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

Abstract

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.

Particle Focusing in Curved Microfluidic Channels

“…These forces allow for the precise alignment of particles in a flow at throughputs orders of magnitudes higher than in previous microfluidic technologies. The high throughput nature of inertial focusing has enabled a range of microfluidic technologies for biomedical applications from separation technologies [8][9][10][11][12] , to automated sample preparations 13,14 , to novel cell analysis techniques such as cell deformability cytometry 15 and the isolation of circulating tumor cells from blood 16,17 .…”

“…These forces allow for the precise alignment of particles in a flow at throughputs orders of magnitudes higher than in previous microfluidic technologies. The high throughput nature of inertial focusing has enabled a range of microfluidic technologies for biomedical applications from separation technologies [8][9][10][11][12] , to automated sample preparations 13,14 , to novel cell analysis techniques such as cell deformability cytometry 15 and the isolation of circulating tumor cells from blood 16,17 .It is generally accepted that inertial focusing in straight channels is dependent on two main parameters: Reynolds number, defined as Re C 5 rU Max D h /m, where r is the fluid density, m is the fluid viscosity, U Max > 3/2U Avg is the maximum velocity of the fluid and D h is the hydraulic diameter of the channel defined as D h 5 2hw/(h 1 w) where h and w are the height and width of the channel cross section respectively, and the particle confinement ratio, l 5 a/D h , where a, is the particle size. Prior research has determined a minimum threshold for inertial focusing to occur such that l .…”

Engineering opportunities in cancer immunotherapy

“…Although much development has occurred in the last few years, there is still great need for effective, sensitive, and easy-to-use methods to capture and characterize these cells. Many microfluidic approaches are being developed, such as a high-throughput capture chip based on intertial focusing that enables sorting of magnetically labeled rare CTCs from the blood and of subsequent RNA-based single-cell characterization (85). Standard flow cytometry can also be used with fluorescence labels, but the relative scarcity of such cells makes detection difficult; approaches to overcome that includes an imaging flow cytometer that images streaks by adjusting exposure time and that allows very rare cells to be detected (86).…”

Opposing shear-induced forces dominate inertial focusing in curved channels and high Reynolds numbers

“…Applications of inertial focusing soared in the advent of microfluidics especially in curved channels where Dean forces could be used to enhance sorting accuracy 1,2,5 . Current understanding of sorting in curved channels rely on Ho and Leal description of a balance between shear-induced and wall-induced lift forces, suggesting that focusing is limited to low Reynolds (Re) numbers 6,7 .…”