Enhanced Isolation and Release of Circulating Tumor Cells Using Nanoparticle Binding and Ligand Exchange in a Microfluidic Chip

Abstract

The detection of rare circulating tumor cells (CTCs) in the blood of cancer patients has the potential to be a powerful and noninvasive method for examining metastasis, evaluating prognosis, assessing tumor sensitivity to drugs, and monitoring therapeutic outcomes. In this study, we have developed an efficient strategy to isolate CTCs from the blood of breast cancer patients using a microfluidic immune-affinity approach. Additionally, to gain further access to these rare cells for downstream characterization, our strategy allows for easy detachment of the captured CTCs from the substrate without compromising cell viability or the ability to employ next generation RNA sequencing for the identification of specific breast cancer genes. To achieve this, a chemical ligand-exchange reaction was engineered to release cells attached to a gold nanoparticle coating bound to the surface of a herringbone microfluidic chip (NP-^HBCTC-Chip). Compared to the use of the unmodified ^HBCTC-Chip, our approach provides several advantages, including enhanced capture efficiency and recovery of isolated CTCs.

Figure 1

(A) Thickness changes with each surface modification step in the proof-of-principle studies comparing NeutrAvidin binding for NP-mediated and control silica substrates. (B) Thickness changes by NeutrAvidin detachment through ligand exchange as a function of GSH concentration for 30 min.

Figure 2

(A) Comparison of capture efficiencies and nonspecific binding of cancer cells. PC3 and MDA-MB-231 cells were spiked into whole blood and run through the original ^HBCTC-Chip and the NP-^HBCTC-Chip. Cells were captured on the surface of the microfluidic device functionalized with anti-EpCAM. Capture efficiency and nonspecific cell binding were quantified using a previously developed protocol. (B) Capture efficiency of MDA-MB-231 cells with different capture antibodies (cocktail is a combination of EpCAM, Her2, and EGFR). Combined fluorescent and bright-field microscopic images of viable (C) PC3 and (D) MDA-MB-231 cells isolated on the NP-^HBCTC-Chip (scale bar = 100 μm).

Figure 3

(A) Linear correlation (R² = 0.9947) was determined between the number of spiked and captured PC3 cells with NP-^HBCTC-Chip, with cancer cell lines spiked into whole blood at a concentration of 10, 50, 100, 200, 500, and 1000 (n = 3 for each condition). (B) Chip capture performance for ultralow concentrations of cancer cells (5 cells/mL of whole blood, n = 10). Each data point indicates the capture efficiency of an independent experiment for MB-MDA-231 and PC3 cells. Cell capture efficiency at such low cancer cell concentrations varied from 0 to 100%. For spiking concentrations of 1000, 500, and 200 cells per mL, a serial dilution of an initial 100 000 cells/mL was used. A micromanipulator equipped with a microneedle was used for spiking concentrations of 100, 50, 10, and 5 cancer cells/mL of whole blood.

Figure 4

(A) Release efficiency and cell viability of recovered PC3 and MDA-MB-231 CTCs using the NP-^HBCTC-Chip. After whole blood spiked with cancer cells was run, the NP-^HBCTC-Chip was washed with phosphate-buffered saline, and a solution of 1% bovine serum albumin with 1 mg/mL of GSH was incubated for 30 min. Bright-field microscopy images of isolated CTCs on the NP-^HBCTC-Chip (B) before and (C) 3 min after GSH treatment (scale bar = 150 μm). (D) Image of cultured individual CTCs 24 h after recovery from the chip (scale bar = 30 μm).

Figure 5

Immunofluorescence staining of cell-surface receptors of a captured (A) single CTC and (B) CTC cluster from a metastatic breast cancer patient. The images shown include EpCAM/CDH11 staining in Alexa Fluor 488 and DAPI nuclear staining in blue (scale bar = 10 μm). (C) Captured and released CTC counts from breast metastatic patients (Br1–Br4) and healthy controls (C1–C2).

Figure 6

Characterization of the patient-derived breast (Brx) CTC line using imaging flow cytometry. Data compare viability, EpCAM expression, and area of control versus captured/released cells from our NP-^HBCTC-Chip. Representative images of one viable, cluster, and dead Brx cell (A) obtained from culture (control) and (B) captured/released from our microfluidic device. Gate settings of (C) control and (D) captured/released Brx cells. Viable cells are defined as calcein positive and caspase 3/7 negative, whereas dead cells are caspase 3/7 positive. The intensity of EpCAM obtained from (E) control and (F) captured/released Brx cells. The area of (G) control and (H) captured/released Brx cells. (I) Heat map of the C_t values of seven genes obtained by RT-qPCR. Comparisons were across control, released Brx cells, and white blood cells (WBC).

Figure 7

Gene expression profiles for a representative set of breast-cancer-specific genes for CTCs isolated using the NP-^HBCTC-Chip. For each breast cancer patient, two processing conditions were analyzed: a control, on-chip, extraction of RNA from the captured CTCs, and a postrelease “R” extraction of RNA from the CTCs.

Scheme 1. Design of the NP-^HBCTC-Chip for the Capture and Release of CTCs^a

^aConditions: (a) Preparation of NHS-functionalized NPs with carboxylic acid (for enhanced NP solubility in EtOH) and NHS (for the NP immobilization and avidin binding) functional groups via ligand exchange with pentanethiol-functionalized NPs. (b) Schematic illustration of each surface modification process step involved in the fabrication of the NP-^HBCTC-Chip and CTC isolation on the chip and subsequent CTC release by ligand exchange with GSH.