GILA, a Replacement for the Soft‐Agar Assay that Permits High‐Throughput Drug and Genetic Screens for Cellular Transformation

Benjamin Izar1, Asaf Rotem2

1 Department of Medical Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, 2 Center for Cancer Precision Medicine, Dana‐Farber Cancer Institute, Brigham and Women's Hospital, Boston Children's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.8
DOI:  10.1002/cpmb.26
Online Posting Date:  October, 2016
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For the last five decades, measuring the ability of cells to grow in soft agar has served as the gold standard assay for in vitro cellular transformation. Nevertheless, the soft agar colony formation assay is time consuming and ill‐suited for high‐throughput screens. This unit describes an equally qualitative and quantitative assay known as growth in low attachment or GILA. The GILA assay is suitable for high‐throughput pharmacological or genetic screens and allows the simultaneous examination of multiple cell lines and experimental perturbations. GILA conditions are specific and relevant to the transformed state because they depend on a property of cancer cells that is not shared by non‐transformed cells. The GILA assay enables ex vivo drug sensitivity testing of patient‐derived tumor cells to define precise treatments for individual patients. © 2016 by John Wiley & Sons, Inc.

Keywords: sphere; soft agar; anchorage‐independent growth; transformation; cancer; 3D

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Table of Contents

  • Introduction
  • Basic Protocol 1: Assessment of Cellular Tumorigenic Potential by Growth on Low‐Attachment Surfaces
  • Support Protocol 1: Isolation of Primary Patient‐Derived Tumor Cells from Malignant Effusions for Ex Vivo Culture
  • Alternate Protocol 1: Genetic Perturbation Followed by GILA Assay
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Assessment of Cellular Tumorigenic Potential by Growth on Low‐Attachment Surfaces

  • Cell lines
  • Complete growth medium (e.g., DMEM containing 10% (v/v) fetal bovine serum (FBS) and antibiotics)
  • PBS (Thermo Fisher Scientific, cat. no. MT21040CV)
  • Trypsin (Life Technologies, cat. no. 25200114)
  • CellTiter‐Glo kit (Promega, cat. no. G7573)
  • dATP (Cell Signaling Technology, cat. no. 9804S)
  • Traditional tissue culture dishes (Thermo Fisher Scientific, cat. nos. 08772E and 07202000)
  • 384‐well plates, PrimeSurface ultra low‐attachment (ULA; Sumitomo Bakelite Co., cat no. MS‐9384UZ).
  • 384‐well plates, traditional high‐attachment surface (Corning; cat. no. 3704)
  • Cell counter or hemacytometer
  • 96‐well solid white polystyrene plates (Corning, cat. no. 3362)
  • 96‐well ultra‐low attachment multiwell plates (Sigma Aldrich, cat. no. CLS3474‐24EA)
  • 96‐well cell clear flat bottom TC‐treated culture plate (Corning, cat. no. 353072)
  • Aluminum foil cover and roller
  • Multichannel pipettes
  • Luminometer for plates
  • Additional reagents and equipment for tissue culture and counting cells ( appendix 3F; Phelan, )
NOTE: Throughout this protocol, follow traditional culturing procedures (e.g., detachment, seeding, and recovery time from seeding) relevant to the chosen cells or as defined by the user, if the user is familiar with special procedures that are necessary for successful culture of a specific cell type.NOTE: The cell numbers and reagent volumes have been optimized for 96‐well plates; some adjustment will be necessary if using another format.

Support Protocol 1: Isolation of Primary Patient‐Derived Tumor Cells from Malignant Effusions for Ex Vivo Culture

  Additional Materials (also see protocol 1Basic Protocol)
  • Library of ORFs (GE Life Sciences, cat. no. OHS6085), or other gene perturbation method
  • Flask with a traditional high attachment surface (Corning, cat. no. 430641)
  • Flask with an ultra‐low attachment surface (Corning, cat. no. 3814)
  • Illumina DNA sequencing kits, including primers to sequence barcodes (Illumina)
  • Genomic DNA kit: DNeasy Blood and Tissue kit (Qiagen, cat. no. 69504)
  • Additional reagents and equipment for pooled lentiviral library screening (see units 9.14 & 31.5; Cepko and Pear, 28.8; McDade et al., 28.8)
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Literature Cited

Literature Cited
  Cepko, C. and Pear, W. 2001. Retrovirus infection of cells in vitro and in vivo. Curr. Protoc. Mol. Biol. 36:9.14.1‐9.14.6.
  Folkman, J. and Moscona, A. 1978. Role of cell shape in growth control. Nature 273:345‐349. doi: 10.1038/273345a0.
  Hahn, W.C., Counter, C.M., Lundberg, A.S., Beijersbergen, R.L., Brooks, M.W., and Weinberg, R.A. 1999. Creation of human tumour cells with defined genetic elements. Nature 400:464‐468. doi: 10.1038/22780.
  Howes, A.L., Richardson, R.D., Finlay, D., and Vuori, K. 2014. 3‐Dimensional culture systems for anti‐cancer compound profiling and high‐throughput screening reveal increases in EGFR inhibitor‐mediated cytotoxicity compared to monolayer culture systems. PLoS One 9:e108283. doi: 10.1371/journal.pone.0108283.
  Imamura, Y., Mukohara, T., Shimono, Y., Funakoshi, Y., Chayahara, N., Toyoda, M., Kiyota, N., Takao, S., Kono, S., Nakatsura, T., and Minami, H. 2015. Comparison of 2D‐ and 3D‐culture models as drug‐testing platforms in breast cancer. Oncol. Rep. 33:1837‐1843. doi: 10.3892/or.2015.3767.
  MacPherson, I. and Montagnier, L. 1964. Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23:291‐294. doi: 10.1016/0042‐6822(64)90301‐0.
  McDade, J.R., Waxmonsky, N.C., Swanson, L.E., and Fan, M. 2016. Practical considerations for using pooled lentiviral CRISPR libraries. Curr. Protoc. Mol. Biol. 115:31.5.1‐31.5.13. doi: 10.1002/cpmb.8.
  Ozturk, S.S. 1996. Engineering challenges in high density cell culture systems. Cytotechnology 22:3‐16. doi: 10.1007/BF00353919.
  Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1–A.3F.18.
  Phung, Y.T., Barbone, D., Broaddus, V.C., and Ho, M. 2011. Rapid generation of in vitro multicellular spheroids for the study of monoclonal antibody therapy. J. Cancer 2:507‐514. doi: 10.7150/jca.2.507.
  Rotem, A., Janzer, A., Izar, B., Ji, Z., Doench, J.G., Garraway, L.A., and Struhl, K. 2015. Alternative to the soft‐agar assay that permits high‐throughput drug and genetic screens for cellular transformation. Proc. Natl. Acad. Sci. U.S.A. 112:5708‐5713. doi: 10.1073/pnas.1505979112.
  Tanner, K. and Gottesman, M.M. 2015. Beyond 3D culture models of cancer. Sci. Transl. Med. 7:283ps9. doi: 10.1126/scitranslmed.3009367.
   Tirosh, I., Izar, B., Prakadan, S.M., Wadsworth, M.H., Treacy, D., Trombetta, J.J., Rotem, A., Rodman, C., Lian, C., Murphy, G., Fallahi‐Sichani, M., Dutton‐Regester, K., Lin, J.R., Cohen, O., Shah, P., Lu, D., Genshaft, A.S., Hughes, T.K., Ziegler, C.G., Kazer, S.W., Gaillard, A., Kolb, K.E., Villani, A.C., Johannessen, C.M., Andreev, A.Y., Van Allen, E.M., Bertagnolli, M., Sorger, P.K., Sullivan, R.J., Flaherty, K.T., Frederick, D.T., Jané‐Valbuena, J., Yoon, C.H., Rozenblatt‐Rosen, O., Shalek, A.K., Regev, A., and Garraway, L.A. 2016. Dissecting the multicellular ecosystem of metastatic melanoma by single‐cell RNA‐seq. Science 352:189‐196. doi: 10.1126/science.aad0501.
   Yang, X., Boehm, J.S., Yang, X., Salehi‐Ashtiani, K., Hao, T., Shen, Y., Lubonja, R., Thomas, S.R., Alkan, O., Bhimdi, T., Green, T.M., Johannessen, C.M., Silver, S.J., Nguyen, C., Murray, R.R., Hieronymus, H., Balcha, D., Fan, C., Lin, C., Ghamsari, L., Vidal, M., Hahn, W.C., Hill, D.E., and Root, D.E. 2011. A public genome‐scale lentiviral expression library of human ORFs. Nat. Methods 8:659‐661. doi: 10.1038/nmeth.1638.
  Yasumasa, K., Shohei, W., Masaaki, K., Toru, M., Makoto, N., and Mari, D. 2013. Isolation, culture and evaluation of multilineage‐differentiating stress‐enduring (Muse) cells. Nat. Protoc. 8:1391‐1415. doi: 10.1038/nprot.2013.076.
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