/Stat3 signaling as a function of cellular context mediates Ras-induced transformation. Breast Cancer Res 12:R80PubMedCrossRefGoogle Scholar
  109. 109.
    Kobayashi H, Man S, Graham CH et al (1993) Acquired multicellular-mediated resistance to alkylating agents in cancer. Proc Natl Acad Sci USA 90:3294–3298PubMedCrossRefGoogle Scholar
  110. 110.
    Thurber AE, Douglas G, Sturm EC et al (2011) Inverse expression states of the BRN2 and MITF transcription factors in melanoma spheres and tumour xenografts regulate the NOTCH pathway. Oncogene 30:3036–3048PubMedCrossRefGoogle Scholar
  111. 111.
    Howes AL, Chiang GG, Lang ES et al (2007) The phosphatidylinositol 3-kinase inhibitor, PX-866, is a potent inhibitor of cancer cell motility and growth in three-dimensional cultures. Mol Cancer Ther 6:2505–2514PubMedCrossRefGoogle Scholar
  112. 112.
    Wu YM, Tang J, Zhao P et al (2009) Morphological changes and molecular expressions of hepatocellular carcinoma cells in three-dimensional culture model. Exp Mol Pathol 87:133–140PubMedCrossRefGoogle Scholar
  113. 113.
    Xiang X, Phung Y, Feng M et al (2011) The development and characterization of a human mesothelioma in vitro 3D model to investigate immunotoxin therapy. PLoS One 6:e14640PubMedCrossRefGoogle Scholar
  114. 114.
    Wolf K, Mazo I, Leung H et al (2003) Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160:267–277PubMedCrossRefGoogle Scholar
  115. 115.
    Pinco KA, He W, Yang JT (2002) alpha4beta1 integrin regulates lamellipodia protrusion via a focal complex/focal adhesion-independent mechanism. Mol Biol Cell 13:3203–3217PubMedCrossRefGoogle Scholar
  116. 116.
    Keese CR, Wegener J, Walker SR et al (2004) Electrical wound-healing assay for cells in vitro. Proc Natl Acad Sci USA 101:1554–1559PubMedCrossRefGoogle Scholar
  117. 117.
    Lampugnani MG (1999) Cell migration into a wounded area in vitro. Methods Mol Biol 96:177–182PubMedGoogle Scholar
  118. 118.
    Yarrow JC, Perlman ZE, Westwood NJ et al (2004) A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods. BMC Biotechnol 4:21PubMedCrossRefGoogle Scholar
  119. 119.
    Zigmond SH (1977) Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. J Cell Biol 75:606–616PubMedCrossRefGoogle Scholar
  120. 120.
    Zicha D, Dunn GA, Brown AF (1991) A new direct-viewing chemotaxis chamber. J Cell Sci 99(Pt 4):769–775PubMedGoogle Scholar
  121. 121.
    Muinonen-Martin AJ, Veltman DM, Kalna G et al (2010) An improved chamber for direct visualisation of chemotaxis. PLoS One 5:e15309PubMedCrossRefGoogle Scholar
  122. 122.
    Cai G, Lian J, Shapiro SS et al (2000) Evaluation of endothelial cell migration with a novel in vitro assay system. Methods Cell Sci 22:107–114PubMedCrossRefGoogle Scholar
  123. 123.
    Pratt BM, Harris AS, Morrow JS et al (1984) Mechanisms of cytoskeletal regulation. Modulation of aortic endothelial cell spectrin by the extracellular matrix. Am J Pathol 117:349–354PubMedGoogle Scholar
  124. 124.
    Albini A, Iwamoto Y, Kleinman HK et al (1987) A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res 47:3239–3245PubMedGoogle Scholar
  125. 125.
    Ohnishi T, Matsumura H, Izumoto S et al (1998) A novel model of glioma cell invasion using organotypic brain slice culture. Cancer Res 58:2935–2940PubMedGoogle Scholar
  126. 126.
    Deryugina EI, Bourdon MA (1996) Tenascin mediates human glioma cell migration and modulates cell migration on fibronectin. J Cell Sci 109(Pt 3):643–652PubMedGoogle Scholar
  127. 127.
    Rosen EM, Meromsky L, Setter E et al (1990) Quantitation of cytokine-stimulated migration of endothelium and epithelium by a new assay using microcarrier beads. Exp Cell Res 186:22–31PubMedCrossRefGoogle Scholar
  128. 128.
    Hart IR, Fidler IF (1978) An in vitro quantitative assay for tumor cell invasion. Cancer Res 38:3218–3224PubMedGoogle Scholar
  129. 129.
    Armstrong PB, Quigley JP, Sidebottom E (1982) Transepithelial invasion and intramesenchymal infiltration of the chick embryo chorioallantois by tumor cell lines. Cancer Res 42:1826–1837PubMedGoogle Scholar
  130. 130.
    An Z, Gluck CB, Choy ML et al (2010) Suberoylanilide hydroxamic acid limits migration and invasion of glioma cells in two and three dimensional culture. Cancer Lett 292:215–227PubMedCrossRefGoogle Scholar
  131. 131.
    Stein AM, Demuth T, Mobley D et al (2007) A mathematical model of glioblastoma tumor spheroid invasion in a three-dimensional in vitro experiment. Biophys J 92:356–365PubMedCrossRefGoogle Scholar
  132. 132.
    Friedl P, Noble PB, Walton PA et al (1995) Migration of coordinated cell clusters in mesenchymal and epithelial cancer explants in vitro. Cancer Res 55:4557–4560PubMedGoogle Scholar
  133. 133.
    Wartenberg M, Finkensieper A, Hescheler J et al (2006) Confrontation cultures of embryonic stem cells with multicellular tumor spheroids to study tumor-induced angiogenesis. Methods Mol Biol 331:313–328PubMedGoogle Scholar
  134. 134.
    Fjellbirkeland L, Bjerkvig R, Laerum OD (1998) Non-small-cell lung carcinoma cells invade human bronchial mucosa in vitro. In Vitro Cell Dev Biol Anim 34:333–340PubMedCrossRefGoogle Scholar

Copyright information

© SpringerScience+Business Media New York 2013

Authors and Affiliations

  • Miriam Zimmermann
    • 1
  • Carol Box
    • 1
  • Suzanne A. Eccles
    • 1
  1. 1.Tumour Biology and Metastasis, Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, McElwain LaboratoriesThe Institute of Cancer ResearchSurreyUK

Personalised recommendations