High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood

<div><p>Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%–90.0%) and excellent linear performance (R² = 0.8189–0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM− tumor cells. Isolation of single captured tumor cells, followed by detection of <i>EGFR</i> mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.</p></div

Effects of DMOG treatment on postoperative angiogenesis and vasculature in mouse subdermal plexus.

<p>A, Sections of proximal parts of the skin flaps near the pedicles were stained with hematoxylin and eosin (HE) and with anti-CD31 antibody (arrowheads). To evaluate the effect of DMOG on the neovascularization in the proximal part of the flap, the number of CD31-positive (green) vessels was counted and is indicated as vessel density per high-power field. Scale bar indicates 200 µm. B, Intravital microscopic analysis of the vasculature of the subdermal plexus in DMOG-treated mice. After the intraperitoneal injection of DMOG, the subdermal plexus visualized by intravenous injection of FITC-dextran was observed in the dorsal skin-fold chamber by fluorescence microscope. Fluorescent intensity at cross-section of the line 1 showed that the number of detectable vessels was increased 48 hours after DMOG treatment, compared to the untreated control (Indicators of arrowheads in the graph represent the detectable vessels). Magnified images of square frame line 2 and 3 showed that vasodilation of preexisting vessels observed in the border zone of the vascular territory. Yellow arrowheads indicate the relative locations in the vasculature as the landmarks, and white arrowheads the detectable vessels in the border zone of the vascular territory 48 hours after DMOG treatment. Scale bar indicates 250 µm.</p

Effects of DMOG pretreatment on angiogenesis-related factors in the mouse skin flap model.

<p>A, Serum VEGF concentrations before and after surgery on DMOG-treated or untreated mice were measured by ELISA. Pre-OP and Day 3 indicate pre-surgery and on day 3 after the surgery, respectively. B, VEGF proteins in the skin tissues before and after surgery were measured by ELISA. Skin tissues from the proximal, distal, and lateral parts of the skin flaps were prepared for the assay. C and D, Transcript levels of HIF-1 target genes (C) and the expression of FLK-1 proteins (D) in the proximal and distal parts of the skin flaps on postoperative day 1 were assessed by quantitative RT-PCR and Western blot analysis, respectively. N.D. indicates not detectable. Values are means ± SDM. *<i>P</i><0.05.E, Left panel, the representative flow cytometric profiles of peripheral blood cells from DMOG-treated or untreated mice. Peripheral blood cells were stained with anti-CD133, anti-CD45, anti-CD34, and anti-CD31 antibodies and analyzed with a flow cytometer. Pre-OP and Post-OP indicate before the flap operation and 1 day after the flap operation, respectively. Data were representative of at least 4 independent experiments. Right panel, summary of the ratio of the EPCs (gated on CD45-positive cells) in peripheral blood cells. F, Cell proliferation in bone marrow cells (gated on CD45-positive and CD34-positive cells) from untreated or DMOG-treated mice 1 and 2 days after the treatment was monitored by BrdU incorporation and analyzed by flow cytometry. *<i>P</i><0.05, ***<i>P</i><0.001.</p

Effect of DMOG pretreatment on expanding survival area of ischemic skin flaps in mice.

<p>A, BALB/c mice were randomly assigned to the control group or the experimental group with intraperitoneal DMOG pretreatment (400 mg/kg body weight), followed by evaluation of flap survival on postoperative day 7. B, Design of ischemic random pattern skin flap and vascular distribution on the mouse dorsum. The flap was designed not to include any major pedicles arising from the deep circumflex iliac vessels and lateral thoracic vessels (white arrows). The distal parts of the flaps exhibited tissue necrosis due to disruption of the blood supply. C, Percentages of the survival area of the flap and dividing by the total area of the flap. Representative ischemic flaps show grossly better flap survival in the DMOG group. D, Effects of timing of DMOG pretreatment on the expansion of flap survival. DMOG pretreatment was performed 2 days or 1 day before the surgery or on the day of the surgery. E, Effect of dose of DMOG pretreatment on the expansion of flap survival. DMOG pretreatment was performed 2 days prior to surgery. The percentages of flap survival ± SEM were measured on postoperative day 7. *<i>P</i>>0.05; ***<i>P</i>>0.001.</p

Plots of the Number of Detected Cells against the Number of Spiked Cells in the Spike-in Experiments.

<p>Cultured tumor cells (SK-BR-3 (breast), PC-9 (non-small cell lung), PC-14 (non-small cell lung), H69 (small cell lung), and SBC-3 (small cell lung)) were separately spiked, in numbers of 10, 100, and 1000, into 3 mL of blood obtained from healthy donors. After enrichment of mononucleated cells from the blood, the cells were entrapped in microwells, followed by immunofluorescent staining with DAPI, chromophore conjugated anti-CK mAb, and anti-CD45 mAb. Then, fluorescent images of the entire area of the microwell array were captured, and the numbers of tumor cells were measured (solid circles). For PC-14, H69, and SBC-3, a further staining protocol, using Alexa Fluor 488 conjugated secondary antibody, was applied for amplification of the weak CK signal (diamonds). The circles and diamonds represent individual data points. The straight lines are the linear fittings, with their slopes and correlation coefficients (R²) given on the plots. Dotted lines represent the function y = x. The plots of spiked tumor cell numbers from 0 to 100 are magnified for easier viewing (logarithmic axes).</p

Effect of bone marrow cell expression of HIF-1α on skin flap survival.

<p>A, Percentages of skin flap survival were calculated in WT and HET mice reconstituted with bone marrow from either WT or HET donor mice. Values are means ± SEM. *<i>P</i><0.05, ***<i>P</i><0.001 vs. WT reconstituted with bone marrow from WT mice. ^#<i>P</i><0.05, ^##<i>P</i><0.01 vs. HET reconstituted with bone marrow from HET mice. B, Transcript levels of HIF-1 target genes were assessed by qRT-PCR analysis at the ischemic skin flap on postoperative day 1 in bone marrow cell-transplanted mice (n = 6). Values are means ± SDM. *<i>P</i><0.05.</p

Expression of HIF-1α protein in the skin after intraperitoneal administration of DMOG.

<p>Immunoblot analyses were performed on tissue lysates from dorsal skin taken preoperatively (A and C) and from the proximal and distal parts of the skin flaps on postoperative day 1 (B and C). In panel A and C, HIF-1α protein expression in the skin is shown before (Pre) and at 4, 12, and 48 hours after DMOG treatment. *<i>P</i><0.05 and **<i>P</i><0.01 compared with Pre. In panel B and C, *<i>P</i><0.05 and **<i>P</i><0.01 compared with the untreated mice. β-Actin was used as a loading control.</p

Effects of DMOG pretreatment on apoptosis in the mouse skin flap model.

<p>A, The apoptotic cells in the proximal and distal parts of the skin flaps taken on postoperative day 1 from mice with or without DMOG pretreatment were detected by TUNEL staining. Sections of the skin flaps were stained for TUNEL (green) and Hoechst (red). Scale bar indicates 100 µm. B, The ratio of TUNEL-positive cells to cell nuclei in the sections was calculated. Values are means ± SEM. ***<i>P</i><0.001. C and D, Evaluation of expression of apoptosis-related factors in BALB/c mice. HK2, BCL2, and BAX protein expression levels in the proximal and distal parts of the skin flaps harvested on postoperative day 1 were detected by immunoblotting of protein extracts.</p

Effects of loss of HIF-1α function on flap survival.

<p>A, Representative ischemic skin flaps from wild-type (WT), heterozygous HIF-1α deficient (HET), and HET with DMOG pretreatment on postoperative day 7. B, Evaluation of the expression of HIF-1α and HIF-2α proteins in the proximal and distal parts of ischemic skin flaps on postoperative day 1. C, Measurement of EPCs in peripheral blood on postoperative day 1. DMOG pretreatment increased the numbers of EPCs in both WT and HET mice. The percentage of the peripheral blood cells constituted by the EPC population was measured as the percentage of CD45⁺-gated cells positive for the indicated cell surface markers by flow cytometry. D, The apoptotic cells in the distal parts of the skin flaps taken from WT and HET mice with or without DMOG pretreatment on postoperative day 1 were detected by TUNEL staining. Sections of the skin flaps were stained for TUNEL (green) and Hoechst (red). The ratio of TUNEL-positive cells to cell nuclei in the sections was calculated. Values are means ± SEM. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Scale bar indicates 50 µm.</p

Typical Examples of Captured Images of Tumor Cells and White Blood Cells in the Spike-in Experiments.

<p>Pictured are tumor cell-enriched mononucleated cells after immunofluorescent staining with DAPI, CK-FITC, or CD45-PE. Fluorescent images of the respective wavelengths (for DAPI, FITC, and PE) over the entire area of the microwell array were captured. Tumor cells were defined according to the criteria DAPI+ and CK+ and CD45–, while the white blood cells were defined as DAPI+ and CK–and CD45+. (a) An example of captured images of a tumor cell and white blood cells in the spike-in experiments. The SK-BR-3 cell line was spiked into blood, followed by serial procedures. The cell indicated by the solid arrow was defined as a tumor cell, while those indicated by dotted arrows were defined as white blood cells. The images were captured with a 10× objective lens. (b) An example of captured images of debris and white blood cells in the spike-in experiments. Bright-field image cells with black filling (solid arrow) were defined as debris, while the dotted-arrowed cells were defined as white blood cells. The images were captured with a 4× objective lens.</p