Size-based Filtration Research Articles

Abstract B24: Integration of filtration with immunoaffinity for isolating circulating tumor cells from pancreatic and colorectal cancer patients

Abstract We report two microfluidic devices that combine filtration and immunoaffinity for the detection of circulating tumor cells (CTCs) from peripheral blood samples of pancreatic and colorectal cancer patients. Our approach contrasts with most CTC isolation methods that are based either on cells’ physical property (e.g., size-based filtration) or on biologic property (e.g., immunoaffinity). The combined approach addresses the challenges related to CTCs’ heterogeneity in their properties. For instance, immunoaffinity-based methods such as CellSearch® that employ antibodies against epithelial cell adhesion molecule (EpCAM) cannot isolate those CTCs expressing little or no EpCAM. Filtration-based methods cannot detect CTCs of a size smaller than the predefined filter pore diameter while many normal blood cells of a size larger than the pore are retained by the filter. Our microfluidic devices contain a serpentine main channel and an array of lateral microfilters. The key difference of our devices from the conventional filtration platforms is that our filters are not in the direction of the main flow. The unique design of the device layout leads to a two-dimensional flow that allows the majority of a sample to pass by while all cells have opportunities to interact with filters, resulting in a larger throughput, reduced cell clogging, and increased purity of cells isolated. Another advantage of our devices is that a series of filter sizes can be created in one device to accommodate different CTC diameters, which is difficult to achieve in traditional filtration platforms. Our devices are further functionalized by immobilizing antibodies on the surfaces of microfilters to combine filtration with immunoaffinity for CTC isolation. We have designed one device with filter sizes of 6-10 μm and another device with filter sizes of 12-24 μm, and we tested them for the isolation of L3.6pl cells (pancreatic cancer cells) spiked in a buffer or blood. We compared an antibody-immobilized device with the same device containing filters only. Without antibody, the device has a capture efficiency from 69.8% to 83.9%, depending on the flow rate used. With antibody, the device with combined CTC isolation mechanisms can accomplish a capture efficiency of (98.7 ± 1.2)% at a flow rate of 1.8 mL/h. Other tumor cells with different sizes have also been evaluated in the devices. Further, we employed the devices for enumerating CTCs from blood samples of pancreatic and colorectal cancer patients. We compared our integration devices with a previously reported device containing herringbone-based micromixers. We found that the integration devices generally detected a higher number of CTCs. In summary, microfluidic devices have been developed to integrate filtration with immunoaffinity for CTC isolation. The devices offer better performance than those based on one isolation mechanism only, with a potential to address CTC heterogeneity and detect CTCs with different sizes and various expression levels. Citation Format: Hugh Fan, Kangfu Chen, Pablo Dopico, Jacob Amontree, Thomas George. Integration of filtration with immunoaffinity for isolating circulating tumor cells from pancreatic and colorectal cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B24.

Colorimetric Detection of Escherichia coli O157:H7 with Signal Enhancement Using Size-Based Filtration on a Finger-Powered Microfluidic Device.

Although immunomagnetic separation is a useful sample pretreatment method that can be used to separate target pathogens from a raw sample, it is challenging to remove unbound free magnetic nanoparticles (MNPs) for colorimetric detection of target pathogens. Here, size-based filtration was exploited for the rapid on-site detection of pathogens separated by immunomagnetic separation in order to remove unbound free MNPs using a finger-powered microfluidic device. A membrane filter and an absorbent pad were integrated into the device and a mixture of unbound free MNPs and MNP-bound Escherichia coli (E. coli) O157:H7 was dispensed over the membrane filter by pressing and releasing the pressure chamber. A colorimetric signal was generated by MNP-bound E. coli O157:H7 while unbound free MNPs were washed out by the absorbent. Furthermore, the colorimetric signals can be amplified using a gold enhancer solution when gold-coated MNPs were used instead of MNPs. As a result, 102 CFU/mL E. coli O157:H7 could be detected by the enhanced colorimetric signal on a proposed device.

High‑throughput and continuous flow isolation of rare circulating tumor cells and clusters in gastric cancer from human whole blood samples using electromagnetic vibration‑based filtration.

Circulating tumor cells (CTCs) or CTC clusters are considered as suitable and relevant targets for liquid biopsy as they more accurately indicate cancer progression, the therapeutic effects of treatment and allows for monitoring of cancer metastasis in real-time. Among the various methods for isolating CTCs, size-based filtration is one of the most convenient methods. However, cell clogging makes the filtration process less efficient. In the present study, an electromagnetic vibration-based filtration (eVBF) device was developed that efficiently isolated rare CTCs and CTC clusters from clinical blood samples of patients with gastric cancer. Using human blood samples spiked with human gastric cancer cells, the parameters of this device such as vibrating amplitude and flow rate were optimized. Putative CTCs were detected using a conventional filtration method and the eVBF device from the peripheral blood samples of patients with gastric cancer. Continuous flow isolation of CTCs was evaluated by a simulated blood flow system. The eVBF device utilized the electromagnetic force to generate a periodic vibration that prevented the cell clogging and improved the filtering efficiency. The optimized eVBF device with the high-amplitude vibration exhibited a recovery efficiency of 80–90% from whole blood samples spiked with 100 or 1,000 gastric cancer cells per ml. Using the eVBF device, CTCs were detected in 100% of patients (10/10) with gastric cancer, and the positive detection rate of the eVBF device was 30% higher compared with the conventional filtration method. Furthermore, CTC clusters were detected in 40% (4/10) of CTC-positive patient samples, and the integrity of CTC clusters was preserved using the eVBF device. The eVBF device allowed for high-throughput (1 ml/min) and continuous flow isolation of CTCs without the addition of any antibodies, any chemical reagents or any pretreatment processes. Thus, the eVBF device provides an efficient tool for isolating rare CTCs and CTC clusters from patients with cancer, highlighting its potential for use in cancer diagnosis, treatment and cancer biology research.

Opto-thermoelectric speckle tweezers.

Opto-thermoelectric tweezers present a new paradigm for optical trapping and manipulation of particles using low-power and simple optics. New real-life applications of opto-thermoelectric tweezers in areas such as biophysics, microfluidics, and nanomanufacturing will require them to have large-scale and high-throughput manipulation capabilities in complex environments. Here, we present opto-thermoelectric speckle tweezers, which use speckle field consisting of many randomly distributed thermal hotspots that arise from an optical speckle pattern to trap multiple particles over large areas. By further integrating the speckle tweezers with a microfluidic system, we experimentally demonstrate their application for size-based nanoparticle filtration. With their low-power operation, simplicity, and versatility, opto-thermoelectric speckle tweezers will broaden the applications of optical manipulation techniques.

150P - Liquid biopsy assays using combined circulating tumour cells and circulating tumour DNA in the same patients for the diagnosis of primary lung cancer

BackgroundLung cancer is the most common cause of cancer-related mortality worldwide. Early diagnosis and surgery may be one of the most important strategy for the treatment of lung cancer patients. However, the procedure of lung cancer diagnosis from initial suspicion to final confirmation is not efficient due to low sensitivity of current diagnostic methods and difficulties involved in tumor tissue biopsy in lung cancer compared to other cancer types. Therefore, we investigated the feasibility of liquid biopsy using circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) analysis for the better diagnosis of lung cancer. MethodsIn this study, we blindly analyzed both CTCs and ctDNA in the blood samples derived from 109 patients including histology-proven and clinically suspicious lung cancer patients to test the sensitivity of liquid biopsy methods. CTC analysis was done by CytoGen’s liquid biopsy platform, in which viable CTCs were isolated by size-based filtration with gravity and validated by immunostaining of EpCAM or CK, excluding CD45 positive cells and enumerated by CytoGen’s cell imaging software. Lung cancer diagnosis was predicted by a cut-off, 2 ≥ CTC in 5ml peripheral blood. For ctDNA analysis, EDGC F-Can platform was used to analyze single nucleotide variations of very low variant allele frequencies by utilizing unique molecular indexes and a novel read error correction algorithm. For comparison, we also analyzed the levels of conventional tumor markers (CEA, cyfra21-1 and NSE) in the patient cohort. ResultsCompared to the diagnostic sensitivities of conventional tumor markers (CEA 29%; cyfra21-1 41%; NSE 39%), both assays of CTCs and ctDNA showed higher diagnostic sensitivity in predicting primary lung cancer (CTCs 67%; ctDNA 83%). When the assays of CTCs and ctDNA were combined for the diagnosis, the sensitivity was increased up to 98%. ConclusionsCollectively, this study suggests that combined CTCs and ctDNA assay would be useful for the diagnosis of primary lung cancer. Legal entity responsible for the studyCytogen, Inc., EDGC, Inc., Department of Medicine, Samsung Medical Center FundingHas not received any funding DisclosureAll authors have declared no conflicts of interest.

Long-Term Dynamics of Three Dimensional Telomere Profiles in Circulating Tumor Cells in High-Risk Prostate Cancer Patients Undergoing Androgen-Deprivation and Radiation Therapy.

Patient-specific assessment, disease monitoring, and the development of an accurate early surrogate of the therapeutic efficacy of locally advanced prostate cancer still remain a clinical challenge. Contrary to prostate biopsies, circulating tumor cell (CTC) collection from blood is a less-invasive method and has potential as a real-time liquid biopsy and as a surrogate marker for treatment efficacy. In this study, we used size-based filtration to isolate CTCs from the blood of 100 prostate cancer patients with high-risk localized disease. CTCs from five time points: +0, +2, +6, +12 and +24 months were analyzed. Consenting treatment-naïve patients with cT3, Gleason 8-10, or prostate-specific antigen > 20 ng/mL and non-metastatic prostate cancer were included. For all time points, we performed 3D telomere-specific quantitative fluorescence in situ hybridization on a minimum of thirty isolated CTCs. The patients were divided into five groups based on the changes of number of telomeres vs. telomere lengths over time and into three clusters based on all telomere parameters found on diagnosis. Group 2 was classified as non-respondent to treatment and the Cluster 3 presented more aggressive phenotype. Additionally, we compared our telomere results with the PSA levels for each patient at 6 months of ADT, at 6 months of completed RT, and at 36 months post-initial therapy. CTCs of patients with PSA levels above or equal to 0.1 ng/mL presented significant increases of nuclear volume, number of telomeres, and telomere aggregates. The 3D telomere analysis of CTCs identified disease heterogeneity among a clinically homogeneous group of patients, which suggests differences in therapeutic responses. Our finding suggests a new opportunity for better treatment monitoring of patients with localized high-risk prostate cancer.

Abstract 1359: Detection of PD-L1 positive circulating tumor cells from patient with bladder cancer

Abstract Immunotherapy with PD-L1 inhibitors greatly enhanced the clinical outcomes of patients with late stage metastatic bladder cancer. However, a concern has been raised in the front-line clinical use of PD-L1 inhibitors (permbrolizumab and atezolizumab) as a single treatment to PD-L1-low patients, due to a poorer overall survival compared with patients treated with plantinum-based chemotherapy. In accordance, reliable evaluation of PD-L1 expression is required for selecting patients relevant for treatment with PD-L1 inhibitors. However, due to the regional heterogeneity in primary tumor, evaluation of PD-L1 levels in tissue biopsy might be less reliable than the levels in circulating tumor cells (CTCs). Here, we developed the evaluating system of PD-L1 levels in CTCs isolated from the blood of patients with bladder cancer. Initially, CTCs were enriched by Smart Biopsy Cell Isolator (CytoGen), which utilizes the size-based gravity filtration for CTC isolation. The isolated CTCs were then validated with the expression of EpCAM, Vimentin and CK as well as a negative selection with CD45, and were analyzed for PD-L1 expression status by immunocytochemical staining. Using the evaluating system, we show that PD-L1 (+) cancer cell was efficiently recovered from the healthy human blood samples in a spike and recovery experiment. Furthermore, we evaluated PD-L1 expression status in CTCs isolated from ~25 patients with bladder cancer. Collectively, this study implicates the possibility that PD-L1 expression status could be analyzed at the levels of CTCs instead of the matching primary tumor tissue biopsy, probably with a better clinical diagnostic option. Citation Format: Yongjoon Suh, Seong-Kun Kim, Hye Seon Lee, Yun-Gyu Jeong, Jeong-Mi Moon, Myoung Shin Kim, Byung Hee Jeon, U-Syn Ha. Detection of PD-L1 positive circulating tumor cells from patient with bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1359.

PDMS membrane filter with nano-slit array fabricated using three-dimensional silicon mold for the concentration of particles with bacterial size range

Herein we report a microfluidic device integrated with a PDMS membrane nano-filter for the size-based trapping of particles. We propose a novel cost-effective fabrication process to form submicron-sized nano-pore and slit arrays in the PDMS membrane by using a reusable three-dimensional silicon mold with the shape of microneedles and microblades. The PDMS is coated onto the silicon mold, etched, and detached from the mold to form nano-pores and slits in the PDMS membrane filter. The sizes of the nano-pores and slits could be adjusted in the range of several hundreds of nanometers by varying the etching time of the PDMS coated on the sharp silicon mold. The diameter of the pores and the width of the slits were controlled to be 354 ± 79 nm and 389 ± 84 nm, respectively, with PDMS etching times of 60 s. Moreover, it was confirmed that the deposition of a parylene-C thin film on the PDMS membrane filter can be effectively used to control the nano-slit size and improve the trapping efficiency. We have demonstrated size-based filtration of microbeads with diameters of 0.4, 0.8, 1.0, and 2.2 μm using the proposed microfluidic device. In addition, the microfluidic device integrated with the proposed PDMS membrane filter was successfully applied to the trapping of submicron-sized E. coli bacteria with a filter recovery efficiency of 89.1%.

Cancer marker-free enrichment and direct mutation detection in rare cancer cells by combining multi-property isolation and microfluidic concentration.

Genetic analysis, rather than simply counting the number of circulating tumor cells (CTCs), which are rare cancer cells in peripheral blood, has great potential for non-invasive biopsy or "liquid biopsy." However, a practical problem in conventional enrichment of CTCs is that the isolated target cells are mixed with numerous residual leukocytes, and are suspended in a large volume. Hence, further isolation (i.e., cytokeratin (CK)-positive cell picking) or DNA purification is required for downstream genetic analysis after isolation. Here, we propose a novel cancer marker-free method of CTC enrichment by size-based Filtration and Immunomagnetic Negative selection followed by Dielectrophoretic concentration (CTC-FIND) for direct detection of genetic mutations in rare cancer cells suspended in whole blood. A combination of two independent isolation methods based on physical (filtration) and biochemical properties (immunomagnetic negative selection) in CTC-FIND allowed highly efficient cancer marker-free purification (5.1-log depletion of leukocytes). The isolated cells were trapped and concentrated using a microfluidic step-channel device using dielectrophoresis for discrimination and downstream genetic analysis. The feasibility of cancer marker-free enrichment by CTC-FIND was successfully demonstrated by directly detecting mutations in various cancer cells with a very high sensitivity of 1 cell per mL, including EpCAM and CK-negative cells, which were used to spike 8 mL of whole blood. Thus, CTC-FIND can be used with liquid biopsy to detect genetic mutations in wide-ranging CTC subsets, independent of cancer cell-specific marker expression.

Fabrication of perforated polyethylene microfiltration membranes for circulating tumor cells separation by thermal nanoimprint method

Size-based filtration has been proved an efficient and quick approach to separate circulating tumor cells (CTCs) from other blood cells for cancer diagnosis and therapy monitoring. In this work, we proposed a simple, cost efficient and scalable approach to fabricate microporous polyethylene (PE) membranes for CTC filtration by thermal nanoimprint method. PE was selected as the material for the membrane due to its commercially available films with a thickness as thin as ~ 10 µm, which is matched well with the size of the micropores for CTCs and critical to a successful nanoimprint with a low and uniform residual layer. A thermal imprint process with a nickel mold was applied to fabricate periodic microporous PE membranes with a pitch of 20 µm and diameter of 10 µm. The perforated micropores were obtained by a short time O2 plasma-etching to remove the imprint residual layer. This PE membrane microfilter achieved 84% average capture efficiency for lung cancer cells spiked in blood samples.

Abstract 1030: Circulating tumor cells from patients' blood induce tumors in a personalized xenograft mouse model: A functional approach to assess the patients' premetastatic disease state

Abstract Background: Circulating tumor cells (CTCs) originate from both primary and metastatic solid tumors and infiltrate the blood system. Although the metastatic potential of the single CTC cell is unknown, CTCs in circulation have been reported to have prognostic value as early predictors of relapse in cancers including ovarian, breast, colorectal, prostate, lung, bladder, head and neck and melanoma. As metastatic spread remains the major problem in cancer treatment, it is of great importance to characterize the subpopulations of CTCs with the highest metastatic potential. Methods: Peripheral blood mononuclear cells (MNCs) or bone marrow MNCs, were collected from each of over 60 cancer patients and grafted separately to immunodeficient NOD.Cg-Prkdc.scid.Il2rgtm1Wjl/SzJ (NSG) mice. Additionally, CTCs were enriched by either CD8 T-cell depletion or by a size-based filtration system. Tumors that developed in the mice were confirmed to be human tumors by demonstrating that they have human HLA expression and by comparing their tumor type by an independent pathologist to that of the donor patient's. The CTC-derived tumors were passaged to naive NSG mice to establish the scale up potential for treatment-groups. To facilitate future research of CTC-derived tumors, we have established a Biobank for CTC-derived tumors with matched samples of the patient's MNCs, plasma, tumor biopsy(s) and patient-biopsy derived xenograft (PDX) tumors. Additional characterization of the CTC-derived tumors was performed, such as comparison to the driver-mutations of the original tumor-biopsies and expression of epithelial or mesenchymal markers. The clinical relevance of the CTC-derived tumors was assessed by long term follow-up of each patient's disease progression. Results: CTC-derived tumors were detected in xenografts in 15 cases, within an average of eight months post grafting, representing a 25% success rate. The CTC-derived tumors were from a variety of cancers including: breast (6), colon (1), lung (2), pancreatic (1), ovarian (1), malignant-mesothelioma (1) head and neck (1) hepatocellular carcinoma (1) and sarcoma (1). The patients, whose blood or marrow contained tumorigenic CTCs, developed metastasis. Interestingly, there was a correlation between sites of CTC-derived tumors in the mice and sites of metastatic spread in the patients. CTC-derived tumors were successfully transferred to subsequent PDX mice, enabling the establishment of large treatment groups to study drug reactivity of these tumors. Summary: This study describes a functional assay to characterize the CTCs subtypes with a high metastatic potential, which utilizes the capacity of CTCs to form tumors in a permissive immune-free environment. CTC-derived tumors may offer a new tool to predict the significance of these malignant cells and prevent metastatic spread. Citation Format: Netta R. Shraga, Amihai Lieberman, Neta Moskovits, Alejandro Livoff, Evgeny Solomonov, Inbar Ben Shachar, Aron Popovtzer, Salomon M. Stemmer*, Izhak Haviv*. Circulating tumor cells from patients' blood induce tumors in a personalized xenograft mouse model: A functional approach to assess the patients' premetastatic disease state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1030.

Cancer panel analysis of circulating tumor cells in patients with breast cancer.

Liquid biopsy using circulating tumor cells (CTCs) is a noninvasive and repeatable procedure, and is therefore useful for molecular assays. However, the rarity of CTCs remains a challenge. To overcome this issue, our group developed a novel technology for the isolation of CTCs on the basis of cell size difference. The present study isolated CTCs from patients with breast cancer using this method, and then used these cells for cancer gene panel analysis. Blood samples from eight patients with breast cancer were collected, and CTCs were enriched using size-based filtration. Enriched CTCs were counted using immunofluorescent staining with an epithelial cell adhesion molecule (EpCAM) and CD45 antibodies. CTC genomic DNA was extracted, amplified, and screened for mutations in 400 genes using the Ion AmpliSeq Comprehensive Cancer Panel. White blood cells (WBCs) from the same patient served as a negative control, and mutations in CTCs and WBCs were compared. EpCAM+ cells were detected in seven out of eight patients, and the average number of EpCAM+ cells was 8.6. The average amount of amplified DNA was 32.7 µg, and the percentage of reads mapped to any targeted region relative to all reads mapped to the reference was 98.6%. The detection rate of CTC-specific mutations was 62.5%. The CTC-specific mutations were enhancer of zeste polycomb repressive complex 2 subunit, notch 1, AT-rich interaction domain 1A, serine/threonine kinase 11, fms related tyrosine kinase 3, MYCN proto-oncogene, bHLH transcription factor, APC, WNT signaling pathway regulator, and phosphatase and tensin homolog. The technique used by the present study was demonstrated to be effective at isolating CTCs at a sufficiently high purity for genomic analysis, and supported the use of comprehensive cancer panel analysis as a potential application for precision medicine.

Isolation and Characterization of CTCs from Patients with Cancer of a Urothelial Origin.

Monitoring of circulating tumor cells' (CTCs) presence has the potential to improve therapeutic management of oncological diseases at an early stage and also to identify patients with increased risk of tumor progression or recurrence before the onset of clinically detected metastasis. Here we describe a new simplified efficient methodology for the separation and in vitro culturing of viable CTCs from peripheral blood by size-based filtration (MetaCell®). The isolation protocol yields preferentially cells bigger than 8μm enabling further cytomorphological and molecular analysis.

Abstract 1737: ALK rearrangement analysis in circulating tumor cells of lung cancer patients

Abstract The evaluation of ALK rearrangement in non-small-cell lung cancer (NSCLC) is a significant tool when considering chemotherapy. It is not always possible to perform a tumor biopsy in patients. We suggest isolation and culturing of circulating tumor cells (CTCs) as an alternative tool to a tumor biopsy for the diagnosis of ALK rearrangement. From 22 patients with NSCLC harboring ALK rearrangement, blood samples were collected and divided into two parts: one for immunofluorescence staining of CTC marker and the other for culturing of CTCs. Both samples were processed by size-based filtration, and Cultured CTCs were analyzed for EML4-ALK translocation by fluorescence in situ hybridization (FISH) using Vysis ALK break apart FISH probe kit. CTC culturing was successful in 18 of 22 cases (81.8%). Among 18 cases of successful CTC cultures, 13 cases showed ALK rearrangement positivity (72.2%). Therefore, we suggest that the CTCs can be used as an alternative method to tissue biopsy for diagnosing ALK rearrangement. In addition, this method may have clinical applications including serial blood sampling for the development of personalized cancer therapy based on individual genomic information. Citation Format: Min Kyung Jeon, Young Hun Kim, Eunjoo Hwang, Hye Seon Lee, Ji-hyun Uh, Myoung Shin Kim, JooKyung Park, Byung Hee Jeon, Se-Hoon Lee. ALK rearrangement analysis in circulating tumor cells of lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1737. doi:10.1158/1538-7445.AM2017-1737

Abstract 3919: Isolation and characterization of viable pancreatic cancer circulating tumor cells using size-based filtration system

Abstract Introduction: Circulating tumor cells (CTCs) are important actors in metastasis, a key feature of pancreatic ductal adenocarcinoma (PDAC). The ability to isolate, characterize and culture viable CTCs holds promise for understanding biology, treatment response and resistance. Methods: Peripheral blood samples from healthy donors and patients with advanced PDAC were processed using an innovative size filtration system (Viatar LLC, Lowell MA). Normal human blood samples were spiked with varying numbers of human PDAC organoid cells. The impact of buffy coat separation was studied. Isolated CTCs were cultured in Matrigel to generate organoids as previously described. Cells were imaged with fluorescently labeled antibody to EpCAM. Data: Using spiked normal human blood samples, viable CTCs can be isolated and expanded reliably in 3D organoid culture from as few as 10 CTCs/mL. Separation and processing of the buffy coat enriched for the number of viable CTCs isolated. EpCAM positive CTCs were isolated from 6/6 blood samples from patients with advanced PDAC. Updated results will be presented. Conclusions: The current size-based filtration system allows for collection of viable CTCs from human PDAC blood samples. Current work will use this system to grow, expand and characterize human PDAC CTCs in organoid culture. Genetic, transcriptional, and epigenetic characteristics will be compared with the primary tumor. CTC characteristics will be studied as biomarker of treatment response and resistance. Citation Format: Kenneth H. Yu, Benjamin D. Krempley, Brian McCarthy. Isolation and characterization of viable pancreatic cancer circulating tumor cells using size-based filtration system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3919. doi:10.1158/1538-7445.AM2017-3919

Development of an automated size-based filtration system for isolation of circulating tumor cells in lung cancer patients.

Circulating tumor cells (CTCs), defined as tumor cells circulating in the peripheral blood of patients with solid tumors, are relatively rare. Diagnosis using CTCs is expected to help in the decision-making for precision cancer medicine. We have developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Herein, we evaluated the system using blood samples from non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. To evaluate the recovery of CTCs, preclinical experiments were performed by spiking NSCLC cell lines (NCI-H820, A549, NCI-H23 and NCI-H441) into peripheral whole blood samples from healthy volunteers. The recovery rates were 70% or more in all cell lines. For clinical evaluation, 6 mL of peripheral blood was collected from 50 patients with advanced lung cancer and from 10 healthy donors. Cells recovered on the filter were stained. We defined CTCs as DAPI-positive, cytokeratin-positive, and CD45-negative cells under the fluorescence microscope. The 50 lung cancer patients had a median age of 72 years (range, 48–85 years); 76% had NSCLC and 20% had SCLC, and 14% were at stage III disease whereas 86% were at stage IV. One or more CTCs were detected in 80% of the lung cancer patients (median 2.5). A comparison of the CellSearch system with our MCA system, using the samples from NSCLC patients, confirmed the superiority of our system (median CTC count, 0 versus 11 for CellSearch versus MCA; p = 0.0001, n = 17). The study results suggest that our MCA system has good clinical potential for diagnosing CTCs in lung cancer.

A computational approach to the characterization of a microfluidic device for continuous size-based inertial sorting

The application of fully-inertial size-based microfluidic filtration technologies for particle separation is an attractive tool, which not only offers label-free control of the microenvironment during separation, but also facilitates integration and automation for high throughput sample processing.In this work, we exploit 3D computational fluid dynamics (CFD) simulations based on the lattice Boltzmann method to evaluate the performance of a microfluidic device specifically designed to trap and extract particles by inertial focusing and microscale vortices. The device geometry consists of a straight microchannel, followed downstream by a microchamber with outlets for continuous size-based separation.Simulations were carried out to characterize the flow properties of the microfluidic device. Here, the influence of the Reynolds number (Re), the chamber dimensions and the outlet channels aspect ratio on the streamtracer distribution were studied. In order to support the simulation results, some preliminary experimental validations have been conducted, finding that the model can accurately characterize the flow in the studied geometry. The results of the simulations and experiments presented in this paper can be very useful to support the design of continuous-flow particle sorting lab-on-a-chip (LOC) devices.

Label-free isolation of prostate circulating tumor cells using Vortex microfluidic technology

There has been increased interest in utilizing non-invasive “liquid biopsies” to identify biomarkers for cancer prognosis and monitoring, and to isolate genetic material that can predict response to targeted therapies. Circulating tumor cells (CTCs) have emerged as such a biomarker providing both genetic and phenotypic information about tumor evolution, potentially from both primary and metastatic sites. Currently, available CTC isolation approaches, including immunoaffinity and size-based filtration, have focused on high capture efficiency but with lower purity and often long and manual sample preparation, which limits the use of captured CTCs for downstream analyses. Here, we describe the use of the microfluidic Vortex Chip for size-based isolation of CTCs from 22 patients with advanced prostate cancer and, from an enumeration study on 18 of these patients, find that we can capture CTCs with high purity (from 1.74 to 37.59%) and efficiency (from 1.88 to 93.75 CTCs/7.5 mL) in less than 1 h. Interestingly, more atypical large circulating cells were identified in five age-matched healthy donors (46–77 years old; 1.25–2.50 CTCs/7.5 mL) than in five healthy donors <30 years old (21–27 years old; 0.00 CTC/7.5 mL). Using a threshold calculated from the five age-matched healthy donors (3.37 CTCs/mL), we identified CTCs in 80% of the prostate cancer patients. We also found that a fraction of the cells collected (11.5%) did not express epithelial prostate markers (cytokeratin and/or prostate-specific antigen) and that some instead expressed markers of epithelial–mesenchymal transition, i.e., vimentin and N-cadherin. We also show that the purity and DNA yield of isolated cells is amenable to targeted amplification and next-generation sequencing, without whole genome amplification, identifying unique mutations in 10 of 15 samples and 0 of 4 healthy samples.

Evaluation of a novel approach to circulating tumor cell isolation for cancer gene panel analysis in patients with breast cancer.

Liquid biopsy isolation of circulating tumor cells (CTCs) allows the genomic analysis of CTCs, which is useful in the determination of personalized cancer therapy. In the present study, CTCs from patients with breast cancer were enriched and successfully analyzed using cancer gene panel analysis. Blood samples from 11 patients with breast cancer were collected and CTCs enriched for using size-based filtration. The enriched CTCs were analyzed using immunofluorescence staining with antibodies directed against epithelial cell adhesion molecule (EpCAM) and cluster of differentiation 45. The genomic DNA of CTCs was extracted, amplified and 50 genes screened for mutations using the Ion AmpliSeq™ Cancer Hotspot Panel v2. EpCAM staining detected CTCs in 10/11 patients and the average CTC count was 3.9 in 5 ml blood. The average purity of enriched CTCs was 14.2±29.4% and the average amount of amplified DNA was 28.6±11.9 µg. Catalogue Of Somatic Mutations In Cancer mutations were detected in the CTCs and included IDH2, TP53, NRAS, IDH1, PDGFRA, HRAS, STK11, EGFR, PTEN, MLH1, PIK3CA, CDKN2A, KIT and SMARCB1. In conclusion, a novel size-based filtration approach for the isolation of CTCs was evaluated and successfully applied for the genomic analysis of CTCs from patients with breast cancer.

Cytologic characteristics of circulating epithelioid cells in pancreatic disease.

Circulating epithelioid cells (CECs), also known as circulating tumor, circulating cancer, circulating epithelial, or circulating nonhematologic cells, are a prognostic factor in various malignancies that can be isolated via various protocols. In the current study, the authors analyzed the cytomorphologic characteristics of CECs isolated by size in a cohort of patients with benign and malignant pancreatic diseases to determine whether cytomorphological features could predict CEC origin. Blood samples were collected from 9 healthy controls and 171 patients with pancreatic disease who were presenting for surgical evaluation before treatment. Blood was processed with the ScreenCell size-based filtration device. Evaluable CECs were analyzed in a blinded fashion for cytomorphologic characteristics, including cellularity; nucleoli; nuclear size, irregularity, variability, and hyperchromasia; and nuclear-to-cytoplasmic ratio. Statistical differences between variables were analyzed via the Fisher exact test. No CECs were identified among the 9 normal healthy controls. Of the 115 patients with CECs (positive or suspicious for), 25 had nonmalignant disease and 90 had malignancy. There were no significant differences in any of the cytologic criteria noted between groups divided by benign versus malignant, neoplastic versus nonneoplastic, or pancreatic ductal adenocarcinoma versus neuroendocrine tumor. CECs were observed in patients with malignant and nonmalignant pancreatic disease, but not in healthy controls. There were no morphologic differences observed between cells from different pancreatic diseases, suggesting that numerous conditions may be associated with CECs in the circulation and that care must be taken not to overinterpret cells identified by cytomorphology as indicative of circulating tumor cells of pancreatic cancer. Additional studies are required to determine the origin and clinical significance of these cells. Cancer Cytopathol 2017;125:332-340. © 2017 American Cancer Society.