Technical validation of a new microfluidic device for enrichment of CTCs from large volumes of blood by using buffy coats to mimic diagnostic leukapheresis products.

Diagnostic leukapheresis (DLA) enables to sample larger blood volumes and increases the detection of circulating tumor cells (CTC) significantly. Nevertheless, the high excess of white blood cells (WBC) of DLA products remains a major challenge for further downstream CTC enrichment and detection. To address this problem, we tested the performance of two label-free CTC technologies for processing DLA products. For the testing purposes, we established ficollized buffy coats (BC) with a WBC composition similar to patient-derived DLA products. The mimicking-DLA samples (with up to 400 × 106 WBCs) were spiked with three different tumor cell lines and processed with two versions of a spiral microfluidic chip for label-free CTC enrichment: the commercially available ClearCell FR1 biochip and a customized DLA biochip based on a similar enrichment principle, but designed for higher throughput of cells. While the samples processed with FR1 chip displayed with increasing cell load significantly higher WBC backgrounds and decreasing cell recovery, the recovery rates of the customized DLA chip were stable, even if challenged with up to 400 × 106 WBCs (corresponding to around 120 mL peripheral blood or 10% of a DLA product). These results indicate that the further up-scalable DLA biochip has potential to process complete DLA products from 2.5 L of peripheral blood in an affordable way to enable high-volume CTC-based liquid biopsies.

Tumor cell capture from blood by flowing across antibody-coated surfaces.

The load of circulating tumor cells (CTC) is related to poor outcomes in cancer patients. A sufficient number of these cells would enable a full characterization of the cancer. An approach to probe larger blood volumes, allowing for the detection of more of these very rare CTC, is the use of leukapheresis. Currently available techniques allow only the analysis of a small portion of leukapheresis products. Here, we present a method that uses flow rather than static conditions which allows processing of larger volumes. We evaluated the conditions needed to isolate tumor cells from blood while passing antibody coated surfaces. Results show that our set-up efficiently captures cancer cells from whole blood. Results show that the optimal velocity at which cells are captured from blood is 0.6 mm s-1. Also, it can be concluded that the VU1D9 antibody targeting the EpCAM antigen has very high capture efficiency. When using an antibody that does not capture 100% of all cells, combining multiple antibodies on the capture surface is very beneficial leading to an increase in cell capture and is therefore worthwhile considering in any cancer cell capture methodology.

Improving the CellSearch® system.

INTRODUCTION: The CellSearch® CTC test enumerates tumor cells present in 7.5 ml blood of cancer patients. improvements, extensions and different utilities of the cellsearch system are discussed in this paper. Areas covered: This paper describes work performed with the CellSearch system, which go beyond the normal scope of the test. All results from searches with the search term 'CellSearch' from Web of Science and PubMed were categorized and discussed. Expert commentary: The CellSearch Circulating Tumor Cell test captures and identifies tumor cells in blood that are associated with poor clinical outcome. How to best use CTC in clinical practice is being explored in many clinical trials. The ability to extract information from the CTC to guide therapy will expand the potential clinical utility of CTC.

Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents.

BACKGROUND: Abiraterone and enzalutamide are novel endocrine treatments that abrogate androgen receptor (AR) signalling in castration-resistant prostate cancer (CRPC). Here, we developed a circulating tumour cells (CTCs)-based assay to evaluate AR expression in real-time in CRPC and investigated nuclear AR expression in CTCs in patients treated with enzalutamide and abiraterone. METHODS: CTCs were captured and characterised using the CellSearch system. An automated algorithm to identify CTCs and quantify AR expression was employed. The primary aim was to evaluate the association between CTC AR expression and prior treatment with abiraterone or enzalutamide. RESULTS: AR expression in CTCs was evaluated in 94 samples from 48 metastatic CRPC patients. We observed large intra-patient heterogeneity of AR expression in CTCs. Prior exposure to abiraterone or enzalutamide was not associated with a change in CTCs AR expression (median intensity and distribution of AR-positive classes). In support of this, we also confirmed maintained nuclear AR expression in tissue samples collected after progression on abiraterone. AR staining also identified additional AR-positive CD45-negative circulating cells that were CK-negative/weak and therefore missed using standard protocols. The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis. CONCLUSIONS: We developed a non-invasive method to monitor AR nuclear expression in CTCs. Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments. Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.