Evaluation of a marker independent isolation method for circulating tumor cells in esophageal adenocarcinoma

Abstract

Objective The enrichment of circulating tumor cells (CTCs) from blood provides a minimally invasive method for biomarker discovery in cancer. Longitudinal interrogation allows monitoring or prediction of therapy response, detection of minimal residual disease or progression, and determination of prognosis. Despite inherent phenotypic heterogeneity and differences in cell surface marker expression, most CTC isolation technologies typically use positive selection. This necessitates the optimization of marker-independent CTC methods, enabling the capture of heterogenous CTCs. The aim of this report is to compare a size-dependent and a marker-dependent CTC-isolation method, using spiked esophageal cells in healthy donor blood and blood from patients diagnosed with esophageal adenocarcinoma. Methods Using esophageal cancer cell lines (OE19 and OE33) spiked into blood of a healthy donor, we investigated tumor cell isolation by Parsortix post cell fixation, immunostaining and transfer to a glass slide, and benchmarked its performance against the CellSearch system. Additionally, we performed DEPArray cell sorting to infer the feasibility to select and isolate cells of interest, aiming towards downstream single-cell molecular characterization in future studies. Finally, we measured CTC prevalence by Parsortix in venous blood samples from patients with various esophageal adenocarcinoma tumor stages. Results OE19 and OE33 cells were spiked in healthy donor blood and subsequently processed using CellSearch (n = 16) or Parsortix (n = 16). Upon tumor cell enrichment and enumeration, the recovery rate ranged from 76.3 ± 23.2% to 21.3 ± 9.2% for CellSearch and Parsortix, respectively. Parsortix-enriched and stained cell fractions were successfully transferred to the DEPArray instrument with preservation of cell morphology, allowing isolation of cells of interest. Finally, despite low CTC prevalence and abundance, Parsortix detected traditional CTCs (i.e. cytokeratin+/CD45-) in 8/29 (27.6%) of patients with esophageal adenocarcinoma, of whom 50% had early stage (I-II) disease. Conclusions We refined an epitope-independent isolation workflow to study CTCs in patients with esophageal adenocarcinoma. CTC recovery using Parsortix was substantially lower compared to CellSearch when focusing on the traditional CTC phenotype with CD45-negative and cytokeratin-positive staining characteristics. Future research could determine if this method allows downstream molecular interrogation of CTCs to infer new prognostic and predictive biomarkers on a single-cell level.

Cancer nanotechnology: current status and perspectives

“…Microfluidics is another area of technology applicable to CTC detection due to portability, high-throughput capability, and precise control within the microchannel. Certain microfluidics platforms (Parsortix® and Vycap systems) can recover CTCs based upon size and deformability instead of EMT status, and transcriptomic analysis of CTCs can be performed at the scale of a cell after isolation [ 267 , 268 ]. Transcriptome analysis then provides information on the state of the cell as to its position in the EMT thanks to a molecular signature by phenotype [ 269 ].…”

Clinical value of folate receptor-positive circulating tumor cells in patients with esophageal squamous cell carcinomas: a retrospective study

“…CTCs shed and relocate at a new site, in a process often referred to as tumor metastasis [ 5 ]. In recent decades, the CTCs in peripheral blood have been demonstrated to be a diagnostic biomarker and prognostic factor and offer new strategies for treating cancer, especially lung cancer [ 6 ]. For ESCC patients, CTCs were explored as a biomarker for diagnosing, staging and evaluating treatment efficacy [ 7 – 10 ].…”

Single cell genomics applications in forensic science: Current state and future directions

“… 2 In contrast scDNA enables the detection and characterization of rare cell types, which can be crucial for not only understanding disease progression but informing potential therapeutic intervention. 3 Forensic genetic analysis relies on the use of technologies such as DNA profiling to facilitate human identification (HID) in criminal and other medical legal matters, including humanitarian efforts with unidentified human remains from victims of accident, natural disasters, or other mass fatality events. In a forensic setting, scDNA has the potential to aid in investigations where limited DNA samples are available from persons of interest as well as deconvoluting the DNA profiles of multiple contributors (e.g., victims and perpetrators) to crime scene stains.…”