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

Area of Science:

• Oncology
• Radiotherapy
• Cancer Biology

Background:

• Radiotherapy is a cornerstone cancer treatment, but radioresistance limits its effectiveness.
• Current methods for identifying radiosensitizing agents are not suitable for high-throughput screening.
• New molecular targets and drugs are needed to enhance cancer cell sensitivity to radiation.

Purpose of the Study:

• To develop a novel high-throughput screening assay for assessing radiation susceptibility in cancer cells.
• To validate the assay's performance against established methods like the colony formation assay (CFA).
• To demonstrate the assay's utility in identifying radiosensitizing targets.

Main Methods:

• A new method for uniform, high-throughput irradiation of microplates using a linear accelerator.
• Automated counting of fluorescently stained nuclei for readout.
• Comparison with CFA and cell viability assays, and validation using a whole-genome siRNA library in PC-3 prostate cancer cells.

Main Results:

• The automated assay generated radiation dose-response curves correlating well with CFA results.
• The assay successfully detected radiosensitization effects from DNA-PKcs silencing and caffeine treatment.
• Clear discrimination between DNA-PKcs siRNA and control siRNA effects was achieved in a high-throughput setting.

Conclusions:

• A simple, high-throughput assay for radiation susceptibility has been developed.
• This assay facilitates the identification of molecular targets for radiosensitization.
• The findings contribute to improving radiotherapy efficacy by overcoming cancer resistance.