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Front Oncol. 2016 Jan 25;5:305. doi: 10.3389/fonc.2015.00305. eCollection 2015.

Applications of High-Throughput Clonogenic Survival Assays in High-LET Particle Microbeams.

Author information

1
Department of Oncology, Addenbrooke's Hospital, University of Cambridge , Cambridge , UK.
2
Manchester Academic Health Science Centre, Institute of Cancer Sciences, University of Manchester, The Christie NHS Foundations Trust , Manchester , UK.
3
Centre for Biomedical Modelling and Analysis, University of Exeter , Exeter , UK.
4
Ion Beam Centre, University of Surrey , Guildford , UK.
5
Department of Microbial and Cellular Sciences, University of Surrey , Guildford , UK.

Abstract

Charged particle therapy is increasingly becoming a valuable tool in cancer treatment, mainly due to the favorable interaction of particle radiation with matter. Its application is still limited due, in part, to lack of data regarding the radiosensitivity of certain cell lines to this radiation type, especially to high-linear energy transfer (LET) particles. From the earliest days of radiation biology, the clonogenic survival assay has been used to provide radiation response data. This method produces reliable data but it is not optimized for high-throughput microbeam studies with high-LET radiation where high levels of cell killing lead to a very low probability of maintaining cells' clonogenic potential. A new method, therefore, is proposed in this paper, which could potentially allow these experiments to be conducted in a high-throughput fashion. Cells are seeded in special polypropylene dishes and bright-field illumination provides cell visualization. Digital images are obtained and cell detection is applied based on corner detection, generating individual cell targets as x-y points. These points in the dish are then irradiated individually by a micron field size high-LET microbeam. Post-irradiation, time-lapse imaging follows cells' response. All irradiated cells are tracked by linking trajectories in all time-frames, based on finding their nearest position. Cell divisions are detected based on cell appearance and individual cell temporary corner density. The number of divisions anticipated is low due to the high probability of cell killing from high-LET irradiation. Survival curves are produced based on cell's capacity to divide at least four to five times. The process is repeated for a range of doses of radiation. Validation shows the efficiency of the proposed cell detection and tracking method in finding cell divisions.

KEYWORDS:

bright-field imaging; cell tracking; clonogenic survival assay; high-LET radiation; microbeam

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