Prognostic impact of circulating tumor cells detected with the microfluidic "universal CTC-chip" for primary lung cancer.

Detecting rare circulating tumor cells (CTCs) in the bloodstream is extremely challenging. We had previously developed a novel polymeric microfluidic device, "CTC-chip," for capturing CTCs and have shown high capture efficiency in lung cancer cell lines by conjugating Abs against epithelial cell adhesion molecules (EpCAM). This study aimed to optimize the EpCAM-chip and clarify the prognostic impact of CTCs in lung cancer patients. Of 123 patients with pathologically proven lung cancer, both progression-free survival (P = .037) and cancer-specific survival (P = .0041) were predominantly poor when CTCs were detected before treatment. After classification into surgical and chemotherapy groups, progression-free survival was worse in CTC-positive patients in both groups (surgery, P = .115; chemotherapy, P = .012), indicating that the detection of baseline CTCs is a risk factor for recurrence and progression. Furthermore, we recovered captured CTCs using micromanipulators and undertook mutation analysis using PCR. Thus, the EpCAM-chip is a highly sensitive system for detecting CTCs that contributes to the prediction of recurrence and progression and enables genetic analysis of captured CTCs, which could open new diagnostic, therapeutic, and prognostic options for lung cancer patients.

Novel circulating tumor cell-detection chip combining conventional podoplanin and EGFR antibodies for all histological malignant pleural mesothelioma.

In our previous study, a microfluidic system was developed based on podoplanin detection for capturing circulating tumor cells (CTCs), derived from malignant pleural mesothelioma (MPM). However, non-epithelioid MPM shows low podoplanin protein expression compared with that in epithelioid MPM; thus, some CTC populations may be missed. To overcome this limitation, a new CTC-detection chip was developed by combining the conventional podoplanin antibody (clone: NZ-1.2) with an epidermal growth factor receptor (EGFR)-targeted antibody (cetuximab). The cell-capture efficiency of the Cocktail-chip reached 100% in all the histological MPM cell lines. The median CTC-counts from 19 patients with MPM (epithelioid/non-epithelioid: 10/9) with the NZ-1.2- and Cocktail-chips were 1 and 3 (P=0.311) in 1 ml peripheral blood, 1.5 and 2 (P=0.332) in epithelioid MPM, and 1 and 3 (P=0.106) in non-epithelioid MPM, respectively. Overall, the Cocktail-chip showed an improved ability to detect more CTCs in patients with non-epithelioid MPM compared with that in the conventional NZ-1.2-chip, showing non-significant, but higher CTC detection. Furthermore, CTC-counts, determined using the Cocktail-chip were significantly correlated with the clinical stage of non-epithelioid MPM. In epithelioid MPM, the Cocktail-chip achieved a CTC-detection efficiency equivalent to that in the conventional NZ-1.2-chip. The Cocktail-chip enabled sensitive CTC detection of all histological MPM, including the non-epithelioid subtype, which may provide a foundation for the diagnosis, treatment, and prognosis of MPM progression.

Using the polymeric circulating tumor cell chip to capture circulating tumor cells in blood samples of patients with colorectal cancer.

The current study clarified the accuracy of a circulating tumor cell (CTC) detection system to diagnose colorectal cancer using blood samples. The system uses the 'polymeric CTC-chip,' (CTC-chip), which is a microfluidic device that is used for CTC isolation. CTCs are considered sensitive diagnostic biomarkers. However, their concentration in the peripheral blood is low and requires highly sensitive and specific capturing techniques. The capture efficiency of the polymeric CTC-chip was first assessed using cell suspensions of the colorectal cancer cell line HCT-116, which was reported as 90.9% in a phosphate-buffered saline suspension and 65.0% in the blood. The CTC-chip was then used to detect CTCs in blood samples obtained from 13 patients with stage II-IV colorectal cancer. On average, the CTCs/ml was lower in patients with stages II and III colorectal cancer (3.3±2.3) than in those with stage IV (7.0±6.2). In patients with stages II-IV, 92% had ≥1 CTC per ml, which was significantly higher than the positive rate (15%) detected using the carbohydrate antigen 19-9 test (CA19-9). Furthermore, CTCs were detected in all patients with stage II and III colorectal cancer, including a number of patients with negative results for the carcinoembryonic antigen (CEA) and CA19-9 tests. With the polymeric CTC-chip detection system, CTCs can be effective cancer markers, particularly for patients with stage II and III colorectal cancer who often exhibit negative conventional serum marker test results. The CTC-chip system may also facilitate the detection of cancer progression based on CTC concentration.

Detection of circulating colorectal cancer cells by a custom microfluid system before and after endoscopic metallic stent placement.

Although the detection of circulating tumor cells (CTCs) should be crucial for future personalized medicine, no efficient and flexible methods have been established. The current study established a polymeric custom-made chip for capturing CTCs with a high efficiency and flexibility. As an example of clinical application, the effects of self-expandable metallic stent (SEMS) placement on the release of cancer cells into the blood of patients with colorectal cancer and bowel obstruction were analyzed. This was assessed as the placement of SEMS may cause mechanical damage and physical force to malignant tissue, increasing the risk of cancer cell release into the bloodstream. The present study examined the number of CTCs using a custom-made chip, before, at 24 h after and at 4 days after SEMS placement in patients with colorectal cancer. The results revealed that, among the 13 patients examined, the number of CTCs was increased in three cases at 24 h after SEMS placement. However, this increase was temporary. The number of CTCs also decreased at 4 days after stent placement in most cases. The CTC chip of the current study detected the number of CD133-positive cancer stem-like cells, which did not change, even in the patient whose total number of CTCs temporarily increased. The results indicated that this custom-made microfluid system can efficiently and flexibly detect CTCs, demonstrating its potential for obtaining information during the management of patients with cancer.

Initial detection of circulating tumor cells from metastatic prostate cancer patients with a novel small device.

BACKGROUND: Various devices for isolating and detecting circulating tumor cells (CTCs) have been developed, whereas the CellSearch® system has been clinically used in numerous prostate CTC studies. CTCs might become more useful surrogate markers of prostate cancer, and they should be measured in all settings, but a smaller, low-cost CTC capture system is required. METHODS: An inexpensive and highly sensitive microfluidic CTC-capture polymeric chip, developed by the Toyama Industrial Technology Center, as described in the following text, was used to assess the number of CTCs from patients with metastatic prostate cancer. After verifying that cultured human prostate cancer cells (PC3 and LNCaP) could be captured with the chip coated with anti-epithelial cell adhesion molecule (CD326) antibody, whole blood samples of 14 patients with prostate cancer were screened. RESULTS: The average capture efficacy of PC3 cells was 94.60% in phosphate-buffered saline (PBS) and 83.82% in whole blood. The average capture efficacy of LNCaP cells was 82.73% in PBS and 75.78% in whole blood. CTCs were detected by the chip device in all 14 patients with metastatic prostate cancer using 2-mL blood samples. Although fewer CTCs were detected in patients with oligometastases, all patients with multiple distant metastases had CTCs. The average CTC count was 48 cells/mL (range 1-81 cells/mL). CONCLUSION: This CTC-chip will be able to capture CTCs and be useful to check CTCs as a surrogate marker in prostate cancer with smaller samples and lower cost in any small institution.

Detection of circulating tumor cells with a novel microfluidic system in malignant pleural mesothelioma.

Detection of rare tumor cells circulating in the blood (CTCs) presents technical challenges. CellSearch, the only approved system for clinical use, fails to capture epithelial cell adhesion molecule-negative CTCs such as malignant pleural mesothelioma (MPM). We have developed a novel microfluidic device (CTC-chip) in which any Ab to capture CTCs is conjugated. The CTC-chip was coated with an Ab against podoplanin that is abundantly expressed on MPM. Circulating tumor cell-detection performance was evaluated in experimental models in which MPM cells were spiked in blood sampled from a healthy volunteer and in clinical samples drawn from MPM patients. The CTC-chip showed superior CTC-detection performance over CellSearch in experimental models (sensitivity, 63.3%-64.5% vs 0%-1.1%; P < .001) and in clinical samples (CTC-positivity, 68.8% vs 6.3%; P < .001). A receiver operating characteristic (ROC) analysis showed that the CTC test provided a significant diagnostic performance in discrimination of unresectable disease from resectable disease (area under the ROC curve, 0.851; P = .003). The higher CTC count (≥2 cells/mL) was significantly associated with a poor prognosis (P = .030). The novel CTC-chip enabled sensitive detection of CTCs, which provided significant diagnostic and prognostic information in MPM.

Highly efficient capture of cancer cells expressing EGFR by microfluidic methods based on antigen-antibody association.

Epidermal growth factor receptor (EGFR) was evaluated as a target antigen for cancer cell capture by microfluidic methods based on antigen-antibody association. A polymer CTC-chip microfluidic device was surface-functionalized with three different anti-EGFR antibodies and used to capture EGFR-expressing cancer cells. Capture efficacy depended on the type of antibody used, and cetuximab efficiently captured cancer cell lines that had a wide range of EGFR expression. Capture efficiency was analyzed from the viewpoint of antigen-antibody association in a kinetic process, i.e., cell rolling well-known in leukocyte adhesion, and antibodies with a smaller dissociation constant were shown to result in more efficient capture. Moreover, a lower limit of cellular EGFR expression level for the capture was estimated and methods to decrease the limit were discussed based on densities of anti-EGFR antibody on the device surface.

Capture of mesothelioma cells with 'universal' CTC-chip.

Malignant mesothelioma (MM) is a highly aggressive malignant tumor, predominantly associated with job-related exposure to asbestos. Development of effective and non-invasive modalities for diagnosis is an important issue in occupational medicine. Circulating tumor cells (CTCs), which are tumor cells that are shed from primary tumors and circulate in the peripheral blood, may be detected at an earlier stage than malignant tumors, and detection of CTCs may provide a novel insight into the diagnosis of MM. In a previous study evaluating clinical utility of CTCs, detected with a widely used system 'CellSearch', the authors indicated a significant however insufficient capability in the diagnosis of MM, suggesting need for a more sensitive system. Accordingly, the authors developed a novel microfluidic system to capture CTCs (CTC-chip), and demonstrated that the CTC-chip effectively captured MM cells (ACC-MESO-4) spiked in the blood by conjugating an anti-podoplanin antibody. The results of the present study demonstrated that the CTC-chip coated with the anti-podoplanin antibody captured another MM cell (ACC-MESO-1). However, the capture efficiencies were lower than those for ACC-MESO-4. In addition, an anti-mesothelin antibody was used to capture CTCs, however the CTC-chip coated with the anti-mesothelin antibody failed to effectively capture MM cells, possibly due to low mesothelin expression. Overall, the CTC-chip may capture specific types of CTCs by conjugating any antibody against an antigen expressed on CTCs, and may be a useful system for the diagnosis of malignant tumors, including MM.

High expression of carcinoembryonic antigen and telomerase reverse transcriptase in circulating tumor cells is associated with poor clinical response to the immune checkpoint inhibitor nivolumab.

The present study aimed to enrich circulating tumor cells (CTCs) from blood samples using a new size-sorting CTC chip. The present study also set out to identify a blood sensitivity marker for the immune checkpoint inhibitor nivolumab in patients with advanced, pre-treatment lung cancer. The CTC sorting efficacy of the chip was investigated and the large cell fraction of blood samples from 15 patients with pre-treatment lung cancer who were later administered nivolumab were purified. The expression levels of carcinoembryonic antigen (CEA), human Telomerase Reverse Transcriptase (hTERT), cytokeratin19 (CK19), and programmed death ligand-1 (PD-L1) were investigated to clarify the association between these CTC markers and the clinical response to nivolumab. The CTC chip effectively enriched cells from lung cancer cell line PC-9. The large cell fraction had a high expression of CEA and hTERT, with the former being significantly associated with the clinical response to nivolumab. The expression of CEA and hTERT in CTCs derived from the blood of a patient with lung cancer were also validated. The evaluation of CEA and possibly hTERT in CTCs collected by the CTC chip may represent a promising predictive blood marker for sensitivity to nivolumab. To the best of our knowledge this is the first report to describe the predictive CTC marker for nivolumab in pre-treatment patients.

EpCAM-independent capture of circulating tumor cells with a 'universal CTC-chip'.

Capture of circulating tumor cells (CTCs), which are shed from the primary tumor site and circulate in the blood, remains a technical challenge. CellSearch® is the only clinically approved CTC detection system, but has provided only modest sensitivity in detecting CTCs mainly because epithelial cell adhesion molecule (EpCAM)-negative tumor cells may not be captured. To achieve more sensitive CTC­capture, we have developed a novel microfluidic platform, a 'CTC-chip' comprised of light-curable resins that has a unique advantage in that any capture antibody is easily conjugated. In the present study, we showed that EpCAM-negative tumor cells as well as EpCAM-positive cells were captured with the novel 'universal CTC-chip' as follows: i) human lung cancer cells (PC-9), with strong EpCAM expression, were efficiently captured with the CTC-chip coated with an anti-EpCAM antibody (with an average capture efficiency of 101% when tumor cells were spiked in phosphate­buffered saline (PBS) and 88% when spiked in blood); ii) human mesothelioma cells (ACC-MESO-4), with no EpCAM expression but with podoplanin expression, were captured with the CTC-chip coated with an anti-podoplanin antibody (average capture efficiency of 78% when tumor cells were spiked in PBS and 38% when spiked in blood), whereas ACC-MESO-4 cells were not captured with the CTC-chip coated with the anti-EpCAM antibody. These results indicate that the novel 'CTC-chip' can be useful in sensitive EpCAM-independent detection of CTCs, which may provide new insights into personalized medicine.

Capture of esophageal and breast cancer cells with polymeric microfluidic devices for CTC isolation.

The present study evaluated the capture efficiency of esophageal and breast cancer cells with a modified 'polymeric circulating tumor cells (CTC)-chip' microfluidic device, which was developed for the isolation of circulating tumor cells. Esophageal cancer cell lines KYSE150, KYSE220 and KYSE510, and breast cancer cell lines MCF7, SKBR3 and MDA-MB-231 were used for evaluation. The capture efficiencies of the esophageal cancer cell lines in phosphate-buffered saline (PBS) were ~0.9, irrespective of epithelial cell adhesion molecule (EpCAM) expression, which was represented as the mean fluorescent intensity from 528 to 76. In the breast cancer cell lines, efficient capture was observed for MCF7 and SKBR3 in PBS; however, a low value of ~0.1 was obtained for MDA-MB-231. Fluorescent imaging of immunolabeled cells revealed marginal EpCAM expression in MDA-MB-231. Using whole blood, no clogging occurred in the microstructure-modified CTC-chip and efficiency of capture was successfully evaluated. Capture efficiencies for KYSE220 and MCF7 in whole blood were >0.7, but were of either equal or lesser efficiency in comparison to PBS. Therefore, the modified CTC-chip appears useful for clinical application due to its cost, practicality of use, and efficient cancer cell capture.

[Identification of Circulating Tumor Cell（CTC）in Breast Cancer Patients Using a Newly Established CTC Detecting System].

We developed a new circulating tumor cell (CTC) chip in order to identify CTCs in the peripheral blood of cancer patients. In this study, we aimed to identify CTCs in the blood of breast cancer patients by using this CTC detecting system. In addition, we used this system to evaluate the response to anticancer agents. We were able to identify CTCs in 5 of 6 patients. In addition, the system showed that the number of CTCs had decreased after chemotherapy. Thus, the CTC detecting system was useful in the identification of CTCs in the breast cancer patients and in the early prediction of response to anticancer agents.

Polymeric microfluidic devices exhibiting sufficient capture of cancer cell line for isolation of circulating tumor cells.

Here, we developed polymeric microfluidic devices for the isolation of circulating tumor cells. The devices, with more than 30,000 microposts in the channel, were produced successfully by a UV light-curing process lasting 3 min. The device surface was coated with anti-epithelial cell adhesion molecule antibody by just contacting the antibody solution, and a flow system including the device was established to send a cell suspension through it. We carried out flow tests for evaluation of the device's ability to capture tumor cells using an esophageal cancer cell line, KYSE220, dispersed in phosphate-buffered saline or mononuclear cell separation from whole blood. After the suspension flowed through the chip, many cells were seen to be captured on the microposts coated with the antibody, whereas there were few cells in the device without the antibody. Owing to the transparency of the device, we could observe the intact and the stained cells captured on the microposts by transmitted light microscopy and phase contrast microscopy, in addition to fluorescent microscopy, which required fluorescence labeling. Cell capture efficiencies (i.e., recovery rates of the flowing cancer cells by capture with the microfluidic device) were measured. The resulting values were 0.88 and 0.95 for cell suspension in phosphate-buffered saline, and 0.85 for the suspension in the mononuclear cell separation, suggesting the sufficiency of this device for the isolation of circulating tumor cells. Therefore, our device may be useful for research and treatments that rely on investigation of circulating tumor cells in the blood of cancer patients.