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Miniaturization of an Anoikis assay using non-adhesive micromolded hydrogels

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Abstract

Anoikis is a specific form of apoptosis resulting from the loss of cellular attachment to extracellular matrix or other cells. Challenges in simulating these conditions in vitro make it difficult to generate a controlled, efficient assay to study anoikis. We developed a microscale method for analysis and quantification of anoikis using micromolded, non-adhesive hydrogels. These hydrogels allow for isolation and observation of single, unattached cells in an ordered array, and controlled distribution. Cell distributions resulting from multiple seeding densities were compared to a mathematical probability model. Normal human fibroblasts, human umbilical vein endothelial cells, and Mandin-Darby canine kidney epithelial cells were seeded at low densities of approximately one cell/well. Because the hydrogel is made of non-adhesive agarose, attachment was negligible. Survival was monitored using fluorescent microscopy, and quantified by image analysis. The attachment and proliferative potential of cells after being held in a non-adherent environment was assessed with a companion attachment assay. The data from both methods revealed that cells were able to survive much longer than expected without attachment. When tested with H35 rat hepatoma cells we showed that single cancer cells could grow into three-dimensional spheroids, demonstrating the utility of this method in understanding the role of anoikis in cancer.

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Abbreviations

NHF:

Normal human fibroblast

MDCK:

Mandin-Darby canine kidney epithelial cells

HUVEC:

Human umbilical vein endothelial cells

DMEM:

Dulbecco’s modified eagle medium

FBS:

Fetal bovine serum

PBS:

Phosphate buffered saline

References

  • Aoudijit F, Vuori K (2001) Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for C-flip and implications for anoikis. J Cell Biol 152(3):633–643

    Article  Google Scholar 

  • Boudrea N, Sympson CJ, Werb Z, Bissell MJ (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267:891–893

    Article  Google Scholar 

  • Cardone MH, Salvesen GS, Widmann C, Johnson G, Frisch SM (1997) Regulation of anoikis: MEKK-1 activations requires cleavage by caspases. Cell 90:315–323

    Article  CAS  Google Scholar 

  • Cheng T, Symons M, Jou T (2004) Regulation of Anoikis by Cdc42 and Rac1. Exp. Cell Res 295:497–511

    Article  CAS  Google Scholar 

  • Dourma S, Laar T, Zevenhoven J, Meuwissen R, Garderen E, Peeper DS (2004) Suppression of anoikis and induction of metastasis by the neurotropic receptor TrkB. Nature 430:1034–1040

    Article  CAS  Google Scholar 

  • Fouquet S, Lugo-Martinez V-H, Faussat A-M, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S (2004) Early loss of e-cadherin from cell–cell contacts in involved in the onset of anoikis in enterocytes. J Biol Chem 41(8):43061–43069

    Article  CAS  Google Scholar 

  • Frisch SM, Francis HJ (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. Cell Biol 124(4):619–626

    Article  CAS  Google Scholar 

  • Grossman J (2002) Molecular mechanisms of detachment-induced apoptosis—Anoikis. Apoptosis 7:247–260

    Article  Google Scholar 

  • Hall P, Coates P, Ansari B, Hopwood D (1994) Regulation of cell number in the gastrointestinal tract: the importance of apoptosis. J Cell Sci 107:3569–3577

    CAS  Google Scholar 

  • Hermiston ML, Gordon JI (1995) In vivo analysis of cadherin function in the mouse intestinal epithelium: essential roles in adhesion, maintenance of differentiation, and regulation of programmed cell death. J Cell Biol 129:489–506

    Article  CAS  Google Scholar 

  • Hu Z, Sanchez-Sweatman O, Huang X, Wiltrout R, Khoka R, Zhao Q, Gorelik E (2001) Anoikis and metastatic potential of cloudman S91 melanoma cells. Cancer Res 61:1707–1716

    Google Scholar 

  • Hungerford J, Compton J, Matter J, Hoffsrom B, Otey C (1996) Inhibition of pp125FAK in culture fibroblasts results in apoptosis. J Cell Biol 135:1383–1390

    Article  CAS  Google Scholar 

  • Ito H, Zinner MJ, Ashley SW, Whang EE (2004) Focal adhesion kinase gene silencing promotes anoikis and suppresses metastasis of human pancreatic adenocarcinoma cells. Surgery 555–562

  • Khademhosseini A, Yeh J, Eng G, Karp J, Kaji H, Borenstein J, Farokhzad OC, Langer R (2005) Cell docking inside microwells within reversibly sealed microfluidic channels for fabricating multiphenotype cell arrays. Lab Chip 5:1380–1386

    Article  CAS  Google Scholar 

  • Korff T, Augustin HG (1998) Integration of endothelial cells in multicellular spherois prevents apoptosis and induces differentiation. J Cell Biol 143(5):1341–1352

    Article  CAS  Google Scholar 

  • Lovchik R, Arx CV, Viviani A, Delamarche E (in press) Cellular microarrays for use with capillary-driven microfluidics. Anal Bioanal Chem. doi:10.1007/s00216-007-1436-3

  • McGill G, Simamura A, Bates RC, Savage RE, Fisher DE (1997) Loss of matrix adhesion triggers rapid transformation—selective apoptosis in fibroblasts. J Cell Biol 138:901–911

    Article  CAS  Google Scholar 

  • Meredith J, Schwartz M (1997) Integrins, adhesion and apoptosis. Trends Cell Biol 7:146–150

    Article  CAS  Google Scholar 

  • Meredith JE, Fazeli B, Schwartz M (1993) The extracellular matrix as a cell survival factor. Mol Biol Cell 4:953–961

    CAS  Google Scholar 

  • Montgomery A, Reisfeld R, Cheresh D (1994) Integrin rescues melanoma cells from apoptosis in three-dimensional dermal collagen. Proc Natl Acad Sci USA 91:8856–8860

    Article  CAS  Google Scholar 

  • Napolitano A, Chai P, Morgan J (2007) Dynamics of the self-assembly of complex cellular aggregates on micromolded non-adhesive hydrogels. Tiss Eng 13(8):2087–2094

    Article  CAS  Google Scholar 

  • Napolitano AP, Dean DM, Man AJ, Youssef J, Ho DN, Rago AP, Lech MP, Morgan JR (2007) Scaffold-free three-dimensional cell culture utilizing nonadhesive hydrogels. Biotechniques 43:494–500

    Article  CAS  Google Scholar 

  • Polakowska R, Piacentini M, Bartlett R, Goldsmith L, Haake A (1994) Apoptosis in human skin development: morphogenesis, periderm, and stem cells. Devel Dynamics 3:176–188

    Google Scholar 

  • Rettig JR, Folch A (2005) Large-Scale Single-Cell Trapping and Imaging Using Microwell Arrays. Anal Chem 77:5628–5634

    Article  CAS  Google Scholar 

  • Rosen K, Rak J, Dean N, Kerbel R, Filmus J (2000) Activated ras prevents down regulation of Bcl-XL triggered by detachment from the extracellular matrix: a mechanism of ras-induced resistance to anoikis in intestinal epithelial cells. J Cell Biol 149(2):447–455

    Article  CAS  Google Scholar 

  • Sims C, Allbritton NL (2007) Analysis of Single mammalian Cells On-Chip. Lab Chip 7:423–440

    Article  CAS  Google Scholar 

  • Vachon P, Loechel F, Xu H, Wewer U, Engvall E (1996) Merosin and Laminin in myogenesis; specific requirement for merosin in myotube stability and survival. J Cell Biol 134:1483–1487

    Article  CAS  Google Scholar 

  • Voldman J (2006) Engineered systems for the Physical manipulation of single cells. Curr Opin Biotech 17(5):532–537

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded, in part by the Brown University MD/PhD and Undergraduate Teaching and Research Award programs, the MRSEC Program of the National Science Foundation under award DMR-0520651 and the NIRT Program under award DMI-0506661.

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Authors and Affiliations

  1. Department of Molecular Pharmacology, Physiology and Biotechnology, Center for Biomedical Engineering, Brown University, G-B393, Biomedical Center, 171 Meeting Street, Providence, RI, 02912, USA

    Adam P. Rago, Anthony P. Napolitano, Dylan M. Dean, Peter R. Chai & Jeffrey R. Morgan

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  1. Adam P. Rago
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  2. Anthony P. Napolitano
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  3. Dylan M. Dean
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  4. Peter R. Chai
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  5. Jeffrey R. Morgan
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Correspondence to Jeffrey R. Morgan.

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Rago, A.P., Napolitano, A.P., Dean, D.M. et al. Miniaturization of an Anoikis assay using non-adhesive micromolded hydrogels. Cytotechnology 56, 81–90 (2008). https://doi.org/10.1007/s10616-007-9116-x

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  • DOI: https://doi.org/10.1007/s10616-007-9116-x

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