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.

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.

Graphene Oxide Nanoparticles Having Long Wavelength Absorbing Chlorins for Highly-Enhanced Photodynamic Therapy with Reduced Dark Toxicity.

The long wavelength absorbing photosensitizer (PS) is important in allowing deeper penetration of near-infrared light into tumor tissue for photodynamic therapy (PDT). A suitable drug delivery vehicle is important to attain a sufficient concentration of PS at the tumor site. Presently, we developed graphene oxide (GO) nanoparticles containing long wavelength absorbing PS in the form of the chlorin derivative purpurin-18-N-ethylamine (maximum absorption wavelength [λmax] 707 nm). The GO-PS complexes comprised a delivery system in which PS was loaded by covalent and noncovalent bonding on the GO nanosheet. The two GO-PS complexes were fully characterized and compared concerning their synthesis, stability, cell viability, and dark toxicity. The GO-PS complexes produced significantly-enhanced PDT activity based on excellent drug delivery effect of GO compared with PS alone. In addition, the noncovalent GO-PS complex displayed higher photoactivity, corresponding with the pH-induced release of noncovalently-bound PS from the GO complex in the acidic environment of the cells. Furthermore, the noncovalently bound GO‒PS complex had no dark toxicity, as their highly organized structure prevented GO toxicity. We describe an excellent GO complex-based delivery system with significantly enhanced PDT with long wavelength absorbing PS, as well as reduced dark toxicity as a promising cancer treatment.

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.

Isolation of nucleated red blood cells in maternal blood for Non-invasive prenatal diagnosis.

This paper introduces a two-step cascade enrichment method for isolating nucleated red blood cells (NRBCs) in maternal blood. The two-step enrichment platform consists of a positive enrichment process based on a red blood cell (RBC) hyperaggregation method and a negative enrichment process using microfluidic technology. An analytical evaluation using blood samples from patients with leukemia showed that the while blood cell (WBC) depletion and NRBC loss rates of the positive enrichment process were 93.98 % and 6.02 %, respectively. Through the two-step cascade enrichment method, 1-396 NRBCs and only 0-6 WBCs were isolated from 1 mL of 18 maternal blood samples. Experimental results also showed that the WBC depletion rate of the proposed two-step method was more than 625,000-fold, and the purity of enriched NRBCs ranged from 20 % to 100 %. Furthermore, SRY (the sex-determining region of the Y chromosome) genes were detected in enriched NRBCs, thereby demonstrating that enriched NRBCs contain fetus-derived NRBCs.

Electrical Detection Method for Circulating Tumor Cells Using Graphene Nanoplates.

This paper presents a microfluidic device for electrical discrimination of circulating tumor cells (CTCs) using graphene nanoplates (GNPs) as a highly conductive material bound to the cell surface. For two-step cascade discrimination, the microfluidic device is composed of a CTC-enrichment device and an impedance cytometry. Using lateral magnetophoresis, the CTC-enrichment device enriches rare CTCs from millions of background blood cells. Then, the impedance cytometry electrically identifies CTCs from the enriched sample, containing CTCs and persistent residual blood cells, based on the electrical impedance of CTCs modified by the GNPs. GNPs were used as a highly conductive material for modifying surface conductivity of CTCs, thereby improving the accuracy of electrical discrimination. The experimental results showed that a colorectal cancer cell line (DLD-1) spiked into peripheral blood was enriched by nearly 500-fold by the CTC-enrichment device. The phase of the electrical signal measured from DLD-1 cells covered by GNPs shifted by about 100° in comparison with that from normal blood cells, which allows the impedance cytometry to identify CTCs at a rate of 94% from the enriched samples.

Circulating tumor cell microseparator based on lateral magnetophoresis and immunomagnetic nanobeads.

This paper presents a circulating tumor cell (CTC) microseparator for isolation of CTCs from human peripheral blood using immunomagnetic nanobeads with bound antiepithelial cell adhesive molecule (EpCAM) antibodies that specifically bind to epithelial cancer cells. The isolation is performed through lateral magnetophoresis, which is induced by high-gradient magnetic separation technology, involving a ferromagnetic wire array inlaid in the bottom substrate of a microchannel. Experimental results showed that the CTC microseparator isolates about 90% of spiked CTCs in human peripheral blood at a flow rate of up to 5 mL/h and purifies to approximately 97%. The overall isolation procedure was completed within 15 min for 200 µL of peripheral blood. CTCs from peripheral blood of patients with breast and lung cancers were isolated with the CTC microseparator, and the results were compared with those of healthy donors. Using a fluorescence-based viability assay, the viability of CTCs isolated from peripheral blood of patients with cancer was observed. In addition, the usefulness of the CTC microseparator for subsequent genetic assay was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of cancer-specific genes using CTCs isolated from patients with cancer.

An electrorotation technique for measuring the dielectric properties of cells with simultaneous use of negative quadrupolar dielectrophoresis and electrorotation.

This paper presents an effective electrorotation technique for measuring the dielectric properties of cells using a superposed electrical signal, which can simultaneously generate negative quadrupolar dielectrophoretic (nQDEP) force and electrorotational (ROT) torque. The proposed technique involves a three-dimensional (3D) octode, which includes four electrodes arranged in a crisscross pattern on the top and bottom of a microchannel, respectively. A single cell was trapped in the center of the 3D octode by the nQDEP force and simultaneously rotated by the ROT torque. Using the proposed electrorotation technique, ROT spectra of human leukocyte subpopulations (T and B lymphocytes, granulocytes, and monocytes) and metastatic human breast (SkBr3) and lung (A549) cancer cell lines were accurately measured without any disturbance. Torque on the cells generated by the ROT signal was analyzed theoretically based on the single-shell dielectric model for the cells. Furthermore, the dielectric properties of the cells, such as area-specific membrane capacitance and cytoplasm conductivity, were extracted using the measured ROT spectra and the analyzed torque.

Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture.

Without using biochemical agents, in this study, we sought to investigate the potential of controlling the differentiation of mesenchymal stem cells (MSCs) into a specific cell type through the use of 3D co-culturing and mechanical stimuli. MSCs and primary cultured chondrocytes were separately encapsulated into alginate beads, and the two types of beads were separated by a membrane. For the investigation a computer-controllable bioreactor was designed and used to engage intermittent hydrostatic pressure (IHP). Five different magnitudes (0.20, 0.10, 0.05, 0.02 MPa and no stimulation) of IHP were applied. The stimulation pattern was the same for all groups: 2 h/day for 7 days starting at 24 h after seeding; 2 and 15 min cycles of stimulating and resting, respectively. Biochemical (DNA and GAG contents), histological (Alcian blue), and RT-PCR (Col II, SOX9, AGC) analyses were performed on days 1, 5, 10, and 20. The results from these analyses showed that stimulation with higher magnitudes of IHP (≥0.10 MPa) were more effective on the proliferation and differentiation of co-cultured MSCs. Together, these data demonstrate the potential of using mechanical stimulation and co-culturing for the proliferation and differentiation of MSCs, even without biochemical agents.

Lateral dielectrophoretic microseparators to measure the size distribution of blood cells.

Lateral displacement of blood cells occurred when they were passed over a planar interdigitated electrode array placed at an angle to the direction of flow, and was determined to be a function of cell size. A simplified line charge model was used to estimate numerically the lateral displacement. Based on the size-specific lateral displacement, a lateral dielectrophoretic (DEP) microseparator was developed to measure the size distribution of blood cells using fluorescence microscopy. To determine whether the lateral DEP microseparator was useful, it was used to detect acute leukemia by measuring the size distribution of blood cells. The lateral DEP microseparator provided a practical method for continuously and simultaneously separating multi-cell populations by size from a heterogeneous cell population. In the future, sensitivity of the lateral DEP microseparator could be improved and it could be automated by integrating subsequent advanced detection technologies in a micro-format.

Upregulation of TLR2 expression on G-CSF-mobilized peripheral blood stem cells is responsible for their rapid engraftment after allogeneic hematopoietic stem cell transplantation.

Granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSCs) are more frequently used as the cellular source in allogeneic hematopoietic stem cell transplantation (HSCT) than bone marrow stem cells (BMSCs) because they promote more rapid engraftment and immune reconstitution. However, the underlying mechanism for this is not fully understood. Here, we investigated the role of Toll-like receptor 2 (TLR2) on PBSCs in promoting rapid engraftment after allogeneic HSCT. We found that PBSCs highly expressed TLR2 in comparison to BMSCs, and TLR2 was directly induced by G-CSF signaling. Treatment with the TLR2 ligand, Pam(3)CSK(4) (PAM), more efficiently induced myeloid differentiation of PBSCs than BMSCs. Similarly, endogenous TLR2 ligands from the serum of recipients of allogeneic transplantation more rapidly stimulated myeloid differentiation of PBSCs compared with BMSCs. PAM treatment of TLR2(-/-) syngeneic recipient mice transplanted with PBSCs resulted in significantly elevated numbers of PBSC-derived myeloid cells and spleen colony formation compared with controls. Our results demonstrate that TLR2 signaling in PBSCs correlates with their ability to rapidly differentiate into myeloid cells, resulting in improved engraftment. Thus, TLR2 may be a novel target for increasing the efficiency of allogeneic HSCT by overcoming engraftment failure or delayed engraftment.

Microsystems for isolation and electrophysiological analysis of breast cancer cells from blood.

This paper presents the development of a microsystem for separating suspended breast cancer cells in peripheral blood and for sorting them based on their electrophysiological characteristics. A continuous paramagnetic capture mode (PMC) magnetophoretic microseparator was utilized for the isolation of suspended breast cancer cells in peripheral blood based on the native magnetic properties of blood cells without any tagging such as with magnetic probes. A micro-electrical impedance spectroscopy (mu-EIS) system was used as a downstream cell analysis tool to extract the pathological characteristics from the breast cancer cells. The system was fabricated on silicon and glass substrates utilizing microfabrication and stereolithography technologies. The experimental results of the PMC microseparator show that 94.8% of the breast cancer cells could be continuously separated out from a spiked blood sample with a 0.2 T external magnetic flux. The electrical impedances of human breast cancer cell lines of different pathological stages (MCF-7, MDA-MB-231, and MDA-MB-435) were measured using mu-EIS and compared to those of normal human breast tissue cell line MCF-10A.