High Throughput Screening Assay Development and Performance of the Oris™ Pro 384 Cell Migration Assay Jennifer A. Fronczak*, Laura Vollmer§, Gopal Krishnan*, and Andreas Vogt§ §University of Pittsburgh Drug Discovery Institute, 10047 BST‐3, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA *Platypus Technologies, LLC, 5520 Nobel Drive, Suite 100, Madison, WI 53711, USA Abstract Establishment of Minimum and Maximum Migration Controls The discovery of anti‐metastatic agents that inhibit cancer cell motility has been hindered by a dearth of high‐throughput screening (HTS) ‐compatible cell motility assays. The Oris™ Pro 384 well cell migration assay, developed by Platypus Technologies, is an innovative cell motility assay designed to enable HTS for the discovery of potential anti‐cancer agents on adherent tumor and endothelial cell lines. The assay utilizes a centrally located, non‐toxic, biocompatible gel (BCG) to form a uniformly sized, cell‐free detection zone into which cells migrate. The assay is logistically simple and does not require any mechanical processing steps, such as cell wounding or removal of physical barriers. The assay is fully compatible with laboratory automation, including robotic liquid handlers, plate washers, and high‐content screening (HCS) readers. A A formal investigation of the accuracy, robustness, and HTS performance of the assay was conducted following guidelines in place at the University of Pittsburgh Drug Discovery Institute (UPDDI). The assay was optimized for migration kinetics, cell seeding density, and DMSO tolerance. In multi‐day variability studies, the assay delivered signal‐to‐background ratios > 10 and Z‐ factors above 0.5 on three consecutive days, was insensitive to process errors or edge‐effects, and passed UPDDI Assay Protocol Approval Committee (APAC) criteria. The assay delivered equal HTS performance on two different HCS platforms – the Thermo Scientific Cellomics ArrayScan II and the Molecular Devices ImageXpress®Ultra. Results from this investigation validate the Oris™ Pro 384 cell migration assay as a logistically l i ti ll simple i l and d information‐rich i f ti i h cellll motility tilit analysis l i platform l tf th t that meets the demands of large scale compound screening. Multi‐day Variability Studies A B C B Assay Schematic A B Figure 5: Multi‐day variability and reproducibility studies. Using two Oris™ Pro 384 plates of maximum and minimum controls on three separate days, HTS performance parameters were calculated. The assay delivered signal‐to‐background ratios > 10, Z‐factors above 0.5, and low intra‐ and inter‐plate variability on all 3 days (data not shown). A. Scatter plots document that the assay performed well with full automation, with few process errors and tight distribution of positive and negative control samples. B. Dose‐response reproducibility and activity profiling of compound activity. The control inhibitor, Cytochalasin D, gave essentially identical IC50 values of 65 nM, 90 nM, and 80 nM on three consecutive days. C. Multiparametric profiling of motility and toxicity, derived from single channel measurements of nuclear morphology, revealed that Cytochalasin D is a selective inhibitor of cell migration that slightly enlarges nuclei but does not cause apoptotic cell death or excessive cell loss. D C Compatibility With Multiple Instruments Figure 1: Schematic of Oris™ Pro 384 Cell Migration Assay. A. Cells are seeded and allowed to adhere in an annular monolayer surrounding the Biocompatible Gel (BCG). B. BCG dissolves to reveal a cell‐free Detection Zone. C. Cells migrate into the Detection Zone. D. Cells are imaged via microscopy or High Content Imagers. Images are then analyzed for cell migration as well as phenotypical changes. HTS Workflow Figure 3: Establishment of minimum and maximum migration controls. MDA‐MB‐231 cells were plated at different densities in duplicate Oris™ Pro 384 plates. After 2h, cell culture medium was removed and cells were treated with vehicle (0.5% DMSO) or 1μM Cytochalasin D. The “Pre‐migration” plate was immediately fixed and stained with Hoechst 33342, while cells on the other plate (“2‐day migration”) were allowed to migrate for 48h at 37°C. Using a 5X objective on the ArrayScan II, an image was acquired in the center of each well, and nuclei enumerated by the Thermo Scientific Cellomics Target Activation Bioapplication. A. Images illustrating migratory phenotype. (Top row) Premigration plate with cell free detection zone. (Middle row) 2‐day migration plates showing cell migration into detection zone, with optimal gap closure observed at 15,000 cells/well. (Bottom row) Cytochalasin D fully inhibited cell migration. B. Quantification of cell migration. HTS assay performance parameters were calculated based on number of cells in the exclusion zone comparing 2‐day migration and Pre‐ migration or Cytochalasin D‐treated D treated wells as maximum and minimum controls, controls respectively. respectively Reproducibility of the assay was determined by computing Coefficient of Variance (CV) and Optimum assay parameters ‐(i) Signal‐to‐ Background (S/B) ratios and (ii) Z‐factors assay performance, when cells were seeded at a density of 15,000 cells/ well, based on 48 wells per condition. ArrayScan ImageXpress® Ultra Day 1 CV min (%) 32.7 44.1 Day 2 CV max (%) S/B Z‐factor CV min (%) 6.3 19.1 0.74 35.1 8.6 26.7 0.68 44.6 Day 3 y CV max (%) S/B Z‐factor CV min (%) 6.5 14.1 0.71 44.4 7.7 31.7 0.68 58.6 ALL DAYS CV max (%) S/B Z‐factor CV min (%) 10.4 17.2 0.57 37.4 12.9 25.0 0.53 49.1 CV max (%) Optimization of DMSO Tolerance Cell seeding, media removal, washing Titertek MAP‐C2 Compound retrieval Matrical MiniStore A B 7.7 9.7 16.8 0.67 27.8 0.63 Figure 6: The Oris™ Pro 384 Cell Migration Assay is compatible with multiple HCS readers. The identical microplates from the three‐day variability studies were rescanned on a Molecular Devices ImageXpress® Ultra confocal point scanning HCS reader. A custom journal was generated that detected migrated cells in a circular region of interest. The assay delivered essentially equivalent results on both HCS platforms. Compound treatment Perkin Elmer EP3 Incubation S/B Z‐factor Conclusions Fixation & Staining Titertex MAP‐C2 C • Results from a formal HTS development and implementation study indicate that the Oris™ Pro 384 Cell Migration Assay is a robust and convenient 2D cell migration assay, that is compatible with robotic liquid handlers and HCS equipment for multi‐parametric analysis • In stringent multi‐day variability assessment studies, the assay delivered Z‐factors > 0.5, signal‐to‐background ratios > 10, and low intra‐plate and inter‐plate variability, thus documenting HTS readiness • The Oris™ Pro 384 Cell Migration Assay passed the University of Pittsburgh Drug Discovery Institute Assay Protocol Approval Committee criteria and is currently used for chemical library screening Imaging & Analysis Cellomics ArrayScan II Multiplexing Figure 2: HTS workflow for laboratory automation. The Oris™ Pro 384 Cell Migration Assay is compatible with automated liquid handling equipment and meets HTS performance parameters. The HTS workflow was developed and setup using laboratory automation equipment as shown: Titertek MAP‐C2 bulk liquid handlers, Perkin Elmer EP3 workstation for compound treatment, pipetting robots, TC incubator and Cellomics ArrayScan II automated microscopy. Figure 4: Optimization of DMSO Tolerance. Cells were treated with a ten‐point, two‐fold dilution gradient of dimethyl sulfoxide (DMSO), and fixed and stained with Hoechst 33342 either immediately after treatment or after 2 days in culture. A. Concentrations higher than 0.3% DMSO had anti‐migratory activity, causing a drop in assay performance. B. Inhibition of cell migration by DMSO did not appear to be a result of DMSO toxicity. C. Quantification of assay performance parameters indicated that assay performance was stable up to 0.6% DMSO and fell sharply thereafter. Results are based on 16 wells per condition. Based on these results, a concentration of 0.1% DMSO was chosen for further experiments. Acknowledgements This work was partially supported by IMAT grant # 5R21CA147985‐02 from the National Cancer Institute.