Palmitate and glucose increase amyloid precursor protein in extracellular vesicles: Missing link between metabolic syndrome and Alzheimer's disease.

The metabolic syndrome (MetS) and Alzheimer's disease share several pathological features, including insulin resistance, abnormal protein processing, mitochondrial dysfunction and elevated inflammation and oxidative stress. The MetS constitutes elevated fasting glucose, obesity, dyslipidaemia and hypertension and increases the risk of developing Alzheimer's disease, but the precise mechanism remains elusive. Insulin resistance, which develops from a diet rich in sugars and saturated fatty acids, such as palmitate, is shared by the MetS and Alzheimer's disease. Extracellular vesicles (EVs) are also a point of convergence, with altered dynamics in both the MetS and Alzheimer's disease. However, the role of palmitate- and glucose-induced insulin resistance in the brain and its potential link through EVs to Alzheimer's disease is unknown. We demonstrate that palmitate and high glucose induce insulin resistance and amyloid precursor protein phosphorylation in primary rat embryonic cortical neurons and human cortical stem cells. Palmitate also triggers insulin resistance in oligodendrocytes, the supportive glia of the brain. Palmitate and glucose enhance amyloid precursor protein secretion from cortical neurons via EVs, which induce tau phosphorylation when added to naïve neurons. Additionally, EVs from palmitate-treated oligodendrocytes enhance insulin resistance in recipient neurons. Overall, our findings suggest a novel theory underlying the increased risk of Alzheimer's disease in MetS mediated by EVs, which spread Alzheimer's pathology and insulin resistance.

Emerging micro-nanotechnologies for extracellular vesicles in immuno-oncology: from target specific isolations to immunomodulation.

Extracellular vesicles (EVs) have been hypothesized to incorporate a variety of crucial roles ranging from intercellular communication to tumor pathogenesis to cancer immunotherapy capabilities. Traditional EV isolation and characterization techniques cannot accurately and with specificity isolate subgroups of EVs, such as tumor-derived extracellular vesicles (TEVs) and immune-cell derived EVs, and are plagued with burdensome steps. To address these pivotal issues, multiplex microfluidic EV isolation/characterization and on-chip EV engineering may be imperative towards developing the next-generation EV-based immunotherapeutics. Henceforth, our aim is to expound the state of the art in EV isolation/characterization techniques and their limitations. Additionally, we seek to elucidate current work on total analytical system based technologies for simultaneous isolation and characterization and to summarize the immunogenic capabilities of EV subgroups, both innate and adaptive. In this review, we discuss recent state-of-art microfluidic/micro-nanotechnology based EV screening methods and EV engineering methods towards therapeutic use of EVs in immune-oncology. By venturing in this field of EV screening and immunotherapies, it is envisioned that transition into clinical settings can become less convoluted for clinicians.

Isolation of circulating tumor cells to diagnose melanoma and evaluate the efficacy of surgical resection using melanoma-specific microsystem.

Melanoma is one of the most aggressive skin cancers due to its potential to metastasize widely in the body. The risk of metastasis is increased with later detection and increased thickness of the primary lesion, thus early identification and surgical removal is critical for higher survival rates for patients. However, even with appropriate treatment, some patients will develop recurrence which may be difficult to identify until advanced or causing symptoms. Recent advances in liquid biopsy have proposed less-invasive alternatives for cancer diagnosis and monitoring using minimal/zero invasion at sample collection, and circulating tumor cells(CTCs) have been considered a promising blood-based surrogate marker of primary tumors. However, previous CTC technologies relying on epithelial-cell adhesion molecules have limited to epithelial cells, thus hampering use of CTCs for non-epithelial cancers such as melanoma. Here, we used the Melanoma-specific OncoBean platform(MelanoBean) conjugated with melanoma specific antibodies(MCAM and MCSP). The device was used in comprehensive studies for diagnosing melanoma and evaluating surgery efficacy based on change in the number and characteristics of CTCs and CTC-clusters pre- and post-surgical treatment. Our study demonstrated that melanoma patients(n=45) at all stages(I-IV) have a noticeable number of MCTCs as well as MCTC-clusters compared to healthy donors(n=9)(P=0.0011), and surgical treatment leads to a significant decrease in the number of CTCs(P<0.0001). The CTCs recovered from the device underwent molecular profiling for melanoma-associated genes expression using multiplexed qRT-PCR, demonstrating the ability to monitor molecular signature through treatment. The presented MelanoBean and the comprehensive approach will empower prognostic value of CTCs in melanoma in much larger cohort studies.

On-Chip Biogenesis of Circulating NK Cell-Derived Exosomes in Non-Small Cell Lung Cancer Exhibits Antitumoral Activity.

As the recognition between natural killer (NK) cells and cancer cells does not require antigen presentation, NK cells are being actively studied for use in adoptive cell therapies in the rapidly evolving armamentarium of cancer immunotherapy. In addition to utilizing NK cells, recent studies have shown that exosomes derived from NK cells also exhibit antitumor properties. Furthermore, these NK cell-derived exosomes exhibit higher stability, greater modification potentials and less immunogenicity compared to NK cells. Therefore, technologies that allow highly sensitive and specific isolation of NK cells and NK cell-derived exosomes can enable personalized NK-mediated cancer therapeutics in the future. Here, a novel microfluidic system to collect patient-specific NK cells and on-chip biogenesis of NK-exosomes is proposed. In a small cohort of non-small cell lung cancer (NSCLC) patients, both NK cells and circulating tumor cells (CTCs) were isolated, and it is found NSCLC patients have high numbers of NK and NK-exosomes compared with healthy donors, and these concentrations show a trend of positive and negative correlations with bloodborne CTC numbers, respectively. It is further demonstrated that the NK-exosomes harvested from NK-graphene oxide chip exhibit cytotoxic effect on CTCs. This versatile system is expected to be used for patient-specific NK-based immunotherapies along with CTCs for potential prognostic/diagnostic applications.

Dual-Isolation and Profiling of Circulating Tumor Cells and Cancer Exosomes from Blood Samples with Melanoma Using Immunoaffinity-Based Microfluidic Interfaces.

Melanoma is among the most aggressive cancers, and its rate of incidence continues to grow. Early detection of melanoma has been hampered due to the lack of promising markers for testing. Recent advances in liquid biopsy have proposed noninvasive alternatives for cancer diagnosis and monitoring. Circulating tumor cells (CTCs) and cancer-exosomes are gaining influence as promising biomarkers because of their cancer-associated molecular markers and signatures. However, technologies that offer the dual-isolation of CTCs and exosomes using a single sample have not been thoroughly developed. The dual-utilization OncoBean (DUO) device is conjugated with melanoma specific antibodies, MCAM and MCSP, enabling simultaneous CTC and exosome isolations. Using blood samples from patients, CTCs and exosomes are specifically isolated from a single sample and then undergo molecular profiling for comprehensive study. Melanoma patients have 0-17CTCs mL-1 and 299 µg exosomal protein mL-1 while healthy donors display fewer than 2CTCs and 75.6 µg of exosomes mL-1, respectively. It is also demonstrated that both markers express melanoma-associated genes using multiplex qRT-PCR to test for expression pattern of a 96 gene panel. The dual isolation and molecular characterization will allow for further research into melanoma to identify viable markers for disease progression and treatment efficacy.

Extracellular vesicles on demand (EVOD) chip for screening and quantification of cancer-associated extracellular vesicles.

While significant advancements have been made in cancer therapeutics and treatments, early disease detection and diagnosis remains critical to ensuring favorable outcomes for patients. To that end, we propose a microfluidic based approach to the sensitive detection of an intriguing cancer biomarker, extracellular vesicles (EVs). Our extracellular vesicles on demand (EVOD) chip utilizes a catalyst-free click chemistry to rapidly and specifically isolate EVs of interest. This specific isolation is followed by subsequent dithiothreitol release of the isolated EVs for downstream functional analysis. This joint isolation and release provide a powerful tool for the screening and quantification of EVs of interest. By incorporating antibodies against cancer associated surface proteins into the click-chemistry, we were able to selectively recover cancer-associated exosomes, allowing for important insights into patient disease. This platform was also tested using non-small cell lung cancer (NSCLC) patient samples, where anti-epidermal growth factor receptor (EGFR) assisted platform were able to selectively isolate and release 76% more exosomes from NSCLC patients than from healthy donors. This matches the previously reported higher EGFR expression commonly found in NSCLC EVs. Through its rapid isolation kinetics and adaptability in marker targeting, the EVOD device provides a highly versatile liquid biopsy platform for clinicians to use in the fight against cancer.

Microfluidic device for high-throughput affinity-based isolation of extracellular vesicles.

Immunoaffinity based EV isolation technologies use antibodies targeting surface markers on EVs to provide higher isolation specificity and purity compared to existing approaches. One standing challenge for researchers is how to release captured EVs from the substrate to increase downstream and biological studies. The strong binding between the antibody and antigen or the antibody and substrate is commonly unbreakable without operating at conditions outside of the critical physiological range, making the release of EVs problematic. Additionally, immuno-affinity approaches are usually low-throughput due to their low flow velocity to ensure adequate time for antibody-antigen binding. To overcome these limitations, we modified the OncoBean chip, a previously reported circulating tumor cell isolation microfluidic device. The OncoBean chip is a radial flow microfluidic device with bean-shape microposts functionalized with biotin-conjugated EPCAM antibody through biotin-avidin link chemistry. It was demonstrated that the high surface area and varying shear rate provided by the bean-shaped posts and the radial flow design in the chip, enabled efficient capture of CTCs at high flow rate. We replace the anti-EPCAM with antibodies that recognize common EV surface markers to achieve high-throughput EV isolation. Moreover, by incorporating desthiobiotin-conjugated antibodies, EVs can be released from the device after capture, which offers a significant improvement over the existing isolation. The released EVs were found to be functional by confirming their uptake by cells using flow cytometry and fluorescent microscopy. We believe the proposed technology can facilitate both the study of EVs as cell-to-cell communicators and the further identification of EV markers.

Isolation and Profiling of Circulating Tumor-Associated Exosomes Using Extracellular Vesicular Lipid-Protein Binding Affinity Based Microfluidic Device.

Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on-chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity-based microfluidic EV isolation using anti-CD63 is widely used; however, anti-CD63 is not specific to cancer-EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer-associated EVs. A new exosome isolation microfluidic device (new ExoChip), conjugated with a PS-specific protein, to isolate cancer-associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549-derived exosomes than an anti-CD63-conjugated device. Immobilized exosomes are then easily released using Ca2+ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western-blot analysis, revealing exosomal shapes and exosomal protein expressions. The new ExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.

Multiplex isolation and profiling of extracellular vesicles using a microfluidic DICE device.

Profiling of extracellular vesicles (EVs) is an emerging area in the field of liquid biopsies because of their innate significance in diseases and abundant information reflecting disease status. However, unbiased enrichment of EVs and thorough profiling of EVs is challenging. In this paper, we present a simple strategy to immobilize and analyze EVs for multiple markers on a single microfluidic device and perform differentiated immunostaining-based characterization of extracellular vesicles (DICE). This device, composed of four quadrants with a single inlet, captures biotinylated EVs efficiently and facilitates multiplexed immunostaining to profile their extracellular proteins, allowing for a multiplexed approach for non-invasive cancer diagnostics in the future. From controlled sample experiments using cancer cell line derived EVs and specific fluorescence staining with lipophilic dyes, we identified that the DICE device is capable of isolating biotinylated EVs with 84.4% immobilization efficiency. We extended our study to profile EVs of 9 clinical samples from non-small cell lung cancer (NSCLC) patients and healthy donors and found that the DICE device successfully facilitates immunofluorescent staining for both the NSCLC patients and the healthy control. This versatile and simple method to profile EVs could be extended to EVs of any biological origin, promoting discoveries of the role of EVs in disease diagnostics and monitoring.

Post-debulking circulating tumor cell as a poor prognostic marker in advanced stage ovarian cancer: A prospective observational study.

Circulating tumor cells (CTCs) have received enormous attention as a novel biomarker in various malignant diseases. We investigated the clinical association between the presence of perioperative CTCs and survival outcomes in women with ovarian cancer. In a total of 30 women who were scheduled to undergo a surgical treatment for ovarian cancer, peripheral blood samples were obtained before and after surgery. CTCs were isolated and counted using the optimized tapered-slit filter (TSF) platform. The association between the presence of perioperative CTCs and tumor features was evaluated. The impact of the presence of perioperative CTCs on progression-free survival (PFS) and overall survival (OS) rates were analyzed using a Kaplan-Meier method. The median age was 58 (range, 24-77) years, and the median follow-up period was 31.5 (range, 1-41) months. Overall, the CTC detection rate was not significantly different before and after surgery (76.7% vs 57.1%, P = .673). The presence of postoperative CTCs was not significantly associated with 3-year PFS (29.1% vs 58.3%, P = .130) and OS (84.4% vs 80.0%, P = .559) rates in the whole study population. In advanced stage, PFS rate in patients with postoperative CTCs had lower PFS rates than those without postoperative CTCs, although there was no statistical significance (18.8% vs 57.1%, P = .077). Postoperative CTC was more frequently detected in women who had lymph node involvement than those who did not (7/7 [100%] vs 3/10 [30.0%], P = .010). The presence of postoperative CTCs as detected using the TSF platform seems to be associated with poorer PFS rates in women with ovarian cancer of advanced stage. Further study with a larger population is warranted to validate our study findings.

Detection of circulating tumour cells and their potential use as a biomarker for advanced renal cell carcinoma.

INTRODUCTION: The aim of this study was to detect circulating tumour cells (CTCs) in patients with advanced renal cell carcinoma (RCC) using a novel CTC detection platform. Furthermore, we evaluated the clinical outcomes associated with a CTC-positive status. METHODS: A total of 34 patients with advanced RCC (stage III or IV) were prospectively enrolled, and 104 peripheral blood samples were analyzed for the presence of CTCs at various time points. CTCs were isolated using a tapered-slit filter, which captures CTCs based on size and deformability. The presence of CTCs was confirmed using both staining and morphological criteria. CTC status was then correlated with clinical characteristics and survival outcomes. RESULTS: CTCs were detected in 62% of patients during the pre-treatment period, and the median CTC count was 2 (interquartile range 1-3). During the followup period, CTCs were detected in 56% (18/32), 65% (20/31), and 41% (7/17) of patients at one week, one month, and three months after treatment, respectively. Overall, CTCs were found in 57.9% (66/114) of blood samples in the range of 1-7 cells. Although no statistical significance was found, CTC detection in patients with stage IV disease was more common than in patients with stage III disease (68.4% vs. 53.3%). Two-year progression-free survival and cancer-specific survival tended to be lower in CTC-positive patients compared with CTC-negative patients. CONCLUSIONS: The tapered-slit filter is an efficient technique to detect CTCs in advanced RCC.

Cytopathological Study of the Circulating Tumor Cells filtered from the Cancer Patients' Blood using Hydrogel-based Cell Block Formation.

Circulating tumor cells have emerged as biomarkers for estimating the tumor burden and metastatic potential of cancer patients. However, to date, most of studies and applications of circulating tumor cells have been conducted and applied to epithelial cancers such as breast, colorectal, and prostate tumor. The only FDA-cleared method, CellSearch, makes use of antibody against epithelial surface protein expressed on CTCs, thus obstructing wide application for various cancers with non-epithelial and semi-epithelial characteristics including renal cell carcinoma. Due to rarity and ambiguity of CTCs, designed experiment including non-biased CTC isolation and subsequent cytopathological study for finding applicable immunomarkers are urgently needed for clinical use of CTCs for less-studied cancers. Here, in order to construct the fundamental step for CTC diagnosis without limitation of its epithelial characteristics, we present the simple and novel method which incorporate both label-free CTC isolation and pathological study using hydrogel-based cell block formation. Six cell lines from lung, ovarian, kidney cancers were used to make cell block and analyzed by conventional immunocytochemical staining method to find the candidate markers for CTC. Especially for renal cancer, the physically isolated CTCs were further immunocytochemically examined with the screened candidate markers by cell block construction, and verified their clinical utility using blood samples from patients with renal cell carcinoma. This comprehensive study demonstrates that the present approach can be used to find the potential markers for any type of cancers regardless of their epithelial characteristics and isolate the specific type of CTCs in label-free manners.

Circulating tumor cells in the differential diagnosis of adnexal masses.

The aim of this study was to evaluate circulating tumor cell (CTC) detection in the differential diagnosis of adnexal masses. A total of 87 preoperative women with an indeterminate adnexal mass were prospectively enrolled. Preoperative diagnostic modalities including CTC detection, risk of ovarian malignancy algorithm, risk of malignancy index, and computed tomography or magnetic resonance imaging were compared. Forty-three (49.4%) benign tumors, 13 (14.9%) borderline malignant masses, and 31 (35.7%) cancers were pathologically confirmed. Forty-nine (56.3%) cases were positive for CTCs: 19/43 (44.2%) benign, 10/10 (100%) early-stage, and 14/21 (66.7%) advanced-stage cancer. CTC detection had sensitivities of 77.4%, 100%, and 100% for benign vs. all stage cancer (n = 74), benign vs. stage I-II cancer (n = 53), and benign vs. stage I cancer (n = 49), respectively. CTC detection had a specificity of 55.8% across all comparisons. The sensitivities of the other modalities assayed were decreased in stage I-II cancer and stage I cancer vs. benign masses. Receiver operating characteristic curves showed that CTCs, of which the area under the curve was modest in all stage cancer (0.655), had the widest area under the curve among the evaluated modalities in stage I-II cancer and stage I cancer (0.768 for both). In conclusion, our study findings suggest that preoperative CTCs could have a substantial role in differentiating early stage cancer from benign tumors for adnexal masses.

High-purity capture and release of circulating exosomes using an exosome-specific dual-patterned immunofiltration (ExoDIF) device.

We present a microfluidic device for the capture and release of circulating exosomes from human blood. The exosome-specific dual-patterned immunofiltration (ExoDIF) device is composed of two distinct immuno-patterned layers, and is capable of enhancing the chance of binding between the antibody and exosomes by generating mechanical whirling, thus achieving high-throughput exosome isolation with high specificity. Moreover, follow-up recovery after the immuno-affinity based isolation, via cleavage of a linker, enables further downstream analysis. We verified the performance of the present device using MCF-7 secreted exosomes and found that both the concentration and proportion of exosome-sized vesicles were higher than in the samples obtained from the conventional exosome isolation kit. We then isolated exosomes from the human blood samples with our device to compare the exosome level between cancer patients and healthy donors. Cancer patients show a significantly higher exosome level with higher selectivity when validating the exosome-sized vesicles using both electron microscopy and nanoparticle tracking analysis. The captured exosomes from cancer patients also express abundant cancer-associated antigens, the epithelial cell adhesion molecule (EpCAM) on their surface. Our simple and rapid exosome recovery technique has huge potential to elucidate the function of exosomes in cancer patients and can thus be applied for various exosome-based cancer research studies.

Label-free Rapid Viable Enrichment of Circulating Tumor Cell by Photosensitive Polymer-based Microfilter Device.

We present a clinical device for simple, rapid, and viable isolation of circulating tumor cells (CTCs) from cancer patient bloods. In spite of the clinical importance of CTCs, the lack of easy and non-biased isolation methods is a big hurdle for implementing CTC into clinical use. The present device made of photosensitive polymer was designed to attach to conventional syringe to isolate the CTCs at minimal resources. Its unique tapered-slits on the filter are capable not only to isolate the cell based on their size and deformability, but also to increase sample flow rate, thus achieving label-free rapid viable CTC isolation. We verified our device performance using 9 different types of cancer cells at the cell concentration from 5 to 100cells/ml, showing that the device capture 77.7% of the CTCs while maintaining their viability of 80.6%. We extended our study using the 18 blood samples from lung, colorectal, pancreatic and renal cancer patients and captured 1-172 CTCs or clustered CTCs by immunofluorescent or immunohistochemical staining. The captured CTCs were also molecularly assayed by RT-PCR with three cancer-associated genes (CK19, EpCAM, and MUC1). Those comprehensive studies proved to use our device for cancer study, thereby inaugurating further in-depth CTC-based clinical researches.

Polyester fabric sheet layers functionalized with graphene oxide for sensitive isolation of circulating tumor cells.

The metastasis of cancer is strongly associated with the spread of circulating tumor cells (CTCs). Based on the microfluidic devices, which offer rapid recovery of CTCs, a number of studies have demonstrated the potential of CTCs as a diagnostic tool. However, not only the insufficient specificity and sensitivity derived from the rarity and heterogeneity of CTCs, but also the high-cost fabrication processes limit the use of CTC-based medical devices in commercial. Here, we present a low-cost fabric sheet layers for CTC isolation, which are composed of polyester monofilament yarns. Fabric sheet layers are easily functionalized with graphene oxide (GO), which is beneficial for improving both sensitivity and specificity. The GO modification to the low-cost fabrics enhances the binding of anti-EpCAM antibodies, resulting in 10-25% increase of capture efficiency compared to the surface without GO (anti-EpCAM antibodies directly onto the fabric sheets), while achieving high purity by isolating only 50-300 leukocytes in 1 mL of human blood. We investigated CTCs in ten human blood samples and successfully isolated 4-42 CTCs/mL from cancer patients, while none of cancerous cells were found among healthy donors. This remarkable results show the feasibility of GO-functionalized fabric sheet layers to be used in various CTC-based clinical applications, with high sensitivity and selectivity.

Lab on a fabric: Mass producible and low-cost fabric filters for the high-throughput viable isolation of circulating tumor cells.

Circulating tumor cells (CTCs) play an important role in estimating the presence and the metastatic relapse of tumor. Despite of their importance, isolation of viable CTCs is still struggling, since chemical or mechanical damages are unavoidable when separating less than 1000 of CTCs out of billions of other blood components. Furthermore, the current CTC isolation devices show low productivity, since they are produced after a series of complicated fabrication processes. Here, we present a low-cost and mass-producible fabric filters for the viable CTC isolation and the further molecular assay for profiling cancer-associated markers. The fabric filter, produced by polyester monofilament yarns, can be massively produced at extremely low-cost, by showing productivity of ~22filters/s at ~59filters/USD. By utilizing size-based sorting method, the fabric filter is capable to isolate both epithelial and mesenchymal CTCs, while slots with curved walls are beneficial for preventing the cell rupture by reducing 21.6% of mechanical stress compared to the conventional straight-walled slots. We applied our filter to 11 human blood samples and found that the number of CTCs was closely related to the expression level of Ki-67, which is highly overexpressed in proliferative tumors. The fabric filter might be an appropriate caner-screening tool in developing countries, where people suffer from insufficient healthcare services.

Dual-patterned immunofiltration (DIF) device for the rapid efficient negative selection of heterogeneous circulating tumor cells.

The analysis of circulating tumor cells (CTCs) is an emerging field for estimating the metastatic relapse and tumor burden of cancer patients. However, the isolation of CTCs is still challenging due to their ambiguity, rarity, and heterogeneity. Here, we present an anti-CD45 antibody based dual-patterned immunofiltration (DIF) device for the enrichment of heterogeneous CTC subtypes by effective elimination of leukocytes. Our uniquely designed dual-patterned layers significantly enhance the binding chance between immuno-patterns and leukocytes due to the fluidic whirling and the increased binding sites, thus achieving superior negative selection in terms of high-throughput and high purity. From the experiments using lung cancer cells, 97.07 ± 2.79% of leukocytes were eliminated with less than 10% loss of cancerous cells at the flow rate of 1 mL h-1. To verify the device as a potential diagnostic tool, CTCs were collected from 11 cancer patients' blood and an average of 283.3 CTC-like cells were identified while less than 1 CTC-like cells were found from healthy donors. The samples were also analyzed by immunohistochemistry and the reverse transcription polymerase chain reaction to identify their heterogeneous characteristics. These remarkable results demonstrate that the present device could help to understand the unknown properties or undiscovered roles of CTCs with a non-biased view.

Poly(ethylene glycol)-Modified Tapered-Slit Membrane Filter for Efficient Release of Captured Viable Circulating Tumor Cells.

The grafting of poly(ethylene glycol) (PEG) onto an SU8 microfilter has been demonstrated for efficient capture and release of circulating tumor cells (CTCs). Previous CTC filters showed low cell release efficiency due to hydrophobic surfaces, even though their capture efficiency was considerable. PEG, a hydrophilic polymeric compound mainly used to form nonfouling thin films on silicon surfaces, induces repulsive force so that the nonspecific adsorption of the surface is incomparably reduced in comparison with unmodified filter surfaces. The effectiveness of PEG-modified CTC filters was verified through lung (H358) and colorectal (SW620) cancer cells spiked, respectively, in phosphate-buffered saline (PBS) and unprocessed whole blood. The modified SU8 filters achieved approximately 37.7% and 22.8% improvement in release efficiency without significant changes in cell viability and capture efficiency. In order to verify the filter's potential for clinical applications, we extended our experiments using cancer patient blood samples. Six blood samples from colorectal and lung cancer patients were processed, and captured CTCs were efficiently released. From these experiments, the present PEG-modified filter captures and releases on average 14 ± 7.4 CTCs/mL, including EpCAM-negative CTCs, which could not be captured by previous single antibody-based methods. The antibody-free isolation with enhanced release efficiency facilitates viable cell retrieval, which is significant to CTC culture and comprehensive molecular study for verifying the mechanism of metastasis and cancer.

Tapered-slit membrane filters for high-throughput viable circulating tumor cell isolation.

This paper presents tapered-slit membrane filters for high-throughput viable circulating tumor cell (CTC) isolation. The membrane filter with a 2D array of vertical tapered slits with a gap that is wide at the entrance and gradually decreases with depth, provide minimal cell stress and reduce 82.14% of the stress generated in conventional straight-hole filters. We designed two types of tapered-slit filters, Filters 6 and 8, respectively, containing the tapered slits with outlet widths of 6 µm and 8 µm at a slit density of 34,445/cm(2) on the membrane. We fabricated the vertical slits with a tapered angle of 2 ° on a SU8 membrane by adjusting the UV expose dose and the air gap between the membrane and the photomask during lithography. In the experimental study, the proposed tapered-slit filter captured 89.87% and 82.44% of the cancer cells spiked in phosphate buffered saline (PBS) and diluted blood (blood: PBS = 1:4), respectively, at a sample flow rate of 5 ml per hour, which is 33.3 times faster than previous lateral tapered-slit filters. We further verified the capability to culture on chip after capturing: 72.33% of cells among the captured cells still remained viable after a 5-day culture. The proposed tapered-slit membrane filters verified high-throughput viable CTC isolation capability, thereby inaugurating further advanced CTC research for cancer diagnosis and prognosis.