29 marzo 2018

Microfluidic assays for mobile tumor cells in human body fluids: rethinking liquid biopsy

Liquid biopsy is a blood test involving the harvesting of tumor materials from peripheral blood. Tumor cells from non-blood body fluids have always been clinically available in cytological examinations but limited for use in differential diagnosis due to the low sensitivity of conventional cytopathology. With the recent significant progress in microfluidic and downstream molecular technologies, liquid biopsies have now evolved to include harvesting tumor cells and DNA fragments in all kinds of non-blood body fluids. This expansion into general body fluids presages the notion that liquid biopsy could soon be used in competition, as well as, in complementarity with tissue biopsy.


As the concept of precision medicine in the field of cancer management continues to evolve, so too do the challenges and demands with regards to diagnosis, prognosis, and prediction of treatment resistance (Ashley EA., 2016; Prasad V et al., 2016). Tissue biopsies, which still currently represent the standard of tumor diagnosis, unfortunately only reflect a single point in time of a single site of the tumor. Such a sampling method is inadequate for the comprehensive characterization of a patient’s tumor, as it has been demonstrated that various areas within the primary tumor or metastases can in fact harbor different genomic profiles (Gerlinger M et al., 2012). Furthermore, a surgical biopsy procedure is hampered by limited repeatability, patient age and comorbidity, costs, and time, potentially leading to clinical complications (Perakis S & Speicher MR, 2017). Compared with traditional ‘solid biopsy’, liquid biopsy (which includes circulating tumor cells, circulating tumor DNA, and tumor-derived exosomes) is minimally invasive, less risky and significantly cheaper; hence, it can be sampled a lot more frequently to achieve a better diagnostic and monitoring accuracy for a more effective treatment (Neoh KH et al., 2017).

With the recent significant progress in microfluidic and downstream molecular technologies, liquid biopsies have now evolved to include harvesting tumor cells and DNA fragments not only from peripheral blood but also in all kinds of non-blood body fluids (ascites, pleural effusion, urine, cerebrospinal fluid, etc.). With <1% of cancer cells released into blood circulation that eventually lead to metastases (Fidler LJ, 1970; Luzzi KJ et al., 1998), it is clear that cancer metastasis via nonblood body fluids is more important than the hematogenous route for many types of cancer. Conventional cytological examination of nonblood body fluids is plagued with low sensitivity, albeit having high specificity (Che J et al., 2013). Multiple centrifugation steps are involved in cytopreparation which may contribute to the loss of tumor cells (Mach AJ et al., 2011) and the presence of blood cells can obscure MTCs too (Che J et al., 2013). In contrast, a microfluidic platform offers many advantages over conventional benchtop techniques: low volume requirement of sample and reagent, high sensitivity, controllable flow pattern, easy to operate, low cost and the ability to perform multiple processes in a single device.

Main microfluidic enrichment technologies rely on physical size-based isolation of cancer cells (e.g. ATC Chip, Peterson et al., 2013), inertia-based isolation (e.g. pinched flow fractionation (PFF), deterministic lateral displacement (DLD), Dean flow fractionation (DFF)), affinity-based isolation. In addition, most popular strategies for downstream tumor cell identification are based on impedance cytometry, Deformability cytometry, immunofluorescence staining, FISH and Next-Generation-sequencing. In this context, CellDynamics paves the way to the development of a cutting-edge technology (i.e. CellViewer) for live tumor cells isolation, time-lapse monitoring and real-time analysis. Indeed, CellViewer is an automated Lab-on-Chip Platform that combines a 3D culture system with time-lapse microscopy, electronics and microfluidic technologies and it allows to isolate viable rare single cells and real-time monitor cell responses to drug conditioned stimuli over extended periods of time. CellViewer is expected to play a worldwide considerable role in understanding cancer heterogeneity and unveil molecular mechanisms involved in EMT and metastasis.



Fig. 6. Microfluidic strategies for detecting cancer cells (from K.H. Neoh et al. Rethinking liquid biopsy: Microfluidic assays for mobile tumor cells in human body fluids. Biomaterials 150 (2018) 112-124). A: Schematic of the Nanoelectromechanical-Chip. B: Schematic of the CTC-eChip. C: Schematic of a cell being stretched at the center of an extensional flow junction, whereby the cell experiences a sudden opposing wall of fluid. D: Schematic of the vortex-mediated deformability cytometry (VDC) chip; HT: high throughput; DC: deformability cytometry. A: adapted with permission from Ref. (Hosseini SA, et al., 2016). Copyright John Wiley and Sons, 2016. B: adapted with permission from Ref. (Han SI & Han KH, 2015). Copyright American Chemical Society, 2015. C: adapted from Ref. (Gossett DR et al., 2012). Copyright National Academy of Sciences, 2012. D: adapted with permission from Ref. (Che J et al., 2017). Copyright Royal Society of Chemistry, 2017.



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