Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry

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.

Microfluidic isolation of platelet-covered circulating tumor cells

“…To this end, our group pioneered the development of microfluidic technologies that isolate CTCs by immunoaffinity-based positive enrichment 18,19 or “negative” selection (i.e., by depleting hematologic cells) 20 , or by taking advantage of the size and asymmetry of CTC clusters 21,22 . However, the isolation of platelet-covered CTCs is extremely difficult and current technologies have not been designed to detect this particular CTC population.…”

Molecular genetic testing methodologies in hematopoietic diseases: current and future methods

“…However, these methods cannot retain spatial information/original architecture of the cells, information which can thus be recovered using laser‐capture‐microdissection (LCM). On the other hand, rare cells also can be isolated using a bead‐based enrichment platform, dielectrophoretic (DEP) arrays, size exclusion based microfluidics, magnetic nanoparticle‐based labeling, and optical tweezers . For single cell DNA sequencing, whole genome amplification (WGA) remains the most widely used protocol to amplify the genome prior to library generation.…”

Clusters of circulating tumor cells: A biophysical and technological perspective

“…Second, the majority of clusters exist as small 2-4 cell aggregates[37], reducing the average size disparities between large single cells and most clusters. Third, cells and clusters often assume alignments that “mask” their longest axes during size-based sorting by microfluidics[38-40] or filtration[41]. Therefore, strategies that rely solely on size-based separation likely have difficulty isolating a significant fraction of CTC clusters from normal blood cells and single CTCs.…”

“…To address this limitation, we developed a continuous-flow microfluidic strategy for isolating CTC clusters that relies on a two-stage deterministic lateral displacement (DLD) approach, integrated into a single device[40]. The first stage separates larger clusters based solely on size-based separation using standard cylindrical DLD micropillar arrays, while the second stage uses asymmetric pillars to discriminate smaller clusters from single CTCs based on their inherent asymmetry.…”