Abstract
AbstractThere is increasing evidence that, in addition to their presence, the propensity of circulating tumour cells to form multi-cellular clusters bears significant information about both cellular resistance to chemotherapy and overall prognosis. We present a novel two-stage, stopped-flow, continuous centrifugal sedimentation strategy to measure the size distributions of events (defined here as cells or clusters thereof) in a blood sample. After off-chip removal of red blood cells, healthy white blood cells are sequestered by negative-immunocapture. The purified events are then resolved along a radially inclined rail featuring a series of gaps with increasing width, each connected to a designated outer collection bin. The isolation of candidate events independent of target-specific epitopes is successfully demonstrated for HL60 (EpCAM positive) and sk-mel28 (EpCAM negative) cells using identical protocols and reagents. The propensity to form clusters was quantified for a number of cell lines, showing a negligible, moderate or elevated tendency towards cluster formation. We show that the occupancy distribution of the collection bins closely correlates with the range of cluster sizes intrinsic to the specific cell line.
Highlights
The presence of circulating tumour cells (CTCs) in the blood of a patient, as found in most forms of epithelial cancer1–5, has been identified as a reliable indicator of the prognosis of various cancer types
Recent literature has provided strong evidence that the cluster size distribution in an oncological sample represents a marker of high diagnostic value
We developed a novel centrifugal size-exclusion rail scheme for the examination of the cluster load in a blood sample, and we demonstrated that samples harbouring single-cell candidate events will exclusively resolve to a given collection bin
Summary
The presence of circulating tumour cells (CTCs) in the blood of a patient, as found in most forms of epithelial cancer, has been identified as a reliable indicator of the prognosis of various cancer types. CTCs within a cluster have been shown to resist anoikis/apoptosis, possibly due to the maintenance of cell–cell junctions within the cluster and protection from shear forces by virtue of the outer cells. Molecular investigations of the cells within a cluster have shown elevated levels of pro-survival and pro-epithelial-to-mesenchymal transition signals. Molecular investigations of the cells within a cluster have shown elevated levels of pro-survival and pro-epithelial-to-mesenchymal transition signals11 These clusters’ genesis is still under investigation, but studies have proposed that rather than emerging from the proliferation of a single CTC or from aggregation in the bloodstream, clusters may represent tissue that breaks away from the site of a primary tumour. CTCs’ lack of proliferation (theoretically) renders these cells resistant to chemotherapies that actively target rapidly dividing cells, increasing the diagnostic and prognostic potential of identifying the cluster load in a blood sample
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