Background: An increasing array of treatments are available for prostate cancer (PCa), with only subsets of patients responding effectively to treatment. Accordingly, the critical challenges now lie in optimally pairing the patient with the right treatment and detecting the onset of resistance in a cancer which is continually changing. Liquid biopsy, specifically the minimally invasive extraction of circulating tumor cells (CTCs) from blood, allows for longitudinal tracking of the cancers continually changing molecular state. Currently CTCs are immunofluorescently labelled with a maximum of ~6 markers, which is insufficient to track the many pathways that can lead to resistance.
Aim: To develop imaging methodologies to characterize the molecular profile of CTCs, to identify critical markers of resistance and provide actionable insight for treatment.
Materials & Methods: Here, we implement cyclic immunofluorescence on CTCs. Liquid biopsies were taken from PCa patients and processed to enrich the population of CTCs, which were then stained via cyclic immunofluorescence, a process of repeatedly blocking, staining with fluorescently conjugated primary antibodies, confocal microscopy and bleaching.
To validate this method for longitudinal tracking of resistance, PCa cell line LNCaP was compared with its castrate-resistant derivative V16D and its enzalutamide-resistant derivative MR49F. This protocol was then implemented on samples from patients with advanced PCa.
Results: Pairing unbiased negative selection of CTCs with multiplexed imaging of >50 markers produced 10 000 features per cell which enabled extensive single-cell characterisation by a variety of statistical and machine learning methods, which identified the signalling pathways and cell states associated with multiple stages of PCa therapy resistance. Using the cell line model, cells were able to be classified as ‘castrate sensitive’, ‘castrate resistant’ and ‘enzalutamide resistant’ with 98% accuracy. Markers were ranked by importance in conferring resistance. In patient samples, cyclic immunofluorescence improved CTC identification, with known markers of resistance identified.
Conclusions: This methodology massively improves the diagnostic and prognostic potential of CTCs in precision medicine, by increasing the number of detectable markers from 6 to >50, the utility of CTCs has been transformed from enumeration to potentially adaptively guiding therapy by identifying resistance mechanisms and potential responsiveness to treatments.