Abstract
Simple SummaryCells that escape the primary tumour and have the potential ability to colonise distant organs through metastasis are called circulating tumour cells (CTCs). The study of CTCs in colorectal cancer (CRC) has demonstrated their prognostic utility, although current methodologies only allow the evaluation of CTC numbers and a maximum of two markers. Here, we developed a novel protocol for the isolation and characterisation of CTCs by combining two existing technologies. This new methodology allows the simultaneous evaluation of multiple markers and parameters. In particular, we evaluated the expression of a mutant protein (BRAFV600E) associated with poor response to therapies against EGFR and the expression of PD-L1, a marker for immunotherapy. Based on these markers, we evaluated the CTCs (positive for cytokeratin) of 16 early CRC patients and demonstrated the suitability of our protocol to classify patients into potential responders and non-responders.The isolation of circulating tumour cells (CTCs) in colorectal cancer (CRC) mostly relies on the expression of epithelial markers such as EpCAM, and phenotypic characterisation is usually performed under fluorescence microscopy with only one or two additional markers. This limits the ability to detect different CTC subpopulations based on multiple markers. The aim of this work was to develop a novel protocol combining two platforms (IsoFluxTM and ImageStream®X) to improve CTC evaluation. Cancer cell lines and peripheral blood from healthy donors were used to evaluate the efficiency of each platform independently and in combination. Peripheral blood was extracted from 16 early CRC patients (before loco-regional surgery) to demonstrate the suitability of the protocol for CTC assessment. Additionally, peripheral blood was extracted from nine patients one month after surgery to validate the utility of our protocol for identifying CTC subpopulation changes over time. Results: Our protocol had a mean recovery efficiency of 69.5% and a limit of detection of at least four cells per millilitre. We developed an analysis method to reduce noise from magnetic beads used for CTC isolation. CTCs were isolated from CRC patients with a median of 37 CTCs (IQ 13.0–85.5) at baseline. CTCs from CRC patients were significantly (p < 0.0001) larger than cytokeratin (CK)-negative cells, and patients were stratified into two groups based on BRAFV600E and PD-L1 expression on CK-positive cells. The changes observed over time included not only the number of CTCs but also their distribution into four different subpopulations defined according to BRAFV600E and PD-L1 positivity. We developed a novel protocol for semi-automatic CTC isolation and phenotypic characterisation by combining two platforms. Assessment of CTCs from early CRC patients using our protocol allowed the identification of two clusters of patients with changing phenotypes over time.
Highlights
Circulating tumour cells (CTCs) are one of the most widely studied types of liquid biopsy and have been used as prognostic and monitoring biomarkers [1] in most solid tumours, such as colorectal [2], lung [3] and breast [4] cancers
circulating tumour cells (CTCs) isolated from oesophageal, thyroid and ovarian cancer patients were characterised using the ISx [19], and the results demonstrated the potential of this technology for multi-parametric phenotypic characterisation
We identified that BRAFV600E expression was highly correlated with CK expression only in the BRAF+/PD-L1− population (p = 0.035), whereas PD-L1 expression was independent of either CK or BRAFV600E in all four populations
Summary
Circulating tumour cells (CTCs) are one of the most widely studied types of liquid biopsy and have been used as prognostic and monitoring biomarkers [1] in most solid tumours, such as colorectal [2], lung [3] and breast [4] cancers. Evaluation of CTCs requires two essential steps: enrichment (or isolation) and enumeration (with or without characterisation). The IsoFluxTM system (Fluxion Biosciences, Inc., Alameda, CA, USA) is a semiautomatic platform for the enrichment of CTCs using a combination of immunomagnetic positive selection and microfluidics, and it significantly increases isolation efficiencies compared to the FDA-approved CellSearch® (Menarini, Italy) system [5]. An improvement in the recovery rate from 40% to 90% using cancer cell lines was achieved compared with CellSearch® [6], and this technology was used to successfully isolate CTCs in bladder [7], prostate [8], ovarian [9], liver [10,11,12,13] and pancreatic [14] cancer patients
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