Liposome-tethered supported lipid bilayer platform for capture and release of heterogeneous populations of circulating tumor cells.

Because of scarcity, vulnerability, and heterogeneity in the population of circulating tumor cells (CTCs), the CTC isolation system relying on immunoaffinity interaction exhibits inconsistent efficiencies for all types of cancers and even CTCs with different phenotypes in individuals. Moreover, releasing viable CTCs from an isolation system is of importance for molecular analysis and drug screening in precision medicine, which remains a challenge for current systems. In this work, a new CTC isolation microfluidic platform was developed and contains a coating of the antibody-conjugated liposome-tethered-supported lipid bilayer in a developed chaotic-mixing microfluidic system, referred to as the "LIPO-SLB" platform. The biocompatible, soft, laterally fluidic, and antifouling properties of the LIPO-SLB platform offer high CTC capture efficiency, viability, and selectivity. We successfully demonstrated the capability of the LIPO-SLB platform to recapitulate different cancer cell lines with different antigen expression levels. In addition, the captured CTCs in the LIPO-SLB platform can be detached by air foam to destabilize the physically assembled bilayer structures due to a large water/air interfacial area and strong surface tension. More importantly, the LIPO-SLB platform was constructed and used for the verification of clinical samples from 161 patients with different primary cancer types. The mean values of both single CTCs and CTC clusters correlated well with the cancer stages. Moreover, a considerable number of CTCs were isolated from patients' blood samples in the early/localized stages. The clinical validation demonstrated the enormous potential of the universal LIPO-SLB platform as a tool for prognostic and predictive purposes in precision medicine.

Proliferative ability of circulating tumor cells is a prognostic factor in Early-Stage lung adenocarcinoma.

INTRODUCTION: Circulating tumor cells (CTCs) and their proliferative ability in lung adenocarcinoma (LUAD) were not well-investigated. We developed a protocol combining an efficient viable CTC isolation and in-vitro cultivation for the CTC enumeration and proliferation to evaluate their clinical significance. METHOD: The peripheral blood of 124 treatment-naïve LUAD patients were processed by a CTC isolation microfluidics, DS platform, followed by in-vitro cultivation. LUAD-specific CTCs were defined by immunostaining of DAPI+/CD45-/(TTF1/CK7)+ and were enumerated upon isolation and after 7-day cultivation. The CTC proliferative ability was evaluated by both the cultured number and the culture index, a ratio of cultured CTC number to the initial CTC number in 2 mL of blood. RESULT: All but two LUAD patients (98.4%) were detected with at least one CTC per 2 mL of blood. Initial CTC numbers did not correlate with metastasis (75 ± 126 for non-metastatic, 87 ± 113 for metastatic groups; P = 0.203). In contrast, both the cultured CTC number (mean: 28, 104, and 185 in stage 0/I, II/III, and IV; P < 0.001), and the culture index (mean: 1.1, 1.7 and 9.3 in stage 0/I, II/III, and IV; P = 0.043) were significantly correlated with the stages. Overall survival analysis within the non-metastatic group (N = 53) showed poor prognosis for patients with elevated cultured counts (cutoff ≥ 30; P = 0.027). CONCLUSION: We implemented a CTC assay in clinical LUAD patients with a high detection rate and cultivation capability. Cultured CTC count and proliferative ability, rather than the crude CTC numbers, highly associated with cancer prognosis.

Multiomic characterization and drug testing establish circulating tumor cells as an ex vivo tool for personalized medicine.

Matching the treatment to an individual patient's tumor state can increase therapeutic efficacy and reduce tumor recurrence. Circulating tumor cells (CTCs) derived from solid tumors are promising subjects for theragnostic analysis. To analyze how CTCs represent tumor states, we established cell lines from CTCs, primary and metastatic tumors from a mouse model and provided phenotypic and multiomic analyses of these cells. CTCs and metastatic cells, but not primary tumor cells, shared stochastic mutations and similar hypomethylation levels at transcription start sites. CTCs and metastatic tumor cells shared a hybrid epithelial/mesenchymal transcriptome state with reduced adhesive and enhanced mobilization characteristics. We tested anti-cancer drugs on tumor cells from a metastatic breast cancer patient. CTC responses mirrored the impact of drugs on metastatic rather than primary tumors. Our multiomic and clinical anti-cancer drug response results reveal that CTCs resemble metastatic tumors and establish CTCs as an ex vivo tool for personalized medicine.

Polycarboxybetaine-Based Hydrogels for the Capture and Release of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are indicators for the detection, diagnosis, and monitoring of cancers and offer biological information for the development of personalized medicine. Techniques for the specific capture and non-destructive release of CTCs from millions of blood cells remain highly desirable. Here, we present a CTC capture-and-release system using a disulfide-containing poly(carboxybetaine methacrylate) (pCB) hydrogel. The non-fouling characteristic of pCB prevents unwanted, nonspecific cell binding, while the carboxyl functionality of pCB is used for the conjugation of anti-epithelial cell adhesion molecule (anti-EpCAM) antibodies for the capture of CTCs. The results demonstrated that the anti-EpCAM-conjugated pCB hydrogel captured HCT116 cells from blood, and the capture ratio reached 45%. Furthermore, the captured HCT116 cells were released within 30 min from the dissolution of the pCB hydrogel by adding cysteine, which breaks the disulfide bonds of the crosslinkers. The cells released were viable and able to grow. Our system has potential in the development of a device for CTC diagnosis.

Early Detection and Dynamic Changes of Circulating Tumor Cells in Transgenic NeuN Transgenic (NTTg) Mice with Spontaneous Breast Tumor Development.

BACKGROUND: This study used NeuN transgenic (NTTg) mice with spontaneous breast tumor development to evaluate the dynamic changes of circulating tumor cells (CTCs) prior to and during tumor development. METHODS: In this longitudinal, clinically uninterrupted study, we collected 75 µL of peripheral blood at the age of 8, 12, 16, and 20 weeks in the first group of five mice, and at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability in the second group of four mice. Diluted blood samples were run through a modified mouse-CMx chip to isolate the CTCs. RESULTS: The CTC counts of the first group of mice were low (1 ± 1.6) initially. The average CTC counts were 16 ± 9.5, 29.0 ± 18.2, and 70.0 ± 30.3 cells per 75 µL blood at the age of 32 weeks, the time of tumor palpability, and one week after tumor palpability, respectively. There was a significant positive correlation between an increase in CTC levels and tumor vascular density (p-value < 0.01). This correlation was stronger than that between CTC levels and tumor size (p-value = 0.076). The captured CTCs were implanted into a non-tumor-bearing NTTg mouse for xenografting, confirming their viability and tumorigenesis. CONCLUSION: Serial CTCs during an early stage of tumor progression were quantified and found to be positively correlated with the later tumor vascular density and size. Furthermore, the successful generation of CTC-derived xenografts indicates the tumorigenicity of this early onset CTC population.

Anticlogging Hemofiltration Device for Mass Collection of Circulating Tumor Cells by Ligand-Free Size Selection.

A new hemofiltration system was developed to continuously capture circulating tumor cells (CTCs) from a large volume of whole blood using a column that was packed with antifouling zwitterionized silica microspheres. The silica microspheres were modified with sulfobetaine silane (SBSi) to inhibit fouling, resist clogging, and give a high surface wettability and prolonged operation time. Packed microspheres with different diameters formed size-controllable interstitial pores that effectively captured CTCs by ligand-free size selection. For optimized performance of the hemofiltration system, operational factors, including the size of microspheres, flow rate, and cross-sectional area of the column, were considered with respect to the removal rate for colorectal cancer cells and the retention rate for white blood cells and red blood cells. The captured CTCs were collected from the column by density sedimentation. A large quantity of colorectal cancer cells was spiked into sheep blood, and the sample was circulated for 5 h with a total operational volume of 2 L followed by collection and culture in vitro. The results showed that the proposed hemofiltration device selectively removed abundant CTCs from in vitro circulatory blood. The viable cells were harvested for amplification and potential applications for precision medicine.

A two-dimensional immunomagnetic nano-net for the efficient isolation of circulating tumor cells in whole blood.

An immunomagnetic "nano-net" was designed and synthesized for specifically capturing rare cells of interest from mixtures. The nano-net, Ab@Lipo-MNP-GO, consists of conjugated antibody molecules on a lipid coated magnetic nanoparticle-graphene oxide sheet complex. The magnetism, chemical composition, and the morphology of the construct and its precursors were characterized by SQUID, FTIR, TGA, DLS and SEM, to confirm the feasibility of the synthetic steps and the resulting properties suitable for solution phase immuno-recognition for cell capture. When applied to capturing circulating tumor cells (CTCs) in oral, colon and lung cancer clinical patients' blood samples, the nano-net construct exhibited far superior ability whereas conventional immunomagnetic beads in some cases were unable to capture any CTCs, even by increasing the bead concentration. Confocal images showed that the nano-net wrapped around the CTCs while the immunomagnetic beads attached them with point contacts. A stable, patch-like multivalent matrix nano-net was demonstrated to tackle the shortcomings of single point contact of immunomagnetic beads to the target cell. This strategy is universal for any cell separation in complex fluids.

Random and aligned electrospun PLGA nanofibers embedded in microfluidic chips for cancer cell isolation and integration with air foam technology for cell release.

BACKGROUND: Circulating tumor cells (CTCs) comprise the high metastatic potential population of cancer cells in the blood circulation of humans; they have become the established biomarkers for cancer diagnosis, individualized cancer therapy, and cancer development. Technologies for the isolation and recovery of CTCs can be powerful cancer diagnostic tools for liquid biopsies, allowing the identification of malignancies and guiding cancer treatments for precision medicine. METHODS: We have used an electrospinning process to prepare poly(lactic-co-glycolic acid) (PLGA) nanofibrous arrays in random or aligned orientations on glass slips. We then fabricated poly(methyl methacrylate) (PMMA)-based microfluidic chips embedding the PLGA nanofiber arrays and modified their surfaces through sequential coating with using biotin-(PEG)7-amine through EDC/NHS activation, streptavidin (SA), and biotinylated epithelial-cell adhesion-molecule antibody (biotin-anti-EpCAM) to achieve highly efficient CTC capture. When combined with an air foam technology that induced a high shear stress and, thereby, nondestructive release of the captured cells from the PLGA surfaces, the proposed device system operated with a high cell recovery rate. RESULTS: The morphologies and average diameters of the electrospun PLGA nanofibers were characterized using scanning electron microscopy (SEM) and confocal Raman imaging. The surface chemistry of the PLGA nanofibers conjugated with the biotin-(PEG)7-amine was confirmed through time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. The chip system was studied for the effects of the surface modification density of biotin-(PEG)7-amine, the flow rates, and the diameters of the PLGA nanofibers on the capture efficiency of EpCAM-positive HCT116 cells from the spiked liquid samples. To assess their CTC capture efficiencies in whole blood samples, the aligned and random PLGA nanofiber arrays were tested for their abilities to capture HCT116 cells, providing cancer cell capture efficiencies of 66 and 80%, respectively. With the continuous injection of air foam into the microfluidic devices, the cell release efficiency on the aligned PLGA fibers was 74% (recovery rate: 49%), while it was 90% (recovery rate: 73%) on the random PLGA fibers, from tests of 200 spiked cells in 2 mL of whole blood from healthy individuals. Our study suggests that integrated PMMA microfluidic chips embedding random PLGA nanofiber arrays may be suitable devices for the efficient capture and recovery of CTCs from whole blood samples.

Snail-induced claudin-11 prompts collective migration for tumour progression.

Epithelial-mesenchymal transition (EMT) is a pivotal mechanism for cancer dissemination. However, EMT-regulated individual cancer cell invasion is difficult to detect in clinical samples. Emerging evidence implies that EMT is correlated to collective cell migration and invasion with unknown mechanisms. We show that the EMT transcription factor Snail elicits collective migration in squamous cell carcinoma by inducing the expression of a tight junctional protein, claudin-11. Mechanistically, tyrosine-phosphorylated claudin-11 activates Src, which suppresses RhoA activity at intercellular junctions through p190RhoGAP, maintaining stable cell-cell contacts. In head and neck cancer patients, the Snail-claudin-11 axis prompts the formation of circulating tumour cell clusters, which correlate with tumour progression. Overexpression of snail correlates with increased claudin-11, and both are associated with a worse outcome. This finding extends the current understanding of EMT-mediated cellular migration via a non-individual type of movement to prompt cancer progression.

Scalable Multilayer Cell Collector to Capture Circulating Tumor Cells with an Unlimited Volume Capacity.

Circulating tumor cells (CTCs) have been suggested as the precursors of metastatic cancer. CTC-based characterization has thus been used to monitor tumor status before the onset of metastasis and has shown to be an independent factor. The low abundance of CTCs, however, makes it challenging to employ CTC as a clinical routine, thus making it impossible to address tumor heterogeneity. Here, we present a cell collection prototype for an efficient capture of CTCs from a large volume of body fluids such as blood. An antibody-PEG modified multilayer matrix column is engineered and connected to an apheresis-based circulation system. This setup allows us to capture CTCs repetitively from an unlimited sample volume through the circulation system, thereby increasing the capture count. Compared to conventional CTC capturing devices where the sample handling is generally limited to 1-10 mL, our collector is able to handle a wide range of fluidic sample (40-2000 mL) at a high flow rate (400 mL/min). By processing 90 min in circulation, we obtained an average capture efficiency of at least 75% for the colorectal cancer cell line HCT116 spiked in either 40-200 mL of buffer solution or 40 mL of a whole blood sample. This result highlights a possibility to construct personalized CTC libraries through high-throughput CTC collection for the study of tumor heterogeneity in precision medicine.

Sport Activity for Health!! The Effects of Karate Participants' Involvement, Perceived Value, and Leisure Benefits on Recommendation Intention.

This study intends to discuss the effects of participants’ involvement, perceived value, and leisure benefits on recommendation intention in the sport of karate. The questionnaires were collected online by karate clubs on Facebook and included 369 valid participants. The research findings show that karate participants from different places of residence do not display significant differences in involvement, perceived value, leisure benefits, and recommendation intention. Furthermore, “attraction” in the involvement category reveals the highest mean, “paid spirit and energy being worthy” in perceived value appears as the highest mean, and “physiological benefits” in leisure benefits shows the highest mean. The Pearson correlation analysis result presents significant strong positive correlations between involvement, perceived value, leisure benefits, and recommendation intention. Finally, multiple regression analysis reveals that leisure benefits, except “physiological benefits”, show notably positive effects on recommendation intention. According to the research results, suggestions are proposed for the reference of karate teaching business managers, participants, and future research.

Promoting Multivalent Antibody-Antigen Interactions by Tethering Antibody Molecules on a PEGylated Dendrimer-Supported Lipid Bilayer.

To efficiently isolate maximal quantity of circulating tumor cells (CTCs) and circulating tumor cell microembolis (CTMs) from patient blood by antibody coated microfluidics, a multifunctional, pegylated polyamidoamine-dendrimers conjugated supported lipid bilayer surface construct was proposed to enhance accessibility of antibody molecules to the antigen molecules on target CTCs. The combination of a hydrated, stretchable dendrimer and a laterally mobile supported lipid bilayer (SLB) provide attached antibody molecules with 2.5-dimensional chain movement, achieving multivalency between the surface antibody and cell antigen molecules. An over 170% enhancement is distinctive for Panc-1 cells that expresses low antigen level. Of seven pancreatic ductal adenocarcinoma patients, an average 440 single CTCs and 90 CTMs were collected in 2 mL of peripheral blood, which were 1.6 times and 2.3 times more, than those captured by the SLB-only microfluidics. In summary, we have demonstrated a material design to enhance multivalent antibody-antigen interaction, which is useful for rare cell enrichment and cancer detection.

Microfluidic Capture and Multiplex Immunofluorescence of Circulating Tumor Cells to Identify Cancer of Origin.

Circulating tumor cells (CTCs) are an important biomarker and their analysis can be considered a form of "liquid biopsy." The purpose of this book chapter is to describe the use of the 4-channel CMx (cells captured in maximum) microfluidic chip, containing special micropatterns coated with an antibody-conjugated supported lipid bilayer (SLB) on its surface, to capture and isolate CTCs from the blood of cancer patients. Captured CTCs are subsequently released by an air foam to an immunofluorescence (IF) staining panel that enables further analysis, including the identification of the primary cancer source of the CTCs.

Strategies for Isolation and Molecular Profiling of Circulating Tumor Cells.

Cancer is the leading cause of death by disease worldwide, and metastasis is responsible for more than 90% of the mortality of cancer patients. Metastasis occurs when tumor cells leave the primary tumor, travel through the blood stream as circulating tumor cells (CTCs), and then colonize secondary tumors at sites distant from the primary tumor. The capture, identification, and analysis of CTCs offer both scientific and clinical benefits. On the scientific side, the analysis of CTCs could help elucidate possible genetic alterations and signaling pathway aberrations during cancer progression, which could then be used to find new methods to stop cancer progression. On the clinical side, non-invasive testing of a patient's blood for CTCs can be used for patient diagnosis and prognosis, as well as subsequent monitoring of treatment efficacy in routine clinical practice. Additionally, investigation of CTCs early in the progression of cancer may reveal targets for initial cancer detection and for anti-cancer treatment. This chapter will evaluate strategies and devices used for the isolation and identification of CTCs directly from clinical samples of blood. Recent progress in the understanding of the significance of both single CTCs and circulating tumor microemboli will be discussed. Also, advancements in the use of CTC-based liquid biopsy in clinical diagnosis and the potential of CTC-based molecular characterization for use in clinical applications will be summarized.

Site-specific antibody modification and immobilization on a microfluidic chip to promote the capture of circulating tumor cells and microemboli.

We design and synthesize EpCAM antibodies with Fc-domain site-specific linkers that allow preferential alignment when coated on microfluidic devices for capturing circulating tumor cells (CTCs) from colorectal cancer patients. The aligned coating is shown to increase the capture efficiency of CTCs and microemboli by 1.6 and 3.0-fold, respectively (both P < 0.05).

Circulating Tumor Cell Count Correlates with Colorectal Neoplasm Progression and Is a Prognostic Marker for Distant Metastasis in Non-Metastatic Patients.

Enumeration of circulating tumor cells (CTCs) has been proven as a prognostic marker for metastatic colorectal cancer (m-CRC) patients. However, the currently available techniques for capturing and enumerating CTCs lack of required sensitivity to be applicable as a prognostic marker for non-metastatic patients as CTCs are even more rare. We have developed a microfluidic device utilizing antibody-conjugated non-fouling coating to eliminate nonspecific binding and to promote the multivalent binding of target cells. We then established the correlation of CTC counts and neoplasm progression through applying this platform to capture and enumerate CTCs in 2 mL of peripheral blood from healthy (n = 27), benign (n = 21), non-metastatic (n = 95), and m-CRC (n = 15) patients. The results showed that the CTC counts progressed from 0, 1, 5, to 36. Importantly, after 2-year follow-up on the non-metastatic CRC patients, we found that those who had ≥5 CTCs were 8 times more likely to develop distant metastasis within one year after curable surgery than those who had <5. In conclusion, by employing a sensitive device, CTC counts show good correlation with colorectal neoplasm, thus CTC may be as a simple, independent prognostic marker for the non-metastatic CRC patients who are at high risk of early recurrence.

A High Circulating Tumor Cell Count in Portal Vein Predicts Liver Metastasis From Periampullary or Pancreatic Cancer: A High Portal Venous CTC Count Predicts Liver Metastases.

Circulating tumor cells (CTCs) released from a periampullary or pancreatic cancer can be more frequently detected in the portal than the systemic circulation and potentially can be used to identify patients with liver micrometastases. Aims of this study is to determine if CTCs count in portal venous blood of patients with nonmetastatic periampullary or pancreatic adenocarcinoma can be used as a predictor for subsequent liver metastases. CTCs were quantified in portal and peripheral venous blood samples collected simultaneously during pancreaticoduodenectomy in patients with presumed periampullary or pancreatic adenocarcinoma without image-discernible metastasis. Postoperatively patients were monitored for liver metastasis by abdominal magnetic resonance imaging or computed tomography every 3 months for 1 year. Sixty patients with a pathological diagnosis of periampullary or pancreatic adenocarcinoma were included in the study. Multivariate analysis indicated that portal CTC count was a significant predictor for liver metastases within 6 months after surgery. Eleven of 13 patients with a high portal CTCs count (defined as >112 CMx Platform estimated CTCs in 2 mL blood) developed liver metastases within 6 months after surgery. In contrast, only 6 of 47 patients with a low portal CTC count developed liver metastases (P < 0.0001). A value of 112 CMx Platform estimated CTCs had 64.7% sensitivity and 95.4% specificity to predict liver metastases within 6 months after surgery. We concluded that a high CTC count in portal venous blood collected during pancreaticoduodenectomy in patients with periampullary or pancreatic adenocarcinoma without metastases detected by currently available imaging tools is a significant predictor for liver metastases within 6 months after surgery.

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection.

Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) "smart coating" to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.

Identifying cancer origin using circulating tumor cells.

Circulating tumor cells (CTCs) have become an established clinical evaluation biomarker. CTC count provides a good correlation with the prognosis of cancer patients, but has only been used with known cancer patients, and has been unable to predict the origin of the CTCs. This study demonstrates the analysis of CTCs for the identification of their primary cancer source. Twelve mL blood samples were equally dispensed on 6 CMx chips, microfluidic chips coated with an anti-EpCAM-conjugated supported lipid bilayer, for CTC capture and isolation. Captured CTCs were eluted to an immunofluorescence (IF) staining panel consisting of 6 groups of antibodies: anti-panCK, anti-CK18, anti-CK7, anti-TTF-1, anti-CK20/anti-CDX2, and anti-PSA/anti-PSMA. Cancer cell lines of lung (H1975), colorectal (DLD-1, HCT-116), and prostate (PC3, DU145, LNCaP) were selected to establish the sensitivity and specificity for distinguishing CTCs from lung, colorectal, and prostate cancer. Spiking experiments performed in 2mL of culture medium or whole blood proved the CMx platform can enumerate cancer cells of lung, colorectal, and prostate. The IF panel was tested on blood samples from lung cancer patients (n = 3), colorectal cancer patients (n = 5), prostate cancer patients (n = 5), and healthy individuals (n = 12). Peripheral blood samples found panCK(+) and CK18(+) CTCs in lung, colorectal, and prostate cancers. CTCs expressing CK7(+) or TTF-1(+), (CK20/ CDX2)(+), or (PSA/ PSMA)(+) corresponded to lung, colorectal, or prostate cancer, respectively. In conclusion, we have designed an immunofluorescence staining panel to identify CTCs in peripheral blood to correctly identify cancer cell origin.

Clinical Significance of Circulating Tumor Microemboli as a Prognostic Marker in Patients with Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Characterization of circulating tumor cells (CTCs) has been used to provide prognostic, predictive, and pharmacodynamic information in many different cancers. However, the clinical significance of CTCs and circulating tumor microemboli (CTM) in patients with pancreatic ductal adenocarcinoma (PDAC) has yet to be determined. METHODS: In this prospective study, CTCs and CTM were enumerated in the peripheral blood of 63 patients with PDAC before treatment using anti-EpCAM (epithelial cell adhesion molecule)-conjugated supported lipid bilayer-coated microfluidic chips. Associations of CTCs and CTM with patients' clinical factors and prognosis were determined. RESULTS: CTCs were abundant [mean (SD), 70.2 (107.6)] and present in 81% (51 of 63) of patients with PDAC. CTM were present in 81% (51 of 63) of patients with mean (SD) 29.7 (1101.4). CTM was an independent prognostic factor of overall survival (OS) and progression free survival (PFS). Patients were stratified into unfavorable and favorable CTM groups on the basis of CTM more or less than 30 per 2 mL blood, respectively. Patients with baseline unfavorable CTM, compared with patients with favorable CTM, had shorter PFS (2.7 vs 12.1 months; P < 0.0001) and OS (6.4 vs 19.8 months; P < 0.0001). Differences persisted if we stratified patients into early and advanced diseases. The number of CTM before treatment was an independent predictor of PFS and OS after adjustment for clinically significant factors. CONCLUSIONS: The number of CTM, instead of CTCs, before treatment is an independent predictor of PFS and OS in patients with PDAC.