Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress 2023 EHA Congress 2023 ASCO Annual Meeting
Clinical Collections
Advances in Alzheimer’s

At a glance: The ONWARDS insulin icodec trials

A round-up of the ONWARDS phase 3 clinical trials evaluating the novel weekly insulin icodec in people with diabetes.
ADVANCED SEARCH
Log in
Top
Radiation Oncology
Published in: Radiation Oncology 1/2015

Open Access 01-12-2015 | Methodology

A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

Authors: Jasmina Hodzic, Ilse Dingjan, Mariëlle JP Maas, Ida H van der Meulen-Muileman, Renee X de Menezes, Stan Heukelom, Marcel Verheij, Winald R Gerritsen, Albert A Geldof, Baukelien van Triest, Victor W van Beusechem

Published in: Radiation Oncology | Issue 1/2015

Login to get access

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.
Literature
1.
Auperin A, Le Pechoux C, Rolland E, Curran WJ, Furuse K, Fournel P, et al. Meta-analysis of concomitant versus sequential radiochemotherapy in locally advanced non-small-cell lung cancer. J Clin Oncol. 2010;28:2181–90.CrossRefPubMed
2.
Begg AC, Stewart FA, Vens C. Strategies to improve radiotherapy with targeted drugs. Nat Rev Cancer. 2011;11:239–53.CrossRefPubMed
3.
Green JA, Kirwan JM, Tierney JF, Symonds P, Fresco L, Collingwood M, et al. Survival and recurrence after concomitant chemotherapy and radiotherapy for cancer of the uterine cervix: a systematic review and meta-analysis. Lancet. 2001;358:781–6.CrossRefPubMed
4.
Sauer R, Liersch T, Merkel S, Fietkau R, Hohenberger W, Hess C, et al. Preoperative versus postoperative chemoradiotherapy for locally advanced rectal cancer: results of the German CAO/ARO/AIO-94 randomized phase III trial after a median follow-up of 11 years. J Clin Oncol. 2012;30:1926–33.CrossRefPubMed
5.
Hoppe R, Phillips T, Roach III M. Textbook of radiation oncology. 3rd ed. Philadelphia: Elsevier Health Sciences; 2010.
6.
Bentzen SM, Overgaard J. Clinical normal-tissue radiobiology. In: Tobias JS, Thomas PRM, editors. Current radiation oncology. London: Arnold; 1996. p. 37–67.
7.
Taiakina D, Pra AD, Bristow RG. Intratumoral hypoxia as the genesis of genetic instability and clinical prognosis in prostate cancer. Adv Exp Med Biol. 2014;772:189–204.CrossRefPubMed
8.
Lobrich M, Jeggo PA. The impact of a negligent G2/M checkpoint on genomic instability and cancer induction. Nat Rev Cancer. 2007;7:861–9.CrossRefPubMed
9.
Nordsmark M, Bentzen SM, Rudat V, Brizel D, Lartigau E, Stadler P, et al. Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol. 2005;77:18–24.CrossRefPubMed
10.
Kastan MB. DNA damage responses: mechanisms and roles in human disease: 2007 G.H.A. Clowes Memorial Award Lecture. Mol Cancer Res. 2008;6:517–24.CrossRefPubMed
11.
Verheij M, Vens C, van Triest B. Novel therapeutics in combination with radiotherapy to improve cancer treatment: rationale, mechanisms of action and clinical perspective. Drug Resist Updat. 2010;13:29–43.CrossRefPubMed
12.
Katz D, Ito E, Liu FF. On the path to seeking novel radiosensitizers. Int J Radiat Oncol Biol Phys. 2009;73:988–96.CrossRefPubMed
13.
Ghotra VP, Geldof AA, Danen EH. Targeted radiosensitization in prostate cancer. Curr Pharm Des. 2013;19:2819–28.CrossRefPubMed
14.
Albert JM, Cao C, Kim KW, Willey CD, Geng L, Xiao D, et al. Inhibition of poly(ADP-ribose) polymerase enhances cell death and improves tumor growth delay in irradiated lung cancer models. Clin Cancer Res. 2007;13:3033–42.CrossRefPubMed
15.
Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, Karnitz LM, et al. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res. 1999;59(17):4375–82.PubMed
16.
Moding EJ, Kastan MB, Kirsch DG. Strategies for optimizing the response of cancer and normal tissues to radiation. Nat Rev Drug Discov. 2013;12:526–42.CrossRefPubMedCentralPubMed
17.
Overgaard J. Hypoxic modification of radiotherapy in squamous cell carcinoma of the head and neck–a systematic review and meta-analysis. Radiother Oncol. 2011;100:22–32.CrossRefPubMed
18.
Bartelink H, Roelofsen F, Eschwege F, Rougier P, Bosset JF, Gonzalez DG, et al. Concomitant radiotherapy and chemotherapy is superior to radiotherapy alone in the treatment of locally advanced anal cancer: results of a phase III randomized trial of the European Organization for Research and Treatment of Cancer Radiotherapy and Gastrointestinal Cooperative Groups. J Clin Oncol. 1997;15:2040–9.PubMed
19.
Bartelink H, Schellens JH, Verheij M. The combined use of radiotherapy and chemotherapy in the treatment of solid tumours. Eur J Cancer. 2002;38:216–22.CrossRefPubMed
20.
Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96.CrossRefPubMed
21.
van Hagen P, Hulshof MC, van Lanschot JJ, Steyerberg EW, van Berge Henegouwen MI, Wijnhoven BP, et al. Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med. 2012;366:2074–84.CrossRefPubMed
22.
Blockhuys S, Vanhoecke B, Smet J, De PB, Van CR, Bracke M, et al. Unraveling the mechanisms behind the enhanced MTT conversion by irradiated breast cancer cells. Radiat Res. 2013;179:433–43.CrossRefPubMed
23.
Blockhuys S, Vanhoecke B, Paelinck L, Bracke M, De Wagter C. Development of in vitro models for investigating spatially fractionated irradiation: physics and biological results. Phys Med Biol. 2009;54:1565–78.CrossRefPubMed
24.
Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for Use in evaluation and validation of high throughput screening assays. J Biomol Screen. 1999;4:67–73.CrossRefPubMed
25.
Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ, Shanks E, et al. Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods. 2009;6:569–75.CrossRefPubMedCentralPubMed
26.
Cahill DP, da Costa LT, Carson-Walter EB, Kinzler KW, Vogelstein B, Lengauer C. Characterization of MAD2B and other mitotic spindle checkpoint genes. Genomics. 1999;58:181–7.CrossRefPubMed
27.
Cheung HW, Chun AC, Wang Q, Deng W, Hu L, Guan XY, et al. Inactivation of human MAD2B in nasopharyngeal carcinoma cells leads to chemosensitization to DNA-damaging agents. Cancer Res. 2006;66:4357–67.CrossRefPubMed
28.
Ni XH, Zhang YG, Ribas J, Chowdhury WH, Castanares M, Zhang ZW, et al. Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts. J Clin Invest. 2011;121:2383–90.CrossRefPubMedCentralPubMed
Metadata
Title
A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting
Authors
Jasmina Hodzic
Ilse Dingjan
Mariëlle JP Maas
Ida H van der Meulen-Muileman
Renee X de Menezes
Stan Heukelom
Marcel Verheij
Winald R Gerritsen
Albert A Geldof
Baukelien van Triest
Victor W van Beusechem
Publication date
01-12-2015
Publisher
BioMed Central
Published in
Radiation Oncology / Issue 1/2015
Electronic ISSN: 1748-717X
DOI
https://doi.org/10.1186/s13014-015-0355-2

Other articles of this Issue 1/2015

Radiation Oncology 1/2015 Go to the issue

Research

Dexamethasone administration during definitive radiation and temozolomide renders a poor prognosis in a retrospective analysis of newly diagnosed glioblastoma patients

Research

Positioning accuracy during VMAT of gynecologic malignancies and the resulting dosimetric impact by a 6-degree-of-freedom couch in combination with daily kilovoltage cone beam computed tomography

Research

Intracranial control after Cyberknife radiosurgery to the resection bed for large brain metastases

Research

Can dosimetric parameters predict acute hematologic toxicity in rectal cancer patients treated with intensity-modulated pelvic radiotherapy?

Research

Fast 3D dosimetric verifications based on an electronic portal imaging device using a GPU calculation engine

Research

Double-arc volumetric modulated therapy improves dose distribution compared to static gantry IMRT and 3D conformal radiotherapy for adjuvant therapy of gastric cancer