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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Abstract

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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Materials

Basic Protocol 1: Assessment of Cellular Tumorigenic Potential by Growth on Low‐Attachment Surfaces

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
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