A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells

Abstract

Circulating tumor cells (CTCs) were introduced as biomarkers more than 10 years ago, but capture of viable CTCs at high purity from peripheral blood of cancer patients is still a major technical challenge. Here, we report a novel microfluidic platform designed for marker independent capture of CTCs. The Parsortix™ cell separation system provides size and deformability‐based enrichment with automated staining for cell identification, and subsequent recovery (harvesting) of cells from the device. Using the Parsortix™ system, average cell capture inside the device ranged between 42% and 70%. Subsequent harvest of cells from the device ranged between 54% and 69% of cells captured. Most importantly, 99% of the isolated tumor cells were viable after processing in spiking experiments as well as after harvesting from patient samples and still functional for downstream molecular analysis as demonstrated by mRNA characterization and array‐based comparative genomic hybridization. Analyzing clinical blood samples from metastatic (n = 20) and nonmetastatic (n = 6) cancer patients in parallel with CellSearch® system, we found that there was no statistically significant difference between the quantitative behavior of the two systems in this set of twenty six paired separations. In conclusion, the epitope independent Parsortix™ system enables the isolation of viable CTCs at a very high purity. Using this system, viable tumor cells are easily accessible and ready for molecular and functional analysis. The system's ability for enumeration and molecular characterization of EpCAM‐negative CTCs will help to broaden research into the mechanisms of cancer as well as facilitating the use of CTCs as “liquid biopsies.”

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells

“…As the CellCollector is based on antiEpCAM antibodies for CTC isolation, it may miss several other CTC subpopulations with downregulated epithelial markers (36 ). To evade limitation by EpCAM, we combined the in situ padlock approach with the EpCAM-independent Parsortix platform (20 ), demonstrating that the in situ padlock approach is generally applicable to different isolation platforms. By combining the in situ AR-V7 detection with the Parsortix system, we observed heterogeneous cytokeratin expression of CTCs.…”

“…Two patients donated an additional set of blood samples on a second visit. CTCs were enriched from EDTA blood using the size-dependent, label-free Parsortix system (ANGLE plc) as described previously (19,20 ). A separation pressure of 99 mbar was chosen to process the blood samples.…”

“…To evaluate whether our novel approach can also be applied to samples obtained from other CTC isolation platforms, spiked prostate cancer cell line cells were enriched from whole blood by the EpCAM-independent Parsortix system (20 ). 22VRv1 and PC-3 cells were chosen for these experiments, having high and low AR-V7 expression, respectively.…”

The Novel Association of Circulating Tumor Cells and Circulating Megakaryocytes with Prostate Cancer Prognosis

“…To address this challenge, we have optimized a novel epitopeindependent CTC isolation system, Parsortix, to capture CTCs based on the much larger size and less deformability nature of tumor cells compared with normal blood cells (9). The efficiency of Parsortix in capturing CTCs has been independently validated by other research groups in comparison with CellSearch in patients with small-cell lung cancer (10), and other types of cancers, including breast, colon, and lung (11). Here, we explored its clinical application in prostate cancer prognosis and disease state monitoring.…”

Biology and clinical significance of circulating tumor cell subpopulations in lung cancer

“…Isolation of CTCs from the whole blood can be done using the physical properties of the cell, such as size and density and also migratory properties. (9,(13)(14)(15)(16)(17)(18)(19)(20)(21).…”