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

Jasmina Hodzic, Ilse Dingjan, Mariëlle 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
<span title="">2015</span> <i title="Springer Nature"> <a target="_blank" rel="noopener" href="https://fatcat.wiki/container/2bagfcobavabhds3rtf4xkgv54" style="color: black;">Radiation Oncology</a> </i> &nbsp;
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
more &raquo; ... erprises 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.
<span class="external-identifiers"> <a target="_blank" rel="external noopener noreferrer" href="https://doi.org/10.1186/s13014-015-0355-2">doi:10.1186/s13014-015-0355-2</a> <a target="_blank" rel="external noopener" href="https://www.ncbi.nlm.nih.gov/pubmed/25888875">pmid:25888875</a> <a target="_blank" rel="external noopener" href="https://pubmed.ncbi.nlm.nih.gov/PMC4355372/">pmcid:PMC4355372</a> <a target="_blank" rel="external noopener" href="https://fatcat.wiki/release/qfsek2odube3lo2ocmsiufi3xi">fatcat:qfsek2odube3lo2ocmsiufi3xi</a> </span>
<a target="_blank" rel="noopener" href="https://web.archive.org/web/20170820121850/https://ro-journal.biomedcentral.com/track/pdf/10.1186/s13014-015-0355-2?site=ro-journal.biomedcentral.com" title="fulltext PDF download" data-goatcounter-click="serp-fulltext" data-goatcounter-title="serp-fulltext"> <button class="ui simple right pointing dropdown compact black labeled icon button serp-button"> <i class="icon ia-icon"></i> Web Archive [PDF] <div class="menu fulltext-thumbnail"> <img src="https://blobs.fatcat.wiki/thumbnail/pdf/55/3d/553d0323b48e1996df16fb5108a58fe3e20d0d6b.180px.jpg" alt="fulltext thumbnail" loading="lazy"> </div> </button> </a> <a target="_blank" rel="external noopener noreferrer" href="https://doi.org/10.1186/s13014-015-0355-2"> <button class="ui left aligned compact blue labeled icon button serp-button"> <i class="unlock alternate icon" style="background-color: #fb971f;"></i> springer.com </button> </a> <a target="_blank" rel="external noopener" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355372" title="pubmed link"> <button class="ui compact blue labeled icon button serp-button"> <i class="file alternate outline icon"></i> pubmed.gov </button> </a>