Cells Captured Research Articles

Biosensor with Microchannel for Broadband Dielectric Characterization of Nanoliter Cell Suspensions up to 110 GHz.

Cell dielectric property measurement holds significant potential for application in cell detection and diagnosis due to its label-free and noninvasive nature. In this study, we developed a biosensor designed to measure the permittivity of liquid samples, particularly cell suspensions at the nanoliter scale, utilizing microwave and millimeter wave coplanar waveguides in conjunction with a microchannel. This biosensor facilitates the measurement of scattering parameters within a frequency domain ranging from 1 GHz to 110 GHz. The obtained scattering parameters are then converted into dielectric constants using specific algorithms. A cell capture structure within the microchannel ensures that cell suspensions remain stable within the measurement zone. The feasibility of this biosensor was confirmed by comparison with a commercial Keysight probe. We measured the dielectric constants of three different cell suspensions (HepG2, A549, MCF-7) using our biosensor. We also counted the number of cells captured in multiple measurements for each cell type and compared the corresponding changes in permittivity. The results indicated that the real part of the permittivity of HepG2 cells is 0.2-0.8 lower than that of the other two cell types. The difference between A549 and MCF-7 was relatively minor, only 0.2-0.4. The fluctuations in the dielectric spectrum caused by changes in cell numbers during measurements were smaller than the differences observed between different cell types. Thus, the sensor is suitable for measuring cell suspensions and can be utilized for label-free, noninvasive studies in identifying biological cell suspensions.

Glycosylated stimuli responsive polyacrylamide microspheres with in-situ formed AgNPs for bacterial capture, detection and killing

Infections due to multidrug resistant bacteria are a major concern and cause for human mortality worldwide. Materials that can selectively capture, and eliminate these pathogens have turned into a priority. Development of excellent point-of-care material/system that can help in the visual detection of the captured bacterial cells in addition to their killing capability can be advantageous. In this study, we demonstrate the synthesis of a hitherto unknown glycosylated gel microspheres that can capture, kill and detect pathogenic bacteria. Silver nanoparticles (AgNPs) loaded glucose/mannose/galactose functionalized microspheres namely; PAM-Glc, PAM-Man and PAM-Gal, were synthesized by inverse emulsion polymerization. The sugar functionalities present on the microspheres interacts with the bacterial cell wall leading to capture of the cells while the antibacterial action was brought about by the AgNPs synthesized in-situ within the microspheres. The highlight of the designed system is visual indication of bacterial capture due to aggregation of gel microspheres and an indicative colour change of AgNPs. The capture kinetics of bacterial cells using PAM-Man was faster compared to PAM-Glc and PAM-Gal. Among the AgNPs loaded glyco microspheres, silver loaded mannose microspheres (Ag@PAM-Man) exhibited greater inhibition against E. coli and antibiotic resistant S. aureus cells. Bacterial cells captured by PAM-Man, exhibited pink coloration upon interaction with Resorufin β-glucuronide; suggesting the destruction of bacteria upon adhesion to microspheres. The plausible mechanistic aspects involved in the capture, detection and killing process is also discussed.

Abstract LB291: High-plexy intracellular and surface CITE-Seq offers multiomic insights into cell function

Abstract CITE-Seq allows researchers to combine high-plex protein information with whole transcriptome sequencing to gain insights about individual cell states. Due to the harsh fixation and permeabilization required for intracellular (IC) antibody staining, only a few studies have recovered mRNA expression profiles alongside IC protein detection. This experiment utilized a novel IC CITE-Seq assay that enables robust profiling of multiple IC protein targets in combination with mRNA and high-plex surface proteins. In this study, we stimulated human peripheral blood mononuclear cells (PBMCs) alongside a resting control. After stimulation, we stained the surface proteins with a 40-plex BD® AbSeq Panel, including the Immune Discovery Panel and human Single-Cell Multiplexing Kit (SMK). Resting and stimulated cells were pooled, and a portion of the sample pool immediately underwent cell capture as a live control using a BD Rhapsody™ single-cell microwell system. The other portion of the pool was stored in BD® OMICS-Guard Sample Preservation Buffer for either 5 minutes or 24 hours followed by permeabilization, IC staining with a 10-plex IC AbSeq Panel (50-plex total, surface and IC), and cells captured on the the same system. We show IC protein detection for the expected positive targets, including pH2AX, actCaspase-3, and cPARP in the stimulated population, as well as T-Bet, Granzyme B and Helios in the resting lymphocytes. Analysis shows excellent correspondence between IC CITE-Seq protein and transcriptome for T-Bet, Granzyme B and Helios. The gene expression correlation for surface antigen and mRNA between the IC and live control each had R2 values > 0.9. The mRNA sensitivity for the IC sample compared to live control was within whole transcriptome analysis (WTA) assay variation at 86–89% and 95–97% median molecules and genes per cell. Our data showed that the new IC CITE-Seq assay can provide rich single-cell multiomics information including mRNA and surface and intracellular proteins. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD, the BD Logo and BD Rhapsody are trademarks of Becton, Dickinson and Company or itsaffiliates. © 2023 BD. All rights reserved. NPM-2532 (v1.0) 1023 Citation Format: Tracy L. Campbell, Adam Wright, Ying Wah Lee, Anne Tran, Yan Chen, Joe Olives, Hye-Won Song, Larry Wang. High-plexy intracellular and surface CITE-Seq offers multiomic insights into cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB291.

Flexible and high-throughput approach for capturing large number of single cells simultaneously using microwell-based technology

Abstract Single-cell RNA-seq is a powerful method for studying complex cell populations. As opportunities grow for single-cell multiomics methods in translational research, there is an increased need for high-throughput approaches capable of efficiently capturing and analyzing large numbers of cells simultaneously. Current assays on scRNA-seq platforms have limited throughput due to the low cell recovery and high multiplet rates with high cell inputs. Here we demonstrate the feasibility of profiling the single-cell gene expression of hundreds of thousands of cells in a single experiment using an eight-lane microwell based cartridge. We also increased the cell label diversity of cell capture beads to enable high cell loading with a lower rate of barcode collisions. To show the range of cell capture capabilities of the eight-lane microwell cartridge, we loaded different numbers of cells per lane ranging from 100 to over 40,000 cells. To evaluate the multiplet rates and sensitivity, we labeled two different cell types with antibody-based sample multiplexing prior to cell capture. Cell multiplet rates were also assessed through Sample Tag analysis. Detection of Sample Tags used showed no contamination of Sample Tag between samples. In addition, less than 0.5% barcode collision rate was detected from 360,000 viable cells captured with a bead. Targeted mRNA profiling had high correlation (R 2> 0.95) of gene expression across cell capture lanes. This study demonstrates that the eight-lane microwell-based cartridge technology provides flexibility and high-throughput cell capture of thousands of cells and could be readily extended to other existing single-cell technologies such as surface protein marker detection and immune profiling.

Malignancy Assessment Using Gene Identification in Captured Cells Algorithm for the Prediction of Malignancy in Women With a Pelvic Mass.

To evaluate the detection of malignancy in women with a pelvic mass by using multiplexed gene expression analysis of cells captured from peripheral blood. This was an IRB-approved, prospective clinical study. Eligible patients had a pelvic mass and were scheduled for surgery or biopsy. Rare cells were captured from peripheral blood obtained preoperatively by using a microfluidic cell capture device. Isolated mRNA from the captured cells was analyzed for expression of 72 different gene transcripts. Serum levels for several commonly assayed biomarkers were measured. All patients had a tissue diagnosis. Univariate and multivariate logistic regression analyses for the prediction of malignancy using gene expression and serum biomarker levels were performed, and receiver operating characteristic curves were constructed and compared. A total of 183 evaluable patients were enrolled (average age 56 years, range 19-91 years). There were 104 benign tumors, 17 low malignant potential tumors, and 62 malignant tumors. Comparison of the area under the receiver operating characteristic curve for individual genes and various combinations of genes with or without serum biomarkers to differentiate between benign conditions (excluding low malignant potential tumors) and malignant tumors showed that a multivariate model combining the expression levels of eight genes and four serum biomarkers achieved the highest area under the curve (AUC) (95.1%, 95% CI 92.0-98.2%). The MAGIC (Malignancy Assessment using Gene Identification in Captured Cells) algorithm significantly outperformed all individual genes (AUC 50.2-65.2%; all P <.001) and a multivariate model combining 14 different genes (AUC 88.0%, 95% CI 82.9-93.0%; P =.005). Further, the MAGIC algorithm achieved an AUC of 89.5% (95% CI 81.3-97.8%) for stage I-II and 98.9% (95% CI 96.7-100%) for stage III-IV patients with epithelial ovarian cancer. Multiplexed gene expression evaluation of cells captured from blood, with or without serum biomarker levels, accurately detects malignancy in women with a pelvic mass. ClinicalTrials.gov, NCT02781272. This study was funded by ANGLE Europe Limited (Surrey Research Park, Guildford, Surrey, United Kingdom).

Array of Interdigitated Bipolar Electrodes for the Selective Capture and Analysis of Melanoma Cells

In this work, we develop an array of biosensors for the selective dielectrophoretic capture and electrochemical detection of single to few melanoma cells. Each biosensor in the array consists of a gold thin film interdigitated bipolar electrode (IDBPE) patterned on a glass substrate. Microwells are patterned over the interdigitated end of each IDBPE to restrict the number of cells captured and to retain cells on the electrodes for subsequent analysis. An overlying microfluidic channel controls delivery of the cell sample to the microwells. Cells are attracted to the interdigitated electrode by a positive dielectrophoretic force, thereby pulling them into the microwells, where they remain trapped during the next step – introduction of reagents for electrochemical enzyme-linked immunosorbent assay (eELISA). The dimensions of the wells, solution flow rate, and AC voltage applied all support the retention of the cells on the electrodes once they are captured.A bipolar electrode (BPE) is a conductor, which in the presence of an externally applied electric field, facilitates electrically coupled faradaic reactions at its opposing ends. In the context of analysis, a sensing reaction at one pole of the BPE is coupled to a reporting reaction, which produces a visual signal at the opposite pole. Because of their wireless operation, arrays of tens to thousands of BPEs can be operated in parallel with a single power supply – an advantageous feature for parallel analysis of few or single cells. However, traditional BPEs with electrogenerated chemiluminescence (ECL) as the reporting reaction do not provide sufficient sensitivity for biologically relevant detection limits, and therefore, require a means of signal amplification for such applications. To address this issue, we previously developed IDBPEs, which facilitate redox cycling by interdigitation of the sensing pole of each BPE of an array with a shared driving electrode, thereby yielding more intense ECL at the reporting pole.1 In the present study, the interdigitated end of the IDBPE is utilized for eELISA to quantify the expression of a cell surface antigen, melanoma cell adhesion marker (MCAM). Cells are labelled with biotinylated anti-human MCAM and alkaline phosphatase (ALP) conjugated streptavidin. Once cells are captured, the redox inactive substrate, para-aminophenyl phosphate (PAPP), is flowed through the microchannels and ALP catalyzes its conversion to the redox active species para-aminophenol (PAP). The amplified current obtained at each IDBPE by the redox cycling of PAP and its oxidized form, quinone imine (QI), is reported by an ECL reaction on its opposing pole. The ECL signal obtained at each IDBPE is correlated to the expression level of MCAM on the melanoma cells isolated in the corresponding well. This work is significant because it allows for the sensitive detection of melanoma cells in a device amenable to point-of-care application by combining selective enrichment of malignant cells by dielectrophoresis with the amplification and facile arraying afforded by IDBPEs. Borchers, J.S., Campbell, C.R., Van Scoy, S.B., Clark, M.J. and Anand, R.K. (2021), Redox Cycling at an Array of Interdigitated Bipolar Electrodes for Enhanced Sensitivity in Biosensing. ChemElectroChem. https://doi.org/10.1002/celc.202100523

Recapitulating early human development with 8C-like cells.

In human embryos, major zygotic genome activation (ZGA) initiates at the eight-cell (8C) stage. Abnormal ZGA leads to developmental defects and even contributes to the failure of human blastocyst formation or implantation. An invitro cell model mimicking human 8C blastomeres would be invaluable to understanding the mechanisms regulating key biological events during early human development. Using the non-canonical promoter of LEUTX that putatively regulates human ZGA, we developed an 8C::mCherry reporter, which specifically marks the 8C state, to isolate rare 8C-like cells (8CLCs) from human preimplantation epiblast-like stem cells. The 8CLCs express a panel of human ZGA genes and have a unique transcriptome resembling that of the human 8C embryo. Using the 8C::mCherry reporter, we further optimize the chemical-based culture condition to increase and maintain the 8CLC population. Functionally, 8CLCs can self-organize to form blastocyst-like structures. The discovery and maintenance of 8CLCs provide an opportunity to recapitulate early human development.

Biosensor device for the photo-specific detection of immuno-captured bladder cancer cells using hexaminolevulinate: An ex-vivo study.

This study investigate the relationship between different regulatory factors (time, concentration, temperature and addition of a fluorescent dye) and Hexaminolevulinate (HAL)-mediated photodynamic diagnosis of bladder cancer (PDD) in vitro. We examine the natural photosensitizer Protoporphyrin IX (PpIX) fluorescence induced by HAL in several human bladder cancer cell lines and one non-cancer foreskin fibroblast cell line and identify the processing conditions that maximise the difference in fluorescence intensity between malign and benign cell types. The detection of HAL induced fluorescence at a single cell level by a selective cancer cell capture platform is also tested. Experiments were performed on cultured monolayer cells and cells in suspension. The cell lines examined included the transitional epithelium carcinoma cell lines HT1197, HT1376, EJ138 and RT4, and the non-cancer foreskin fibroblasts HFF. Cells were incubated with HAL in various doses, time and temperature settings. We also used the nuclear red as a tool to study the PpIX subcellular localization. PpIX fluorescence intensities were measured and analysed using fluorescence microscope software. Finally, we evaluated the possibility of using HAL to discriminate between cancer and non-cancer cells from a mixed cell population using a newly developed immunofunctionalized microfluidic platform. The accumulation of PpIX in bladder cancer cells was significantly higher than in non-cancer cells, both cultured monolayer cells and cells in suspension. Effectively, the fluorescence intensity was 4.5 times brighter in bladder cancer cells than non-cancer foreskin fibroblast cells when incubated in the optimum condition, in which the nuclear stain adjuvant acted as a fluorescence enhancer. Cancer cells displayed PpIX accumulated mainly in mitochondria but none or very little PpIX was observed in non-cancer cells. HAL-induced fluorescence could be used to correctly identify bladder cancer cells within the EpCAM conjugated POx based microfluidic sensor with an 88% capture selectivity rate. These findings prove that the application of HAL-induced PpIX fluorescence can successfully distinguish between cancer and non-cancer cells in vitro. This test can provide advanced second level of confidence on the cancerous nature of cells captured by the immunofunctionalized bladder cancer diagnostic platform.

Microfluidic Cell Trapping for Single-Cell Analysis.

The single-cell capture microfluidic chip has many advantages, including low cost, high throughput, easy manufacturing, integration, non-toxicity and good stability. Because of these characteristics, the cell capture microfluidic chip is increasingly becoming an important carrier on the study of life science and pharmaceutical analysis. Important promises of single-cell analysis are the paring, fusion, disruption and analysis of intracellular components for capturing a single cell. The capture, which is based on the fluid dynamics method in the field of micro fluidic chips is an important way to achieve and realize the operations mentioned above. The aim of this study was to compare the ability of three fluid dynamics-based microfluidic chip structures to capture cells. The effects of cell growth and distribution after being captured by different structural chips and the subsequent observation and analysis of single cells on the chip were compared. It can be seen from the experimental results that the microfluidic chip structure most suitable for single-cell capture is a U-shaped structure. It enables single-cell capture as well as long-term continuous culture and the single-cell observation of captured cells. Compared to the U-shaped structure, the cells captured by the microcavity structure easily overlapped during the culture process and affected the subsequent analysis of single cells. The flow shortcut structure can also be used to capture and observe single cells, however, the shearing force of the fluid caused by the chip structure is likely to cause deformation of the cultured cells. By comparing the cell capture efficiency of the three chips, the reagent loss during the culture process and the cell growth state of the captured cells, we are provided with a theoretical support for the design of a single-cell capture microfluidic chip and a reference for the study of single-cell capture in the future.

Simple fabrication of rough halloysite nanotubes coatings by thermal spraying for high performance tumor cells capture

Here, we reported a fast, low-cost, and effective fabrication method of large-area and rough halloysite nanotubes (HNTs) coatings by thermal spraying of HNTs ethanol dispersions. A uniform HNTs coating with high transparence is achieved with tailorable surface roughness and thickness. Compared with normal cells, the tumor cells can be captured effectively with high capture yield by the HNTs coatings (expect HeLa cells), which is attributed to the enhanced topographic interactions between HNTs coating and cancer cells. HNTs coating formed from 2.5% ethanol dispersions shows the highest tumor cells capture yeild (90%), which is related to the appropriate roughness and anti-EpCAM conjugation. The capture yield of HNTs coating towards MCF-7 cells can be further improved to 93% within 2h under dynamic shear using a peristaltic pump. The capture yield increases with the incubation time, and the flow rate with 1.25mL/min leads to the maximum capture yield. The HNTs coatings are also effective for capture of tumor cells spiked in artificial blood samples and blood samples from patients with metastatic breast cancer. More than 90% targeted MCF-7 cells and very small amounts of white blood cells are captured by the anti-EpCAM conjugated HNTs coatings from a blood sample. HNTs are further loaded anticancer drug doxorubicin (DOX) and then thermally sprayed into coatings. The MCF-7 cells captured on DOX loaded HNTs coating display significant membrane rupture characteristic and only 3% cell viability after 16h. The high capture efficiency of tumor cells by HNTs coating fabricated by the thermal spraying method makes them show promising applications in clinical circulating tumor cells capture for early diagnosis and monitoring of cancer patients. The high killing ability of the DOX loaded HNTs coating can also be designed as an implantable therapeutic device for preventing tumor metastasis.

Tumor cell capture patterns around aptamer-immobilized microposts in microfluidic devices.

Circulating tumor cells (CTCs) have shown potential for cancer diagnosis and prognosis. Affinity-based CTC isolation methods have been proved to be efficient for CTC detection in clinical blood samples. One of the popular choices for affinity-based CTC isolation is to immobilize capture agents onto an array of microposts in microchannels, providing high CTC capture efficiency due to enhanced interactions between tumor cells and capture agents on the microposts. However, how the cells interact with microposts under different flow conditions and what kind of capture pattern results from the interactions have not been fully investigated; a full understanding of these interactions will help to design devices and choose experimental conditions for higher CTC capture effeciency. We report our study on their interaction and cell distribution patterns around microposts under different flow conditions. Human acute lymphoblastic leukemia cells (CCRF-CEM) were used as target cancer cells in this study, while the Sgc8 aptamer that has specific binding with CCRF-CEM cells was employed as a capture agent. We investigated the effects of flow rates and micropost shapes on the cell capture efficiency and capture patterns on microposts. While a higher flow rate decreased cell capture efficiency, we found that the capture pattern around microposts also changed, with much more cells captured in the front half of a micropost than at the back half. We also found the ratio of cells captured on microposts to the cells captured by both microposts and channel walls increased as a function of the flow rate. We compared circular microposts with an elliptical shape and found that the geometry affected the capture distribution around microposts. In addition, we have developed a theoretical model to simulate the interactions between tumor cells and micropost surfaces, and the simulation results are in agreement with our experimental observation.

High-Throughput Selective Capture of Single Circulating Tumor Cells by Dielectrophoresis at a Wireless Electrode Array.

We demonstrate continuous high-throughput selective capture of circulating tumor cells by dielectrophoresis at arrays of wireless electrodes (bipolar electrodes, BPEs). The use of BPEs removes the requirement of ohmic contact to individual array elements, thus enabling otherwise unattainable device formats. Capacitive charging of the electrical double layer at opposing ends of each BPE allows an AC electric field to be transmitted across the entire device. Here, two such designs are described and evaluated. In the first design, BPEs interconnect parallel microchannels. Pockets extruding from either side of the microchannels volumetrically control the number of cells captured at each BPE tip and enhance trapping. High-fidelity single-cell capture was achieved when the pocket dimensions were matched to those of the cells. A second, open design allows many non-targeted cells to pass through. These devices enable high-throughput capture of rare cells and single-cell analysis.

Cells capture and antimicrobial effect of hydrophobically modified chitosan coating on Escherichia coli

Hydrophobically modified chitosan (HMCS), prepared by reacting alkyl aldehyde with chitosan was demonstrated to be an effective antimicrobial and transparent coating. The grafted alkyl chains exist as protruded hydrophobic tails on the HMCS coating surface. In contact with E. coli cells, HMCS coating captured and immobilized the cells via these hydrophobic tails. The hydrophobic tails could also kill the cells captured on the coating surface as visualized by fluorescence microscope. More than 50% of the initially loaded cells (2.5×104 CFU) could be killed after 2h contact with HMCS coating. The cells capture and killing effects of the coating surface could be completely neutralized by treating with α-cyclodextrin to sequester the protruded hydrophobic tails. The facile coating of antimicrobial HMCS on surface also enabled the easy fabrication of patterned E. coli cells arrays.

A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells.

Circulating tumor cells (CTCs) were introduced as biomarkers more than 10 years ago, but capture of viable CTCs at high purity from peripheral blood of cancer patients is still a major technical challenge. Here, we report a novel microfluidic platform designed for marker independent capture of CTCs. The Parsortix™ cell separation system provides size and deformability‐based enrichment with automated staining for cell identification, and subsequent recovery (harvesting) of cells from the device. Using the Parsortix™ system, average cell capture inside the device ranged between 42% and 70%. Subsequent harvest of cells from the device ranged between 54% and 69% of cells captured. Most importantly, 99% of the isolated tumor cells were viable after processing in spiking experiments as well as after harvesting from patient samples and still functional for downstream molecular analysis as demonstrated by mRNA characterization and array‐based comparative genomic hybridization. Analyzing clinical blood samples from metastatic (n = 20) and nonmetastatic (n = 6) cancer patients in parallel with CellSearch® system, we found that there was no statistically significant difference between the quantitative behavior of the two systems in this set of twenty six paired separations. In conclusion, the epitope independent Parsortix™ system enables the isolation of viable CTCs at a very high purity. Using this system, viable tumor cells are easily accessible and ready for molecular and functional analysis. The system's ability for enumeration and molecular characterization of EpCAM‐negative CTCs will help to broaden research into the mechanisms of cancer as well as facilitating the use of CTCs as “liquid biopsies.”

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133+ cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Abstract 623: Evaluation of a multiplex biomarker assay for the detection of prostate cancer cells in urine

Abstract Background and Objectives: Over one million men undergo prostate biopsies annually in the US, a majority of them due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of PSA test as well as the biopsy procedure have prompted to develop more precise CaP detection assays in urine- (e.g. PCA3, TMPRSS2-ERG) or blood (e.g. PHI, 4K). Here we describe the development and evaluation of a Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine. Methods: The cellular content of the post-DRE urine, was captured on translucent membrane for direct evaluation by microscopy and immunocytochemistry. The membranes placed on Cytoclear microscope slides were used for characterization of prostate epithelial cell by PSA and Prostein or tumor cells by ERG and AMACR protein markers. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy. Results: Assay was optimized for single cell capture sensitivity and downstream evaluations by spiking known number of cells from established prostate cancer cell lines, LNCaP and VCaP, into cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR, Prostein and PSA, rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with the blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy. Conclusion: This pilot study assessing a minimally invasive prostate cancer detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this novel assay platform. Citation Format: Kristen Nickens, Amina Ali, Tatiana Scoggin, Shyh-han Tan, Lakshmi Ravindranath, David McLeod, David Tacha, Isabell Sesterhenn, Albert Dobi, Shiv Srivastava, Gyorgy Petrovics. Evaluation of a multiplex biomarker assay for the detection of prostate cancer cells in urine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 623. doi:10.1158/1538-7445.AM2015-623

MP66-12 A NOVEL CELL BASED MULTIPLEX BIOMARKER ASSAY FOR PROSTATE CANCER DETECTION IN URINE

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2015MP66-12 A NOVEL CELL BASED MULTIPLEX BIOMARKER ASSAY FOR PROSTATE CANCER DETECTION IN URINE Shyh-Han Tan, Kristen P. Nickens, Amina Ali, Tatiana Scoggin, Lakshmi Ravindranath, David G. McLeod, David Tacha, Shiv Srivastava, Isabell Sesterhenn, and Gyorgy Petrovics Shyh-Han TanShyh-Han Tan More articles by this author , Kristen P. NickensKristen P. Nickens More articles by this author , Amina AliAmina Ali More articles by this author , Tatiana ScogginTatiana Scoggin More articles by this author , Lakshmi RavindranathLakshmi Ravindranath More articles by this author , David G. McLeodDavid G. McLeod More articles by this author , David TachaDavid Tacha More articles by this author , Shiv SrivastavaShiv Srivastava More articles by this author , Isabell SesterhennIsabell Sesterhenn More articles by this author , and Gyorgy PetrovicsGyorgy Petrovics More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2365AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Over one million men undergo prostate biopsies annually in the US, a majority of them due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of PSA test as well as the biopsy procedure have prompted to develop more precise CaP detection assays in urine- (e.g. PCA3, TMPRSS2-ERG) or blood (e.g. PHI, 4K). Here we describe the development and evaluation of a Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post-digital rectal examination (post-DRE) urine. METHODS The cellular content of the post-DRE urine, was captured on translucent membrane for direct evaluation by microscopy and immunocytochemistry. The membranes placed on Cytoclear microscope slides were used for characterization of prostate epithelial cell by PSA and Prostein or tumor cells by ERG and AMACR protein markers. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy. RESULTS Assay was optimized for single cell capture sensitivity and downstream evaluations by spiking known number of cells from established prostate cancer cell lines, LNCaP and VCaP, into cleared control urine. The cells captured from the post-DRE urine of subjects, obtained prior to biopsy procedure, were co-stained for ERG, AMACR, Prostein and PSA, rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post-DRE urine specimens was assessed. Comparison of the UCMP results with the blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy. CONCLUSIONS This pilot study assessing a minimally invasive prostate cancer detection assay with single cell sensitivity cell-capture and characterization from the post-DRE urine holds promise for further development of this novel assay platform. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e820 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shyh-Han Tan More articles by this author Kristen P. Nickens More articles by this author Amina Ali More articles by this author Tatiana Scoggin More articles by this author Lakshmi Ravindranath More articles by this author David G. McLeod More articles by this author David Tacha More articles by this author Shiv Srivastava More articles by this author Isabell Sesterhenn More articles by this author Gyorgy Petrovics More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Prostate cancer marker panel with single cell sensitivity in urine.

BackgroundOver one million men undergo prostate biopsies annually in the United States, a majority of whom due to elevated serum PSA. More than half of the biopsies turn out to be negative for prostate cancer (CaP). The limitations of both the PSA test and the biopsy procedure have led to the development for more precise CaP detection assays in urine (e.g., PCA3, TMPRSS2‐ERG) or blood (e.g., PHI, 4K). Here, we describe the development and evaluation of the Urine CaP Marker Panel (UCMP) assay for sensitive and reproducible detection of CaP cells in post‐digital rectal examination (post‐DRE) urine.MethodsThe cellular content of the post‐DRE urine was captured on a translucent filter membrane, which is placed on Cytoclear slides for direct evaluation by microscopy and immuno‐cytochemistry (ICC). Cells captured on the membrane were assayed for PSA and Prostein expression to identify prostate epithelial cells, and for ERG and AMACR to identify prostate tumor cells. Immunostained cells were analyzed for quantitative and qualitative features and correlated with biopsy positive and negative status for malignancy.ResultsThe assay was optimized for single cell capture sensitivity and downstream evaluations by spiking a known number of cells from established CaP cell lines, LNCaP and VCaP, into pre‐cleared control urine. The cells captured from the post‐DRE urine of subjects, obtained prior to biopsy procedure, were co‐stained for ERG, AMACR (CaP specific), and Prostein or PSA (prostate epithelium specific) rendering a whole cell based analysis and characterization. A feasibility cohort of 63 post‐DRE urine specimens was assessed. Comparison of the UCMP results with blinded biopsy results showed an assay sensitivity of 64% (16 of 25) and a specificity of 68.8% (22 of 32) for CaP detection by biopsy.ConclusionsThis pilot study assessing a minimally invasive CaP detection assay with single cell sensitivity cell‐capture and characterization from the post‐DRE urine holds promise for further development of this novel assay platform. Prostate 75: 969–975, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

Hematopoietic stem cell capture and directional differentiation into vascular endothelial cells for metal stent-coated chitosan/hyaluronic acid loading CD133 antibody.

A series of metal stents coated with chitosan/hyaluronic acid (CS/HA) loading antibodies by electrostatic self-assembled method were prepared, and the types of cells captured by antibodies and their differentiation in vascular endothelial cells (ECs) evaluated by molecular biology and scanning electron microscope. The results showed that CD133 stent can selectively capture hematopoietic stem cells (HSC),which directionally differentiate into vascular ECs in peripheral blood by (CS/HA) induction, and simultaneously inhibit migration and proliferation of immune cells and vascular smooth muscle cells (MCs). CD34 stent can capture HSC, hematopoietic progenitor cells that differentiate into vascular ECs and immune cells, promoting smooth MCs growth, leading to thrombosis, inflammation, and rejection. CD133 stent can be implanted into miniature pig heart coronary and can repair vascular damage by capturing own HSC, thus contributing to the rapid natural vascular repair, avoiding inflammation and rejection, thrombosis and restenosis. These studies demonstrated that CD133 stent of HSC capture will be an ideal coated metal stent providing a new therapeutic approach for cardiovascular and cerebrovascular disease.

Microfluidic antibody arrays for simultaneous cell separation and stimulus.

A microfluidic chip containing stamped antibody arrays was developed for simultaneous cell separation and drug testing. Poly(dimethyl siloxane) (PDMS) stamping was used to deposit antibodies in a microfluidic channel, forming discrete cell-capture regions on the surface. Cell mixtures were then introduced, resulting in the separation of cells when specific antibodies were used. Anti-CD19 antibody regions resulted in 94% capture purity for CD19+ Ramos cells. An antibody that captures multiple cell types, for example anti-CD71, can also be used to capture several cell types simultaneously. Cells could also be loaded onto the arrays with spatial control using laminar streams. Both Ramos B cells and HuT 78T cells were isolated in the chip and exposed to staurosporine in the same channel. Both cell lines had similar responses to the drug, with 2-10% of cells remaining viable after 20h of drug treatment, depending on cell type. The chip can also be used to analyze the efficacy of antibody therapy against cancer cells. Anti-CD95 was deposited on the surface and used for simultaneous cell capture and apoptosis induction via the extrinsic pathway. Cells captured on anti-CD95 surfaces had significant viability loss (15% viability after 24h) when compared with a control anti-CD71 antibody (81% viability after 24h). This chip can be used for a variety of cell separation and/or drug testing studies, enabling researchers to isolate cells and test them against different anti-cancer compounds and to follow cell response using fluorescence or other readout methods.