Circulating Tumor Cell Clusters Are Oligoclonal Precursors of Breast Cancer Metastasis

Circulating tumour cells (CTCs) are the subject of many published papers, but the diversity of assays used for their analysis can be daunting. This Opinion article discusses issues regarding the detection and characterization of CTCs, and poses the major outstanding questions in this field.

Challenges in circulating tumour cell research.

Accurate and high throughput cell sorting is a critical enabling technology in molecular and cellular biology, biotechnology, and medicine While conventional methods can provide high efficiency sorting in short timescales, advances in microfluidics have enabled the realization of miniaturized devices offering similar capabilities that exploit a variety of physical principles We classify these technologies as either active or passive Active systems generally use external fields (eg, acoustic, electric, magnetic, and optical) to impose forces to displace cells for sorting, whereas passive systems use inertial forces, filters, and adhesion mechanisms to purify cell populations Cell sorting on microchips provides numerous advantages over conventional methods by reducing the size of necessary equipment, eliminating potentially biohazardous aerosols, and simplifying the complex protocols commonly associated with cell sorting Additionally, microchip devices are well suited for parallelization, enabling complete lab-on-a-chip devices for cellular isolation, analysis, and experimental processing In this review, we examine the breadth of microfluidic cell sorting technologies, while focusing on those that offer the greatest potential for translation into clinical and industrial practice and that offer multiple, useful functions We organize these sorting technologies by the type of cell preparation required (ie, fluorescent label-based sorting, bead-based sorting, and label-free sorting) as well as by the physical principles underlying each sorting mechanism

Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation

Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor-positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.

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Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility

In the last decade, inertial microfluidics has attracted significant attention and a wide variety of channel designs that focus, concentrate and separate particles and fluids have been demonstrated. In contrast to conventional microfluidic technologies, where fluid inertia is negligible and flow remains almost within the Stokes flow region with very low Reynolds number (Re ≪ 1), inertial microfluidics works in the intermediate Reynolds number range (~1 < Re < ~100) between Stokes and turbulent regimes. In this intermediate range, both inertia and fluid viscosity are finite and bring about several intriguing effects that form the basis of inertial microfluidics including (i) inertial migration and (ii) secondary flow. Due to the superior features of high-throughput, simplicity, precise manipulation and low cost, inertial microfluidics is a very promising candidate for cellular sample processing, especially for samples with low abundant targets. In this review, we first discuss the fundamental kinematics of particles in microchannels to familiarise readers with the mechanisms and underlying physics in inertial microfluidic systems. We then present a comprehensive review of recent developments and key applications of inertial microfluidic systems according to their microchannel structures. Finally, we discuss the perspective of employing fluid inertia in microfluidics for particle manipulation. Due to the superior benefits of inertial microfluidics, this promising technology will still be an attractive topic in the near future, with more novel designs and further applications in biology, medicine and industry on the horizon.

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Fundamentals and applications of inertial microfluidics: a review

Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing–enhanced microfluidic CTC capture platform, termed “CTC-iChip,” that is capable of sorting rare CTCs from whole blood at 107 cells/s. Most importantly, the iChip is capable of isolating CTCs using strategies that are either dependent or independent of tumor membrane epitopes, and thus applicable to virtually all cancers. We specifically demonstrate the use of the iChip in an expanded set of both epithelial and nonepithelial cancers including lung, prostate, pancreas, breast, and melanoma. The sorting of CTCs as unfixed cells in solution allows for the application of high-quality clinically standardized morphological and immunohistochemical analyses, as well as RNA-based single-cell molecular characterization. The combination of an unbiased, broadly applicable, high-throughput, and automatable rare cell sorting technology with generally accepted molecular assays and cytology standards will enable the integration of CTC-based diagnostics into the clinical management of cancer.

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Inertial Focusing for Tumor Antigen–Dependent and –Independent Sorting of Rare Circulating Tumor Cells

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d.

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Bailey Morgan

Papers

Inertial Focusing for Tumor Antigen–Dependent and –Independent Sorting of Rare Circulating Tumor Cells

Microfluidic, marker-free isolation of circulating tumor cells from blood samples