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Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model

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Abstract

Solid tumors consist of genetically and phenotypically diverse subpopulations of cancer cells with unique capacities for growth, differentiation, and invasion. While the molecular and microenvironmental bases for heterogeneity are increasingly appreciated, the outcomes of such intratumor heterogeneity, particularly in the context of tumor invasion and metastasis, remain poorly understood. To study heterotypic cell–cell interactions and elucidate the biological consequences of intratumor heterogeneity, we developed a tissue-engineered multicellular spheroid (MCS) co-culture model that recapitulates the cellular diversity and fully three-dimensional cell–cell and cell–matrix interactions that characterize human carcinomas. We found that “invasion-competent” malignant cells induced the collective invasion of otherwise “invasion-incompetent” epithelial cells, and that these two cell types consistently exhibited distinct leader and follower roles during invasion. Analysis of extracellular matrix (ECM) microarchitecture revealed that malignant cell invasion was accompanied by extensive ECM remodeling including matrix alignment and proteolytic track-making. Inhibition of cell contractility- and proteolysis-mediated matrix reorganization prevented leader-follower behavior and malignant cell-induced epithelial cell invasion. These results indicate that heterogeneous subpopulations within a tumor may possess specialized roles during tumor progression and suggest that complex interactions among the various subpopulations of cancer cells within a tumor may regulate critical aspects of tumor biology and affect clinical outcome.

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References

  1. Bissell MJ, Radisky D (2001) Putting tumours in context. Nat Rev Cancer 1:46–54

    Article  PubMed  CAS  Google Scholar 

  2. Dvorak HF, Weaver VM, Tlsty TD, Bergers G (2011) Tumor microenvironment and progression. J Surg Oncol 103:468–474

    Article  PubMed  CAS  Google Scholar 

  3. Fidler IJ (1978) Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 38:2651–2660

    PubMed  CAS  Google Scholar 

  4. Marusyk A, Polyak K (2010) Tumor heterogeneity: causes and consequences. Biochim Biophys Acta 1805:105–117

    PubMed  CAS  Google Scholar 

  5. Yamada KM, Cukierman E (2007) Modeling tissue morphogenesis and cancer in 3D. Cell 130:601–610

    Article  PubMed  CAS  Google Scholar 

  6. Dong-Le Bourhis X, Berthois Y, Millot G, Degeorges A, Sylvi M, Martin P-M, Calvo F (1997) Effect of stromal and epithelial cells derived from normal and tumorous breast tissue on the proliferation of human breast cancer cell lines in co-culture. Int J Cancer 71:42–48

    Article  PubMed  CAS  Google Scholar 

  7. Guo X, Oshima H, Kitmura T, Taketo MM, Oshima M (2008) Stromal fibroblasts activated by tumor cells promote angiogenesis in mouse gastric cancer. J Biol Chem 283:19864–19871

    Article  PubMed  CAS  Google Scholar 

  8. Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall JF, Harrington K, Sahai E (2007) Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol 9:1392–1400

    Article  PubMed  CAS  Google Scholar 

  9. Jones JL, Shaw JA, Pringle JH, Walker RA (2003) Primary breast myoepithelial cells exert an invasion-suppressor effect on breast cancer cells via paracrine down-regulation of MMP expression in fibroblasts and tumour cells. J Pathol 201:562–572

    Article  PubMed  CAS  Google Scholar 

  10. Goswami S, Sahai E, Wyckoff JB, Cammer M, Cox D, Pixley FJ, Stanley ER, Segall JE, Condeelis JS (2005) Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop. Cancer Res 65:5278–5283

    Article  PubMed  CAS  Google Scholar 

  11. Hanahan D, Weinberg R (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674

    Article  PubMed  CAS  Google Scholar 

  12. Gerlinger M, Rowan A, Horswell S et al (2012) Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366:883–982

    Article  PubMed  CAS  Google Scholar 

  13. Friedl P, Wolf K (2010) Plasticity of cell migration: a multiscale tuning model. J Cell Biol 188:11–19

    Article  PubMed  CAS  Google Scholar 

  14. Provenzano P, Inman D, Eliceiri K, Keely P (2009) Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage. Oncogene 28:4326–4343

    Article  PubMed  CAS  Google Scholar 

  15. Petersen OW, Lind Nielsen H, Gudjonsson T, Villadsen R, Rønnov-Jessen L, Bissell MJ (2001) The plasticity of human breast carcinoma cells is more than epithelial to mesenchymal conversion. Breast Cancer Res 3:213–217

    Article  PubMed  CAS  Google Scholar 

  16. Carey SP, D’Alfonso TM, Shin SJ, Reinhart-King CA (2012) Mechanobiology of tumor invasion: engineering meets oncology. Crit Rev Oncol/Hematol 83:170–183

    Article  Google Scholar 

  17. Weiss L (2000) Heterogeneity of cancer cell populations and metastasis. Cancer Metastasis Rev 19:351–379

    Article  Google Scholar 

  18. Liotta LA, Stetler-Stevenson WG (1991) Tumor invasion and metastasis: an imbalance of positive and negative regulation. Cancer Res 51:5054s–5059s

    PubMed  CAS  Google Scholar 

  19. Pathak A, Kumar S (2011) Biophysical regulation of tumor cell invasion: moving beyond matrix stiffness. Integr Biol 3:267–278

    Article  CAS  Google Scholar 

  20. Sabeh F, Shimizu-Hirota R, Weiss SJ (2009) Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited. J Cell Biol 185:11–19

    Article  PubMed  CAS  Google Scholar 

  21. Wolf K, Wu YI, Liu Y, Geiger J, Tam E, Overall C, Stack MS, Friedl P (2007) Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat Cell Biol 9:893–904

    Article  PubMed  CAS  Google Scholar 

  22. Sabeh F, Ota I, Holmbeck K et al (2004) Tumor cell traffic through the extracellular matrix is controlled by the membrane-anchored collagenase MT1-MMP. J Cell Biol 167:769–781

    Article  PubMed  CAS  Google Scholar 

  23. Nguyen-Ngoc K-V, Cheung KJ, Brenot A, Shamir ER, Gray RS, Hines WC, Yaswen P, Werb Z, Ewald AJ (2012) ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc Natl Acad Sci USA 109:E2595–E2604

    Article  PubMed  CAS  Google Scholar 

  24. Carey SP, Kraning-Rush CM, Williams RM, Reinhart-King CA (2012) Biophysical control of invasive tumor cell behavior by extracellular matrix microarchitecture. Biomaterials 33:4157–4165

    Article  PubMed  CAS  Google Scholar 

  25. Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White JG, Keely PJ (2006) Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Med. doi:10.1186/1741-7015-4-38

    PubMed  Google Scholar 

  26. Levental KR, Yu H, Kass L et al (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139:891–906

    Article  PubMed  CAS  Google Scholar 

  27. Pedersen JA, Swartz MA (2005) Mechanobiology in the third dimension. Ann Biomed Eng 33:1469–1490

    Article  PubMed  Google Scholar 

  28. Friedl P, Locker J, Sahai E, Segall JE (2012) Classifying collective cancer cell invasion. Nat Cell Biol 14:777–783

    Article  PubMed  Google Scholar 

  29. Pampaloni F, Reynaud EG, Stelzer EHK (2007) The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Bio 8:839–845

    Article  CAS  Google Scholar 

  30. Burdett E, Kasper FK, Mikos AG, Ludwig JA (2010) Engineering tumors: a tissue engineering perspective in cancer biology. Tissue Eng Part B Rev 16:351–359

    Article  PubMed  Google Scholar 

  31. Paszek MJ, Zahir N, Johnson KR et al (2005) Tensional homeostasis and the malignant phenotype. Cancer Cell 8:241–254

    Article  PubMed  CAS  Google Scholar 

  32. Bissell MJ, Rizki A, Mian IS (2003) Tissue architecture: the ultimate regulator of breast epithelial function. Curr Opin Cell Biol 15:753–762

    Article  PubMed  CAS  Google Scholar 

  33. Gumbiner BM (2005) Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol 6:622–634

    Article  PubMed  CAS  Google Scholar 

  34. Lorenzo C, Frongia C, Jorand R, Fehrenbach J, Weiss P, Maandhui A, Gay G, Ducommun B, Lobjois V (2011) Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy. Cell Div. doi:10.1186/1747-1028-6-22

    PubMed  Google Scholar 

  35. Ilina O, Bakker G-J, Vasaturo A, Hoffman RM, Friedl P (2011) Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and -independent collective cancer cell invasion. Phys Biol 8:015010

    Article  PubMed  Google Scholar 

  36. 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 28:461–468

    Article  PubMed  CAS  Google Scholar 

  37. Rezakhaniha R, Agianniotis A, Schrauwen J, Griffa A, Sage D, Bouten C, van de Vosse F, Unser M, Stergiopulos N (2012) Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy. Biomech Model Mechanobiol 11:461–473

    Article  PubMed  CAS  Google Scholar 

  38. Wolman SR (1986) Cytogenetic heterogeneity: its role in tumor evolution. Cancer Genet Cytogenet 19:129–140

    Article  PubMed  CAS  Google Scholar 

  39. Nowell PC (1976) The clonal evolution of tumor cell populations. Science 194:23–28

    Article  PubMed  CAS  Google Scholar 

  40. Aubele M, Mattis A, Zitzelsberger H, Walch A, Kremer M, Hutzler P, Höfler H, Werner M (1999) Intratumoral heterogeneity in breast carcinoma revealed by laser-microdissection and comparative genomic hybridization. Cancer Genet Cytogenet 110:94–102

    Article  PubMed  CAS  Google Scholar 

  41. Keller PJ, Lin AF, Arendt LM et al (2010) Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines. Breast Cancer Res 12:R87

    Article  PubMed  Google Scholar 

  42. Debnath J, Muthuswamy SK, Brugge JS (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30:256–268

    Article  PubMed  CAS  Google Scholar 

  43. Soule HD, Maloney TM, Wolman SR, Peterson WD, Brenz R, Mcgrath CM, Russo J, Pauley RJ, Jones RF, Brooks SC (1990) Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res 50:6075–6086

    PubMed  CAS  Google Scholar 

  44. Santner S, Dawson P, Tait L, Soule H, Eliason J, Mohamed A, Wolman S, Heppner G, Miller F (2001) Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells. Breast Cancer Res Treat 65:101–110

    Article  PubMed  CAS  Google Scholar 

  45. Ivascu A, Kubbies M (2007) Diversity of cell-mediated adhesions in breast cancer spheroids. Int J Oncology 31:1403–1413

    CAS  Google Scholar 

  46. Foty RA, Steinberg MS (2004) Cadherin-mediated cell–cell adhesion and tissue segregation in relation to malignancy. Int J Dev Biol 48:397–409

    Article  PubMed  CAS  Google Scholar 

  47. Foty RA, Steinberg MS (2005) The differential adhesion hypothesis: a direct evaluation. Dev Biol 278:255

    Article  PubMed  CAS  Google Scholar 

  48. Vamvakidou AP, Mondrinos MJ, Petushi SP, Garcia FU, Lelkes PI, Tozeren A (2007) Heterogeneous breast tumoroids: an in vitro assay for investigating cellular heterogeneity and drug delivery. J Biomol Screen 12:13–20

    Article  PubMed  CAS  Google Scholar 

  49. Chanson L, Brownfield D, Garbe JC, Kuhn I, Stampfer MR, Bissell MJ, LaBarge MA (2011) Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells. Proc Natl Acad Sci USA 108:3264–3269

    Article  PubMed  CAS  Google Scholar 

  50. Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148:3–15

    Article  PubMed  CAS  Google Scholar 

  51. Kraning-Rush CM, Califano JP, Reinhart-King CA (2012) Cellular traction stresses increase with increasing metastatic potential. PLoS ONE 7:e32572

    Article  PubMed  CAS  Google Scholar 

  52. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S (1980) Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 284:67–68

    Article  PubMed  CAS  Google Scholar 

  53. Baker EL, Srivastava J, Yu D, Bonnecaze RT, Zaman MH (2011) Cancer cell migration: integrated roles of matrix mechanics and transforming potential. PLoS ONE 6:e20355

    Article  PubMed  CAS  Google Scholar 

  54. Wolf K, Alexander S, Schacht V, Coussens LM, von Andrian UH, van Rheenen J, Deryugina E, Friedl P (2009) Collagen-based cell migration models in vitro and in vivo. Semin Cell Dev Biol 20:931–941

    Article  PubMed  CAS  Google Scholar 

  55. Sarrió D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J (2008) Epithelial-mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res 68:989–997

    Article  PubMed  Google Scholar 

  56. Wiercinska E, Naber HPH, Pardali E, van der Pluijm G, van Dam H, ten Dijke P (2011) The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system. Breast Cancer Res Treat 128:657–666

    Article  PubMed  CAS  Google Scholar 

  57. Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS (2002) The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell 111:29–40

    Article  PubMed  CAS  Google Scholar 

  58. Alexander S, Koehl G, Hirschberg M, Geissler EK, Friedl P (2008) Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model. Histochem Cell Biol 130:1147–1154

    Article  PubMed  CAS  Google Scholar 

  59. Inaki M, Vishnu S, Cliffe A, Rørth P (2012) Effective guidance of collective migration based on differences in cell states. Proc Natl Acad Sci USA 109:2027–2032

    Article  PubMed  CAS  Google Scholar 

  60. Khalil A, Friedl P (2010) Determinants of leader cells in collective cell migration. Integr Biol 2:568–574

    Article  Google Scholar 

  61. Thiery JP (2002) Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2:442–454

    Article  PubMed  CAS  Google Scholar 

  62. Friedl P, Wolf K (2008) Tube travel: the role of proteases in individual and collective cancer cell invasion. Cancer Res 68:7247–7249

    Article  PubMed  CAS  Google Scholar 

  63. Shieh AC, Rozansky HA, Hinz B, Swartz MA (2011) Tumor cell invasion is promoted by interstitial flow-induced matrix priming by stromal fibroblasts. Cancer Res 71:790–800

    Article  PubMed  CAS  Google Scholar 

  64. Friedl P, Maaser K, Klein CE, Niggemann B, Krohne G, Zänker KS (1997) Migration of highly aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local matrix reorganization and shedding of alpha2 and beta1 integrins and CD44. Cancer Res 57:2061–2070

    PubMed  CAS  Google Scholar 

  65. Gordon V, Valentine M, Gardel M, Andor-Ardo D, Dennison S, Bogdanov A, Weitz D, Deisboeck T (2003) Measuring the mechanical stress induced by an expanding multicellular tumor system: a case study. Exp Cell Res 289:58–66

    Article  PubMed  CAS  Google Scholar 

  66. Kraning-Rush CM, Carey SP, Califano JP, Smith BN, Reinhart-King CA (2011) The role of the cytoskeleton in cellular force generation in 2D and 3D environments. Phys Biol 8:015009

    Article  PubMed  Google Scholar 

  67. 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 95:5374–5384

    Article  PubMed  CAS  Google Scholar 

  68. Fisher KE, Sacharidou A, Stratman AN, Mayo AM, Fisher SB, Mahan RD, Davis MJ, Davis GE (2009) MT1-MMP- and Cdc42-dependent signaling co-regulate cell invasion and tunnel formation in 3D collagen matrices. J Cell Sci 122:4558–4569

    Google Scholar 

  69. De Smet F, Segura I, De Bock K, Hohensinner PJ, Carmeliet P (2009) Mechanisms of vessel branching: filopodia on endothelial tip cells lead the way. Arterioscler Thromb Vasc Biol 29:639–649

    Article  PubMed  Google Scholar 

  70. Friedl P, Gilmour D (2009) Collective cell migration in morphogenesis, regeneration and cancer. Nat Rev Mol Cell Bio 10:445–457

    Article  CAS  Google Scholar 

  71. Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 3:362–374

    Article  PubMed  CAS  Google Scholar 

  72. Cross SE, Jin Y-S, Rao J, Gimzewski JK (2007) Nanomechanical analysis of cells from cancer patients. Nat Nanotechnol 2:780–783

    Article  PubMed  CAS  Google Scholar 

  73. Wolf K, Mazo I, Leung H, Engelke K, von Andrian UH, Deryugina EI, Strongin AY, Bröcker E-B, Friedl P (2003) Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160:267–277

    Article  PubMed  CAS  Google Scholar 

  74. Sahai E, Marshall CJ (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 5:711–719

    Article  PubMed  CAS  Google Scholar 

  75. Wyckoff JB, Pinner SE, Gschmeissner S, Condeelis JS, Sahai E (2006) ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr Biol 16:1515–1523

    Article  PubMed  CAS  Google Scholar 

  76. Poincloux R, Collin O, Lizárraga F, Romao M, Debray M, Piel M, Chavrier P (2011) Contractility of the cell rear drives invasion of breast tumor cells in 3D matrigel. Proc Natl Acad Sci USA 108:1943–1948

    Article  PubMed  CAS  Google Scholar 

  77. Mierke CT, Rösel D, Fabry B, Brábek J (2008) Contractile forces in tumor cell migration. Eur J Cell Biol 87:669–676

    Article  PubMed  CAS  Google Scholar 

  78. Guiet R, Van Goethem E, Cougoule C, Balor S, Valette A, Al Saati T, Lowell Ca, Le Cabec V, Maridonneau-Parini I (2011) The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells. J Immunol 187:3806–3814

    Article  PubMed  CAS  Google Scholar 

  79. Hotary KB, Allen ED, Brooks PC, Datta NS, Long MW, Weiss SJ (2003) Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix. Cell 114(1):33–45

    Article  PubMed  CAS  Google Scholar 

  80. Hu M, Yao J, Carroll DK et al (2008) Regulation of in situ to invasive breast carcinoma transition. Cancer Cell 13:394–406

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported in part by the Cornell Center on the Microenvironment & Metastasis through Award Number U54CA143876 from the National Cancer Institute (NCI) and award number CMMI-1233827 from the NSF to CAR and a National Science Foundation Graduate Research Fellowship to SPC.

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The authors declare that they have no conflict of interest.

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Correspondence to Cynthia A. Reinhart-King.

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Shawn P. Carey and Alina Starchenko contributed equally to this study.

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Carey, S.P., Starchenko, A., McGregor, A.L. et al. Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model. Clin Exp Metastasis 30, 615–630 (2013). https://doi.org/10.1007/s10585-013-9565-x

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