Abstract
Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm–50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15–20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.
Highlights
Circulating tumor cells (CTCs) are critical rare cell targets as they can be present in extremely low numbers and have been shown to be a root cause of the majority of cancer related deaths
Work used microfilters[31] while more recent studies involve the use of deterministic lateral displacement or DLD32, isolation by size of epithelial tumor cells or ISET33, and inertial focusing[34]. These technologies work on the presumption that CTCs are larger than typical blood cells, which is shown to be true for cancer cell lines but the limited amount of data with patient CTCs do not support this assumption[16, 17]
Another approach that does not rely on any single protein based enrichment of CTCs is the use of high-definition CTC analysis developed by Kuhn and colleagues, where all nucleated cells are panned onto slides for staining and subsequent multi-marker immunofluorescent imaging to identify CTCs37
Summary
Fabio Fachin[1], Philipp Spuhler[1], Joseph M. The CTC-iChip combines hydrodynamic size-based separation of all nucleated cells (leukocytes and CTCs) away from red blood cells, platelets, and plasma, with subsequent inertial focusing of the nucleated cells onto a single streamline to achieve high-efficiency in-line magnetophoretic depletion of white blood cells (WBCs) that are tagged with magnetic beads in whole blood This antigen-independent isolation of CTCs enables the characterization of CTCs with both epithelial and mesenchymal characteristics. We present the culmination of microfluidic and process engineering efforts to develop a high-throughput monolithic CTC-iChip, which uses deterministic lateral displacement, inertial focusing, and magnetophoresis to deplete blood cells at 15–20 million cells per second to sort CTCs. The individual components previously manufactured using deep reactive ion silicon etching and PDMS (polydimethylsiloxane) soft lithography[21, 38] have been integrated on a single mass-produced plastic chip, vastly decreasing both technical requirements and hands on time and improving the accessibility of the technology. This paper details the extensive characterization and rigorous testing of the limits of a technology that is already being utilized for clinical and scientific pursuits
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