Label-Free Enrichment and Molecular Characterization of Viable Circulating Tumor Cells from Diagnostic Leukapheresis Products

Abstract

INTRODUCTION Circulating tumor cells (CTCs) may be used to improve cancer diagnosis, prognosis, and treatment. However, because knowledge regarding CTC biology is limited and the numbers of CTCs and CTC-positive cancer patients are low, progress in this field is slow. We addressed this limitation by combining diagnostic leukapheresis (DLA) and microfluidic enrichment to obtain large numbers of viable CTCs from metastasized breast cancer patients. METHODS DLA was applied to 9 patients, and 7.5 mL of peripheral blood was drawn. CTCs were enriched with the Parsortix™ system. The quality of CTCs from fresh and cryopreserved DLA products was tested, and CTCs were cultured in vitro. Single uncultured and cultured CTCs were isolated by micromanipulation to determine different parameters, such as genomic aberrations and mutation profiles of selected tumor-associated genes. Expression levels of estrogen receptor and HER2/neu were monitored during in vitro culture. RESULTS Viable CTCs from peripheral blood and fresh or frozen DLA products could be enriched. DLA increased the likelihood of successful CTC culture. Cryopreserved DLA products could be stored with minimal CTC loss and no overt reduction in the tumor cell quality and viability during an observation period of up to 3 years. The analyzed parameters did not change during in vitro culture. DLA samples with high CTC numbers and lower ratios of apoptotic CTCs were more likely to grow in culture. CONCLUSIONS The increased CTC numbers from fresh or cryopreserved DLA products facilitate multiple functional and molecular analyses and, thus, could improve our knowledge of their biology.

The Significance of Circulating Tumor Cells in Patients with Hepatocellular Carcinoma: Real-Time Monitoring and Moving Targets for Cancer Therapy

“…CTCs enrichment based on their biophysical properties can be achieved through high-throughput and label-free enrichment, but it faces challenges in enhancing the specificity, as well as the purity of the enriched CTCs [53,54,56]. Unlike "label free" physical attribute-based methods, immunoaffinity-based methods rely on the highly specific interaction between ligands (antibodies or chemical antibodies) and tumor-specific antigens that are present on the cell membrane of CTCs [52].…”

Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells

“…While leukapheresis allows for initial cell density-based sorting of entire blood volumes, current CTC isolation technologies can only process up to 200 million mononuclear cells, or about 3 to 5% of a leukopak, significantly limiting the ultimate benefit of processing leukapheresis products ( 23 – 28 ). Here we describe a microfluidic chip, termed “ LP CTC-iChip,” (LP: leukapheresis product) that can process the entire leukapheresis volume of 65 mL and is capable of recovering thousands of untagged viable CTCs by depleting RBCs, platelets, and white blood cells (WBCs) in a tumor-agnostic manner.…”

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform

“…The viability of captured tumor cells following enrichment or isolation represents a vital prerequisite for successful transient or permanent culture. A cell viability of 99% was demonstrated for cells separated by the Parsortix system in earlier studies indicating that processing of samples using this technology has a negligible effect on cell viability. For this reason, in this study, we focused on assessing the impact of chip processing in itself on cell viability.…”