Impact of Circulating Tumor Cell-Expressed Prostate-Specific Membrane Antigen and Prostate-Specific Antigen Transcripts in Different Stages of Prostate Cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA)-based images, which visually quantify PSMA expression, are used to determine prostate cancer micrometastases. This study evaluated whether a circulating tumor cell (CTC)-based transcript platform, including PSMA mRNA, could help identify potential prognostic markers in prostate cancer. EXPERIMENTAL DESIGN: We prospectively enrolled 21 healthy individuals and 247 patients with prostate cancer [localized prostate cancer (LPCa), n = 94; metastatic hormone-sensitive prostate cancer (mHSPC), n = 44; and metastatic castration-resistant prostate cancer (mCRPC), n = 109]. The mRNA expression of six transcripts [PSMA, prostate-specific antigen (PSA), AR, AR-V7, EpCAM, and KRT 19] from CTCs was measured, and their relationship with biochemical recurrence (BCR) in LPCa and mCRPC progression-free survival (PFS) rate in mHSPC was assessed. PSA-PFS and radiological-PFS were also calculated to identify potential biomarkers for predicting androgen receptor signaling inhibitor (ARSI) and taxane-based chemotherapy resistance in mCRPC. RESULTS: CTC detection rates were 75.5%, 95.3%, and 98.0% for LPCa, mHSPC, and mCRPC, respectively. In LPCa, PSMA [hazard ratio (HR), 3.35; P = 0.028) and PSA mRNA (HR, 1.42; P = 0.047] expressions were associated with BCR. Patients with mHSPC with high PSMA (HR, 4.26; P = 0.020) and PSA mRNA (HR, 3.52; P = 0.042) expressions showed significantly worse mCRPC-PFS rates than those with low expression. Increased PSA and PSMA mRNA expressions were significantly associated with shorter PSA-PFS and radiological PFS in mCPRC, indicating an association with drug resistance. CONCLUSIONS: PSMA and PSA mRNA expressions are associated with BCR in LPCa. In advanced prostate cancer, PSMA and PSA mRNA can also predict rapid progression from mHSPC to mCRPC and ARSI or taxane-based chemotherapy resistance.

Effective Circulating Tumor Cell Isolation Using Epithelial and Mesenchymal Markers in Prostate and Pancreatic Cancer Patients.

Circulating tumor cells (CTCs) display antigenic heterogeneity between epithelial and mesenchymal phenotypes. However, most current CTC isolation methods rely on EpCAM (epithelial cell adhesion molecule) antibodies. This study introduces a more efficient CTC isolation technique utilizing both EpCAM and vimentin (mesenchymal cell marker) antibodies, alongside a lateral magnetophoretic microseparator. The effectiveness of this approach was assessed by isolating CTCs from prostate (n = 17) and pancreatic (n = 5) cancer patients using EpCAM alone, vimentin alone, and both antibodies together. Prostate cancer patients showed an average of 13.29, 11.13, and 27.95 CTCs/mL isolated using EpCAM alone, vimentin alone, and both antibodies, respectively. For pancreatic cancer patients, the averages were 1.50, 3.44, and 10.82 CTCs/mL with EpCAM alone, vimentin alone, and both antibodies, respectively. Combining antibodies more than doubled CTC isolation compared to single antibodies. Interestingly, EpCAM antibodies were more effective for localized prostate cancer, while vimentin antibodies excelled in metastatic prostate cancer isolation. Moreover, vimentin antibodies outperformed EpCAM antibodies for all pancreatic cancer patients. These results highlight that using both epithelial and mesenchymal antibodies with the lateral magnetophoretic microseparator significantly enhances CTC isolation efficiency, and that antibody choice may vary depending on cancer type and stage.

Association of serum prostate-specific antigen (PSA) level and circulating tumor cell-based PSA mRNA in prostate cancer.

BACKGROUND: Prostate-specific antigen (PSA) is used for diagnosing prostate cancer, but does not reflect the characteristics of prostate cancer cells to allow assessment of cancer progression. PSA mRNA and circulating tumor cells (CTCs) could be potential biomarkers. However, the relationship between serum PSA levels and PSA mRNA in CTCs is unclear, and this study aimed to investigate this relationship. METHODS: Healthy donors (HD, n = 9), and patients with local non-metastatic stage prostate cancer (n = 30), metastatic hormone-sensitive prostate cancer (mHSPC, n = 10), and metastatic castration-resistant prostate cancer (mCRPC, n = 75), were included. The expression of PSA mRNA in CTCs was measured by droplet digital PCR. Serum PSA (ng/mL) levels and PSA mRNA (copies/µL) in CTCs were then compared using Spearman correlation coefficients. RESULTS: PSA mRNA expression in CTCs was observed in 30% (9/30) of patients with localized cancer, 60.0% (6/10) among patients with mHSPC, 65.3% (49/75) among patients with mCRPC, and 0% among patients with HD, indicating that the detection rate of PSA mRNA increased with cancer stage. PSA mRNA expression in CTCs also increased from localized to metastatic stages. PSA mRNA levels rapidly increased in the mHSPC and mCRPC stages. Interestingly, PSA mRNA expression in CTCs was not correlated with serum PSA levels at the localized stage (R = 0.064, P = 0.512). However, there were significant correlations between serum PSA levels and PSA mRNA expression in mHSPC (R = 0.532, P = 0.041) and mCRPC (R = 0.566, P = 0.025). The number of CTCs isolated from mHSPC and mCRPC was not proportional to serum PSA and PSA mRNA levels. CONCLUSION: CTC PSA mRNA has the potential to be used as a biomarker to complement serum PSA protein analysis or replace serum PSA in metastatic stages of prostate cancer.

Lateral Degassing Method for Disposable Film-Chip Microfluidic Devices.

It is critical to develop a fast and simple method to remove air bubbles inside microchannels for automated, reliable, and reproducible microfluidic devices. As an active degassing method, this study introduces a lateral degassing method that can be easily implemented in disposable film-chip microfluidic devices. This method uses a disposable film-chip microchannel superstrate and a reusable substrate, which can be assembled and disassembled simply by vacuum pressure. The disposable microchannel superstrate is readily fabricated by bonding a microstructured polydimethylsiloxane replica and a silicone-coated release polymeric thin film. The reusable substrate can be a plate that has no function or is equipped with the ability to actively manipulate and sense substances in the microchannel by an elaborately patterned energy field. The degassing rate of the lateral degassing method and the maximum available pressure in the microchannel equipped with lateral degassing were evaluated. The usefulness of this method was demonstrated using complex structured microfluidic devices, such as a meandering microchannel, a microvortex, a gradient micromixer, and a herringbone micromixer, which often suffer from bubble formation. In conclusion, as an easy-to-implement and easy-to-use technique, the lateral degassing method will be a key technique to address the bubble formation problem of microfluidic devices.

A disposable smart microfluidic platform integrated with on-chip flow sensors.

Microfluidic devices are powerful tools for biological, biomedical, chemical, and pharmaceutical applications, but their commercialization is still hindered by the lack of methods to automatically control fluid flow in a low-cost, simple, accurate, and safe manner. This study introduces a disposable smart microfluidic platform (DIS-µChip), which can be fully automated and utilized for a wide range of applications. On-chip microfluidic flow sensors are integrated with the platform and placed at all inlet and outlet channels, thereby allowing the DIS-µChip to be fully automated with a pressure control system. Furthermore, these confer a self-diagnosis function through monitoring of all the input and output flow rates. The DIS-µChip consists of a disposable polymeric microchannel superstrate and a permanent multifunctional substrate, which could be assembled and disassembled using only vacuum pressure. The superstrate was fabricated by combining a polydimethylsiloxane microchannel structure with a polyethylene terephthalate (PET) thin film. The substrate contains sense electrodes for the on-chip-integrated flow sensors and functional components for creating an energy field, which can penetrate the PET thin film and manipulate the fluid in the microchannels of the superstrate. Owing to the film-chip technique, the superstrate was disposable and could prevent biological cross-contamination, which cannot be realized with conventional flow sensors. The usefulness of the DIS-µChip was demonstrated by using it to isolate circulating tumor cells from the blood of patients with pancreatic cancer and to obtain cancer-specific genetic information from them with droplet digital PCR.

Multigene model for predicting metastatic prostate cancer using circulating tumor cells by microfluidic magnetophoresis.

We aimed to isolate circulating tumor cells (CTCs) using a microfluidic technique with a novel lateral magnetophoretic microseparator. Prostate cancer-specific gene expressions were evaluated using mRNA from the isolated CTCs. A CTC-based multigene model was then developed for identifying advanced prostate cancer. Peripheral blood samples were obtained from five healthy donors and patients with localized prostate cancer (26 cases), metastatic hormone-sensitive prostate cancer (mHSPC, 10 cases), and metastatic castration-resistant prostate cancer (mCRPC, 28 cases). CTC recovery rate and purity (enriched CTCs/total cells) were evaluated according to cancer stage. The areas under the curves of the six gene expressions were used to evaluate whether multigene models could identify mHSPC or mCRPC. The number of CTCs and their purity increased at more advanced cancer stages. In mHSPC/mCRPC cases, the specimens had an average of 27.5 CTCs/mL blood, which was 4.2 × higher than the isolation rate for localized disease. The CTC purity increased from 2.1% for localized disease to 3.8% for mHSPC and 6.7% for mCRPC, with increased CTC expression of the genes encoding prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and cytokeratin 19 (KRT19). All disease stages exhibited expression of the genes encoding androgen receptor (AR) and epithelial cell adhesion molecule (EpCAM), although expression of the AR-V7 variant was relatively rare. Relative to each gene alone, the multigene model had better accuracy for predicting advanced prostate cancer. Our lateral magnetophoretic microseparator can be used for identifying prostate cancer biomarkers. In addition, CTC-based genetic signatures may guide the early diagnosis of advanced prostate cancer.

A Direct Comparison between the Lateral Magnetophoretic Microseparator and AdnaTest for Isolating Prostate Circulating Tumor Cells.

Circulating tumor cells (CTCs) are important biomarkers for the diagnosis, prognosis, and treatment of cancer. However, because of their extreme rarity, a more precise technique for isolating CTCs is required to gain deeper insight into the characteristics of cancer. This study compares the performance of a lateral magnetophoretic microseparator ("CTC-µChip"), as a representative microfluidic device, and AdnaTest ProstateCancer (Qiagen), as a commercially available specialized method, for isolating CTCs from the blood of patients with prostate cancer. The enumeration and genetic analysis results of CTCs isolated via the two methods were compared under identical conditions. In the CTC enumeration experiment, the number of CTCs isolated by the CTC-µChip averaged 17.67 CTCs/mL, compared to 1.56 CTCs/mL by the AdnaTest. The number of contaminating white blood cells (WBCs) and the CTC purity with the CTC-µChip averaged 772.22 WBCs/mL and 3.91%, respectively, whereas those with the AdnaTest averaged 67.34 WBCs/mL and 1.98%, respectively. Through genetic analysis, using a cancer-specific gene panel (AR (androgen receptor), AR-V7 (A\androgen receptor variant-7), PSMA (prostate specific membrane antigen), KRT19 (cytokeratin-19), CD45 (PTPRC, Protein tyrosine phosphatase, receptor type, C)) with reverse transcription droplet digital PCR, three genes (AR, AR-V7, and PSMA) were more highly expressed in cells isolated by the CTC-µChip, while KRT19 and CD45 were similarly detected using both methods. Consequently, this study showed that the CTC-µChip can be used to isolate CTCs more reliably than AdnaTest ProstateCancer, as a specialized method for gene analysis of prostate CTCs, as well as more sensitively obtain cancer-associated gene expressions.

Evaluation of Positive and Negative Methods for Isolation of Circulating Tumor Cells by Lateral Magnetophoresis.

We developed an epithelial cell adhesion molecule (EpCAM)-based positive method and CD45/CD66b-based negative method for isolating circulating tumor cells (CTCs) by lateral magnetophoresis. The CTC recovery rate, white blood cell depletion rate, and purity of CTCs isolated using the positive and negative methods were analyzed using blood samples spiked with cancer cells with different expression levels of EpCAM. The aim was to assess the strengths and weaknesses of the positive and negative isolation methods for CTC-based diagnostics, prognostics, and therapeutics for cancer. The EpCAM-based positive method yielded CTCs of high purity, while the CD45/CD66b-based negative method yielded a large number of CTCs. In conclusion, the positive method shows promise for detecting somatic oncogenic mutations and the negative method shows promise for discovery of cellular and transcriptomic biomarkers of cancer.

Microfluidic technologies for circulating tumor cell isolation.

Metastasis is the main cause of tumor-related death, and the dispersal of tumor cells through the circulatory system is a critical step in the metastatic process. Early detection and analysis of circulating tumor cells (CTCs) is therefore important for early diagnosis, prognosis, and effective treatment of cancer, enabling favorable clinical outcomes in cancer patients. Accurate and reliable methods for isolating and detecting CTCs are necessary to obtain this clinical information. Over the past two decades, microfluidic technologies have demonstrated great potential for isolating and detecting CTCs from blood. The present paper reviews current advanced microfluidic technologies for isolating CTCs based on various biological and physical principles, and discusses their fundamental advantages and drawbacks for subsequent cellular and molecular assays. Owing to significant genetic heterogeneity among CTCs, microfluidic technologies for isolating individual CTCs have recently been developed. We discuss these single-cell isolation methods, as well as approaches to overcoming the limitations of current microfluidic CTC isolation technologies. Finally, we provide an overview of future innovative microfluidic platforms.

A disposable microfluidic device with a reusable magnetophoretic functional substrate for isolation of circulating tumor cells.

We describe an assembly-disposable microfluidic device based on a silicone-coated release polymer thin film. It consists of a disposable polymeric superstrate and a reusable functional substrate and they are assembled simply using vacuum pressure. The disposable polymeric superstrate is manufactured by bonding a silicone-coated release polymer thin film and a microstructured polydimethylsiloxane (PDMS) replica, containing only a simple structured microchannel. The reusable functional substrate generates an intricate energy field that can penetrate the micrometer-thick polymer film into the microchannel and control microfluids. This is the first report to introduce a silicone-coated release polyethylene terephthalate (PET) thin film as a bonding layer on a microstructured PDMS replica. The bonding strength was ∼600 kPa, which is the strongest among bonding methods of PDMS and PET polymer. Additionally, accelerated tests for bond stability and leakage demonstrated that the silicone-coated release PET film can form a very robust bond with PDMS. To demonstrate the usefulness of the proposed assembly-disposable microfluidic device, a lateral magnetophoretic microseparator was developed in an assembly-disposable microfluidic device format and was evaluated for isolating circulating tumor cells (CTCs) from patients with breast cancer.

Analytical evaluation for somatic mutation detection in circulating tumor cells isolated using a lateral magnetophoretic microseparator.

CTCs are currently in the spotlight because provide comprehensive genetic information that enables monitoring of the evolution of cancer and selection of appropriate therapeutic strategies that cannot be obtained from a single-site tumor biopsy. Despite their importance, current techniques for isolating CTCs are limited in terms of their ability to yield high-quality CTCs from peripheral blood for use in profiling cancer genetic mutations by DNA sequencing technologies. This paper introduces a lateral magnetophoretic microseparator (the 'CTC-µChip') for isolating highly pure CTCs from blood, which facilitates the detection of somatic mutations in isolated CTCs. To isolate CTCs from peripheral blood, nucleated cells were first prepared by red blood cell lysis. Then, CTCs were isolated from nucleated cells within 30 min using the CTC-µChip. Analytical evaluation using 5 mL blood samples spiked with 5-50 MCF7 breast cancer cells demonstrated that the average recovery rate of the CTC-µChip was 99.08 %. The average number of residual white blood cells (WBCs) in isolated samples was 53, meaning that the WBC depletion rate is 472,000-fold (5.67 log), assuming that blood contains 5 × 10(6) WBCs per milliliter. The isolated MCF7 cells had a purity of 6.9 - 67.9 %, depending on the spiked MCF7 concentration. Using next-generation sequencing technology, heterozygous somatic mutations (PIK3CA and APC) of MCF7 cells were evaluated in the isolated samples. The results showed that somatic mutations could be detected in as few as two MCF7 cells per milliliter of blood, indicating that the CTC-µChip facilitates the detection of somatic variants in CTCs.

Single-Cell Isolation of Circulating Tumor Cells from Whole Blood by Lateral Magnetophoretic Microseparation and Microfluidic Dispensing.

This paper introduces a single-cell isolation technology for circulating tumor cells (CTCs) using a microfluidic device (the "SIM-Chip"). The SIM-Chip comprises a lateral magnetophoretic microseparator and a microdispenser as a two-step cascade platform. First, CTCs were enriched from whole blood by the lateral magnetophoretic microseparator based on immunomagnetic nanobeads. Next, the enriched CTCs were electrically identified by single-cell impedance cytometer and isolated as single cells using the microshooter. Using 200 µL of whole blood spiked with 50 MCF7 breast cancer cells, the analysis demonstrated that the single-cell isolation efficiency of the SIM-Chip was 82.4%, and the purity of the isolated MCF7 cells with respect to WBCs was 92.45%. The data also showed that the WBC depletion rate of the SIM-Chip was 2.5 × 10(5) (5.4-log). The recovery rates were around 99.78% for spiked MCF7 cells ranging in number from 10 to 90. The isolated single MCF7 cells were intact and could be used for subsequent downstream genetic assays, such as RT-PCR. Single-cell culture evaluation of the proliferation of MCF7 cells isolated by the SIM-Chip showed that 84.1% of cells at least doubled in 5 days. Consequently, the SIM-Chip could be used for single-cell isolation of rare target cells from whole blood with high purity and recovery without cell damage.