Entrapment of Prostate Cancer Circulating Tumor Cells with a Sequential Size-Based Microfluidic Chip

Abstract

Circulating tumor cells (CTCs) are broadly accepted as an indicator for early cancer diagnosis and disease severity. However, there is currently no reliable method available to capture and enumerate all CTCs as most systems require either an initial CTC isolation or antibody-based capture for CTC enumeration. Many size-based CTC detection and isolation microfluidic platforms have been presented in the past few years. Here we describe a new size-based, multiple-row cancer cell entrapment device that captured LNCaP-C4-2 prostate cancer cells with >95% efficiency when in spiked mouse whole blood at ∼50 cells/mL. The capture ratio and capture limit on each row was optimized and it was determined that trapping chambers with five or six rows of micro constriction channels were needed to attain a capture ratio >95%. The device was operated under a constant pressure mode at the inlet for blood samples which created a uniform pressure differential across all the microchannels in this array. When the cancer cells deformed in the constriction channel, the blood flow temporarily slowed down. Once inside the trapping chamber, the cancer cells recovered their original shape after the deformation created by their passage through the constriction channel. The CTCs reached the cavity region of the trapping chamber, such that the blood flow in the constriction channel resumed. On the basis of this principle, the CTCs will be captured by this high-throughput entrapment chip (CTC-HTECH), thus confirming the potential for our CTC-HTECH to be used for early stage CTC enrichment and entrapment for clinical diagnosis using liquid biopsies.

Figure 1.

Micrographs depicting the morphology of prostate cancer cell line LNCaP-C4–2.

Figure 2.

Illustration of high-throughput entrapment chip for CTC (CTC-HTECH).

Figure 3.

(a) Illustration of the configuration of the device (not to scale) with the inlet connected to programmable pressure pump; each row and each outlet was assigned and labeled individually; (b–g) the GFP+ LNCaP-C4–2 prostate cancer cells; (b) image of inlet with a GFP+ cell starting to enter row ①; (c) a GFP+ cell trapped in row ① after the blood flow ceased; (d) image of the waste collection at outlet 1; (e) a GFP+ cell deforming and passing through row ②; (f) two GFP+ cells in row ③ with one cell still passing and one cell exiting this row; (g) one GFP+ cell trapped in the trapping chamber of row ④; (h) the overall capture efficiency of each outlet. The data presented here is from three or four runs on CTC-HTECH device; (i) the percentage of the trapped GFP+ cells in every available row in the configuration where the indicated outlet was open (outlet 1 (green), outlet 2 (pink), outlet 3 (blue), outlet 4 (purple), outlet 5 (orange), and outlet 6 (red)).

Figure 4.

Number of cells captured compared to the number of cells spiked per 0.6−0.7 mL mouse whole blood in different outlet configurations. Results shown are the results testing n = 3−4 different devices at six different outlet configurations.

Figure 5.

Image of the control group mouse whole blood without cancer cells spiked (a,b), and with cancer cells spiked (c,d) through the CTCHTECH: (a) whole blood passing through the microchannels; (b) the inlet switched to LNCaP-C4–2 culture medium to remove the blood cells;(c) image of a trapped cancer cell after 15 min rinsing by LNCaP-C4–2 medium; (d) image of a microchannel with WBCs/lymphocytes trapped after 15 min of rinsing by LNCaP-C4–2 medium.