Deep Phenotypic Characterisation of CTCs by Combination of Microfluidic Isolation (IsoFlux) and Imaging Flow Cytometry (ImageStream)

Abstract

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.

Multiparametric Phenotyping of Circulating Tumor Cells for Analysis of Therapeutic Targets, Oncogenic Signaling Pathways and DNA Repair Markers

“…In the current study, we could extend this limited evidence and show that sensitive and specific CTC detection is feasible by using this platform. Spiking experiments with our ISX protocol showed a median recovery rate of 73%, which was similar to the ISX–based CTC study of Ruiz-Rodríguez et al [ 12 ], and even slightly better compared to other ISX–based studies showing recovery rates from 44% to 55%, respectively [ 13 , 14 ]. We did not compare the performance of the ISX with the CS system on corresponding patient samples.…”

“…To date, only a few studies have assessed the suitability of the ISX system for CTC detection [ 12 , 13 , 14 , 15 , 16 ]. In the current study, we could extend this limited evidence and show that sensitive and specific CTC detection is feasible by using this platform.…”

Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer

“…While membrane-based methods such as MetaCell have several advantages, phenotypic characterisation of CTCs enriched by membrane-based methods are usually performed via immunocytochemistry and fluorescence microscopy with only one or two additional markers [ 45 ]. This limited our use of markers and prompted us to choose the most popular markers used for CTC characterisation, i.e., EpCAM and cytokeratins [ 46 , 47 ].…”

Detection and Characterization of Circulating Tumor Cells Using Imaging Flow Cytometry—A Perspective Study

“…Indeed, imFC enables fast acquisition of images of all the cells in an investigated sample (high-scale analysis), their selection is based on multiple features and gating strategies, as well as subsequent manual verification similar to the one applied during CellSearch ® analysis [ 41 ]. Notably, sensitivity and specificity of CTC detection using imFC are in principle similar to FC [ 35 , 39 , 42 , 43 , 44 , 45 ]. However, the keystone of this technology in this context is its potential for the experimenter to verify the cells, which significantly improves the final outcome, typical for FC.…”