The identification of circulating tumour cells in prostate cancer using imaging flow cytometry

Abstract

The emergence of circulating tumour cells (CTCs) readily available in the blood, renewed hope of developing a tool that may serve as a surrogate biomarker for the identification of metastatic disease in cancer patients, in the form of a ‘liquid biopsy’ (i.e. peripheral blood sample instead of cancer tissue). Examination of CTCs has been attempted for a number of years and been limited by several factors including biological heterogeneity and the small number of CTCs present. Despite these limitations, there is increased evidence of the clinical utility of CTCs as diagnostic, prognostic and predictive markers in cancer patients. In this study, I evaluated the possibility of detecting and distinguishing prostate CTCs from blood cells using the ImageStreamX, a novel device that uses multispectral imaging flow cytometry. In order to conduct this evaluation, PC3 prostate cancer cells were used to mimic CTCs in blood. To accurately define the sensitivity of imaging flow cytometry, immunological methods for CTC quantitation using the Epithelial Cell Adhesion Molecule (EpCAM), CD45 and Zonula Occludens Tight Junctions (ZO-1 TJ) antibodies were examined. Additionally, I have utilised the unique features of the ImageStreamX and examined its ability to distinguish CTCs from blood cells based on morphological and/or physical properties. The immunostaining methods appeared to produce false negative identification of PC3 cells. However, when live PC3 cells pre-stained with a nucleic acid stain (DRAQ5) were analysed using solely physical and morphological parameters such as cell size, the results were a far more robust enumeration of the CTC population, thus increasing markedly the detection sensitivity. The live cell analysis approach was then validated using blood samples collected from metastatic castrate-resistant prostate cancer (CRPC) patients with an informed consent and ethical approval. CTC enumeration in these samples using the live cell analysis approach confirmed findings about the higher sensitivity of this procedure over immunological labelling of CTCs in imaging flow cytometry