Accurate isolation and detection of circulating tumor cells using enrichment-free multiparametric high resolution imaging

Abstract

IntroductionThe reliable and accurate detection of rare circulating tumor cells (CTCs) from cancer patient blood samples promises advantages in both research and clinical applications. Numerous CTC detection methods have been explored that rely on either the physical properties of CTCs such as density, size, charge, and/or their antigen expression profiles. Multiple factors can influence CTC recovery including blood processing method and time to processing. This study aimed to examine the accuracy and sensitivity of an enrichment-free method of isolating leukocytes (AccuCyte® system) followed by immunofluorescence staining and high-resolution imaging (CyteFinder® instrument) to detect CTCs.MethodHealthy human blood samples, spiked with cancer cells from cancer cell lines, as well as blood samples obtained from 4 subjects diagnosed with cancer (2 pancreatic, 1 thyroid, and 1 small cell lung) were processed using the AccuCyte-CyteFinder system to assess recovery rate, accuracy, and reliability over a range of processing times.ResultsThe AccuCyte-CyteFinder system was highly accurate (95.0%) at identifying cancer cells in spiked-in samples (in 7.5 mL of blood), even at low spiked-in numbers of 5 cells with high sensitivity (90%). The AccuCyte-CyteFinder recovery rate (90.9%) was significantly higher compared to recovery rates obtained by density gradient centrifugation (20.0%) and red blood cell lysis (52.0%). Reliable and comparable recovery was observed in spiked-in samples and in clinical blood samples processed up to 72 hours post-collection. Reviewer analysis of images from spiked-in and clinical samples resulted in high concordance (R-squared value of 0.998 and 0.984 respectively).DiscussionThe AccuCyte-CyteFinder system is as an accurate, sensitive, and clinically practical method to detect and enumerate cancer cells. This system addresses some of the practical logistical challenges in incorporating CTCs as part of routine clinical care. This could facilitate the clinical use of CTCs in guiding precision, personalized medicine.

Circulating Tumor Cells as Predictive and Prognostic Biomarkers in Solid Tumors

“…Unlike regular blood cells, CTCs are characterized by the presence of epithelial markers, such as epithelial cell adhesion molecules (EpCAM) and cytokeratin (CK) [38,39]. Specific techniques that leverage this principle include the conjugation of antibodies to magnetic nanoparticles and the use of microfluidics [40,41]. While there is some variability in the phenotypic and functional definitions of CTCs across different studies, it is widely agreed upon that CTCs typically lack the CD45 marker and exhibit the presence of EpCAM and CK [42].…”

Prognostic evaluation of a multi-target magnetic bead-enriched circulating tumor cell-enriched identification system for colorectal cancer

“…This platform specifically captures EpCAM + tumor cells from the peripheral blood (PB) of patients through ferrofluidic nanoparticles. CellSearch ® has demonstrated through numerous clinical trials that CTCs detected by this method have a close relationship with patient prognosis, and some studies have shown that tumor cells enriched by CellSearch ® are of great value in clinical research ( 11 - 15 ). However, CellSearch ® based on an EpCAM-based enrichment strategy has some limitations.…”

“…Firstly, this method may overlook CTCs that poorly express or have a lack of epithelial surface markers ( 16 ). Secondly, CellSearch ® is also unable to interstitially identify tumors that express little or no EpCAM of mesenchymal origin, as well as epithelial-derived CTCs that are partially in EMT; several published reports have highlighted this issue ( 11 , 17 - 21 ), which severely limits the clinical application of CellSearch ® . In this study, we constructed a novel multiplexed magnetic bead enrichment platform in which we used EpCAM-coated immunoliposomal magnetic beads (Ep-IMBs) and vimentin-coated immunoliposomal magnetic beads (Vi-IMBs) sequentially to capture CTCs.…”