Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry

Sam H Au,Mehmet Toner,Jon Edd,Keith H K Wong,Ravi Kapur,Fabio Fachin,Shyamala Maheswaran,Amy E Stoddard,Daniel A Haber,Shannon L Stott

doi:10.1038/s41598-017-01150-3

Abstract

Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2–100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

Highlights

Individual circulating tumor cells (CTCs) are not the only valuable cells for isolation from patient blood
We developed an integrated two-stage strategy for capturing CTC clusters that relies on the combination of two characteristics of CTC clusters, that when probed together, can be used to effectively distinguish them from other cells in whole blood: size and asymmetry (Fig. 1)
The first stage of the microfluidic device, “Stage 1”, was designed to remove large clusters from whole blood (Fig. 1a) by deterministic lateral displacement (DLD), which isolates large clusters based on their greater sizes

Summary

Introduction

Individual CTCs are not the only valuable cells for isolation from patient blood. Given the large variability in reported cluster capture efficiencies and the large shear rates inherent in many devices, it is likely that the majority of CTC clusters in sampled patient blood escape capture or dissociate into single cells or smaller clusters during processing[7]. The second stage, whose input is the undeflected product of Stage 1, uses asymmetrical pillars and height restrictions to extract smaller clusters based on the inherent asymmetric nature of multicellular aggregates This generation cluster chip isolates high integrity CTC clusters from whole blood that experience physiological-or-lower shear stress rates throughout, on-chip residence times on the order of seconds which minimizes damage or processing bias, and the continuous-flow nature of the chip enables in-line integration with other analysis and capture strategies

Methods

Results

Discussion

Conclusion