Isolation of circulating tumor cells using a microvortex-generating herringbone-chip

Abstract

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or "HB-Chip," which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

Fig. 1.

(A) The HB-Chip consists of a microfluidic array of channels with a single inlet and exit. Inset illustrates the uniform blood flow through the device. (B) A micrograph of the grooved surface illustrates the asymmetry and periodicity of the herringbone grooves. Cartoon illustrating the cell-surface interactions in (C) the HB-Chip, and (D) a traditional flat-walled microfluidic device. Flow visualization studies using two paired streams of the same viscosity (one stream is green, the other clear) demonstrate (E) the chaotic microvortices generated by the herringbone grooves, and the lack of mixing in (F) traditional flat-walled devices.

Fig. 2.

(A) Device proof-of-principle studies were conducted using PC3 cells spiked into whole blood at 1,000 cells/mL and processed with a small version of the HB-Chip (Fig. S4, channel height 70 μm, α = 0.43) and a flat-walled device (channel height 100 μm). Capture efficiency is shown for both devices in addition to IgG controls. (B) Relative capture efficiency for head to head comparisons run between the full-sized HB-Chip (channel height 50 μm, α = 0.8) and the silicon CTC-Chip (17). For the data in (B), a single preparation of blood was spiked with PC3 cells at a concentration of 500 cells/mL, divided into equal parts and processed on both platforms. Due to slight errors in spiking concentration, the HB-Chip capture efficiency was determined to be greater than 100% (112.4% ± 1.8%, n = 4). Consequently, the comparison data is normalized against the HB-Chip. (C) Variations in device chamber height showed a minimal impact on capture efficiency, until the chamber height was increased threefold. (D) Variability in device performance is shown for four independent experiments, each with different spiking concentrations of PC3 cells in blood. (E) Micrograph of spiked cancer cells captured on the HB-Chip, representative of capture cell viability (LIVE/DEAD). (Scale bar: 40 μm). (F) Micrograph of spiked cells captured on the herringbone chip and subsequently cultured on the device for 21 d. (G) On-chip FISH of captured LNCaP cells with nuclei stained with DAPI, CEPX (green) and AR gene locus (red). (H) Micrograph of a fluorescently labeled PC3 cell captured on the HB-Chip (top) and subsequent micrograph taken of the same cell stained with H and E. (Scale bar: 10 μm).

Fig. 3.

Blood from metastatic prostate cancer patients and healthy donors was processed using the HB-Chip. (A) Healthy donors (n = 10, red circles) were used to select the signal intensity threshold in our automated imaging system (19). The metastatic prostate cancer patient data (n = 15, green circles) had significantly higher results, with 14∶15 patient samples presenting CTCs above-threshold levels. Additionally, (B) the TMPRSS2-ERG fusion transcript was detected from RNA isolated from the HB-Chip, using RT-PCR. Sequencing of the gel band identified the rare gene fusion of TMPRSS2 exon 1 and ERG exon 5. (C and D) Micrographs of CTCs isolated from patients with metastatic prostate cancer costained using antibodies against PSA (green), a leukocyte marker, CD45 (red), and a nuclear stain (DAPI). Corresponding micrographs of the same cells are shown to the right of the fluorescent images, demonstrating their appearance after subsequent staining with H and E. (Scale bars: 10 μm).

Fig. 4.

(A and B) Micrographs of cells isolated on the HB-Chip from a metastatic prostate cancer patient that were not classified as CTCs due to their dual-positivity for both PSA (green) and CD45 (red). Corresponding H and E images are shown. (C) Micrographs of a CTC cluster isolated from a metastatic prostate cancer patient on the HB-Chip; immunofluoresence staining (DNA (blue), prostate-specific membrane antigen (green), and CD45 (red)) and subsequent immunohistochemical staining (H and E) are shown. A three-dimensional projection of this cluster can be viewed in SI Text. (D) A CTC cluster isolated from a metastatic lung cancer patient on the HB-Chip; immunofluoresence staining (DNA (blue), cytokeratins 7/8 (green), and CD45 (red)) and subsequent immunohistochemical staining (H and E) are shown. All scale bars represent 10 μm.