A Unique High‐Throughput Assay to Identify Novel Small Molecule Inhibitors of Chemotaxis and Migration

Xin‐Hua Liao1, Alan R. Kimmel2

1 Fujian Key Laboratory for Translational Research in Cancer and Neurodegenerative Diseases, Institute for Translational Medicine, and Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, Fuzhou, Fujian, 2 Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.11
DOI:  10.1002/cpcb.17
Online Posting Date:  March, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Novel chemotaxis/migration inhibitors are desirable for developing effective therapeutics and probing molecular mechanisms. We describe a fluorescence‐based phenotypic assay in a 1536‐well plate format for high‐throughput screening of novel inhibitors of chemotaxis/migration within complex libraries of thousands of compounds. Although the assay utilizes the unique cellular response properties of Dictyostelium, the compounds identified are able to inhibit chemotaxis of mammalian cells. In addition, a parallel cell cytotoxicity counter‐screen with an ATP content assay is described that eliminates cytotoxic compounds from the screen. This novel compound screening approach enables rapid identification of novel lead compounds that inhibit chemotaxis in human and other cells for drug development and research tools. © 2017 by John Wiley & Sons, Inc.

Keywords: cell migration; chemotaxis; LOPAC; acumen eX3; Dictyostelium

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Miniaturized Dictyostelium Aggregation Assay for Screening of Chemotaxis/Migration Inhibitors
  • Basic Protocol 2: Dictyostelium Cytotoxicity Assay for Eliminating Cytotoxic Compounds
  • Support Protocol 1: Data Analysis
  • Support Protocol 2: Re‐Confirmation and Validation of Chemotaxis Inhibition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Miniaturized Dictyostelium Aggregation Assay for Screening of Chemotaxis/Migration Inhibitors

  Materials
  • Dictyostelium strain [cotB]:GFP (dictyBase, strain i.d. DBS0236466) (Basu et al., )
  • D3‐T medium (see recipe)
  • DB starvation buffer (see recipe)
  • DMSO (Sigma‐Aldrich, cat. no. D2650)
  • Library of Pharmacologically Active Compounds: LOPAC1280 (Sigma‐Aldrich, #LO1280) or other compound collections
  • Control compound: latrunculin A (Sigma‐Aldrich, cat. no. L5163)
  • 25‐cm² canted‐neck cell culture flask, sterile
  • Incubator with temperature controlled at 21°C
  • 250‐ml or larger glass or plastic Erlenmeyer flask, sterile
  • Orbital shaker at 21°C
  • Centrifuge 5810R (Eppendorf)
  • Evolution P3 system (PerkinElmer)
  • 384‐well plates
  • 1536‐well clear‐bottom plates (Aurora Biotechnologies)
  • Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific)
  • Kalypsys microplate metal lids
  • Peelable aluminum microplate sealers (Agilent Technologies, cat. no. 24210‐001)
  • Agilent PlateLoc thermal microplate sealer
  • Pintool Station (Kalypsys)
  • Acumen eX3 (TTP LabTech)
  • Additional reagents and equipment for counting cells (unit 1.1; Phelan, )

Basic Protocol 2: Dictyostelium Cytotoxicity Assay for Eliminating Cytotoxic Compounds

  Materials
  • Hygromycin B (Sigma‐Aldrich)
  • ATPlite assay kit (PerkinElmer, cat. no. 6016731)
  • CCD‐imager based ViewLux plate reader (PerkinElmer)
  • Additional reagents and equipment for Dictyostelium aggregation assay ( protocol 1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Artemenko, Y., Lampert, T.J., and Devreotes, P.N. 2014. Moving towards a paradigm: Common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes. Cell. Mol. Life Sci. 71:3711‐3747. doi: 10.1007/s00018‐014‐1638‐8.
  Basu, S., Fey, P, Pandit, Y., Dodson, R., Kibbe, W.A., and Chisholm, R.L. 2013. DictyBase 2013: Integrating multiple Dictyostelid species. Nucleic Acids Res. 41:D676‐D683. doi: 10.1093/nar/gks1064. Epub 2012 Nov 20.
  Berthier, E., Lim, F.Y., Deng, Q., Guo, C.J., Kontoyiannis, D.P., Wang, C.C., Rindy, J., Beebe, D.J., Huttenlocher, A., and Keller, N.P. 2013. Low‐volume toolbox for the discovery of immunosuppressive fungal secondary metabolites. PLoS Pathog. 9:e1003289. doi: 10.1371/journal.ppat.1003289.
  Chan, G. and Mooney, D.J. 2008. New materials for tissue engineering: Towards greater control over the biological response. Trends Biotechnol. 26:382‐392. doi: 10.1016/j.tibtech.2008.03.011.
  Djekic, U.V., Gaggar, A., and Weathington, N.M. 2009. Attacking the multi‐tiered proteolytic pathology of COPD: New insights from basic and translational studies. Pharmacol. Ther. 121:132‐146. doi: 10.1016/j.pharmthera.2008.09.008.
  Guckenberger, D.J., Berthier, E., and Beebe, D.J. 2015. High‐density self‐contained microfluidic KOALA kits for use by everyone. J. Lab. Autom. 20:146‐153. doi: 10.1177/2211068214560609.
  Hattori, H., Subramanian, K.K., Sakai, J., Jia, Y., Li, Y., Porter, T.F., Loison, F., Sarraj, B., Kasorn, A., Jo, H., Blanchard, C., Zirkle, D., McDonald, D., Pai, S.Y., Serhan, C.N., and Luo, H.R. 2010. Small‐molecule screen identifies reactive oxygen species as key regulators of neutrophil chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 107:3546‐3551. doi: 10.1073/pnas.0914351107.
  Hulkower, K.I. and Herber, R.L. 2011. Cell migration and invasion assays as tools for drug discovery. Pharmaceutics 3:107‐124. doi: 10.3390/pharmaceutics3010107.
  Inamdar, G.S., Madhunapantula, S.V., and Robertson, G.P. 2010. Targeting the MAPK pathway in melanoma: Why some approaches succeed and other fail. Biochem. Pharmacol. 80:624‐637. doi: 10.1016/j.bcp.2010.04.029.
  Inglese, J., Auld, D.S., Jadhav, A., Johnson, R.L., Simeonov, A., Yasgar, A., Zheng, W., and Austin, C.P. 2006. Quantitative high‐throughput screening: A titration‐based approach that efficiently identifies biological activities in large chemical libraries. Proc. Natl. Acad. Sci. U.S.A. 103:11473‐11478. doi: 10.1073/pnas.0604348103.
  Irimia, D. and Toner, M. 2009. Spontaneous migration of cancer cells under conditions of mechanical confinement. Integr. Biol. (Camb.) 1:506‐512. doi: 10.1039/b908595e.
  Jin, T. 2013. Gradient sensing during chemotaxis. Curr. Opin. Cell Biol. 25:532‐537. doi: 10.1016/j.ceb.2013.06.007.
  Jin, T., Xu, X., and Hereld, D. 2008. Chemotaxis, chemokine receptors and human disease. Cytokine 44:1‐8. doi: 10.1016/j.cyto.2008.06.017.
  Kato, H., Ueki, S., Ito, W., Takeda, M., Chiba, T., Yamaguchi, K., Kayaba, H., and Chihara, J. 2008. Eosinophil chemotaxis assay using novel device EZ‐TAXIScan. Arerugi 57:1317‐1324.
  Liang, C.C., Park, A.Y., and Guan, J.L. 2007. in vitro scratch assay: A convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2:329‐333. doi: 10.1038/nprot.2007.30.
  Liao, X.H., Meena, N.P., Southall, N., Liu, L., Swaroop, M., Zhang, A.L., Xiang, J.J., Parent, C.A., Zheng, W., and Kimmel, A.R. 2016. A high‐throughput, multi‐cell phenotype assay for the identification of novel inhibitors of chemotaxis/migration. Sci. Rep. 6:22273. doi: 10.1038/srep22273.
  Liu, L., Das, S., Losert, W., and Parent, C.A. 2010. mTORC2 regulates neutrophil chemotaxis in a cAMP‐ and RhoA‐dependent fashion. Dev. Cell 19:845‐857. doi: 10.1016/j.devcel.2010.11.004.
  Mackay, C.R. 2008. Moving targets: Cell migration inhibitors as new anti‐inflammatory therapies. Nat. Immunol. 9:988‐998. doi: 10.1038/ni.f.210.
  Mastyugin, V., McWhinnie, E., Labow, M., and Buxton, F. 2004. A quantitative high‐throughput endothelial cell migration assay. J. Biomol. Screen. 9:712‐718. doi: 10.1177/1087057104269495.
  McMains, V.C., Liao, X.H., and Kimmel, A.R. 2008. Oscillatory signaling and network responses during the development of Dictyostelium discoideum. Ageing Res. Rev. 7:234‐248. doi: 10.1016/j.arr.2008.04.003.
  Miret, S., De Groene, E.M., and Klaffke, W. 2006. Comparison of in vitro assays of cellular toxicity in the human hepatic cell line HepG2. J. Biomol. Screen 11:184‐193. doi: 10.1177/1087057105283787.
  Nicol, A., Rappel, W., Levine, H., and Loomis, W.F. 1999. Cell‐sorting in aggregates of Dictyostelium discoideum. J. Cell Sci. 112:3923‐3929.
  Phelan, K. and May, K.M. 2015. Basic techniques in mammalian cell tissue culture. Curr. Protoc. Cell Biol. 66:1.1.1‐1.1.22. doi: 10.1002/0471143030.cb0101s66.
  Ponath, P.D., Wang, J., and Heath, H. 2000. Transwell chemotaxis. Methods Mol. Biol. 138:113‐120.
  Soltaninassab, S.R., Sotos, J.P., Bonds, M.D., Williams, L.A., Williams, J.W., Abbott, N.L., Murphy, C.J., and Held, P.G. 2008. A novel high throughput‐compatible cell migration screening assay. BioTek Appl. Note Rev. 8/12:1‐4.
  Southall, N., Jadhav, A., Huang, R., Nguyen, T., and Wang, Y. 2009. Enabling the Large Scale Analysis of Quantitative High Throughput Screening Data. Taylor and Francis, New York.
  Su, W., Chen, Q., and Frohman, M.A. 2009. Targeting phospholipase D with small‐molecule inhibitors as a potential therapeutic approach for cancer metastasis. Future Oncol. 5:1477‐1486. doi: 10.2217/fon.09.110.
  Swaney, K.F., Huang, C.H., and Devreotes, P.N. 2010. Eukaryotic chemotaxis: A network of signaling pathways controls motility, directional sensing, and polarity. Annu. Rev. Biophys. 39:265‐289. doi: 10.1146/annurev.biophys.093008.131228.
  Timm, D.M., Chen, J., Sing, D., Gage, J.A., Haisler, W.L., Neeley, S.K., Raphael, R.M., Dehghani, M., Rosenblatt, K.P., Killian, T.C., Tseng, H., and Souza, G.R. 2013. A high‐throughput three‐dimensional cell migration assay for toxicity screening with mobile device‐based macroscopic image analysis. Sci. Rep. 3:3000. doi: 10.1038/srep03000.
  Van Haastert, P.J. and Veltman, D.M. 2007. Chemotaxis: Navigating by multiple signaling pathways. Sci. STKE 2007:pe40. doi: 10.1126/stke.3962007pe40.
  Williams, J.G. 2010. Dictyostelium finds new roles to model. Genetics 185:717‐726. doi: 10.1534/genetics.110.119297.
  Wu, J., Wu, X., and Lin, F. 2013. Recent developments in microfluidics‐based chemotaxis studies. Lab Chip 13:2484‐2499. doi: 10.1039/c3lc50415h.
  Wu, X., Lee, V.C., Chevalier, E., and Hwang, S.T. 2009. Chemokine receptors as targets for cancer therapy. Curr. Pharm. Des. 15:742‐757. doi: 10.2174/138161209787582165.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library