title: A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting.
abstract: BACKGROUND
  Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening.
  
  
  METHODS
  We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells.
  
  
  RESULTS
  On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated.
  
  
  CONCLUSIONS
  We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid the identification of molecular targets for radiosensitization, thereby contributing to improving the efficacy of radiotherapy.
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. j.hodzic@vumc.nl.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Hodzic
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. ilsedingjan@gmail.com.
authors.forename: Ilse
authors.initials: I
authors.lastname: Dingjan
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. marielle_maas@hotmail.com.
authors.forename: Mariëlle Jp
authors.initials: MJ
authors.lastname: Maas
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. ih.vandermeulen@vumc.nl.
authors.forename: Ida H
authors.initials: IH
authors.lastname: van der Meulen-Muileman
authors.affiliation: Department of Epidemiology and Biostatistics, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. r.xavierdemenezes@vumc.nl.
authors.forename: Renee X
authors.initials: RX
authors.lastname: de Menezes
authors.affiliation: Department of Radiology & Nuclear Medicine, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. s.heukelom@vumc.nl.
authors.forename: Stan
authors.initials: S
authors.lastname: Heukelom
authors.affiliation: Department of Radiotherapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. m.verheij@nki.nl.
authors.forename: Marcel
authors.initials: M
authors.lastname: Verheij
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. Winald.Gerritsen@radboudumc.nl.|Present address: Department of Medical Oncology, The Radboud University Medical Center, Comeniuslaan 4, 6525 HP, Nijmegen, The Netherlands. Winald.Gerritsen@radboudumc.nl.
authors.forename: Winald R
authors.initials: WR
authors.lastname: Gerritsen
authors.affiliation: Department of Radiology & Nuclear Medicine, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. aa.geldof@vumc.nl.|Department of Urology, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. aa.geldof@vumc.nl.
authors.forename: Albert A
authors.initials: AA
authors.lastname: Geldof
authors.affiliation: Department of Radiotherapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands. b.v.triest@nki.nl.
authors.forename: Baukelien
authors.initials: B
authors.lastname: van Triest
authors.affiliation: Department of Medical Oncology, VU University Medical Center, De Boelelaan 1118, 1081HV, Amsterdam, The Netherlands. VW.vanBeusechem@vumc.nl.
authors.forename: Victor W
authors.initials: VW
authors.lastname: van Beusechem
mesh_terms: D001331:Automation; D002110:Caffeine / Q000494:pharmacology; D002470:Cell Survival / Q000528:radiation effects*; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D003114:Colony-Forming Units Assay; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics; D015894:Genome, Human; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology / Q000532:radiotherapy*; D034741:RNA, Small Interfering / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics*; D011839:Radiation, Ionizing; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D000697:Central Nervous System Stimulants; D034741:RNA, Small Interfering; D011838:Radiation-Sensitizing Agents; D002110:Caffeine; D051747:DNA-Activated Protein Kinase