Label-free isolation of prostate circulating tumor cells using Vortex microfluidic technology

Abstract

There has been increased interest in utilizing non-invasive “liquid biopsies” to identify biomarkers for cancer prognosis and monitoring, and to isolate genetic material that can predict response to targeted therapies. Circulating tumor cells (CTCs) have emerged as such a biomarker providing both genetic and phenotypic information about tumor evolution, potentially from both primary and metastatic sites. Currently, available CTC isolation approaches, including immunoaffinity and size-based filtration, have focused on high capture efficiency but with lower purity and often long and manual sample preparation, which limits the use of captured CTCs for downstream analyses. Here, we describe the use of the microfluidic Vortex Chip for size-based isolation of CTCs from 22 patients with advanced prostate cancer and, from an enumeration study on 18 of these patients, find that we can capture CTCs with high purity (from 1.74 to 37.59%) and efficiency (from 1.88 to 93.75 CTCs/7.5 mL) in less than 1 h. Interestingly, more atypical large circulating cells were identified in five age-matched healthy donors (46–77 years old; 1.25–2.50 CTCs/7.5 mL) than in five healthy donors <30 years old (21–27 years old; 0.00 CTC/7.5 mL). Using a threshold calculated from the five age-matched healthy donors (3.37 CTCs/mL), we identified CTCs in 80% of the prostate cancer patients. We also found that a fraction of the cells collected (11.5%) did not express epithelial prostate markers (cytokeratin and/or prostate-specific antigen) and that some instead expressed markers of epithelial–mesenchymal transition, i.e., vimentin and N-cadherin. We also show that the purity and DNA yield of isolated cells is amenable to targeted amplification and next-generation sequencing, without whole genome amplification, identifying unique mutations in 10 of 15 samples and 0 of 4 healthy samples.

Developments in label‐free microfluidic methods for single‐cell analysis and sorting

“…Renier et al successfully used the Vortex High Throughput chip to isolate and concentrate prostate CTCs from the diluted whole blood of prostate cancer patients (Fig. 4d) (Renier et al, 2017). …”

VTX‐1 Liquid Biopsy System for Fully‐Automated and Label‐Free Isolation of Circulating Tumor Cells with Automated Enumeration by BioView Platform

“…Here, CTC capture is based on cell size, cell shape, and deformability and is the agnostic of tumor biomarkers (Fig. Numerous workflows have been optimized for various CTC research applications, such as immunofluorescence staining and CTC enumeration (23)(24)(25)(26)29), cytopathology and cytogenetics (24), Sanger sequencing (26), next-generation sequencing (27,29), and even Western-blotting on single cells (28) (Fig. The Vortex chip has been characterized and validated for CTC isolation from blood samples of metastatic cancer patients with breast, colon, lung and prostate cancer (23)(24)(25)(26)(27)(28)(29).…”

“…Isolated CTCs are thus unbiased by molecular characteristics, enabling the isolation of the diverse population of CTCs in the patient whether they be epithelial or mesenchymal (29). CTCs have a different shape and deformability and are larger than white and red blood cells, resulting in their stable capture in the micro-vortices created in the microfluidic chip.…”

“…2B) (24,26,29), using a CTC identification criteria previously defined (23). In preliminary clinical studies using the Vortex technology with an early-stage platform, CTCs were isolated with high purity (from 1.4 to 92.5 WBCs/ml blood) from patients with metastatic breast (median 40.7 CTCs per 7.5 ml; n = 22), colorectal (median 12.2 CTCs per 7.5 ml, n = 41), non-small-cell lung (NSCLC) (median 26.2 CTCs per 7.5 ml, n = 15), and prostate (median 5.6 CTCs per 7.5 ml, n = 20) cancers ( Fig.…”

Toward Microfluidic Label‐Free Isolation and Enumeration of Circulating Tumor Cells from Blood Samples

“…At the outlet, CTCs are collected in suspension at high purity, and different downstream assays can be performed such as, immunofluorescence, cytology, cytogenetics, nextgeneration sequencing, qPCR, live cell assays, RNA sequencing, or protein assays (20)(21)(22)(23). Several case studies have validated this technology with patient samples of metastatic breast, lung, colon, and prostate cancer (24).…”