Abstract
Background
Circulating tumor cells (CTCs) are detectable in peripheral blood of metastatic lung cancer patients. In this article, we evaluate a new CTC separation method based on a combination of anti‐EpCAM and immunomagnetic beads with the aim to detect CTCs more conveniently and specifically.
Methods
Lung cancer cells were magnetically labeled by anti‐EpCAM magnetic beads, and subsequently captured by magnetic separation using our novel device. Isolated lung cancer cells were identified by pathomorphological by hematoxylin–eosin staining protocol. The system was used to detect CTCs in 2 ml blood. Blood samples of healthy donors spiked with lung cancer cell line A549 cells were used to determine the sensitivity and specificity of the method. Prevalence of CTCs was examined in samples from 56 patients with lung cancer.
Results
Regression analysis of number of recovered versus spiked A549 cells yielded a coefficient of determination of R 2 = 0.996 (P < 0.001). The average recovery was 68% or more at each spiking level. The coefficient of variation increased as the number of spiked cells decreased, ranging from 6.4% (1,000‐cell spike) to 18.4% (50‐cell spike). Forty‐nine of the fifty‐six patients (87.5%) were found to have CTCs in peripheral blood. None of the 2 ml peripheral blood samples of the 20 healthy subjects analyzed were found to have CTCs.
Conclusions
This novel turbulence device provides a new tool allowing for feasible and specific detection of CTCs in lung cancer patients. It is likely clinically useful in diagnosis and monitoring of lung cancer and may have a role in clinical decision making.
Keywords: circulating tumor cells, anti‐EpCAM antibody, magnetic beads, lung cancer, immunocytochemical methods
INTRODUCTION
The circulating tumor cells (CTCs) in blood originate from both primary and metastatic lesions, which have the properties to migrate into the blood circulation. A small subset of CTCs probably has the capability to establish the metastatic growth after seeding in a niche tissue. CTCs present in the peripheral blood of patients provide a potentially accessible source for the detection, characterization, and monitoring of cancers. The concentration of CTCs in blood may be used as an intermediate biomarker for monitoring the therapy effect of the patients with cancer. Therefore, a variety of methods have been developed to detect these rare CTCs in blood circulation. As the half‐life of CTCs is less than 3 h 1 and the CTCs occur at very low concentration, in the background of millions of normal hematopoietic cells 2, their identification and capturing require extremely sensitive and specific methods. The detection of CTCs remains huge challenge in technique at present.
Immunocytochemical methods have been widely used for CTC detection. The choice of appropriate markers is a challenge due to scarce antigens exclusively expressed by CTCs while not shared by other circulating nontumor or blood cells. As an antibody specific to epithelial antigen, epithelial cell adhesion molecule (EpCAM) is the most widely used marker for epithelial tumor cell detection. The most commonly used immunocytochemistry methods include CellSearch and CTC‐Chip. CellSearch system, with high accuracy, precision, and reproducibility, is the only diagnostic test approved by the U.S. Food and Drug Inspection Agency. Using anti‐EpCAM–coated magnetic beads, CTCs can be extracted from blood, and further be fixed, stained, and manually counted. A validation study conducted by Riethdorf et al. 3 showed that CTC counts could remain stable for 72 h after blood sample collection, even at room temperature. Moreover, there was high reproducibility of CTC counts among different hospitals. Just for this reason, CellSearch technology was used in the majority of the earlier immunocytometric researches. In the United States, clinical trials for the CellSearch System have been performed in the patients with metastatic breast cancer (MBC), metastatic colorectal cancer (MCRC), and metastatic prostate cancer (MPC), confirming that CTC counts are a useful prognostic factor 4, 5, 6. However, this semiautomated technology has some inherent issues, such as complicated sample processing and limited sensitivity. In addition, the expensive cost also limits its popularization.
Another innovative and exciting microfluidic technology, a microfluidic device—the “CTC‐Chip,” has recently been developed and applied for CTC detection, which can effectively and selectively separate the viable CTCs from whole blood samples 7, 8, 9. The chip consists of microscopic posts, which are etched in silicon and coated with specific antibodies to epithelial surface antigens. When whole blood sample flows through the chip, the flow kinetics with minimal shear stress on cells enhances the contacts with the microposts coated by antibodies. Once epithelial CTCs are captured, they can be further visualized by staining with antibodies against cytokeratin or tissue‐specific markers. Using this method, Nagrath et al. 8 successfully isolated the CTCs from the samples of 99% (115/116) of patients, with a range of 5–1,281 CTCs per milliliter and approximately 50% of purity. Thus, the CTC‐Chip provides a new and effective tool to accurately identify and measure the CTCs from the patients with cancer, which has been widely applied in the cancer biology research and clinical cancer management, including the detection, diagnosis, and monitoring of cancers 10.
Recently, a novel method to concentrate rare CTCs has been developed in our lab, which has high reproducibility, standardized operation, and low cost. It makes cell suspension form a bidirectional flow with a shaker, and with the help of a plane magnet and magnetic beads coated by antibodies, the CTCs are retained. In order to reduce the contamination of leukocytes and the loss of CTCs, we make wooden box locate at a 15° slope and make phosphate buffer solution (PBS) solution flow from the bottom of the box. An important advantage of the method is that CTCs can be concentrated on a piece of glass slide, which is helpful to further count the number of CTCs and analyze the cell morphology.
Specifically, we plan to use this novel device to capture CTCs from the patients with lung cancer. Despite recent progress in treatment, lung cancer remains the leading cause of cancer deaths in both women and men throughout the world 6. Not all patients with lung cancer benefit from routine surgery and chemotherapy. However, lung cancer metastasis has already occurred in patients at diagnosis when it is too late to perform surgical treatment. Even though the patients have the opportunity to receive surgical treatment, their prognosis is poor, often with local recurrence and distant metastasis. Of the recurrences, Kotlyarov et al. 11 reported that the focal recurrence rate was 33.8%, and the rate of blood‐born metastasis was 55.2% during the 10 years after radical resection. Furthermore, 69.4% of focal recurrence and 88.7% of blood‐born metastasis occurred within 1 year after surgery [7]. Therefore, searching a valuable tool to diagnose early‐stage cancer and assess tumor invasion is necessary. CTCs have been a marker for the diagnosis of tumor metastasis during the pre‐ or postoperative period since it was found by Ashworth in 1869 12. Researchers have developed a variety of methods to capture CTCs, and mostly applied to capture MBC, MCRC, or MPC. Although the detection of CTCs in lung cancer patients has been described, the related reports are relatively rare. Farace et al. 13 enumerated the numbers of CTCs in patients with metastasis carcinomas of breast (55%), prostate (60%), and lung (20%) origins using the CellSearch system, indicating that the system was limited to capture CTCs, especially in the patients with metastasis lung cancer. Herein, we use the homemade device for the first time to detect the CTCs from the patients with lung cancer based on the magnetic beads coated by anti‐EpCAM antibodies (Miltenyi Biotec, Germany). We investigated the enrichment efficiency of the device and found that the target cells were obtained from 87.5% (49/56) of lung cancer patients.
MATERIALS AND METHODS
Cell Culture
Human pulmonary adenocarcinoma cell line A549 was a generous gift from the University of Science and Technology of China. The cells were cultured in RPMI 1640 (Invitrogen, America) medium (Invitrogen) supplemented with 10% of fetal bovine serum (heat inactivated, GIBCO, America) and 100 U/ml of penicillin and streptomycin, respectively, at 37℃ in an atmosphere of 5% CO2 under bacteria‐free condition with a passage per 2–3 days. Before the experiments, cells were digested by trypsin (Sigma, America) and resuspended with PBS.
Clinical Specimens
Fifty‐six patients with primary nonsmall cell lung cancer (NSCLC) admitted to our hospital between August 2012 and February 2013 were enrolled in this study, including 32 males and 24 females, aged from 38 to 80 with an average age of 68. The grades of tumors were as follows: 19 cases for stageⅠ, 17 for stage Ⅱ, 14 for stage Ⅲ, and 6 for stage Ⅳ. All the patients were confirmed to have NSCLC after surgical inspection, and NSCLC was staged according to the Tumor, Node, and Metastasis (TNM) system. All the patients had no other epithelial tumors and infectious diseases before diagnosed as NSCLC. Additionally, 20 healthy volunteers, including 11 males and 9 females, with an average age of 37 (24–63 years), were served as controls.
Blood Sample Preparation
Two milliliters of blood sample was drawn from each subject and collected into BD vacuum tube containing sodium heparin. After standing for 3 min, 2 ml of anticoagulated peripheral blood sample was centrifuged at 460 × g for 7 min. Then, the supernatant was removed, and the nucleated cells were isolated using RBC lysis buffer. Briefly, 2 ml of blood sample was mixed with 10 ml of RBC lysis buffer, kept at room temperature for 15 min, and then centrifuged at 460 × g for 7 min. The obtained cells were suspended with 5 ml of RBC lysis buffer and centrifuged at 460 × g for 7 min again, followed by rinsing with PBS twice.
Device Fabrication and Operation
The new type of testing device is composed of four main parts: a peristaltic pump (B), a shaker and a homemade separate container (C), and a minitable type of vacuum pump (D, Fig. 1). The peristaltic pump is connected to the centrifuge tube full of 50 ml of PBS (A), which can wash away leucocytes in the separate container. The homemade separate container consists of an inclined support, a powerful magnet, and a box with a hydrophobic surface inside, and its bottom is equipped with a standardized glass slide.
Figure 1.
Design and operation of a microfluidic device to separate and enrich the CTCs. (A) The centrifuge tube full of 50 ml of PBS. (B) The peristaltic pump draws PBS to the separate container for washing away leucocytes. (C) There is a support with 15° angle under the separate container, which makes the glass slide set up a gradient to wash away leucocytes. There is a shaker under the separate container to keep cell suspension bidirectional flow, and prevent from more leucocytes adhering to the glass slide. (D) The vacuum pump was connected to a speed regulator to draw the fluid from the box with constant speed. (E) PBS containing leucocytes was pumped out from the box.
Following the manufacturer's instructions, the obtained cells were first incubated with the magnetic beads coated by anti‐EpCAM antibodies (Miltenyi Biotec) at 4℃ for 30 min. Then, the cell suspension was injected into the box, and the switches of the shaker (40 rpm), peristaltic pump (flow rate: 10 × 10 ml/h), and the minitable type of vacuum pump were in turn turned on.
Hematoxylin–Eosin Staining
The obtained cells were captured on a glass slide and stained as follows. First, the slide was immersed in 95% of ethanol for 15 min, rinsed with distilled water for 3 min, stained with hematoxylin for 10 min, washed in tap water until the fluid clear, immersed in warm water (50℃) for 5 min, and washed in tap water again. Then, the slide was stained with eosin for 2 min, dehydrated in an ascending concentration of ethanol (75%, 95%, 100%), and cleared with xylene for two to three times. Finally, the slide was fixed with neutral balata (Shanghai, Chian) and observed under the microscope.
Determination of Recovery Rate of A549 Cells
To investigate the sensitivity of the CTC detection method, peripheral blood samples from five healthy volunteers were collected and spiked with different concentrations of human lung adenocarcinoma A549 cells (50, 100, 200, 500, and 1,000 cells). Then, the peripheral blood samples were processed according to the sample preparation protocol. Three microliters of magnetic beads with anti‐EpCAM antibodies were added into each specimen. The obtained suspension was mixed for 30 min at 4℃, and then separated using the above‐mentioned device. Enriched tumor cells on the glass slide were air‐dried and stained with hematoxylin–eosin (HE) dye (Hubei, China). After fixed with neutral balata, the cells on the slide were evaluated by light microscopy (40× objective). The procedures were repeated five times for each concentration of A549 cells and their recovery rates were calculated.
Novel Device for the CTC Detection of Lung Cancer Patients
Two milliliters of blood samples were obtained from all patients with lung cancer. As described previously, the blood samples were subjected to a red‐cell lysis step to isolate peripheral blood nucleated cells. The obtained nucleated cells were suspended with PBS, followed by the addition of 3 μl of magnetic beads with anti‐EpCAM antibodies. The mixture was rotated and mixed for 30 min at 4℃ on a shaker. Then, the separation, staining, and observation of CTCs were performed as the above‐mentioned procedures.
Optimization of the Device
In order to improve the recovery rate of CTCs and reduce the contamination of leukocytes, we optimized this device. The flow rate of peristaltic pump was fixed at 10 × 10 ml/h to be consistent with the speed of the vacuum pump, which could keep the balance between the inlet and outlet of PBS in the wooden box. The vibrating speed of the shaker of the device was divided into four, 20, 40, 60, and 80 rpm, and the inclined angle of the wooden box included 5°, 15°, and 25°. In order to select the optimal parameters of the device, 100 A549 cells were spiked into 2 ml of peripheral blood sample of one healthy volunteer and separated under different conditions of parameters. The control for flow velocity is very important. The rapid flow of the transport channels can keep CTCs from adhering to the glass slide, while the low velocity may lead to more leucocytes retained. By multiple tests, the optimal velocity was selected.
RESULTS
Identification of CTCs
Before the experiment, the cytoplasm of A549 cells was strongly stained blue‐violet and their nucleus was intact. After the CTCs were reacted with immunomagnetic beads, their morphology was significantly changed (Fig. 2). The size of A549 cells became large, the cytoplasma was stained pink or uneven, and the irregular nucleus showed fine granula.
Figure 2.
Images of A549 cells stained by HE (optical microscope, 40×). (A and B) A549 cells before the experiment; (C) A549 cells incubated with immunomagnetic beads with anti‐EpCAM antibodies; (D) A549 cells and leukocytes.
CTC Numbers Detected From the Optimized Device
The number of detected CTCs was presented in Table 1. When the vibrating speed of the shaker was 60 or 80 rpm and the inclined angle of the wooden box was 25°, the detected CTCs’ number was low. However, the results of detected CTCs’ counts from the other four groups (20 rpm and 5°, 20 rpm and 15°, 40 rpm and 5°, and 40 rpm and 15°) were similar to each other. After the CTCs were stained with HE, we found that there was significant difference of leukocyte counts among the four groups (Fig. 3). There were many leukocytes in the three groups, except the 40 rpm and 15° group. Therefore, we selected 40 rpm and 15° as the optimal parameters for the detection of CTCs.
Table 1.
CTC Counts Under Different Conditions of Parameters
| CTC count | ||||
|---|---|---|---|---|
| Vibrating speed of the shaker (rpm) | ||||
| Angle of the wooden box | 20 | 40 | 60 | 80 |
| 5° | 86 | 82 | 77 | 63 |
| 15° | 85 | 80 | 70 | 54 |
| 25° | 78 | 71 | 59 | 40 |
The vibrating speed of the shaker was divided into four, 20, 40, 60, and 80 rpm, and the inclined angle of the wooden box included 5°, 15°, and 25°. The number of CTCs was counted under the different conditions of parameters.
Figure 3.
Comparison of detected leukocyte counts under the different conditions of parameters (optical microscope, 10×). (A) The vibrating speed of the shaker is 20 rpm and the inclined angle of the wooden box is 5°. (B) The vibrating speed of the shaker is 40 rpm and the inclined angle of the wooden box is 5°. (C) The vibrating speed of the shaker is 20 rpm and the inclined angle of the wooden box is 15°. (D) The vibrating speed of the shaker is 40 rpm and the inclined angle of the wooden box is 15°.
Recovery Rate, Accuracy, and Specificity of CTC Detection
The results of the recovery tests, which were performed by spiking different amounts of A549 lung cancer cells into blood samples of healthy volunteers, were summarized in Table 2. The recovery rates of A549 cells enriched with immunomagnetic beads ranged from 68% to 82%. The more the number of added spiked tumor cells was, the more the number of recovered tumor cells was (R2 = 0.996, P < 0.001; Fig. 4). As expected, the coefficients of variation (CVs) increased with the reduction of spiked cells, ranging from 6.4% (1,000 spiked cells) to 18.4% (50 spiked cells). In addition, none of the peripheral blood samples from 20 healthy volunteers were found to have CTCs.
Table 2.
Recovery Rates of the Spiked A549 Cells
| No. of detected A549 cells | Recovery% | |||||
|---|---|---|---|---|---|---|
| No. of spiked A549 cells | Mean | SD | 95% CI | Mean | 95% CI | CV% |
| 50 | 36 | 7 | 30–41 | 71 | 68–74 | 18 |
| 100 | 82 | 12 | 72–92 | 82 | 77–87 | 14 |
| 200 | 149 | 14 | 137–162 | 75 | 68–81 | 10 |
| 500 | 368 | 27 | 345–391 | 74 | 62–86 | 7 |
| 1,000 | 684 | 44 | 645–722 | 68 | 49–88 | 6 |
The recovery tests were performed by spiking different amounts of A549 lung cancer cells into blood samples of healthy volunteers.
Figure 4.
Recovery analysis of spiked A549 cells into whole blood samples of healthy volunteers. Different amounts of A549 cells (50, 100, 200, 500, and 1,000 cells) were spiked into 2 ml of blood samples from 20 healthy volunteers. The number of spiked cells (x‐axis) was plotted versus the number of the detected cells (y‐axis).
Detection of CTCs in the Patients With Lung Cancer
The results of CTC detection from 56 patients with lung cancer were listed in Table 3. The number and percentage of patients with 1–5%, 6–10%, and above 10% CTCs in 2 ml of blood samples were displayed. CTCs were detected in the blood samples from 87.5% (49/56) of patients with lung cancer. The positive rate of CTCs was highly correlated with the TNM staging, from 62.5% in stage Ⅰ to 100% in stage Ⅳ, and the number of CTCs detected was also highly correlated with TNM staging, from 3 ± 2 in stage Ⅰ to 13 ± 6 in stage Ⅳ. All the CTCs captured from the samples of the patients with lung cancer were identified according to the introduction of Figure 2C and D and Figure 5.
Table 3.
Detail Information for 56 Patients With Lung Cancer and Their CTC Counts in Peripheral Blood Samples
| Percentage of CTCs in all nucleated cells | ||||||
|---|---|---|---|---|---|---|
| Group | Number of patients | Positive CTCs (n (%)) | 1–5 | 6–10 | >10 | Mean ± SD |
| Tumor diameter (cm) | ||||||
| <3 | 16 | 13 (81.2) | 4 (25.0) | 8 (50.0) | 1 (6.2) | 7 ± 4 |
| 3–7 | 21 | 19 (90.5) | 6 (28.6) | 8 (38.1) | 5 (23.8) | 9 ± 5 |
| >7 | 19 | 17 (89.4) | 2 (10.5) | 5 (26.3) | 10 (52.6) | 13 ± 6 |
| TNM | ||||||
| Stage Ⅰ | 8 | 5 (62.5) | 4 (50.0) | 1 (12.5) | 0 (0) | 3 ± 2 |
| Stage Ⅱ | 13 | 11 (84.6) | 5 (38.5) | 4 (30.7) | 2 (15.4) | 7 ± 4 |
| Stage Ⅲ | 15 | 13 (86.7) | 3 (20.0) | 8 (53.3) | 2 (13.4) | 9 ± 4 |
| Stage Ⅳ | 20 | 20 (100) | 0 (0) | 8 (40.0) | 12 (60.0) | 13 ± 6 |
| Total | 56 | 49 (87.5) | 12 (21.4) | 21 (37.5) | 16 (28.6) | 10 ± 6 |
Fifty‐six lung cancer patients were classified by the maximal tumor diameter and the TNM category.
Figure 5.
CTCs isolated from the peripheral blood samples of the patients with lung cancer by immunomagnetic beads separation (optical microscope, 40×). Three images displayed the captured CTCs and normal leukocytes from the same sample.
DISCUSSION
The poor long‐term survival rate in the patients with lung cancer was related to the spread of tumor cells, which led to the recrudescence of cancer at distant sites or the patient's death. Therefore, the detection of occult metastatic cells was important to predict the recurrence of the cancer and improve its survival. Common technologies for detecting lung cancer metastasis included immunohistochemistry and RT‐PCR. Immunohistochemistry could provide the morphological detail of tumor cells but was not sensitive and lacked methodological standardization 14. Although RT‐PCR was able to find one cancer cell among 106 irrelevant cells 15, it could not exactly quantify the number of tumor cells according to the mRNA levels because mRNA was free in blood. Furthermore, its utility was limited for its low specificity and false‐positive result, which may be explained by the phenomenon of “illegitimate expression” 16. Here, we provide a new method to separate CTCs based on the magnetic cell separation with anti‐EpCAM antibody. EpCAM is a cell‐surface protein with oncogenic features, which is highly expressed in a variety of adenocarcinomas with different origins, such as breast, ovary, colon, and lung. However, the expression of EpCAM is limited in normal tissues 17. In this study, immunomagnetic particles were combined with anti‐EpCAM antibody, which can specifically bind with EpCAM, a specific marker of lung cancer cells 18.
In contrast to other approaches to detect the CTCs, the method established by us was simple, rapid, and low‐cost. First, the device was simple, and its components, such as peristaltic pump, shaker, special container, vacuum pump, and optical microscope, were all commonly used in laboratories, unlike those specialized equipments such as special preservation tube, laser, chip, and analyzer. Second, the cost was very low, because all the reagents used in the method, such as standard slide, PBS buffer, and HE dye, were common‐used supplies. Third, the whole operation process was simple and there were no tedious steps. Above all, the detection results of the method were reliable. HE staining was the gold standard for distinguishing tumor cells from normal cells, so it was used to identify the CTCs in our experiment to increase the reliability of the results. In order to improve the enrichment efficiency of the CTCs, the device was designed with a high surface area to volume ratio, which could increase the collision chance of the target cells with the capture surface. It was shown that a whole glass slide combined with a magnet in the bottom of the special container could quickly and efficiently enrich tumor cells from a background of irrelevant cells.
Usually, detecting the CTCs in peripheral blood required 7.5 ml of blood samples 13, 19, 20. However, the established method in this study only required 2 ml of blood samples, which means it was easier to gather informed consent from the patients and volunteers, and that the blood sample from the same patient could be detected repeatedly.
After A549 cells were spiked into 2 ml of blood samples, their recovery rates were higher than 68% when detected by the established method in this study. In addition, when only 50 A549 cells were spiked into 2 ml of blood samples, at least 27 A549 cells could be detected, indicating the high sensitivity of the method. Moreover, no positive result was obtained from the blood samples of 20 healthy volunteers, indicating 100% of specificity of the method. Further, we determined the peripheral blood samples from 56 patients with lung cancer at various stages 21, and the results showed that the CTCs could be detected in 87.5% of patients with lung cancer, even in patients at early stage or in patients with tumor size of less than 3 cm. The positivity rate and the number of CTCs detected were positively correlated with the disease extent as classified by the TNM category.
In conclusion, we have established a simple method to detect the CTCs in the patients with lung cancer. Although its sensitivity does not seem as high as RT‐PCR, its specificity and accuracy are encouraging. As described above, the detection of CTCs has important implications in the diagnosis, prognosis, and therapy of lung cancer. The novel device may have considerably potential clinical value and further clinical trials are warranted.
CONFLICT OF INTEREST
None.
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