Proteomic screening of 725 antibodies simultaneously using antibody microarray technology to identify proteins associated with radiotherapy resistance in breast cancer cells

Background

Resistance to radiotherapy may be a significant factor in the development of local recurrence following surgical resection and radiotherapy. In addition, if patients with radioresistant breast cancers can be identified, harmful side effects from exposure to unnecessary ionizing radiation could be prevented. We aimed to develop a novel in vitro model of radio-resistance using a breast cancer cell line and to subsequently identify molecular biomarkers that may be associated with the radioresistant phenotype. Antibody microarrays offer a complementary approach for proteomic analysis in conjunction with standard screening methods such as two-dimensional gel electrophoresis/mass spectrometry. We have previously utilised the Panorama Cell Signalling Antibody Microarray Kit (Sigma-Aldrich, Poole, UK) consisting of 224 antibodies [1]. In the present study we assessed a novel high-density 725-antibody microarray to screen for proteins associated with radioresistance.

Methods

We established a novel breast cancer cell subline that was significantly resistant to radiotherapy when compared with the parental cells (T47D). The radioresistant subline was created by irradiating cells in fractionated doses of 2 Gy up to a total dose of 40 Gy. Sufficient time was allowed for the cells to recover between subsequent irradiations. A dose–response curve was assessed at the end of treatment to demonstrate a statistically significant increase in radioresistance for the novel cell subline when compared with parental cells. The radioresistant/parental cell pair was analysed using the Panorama Antibody Microarray XPRESS Profiler725 Kit (Sigma-Aldrich). The microarray comprised 725 different antibodies on nitrocellulose-coated microscope slides. The antibodies were selected from a wide variety of pathways, including apoptotic and cell signalling pathways.

Results

Utilising a Cy3/Cy5 labelling strategy, the antibody microarray approach yielded a number of possible targets for further study. These include zyxin, growth factor independence 1 and lysine-specific demethylase 1, which were differentially expressed between the radioresistant subline and parental cells. Immunoblotting has confirmed the identities and differential expression of some candidate protein targets.

Conclusion

The use of a novel high-density antibody microarray has successfully identified a number of protein targets that may be associated with a radioresistant phenotype. These proteins require further study to validate the results. High-density antibody microarrays potentially offer a powerful new proteomic technique to allow the global analysis of many proteins simultaneously. These could be invaluable in the identification of candidate biomarkers that may be involved in radioresistance and may reveal novel therapeutic targets in breast cancer.