9 research outputs found

    Resolving Low-Expression Cell Surface Antigens by Time-Gated Orthogonal Scanning Automated Microscopy

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    We report a highly sensitive method for rapid identification and quantification of rare-event cells carrying low-abundance surface biomarkers. The method applies lanthanide bioprobes and time-gated detection to effectively eliminate both nontarget organisms and background noise and utilizes the europium containing nanoparticles to further amplify the signal strength by a factor of ∼20. Of interest is that these nanoparticles did not correspondingly enhance the intensity of nonspecific binding. Thus, the dramatically improved signal-to-background ratio enables the low-expression surface antigens on single cells to be quantified. Furthermore, we applied an orthogonal scanning automated microscopy (OSAM) technique to rapidly process a large population of target-only cells on microscopy slides, leading to quantitative statistical data with high certainty. Thus, the techniques together resolved nearly all false-negative events from the interfering crowd including many false-positive events

    Multiplex Flow Cytometry Barcoding and Antibody Arrays Identify Surface Antigen Profiles of Primary and Metastatic Colon Cancer Cell Lines

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    <div><p>Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116). Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.</p> </div

    CD10 expression in SW480 versus SW620.

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    <p>Histogram plots from antibody array for the CD10 antigen in SW480 (<b>A</b>) and SW620 (<b>B</b>). Red indicates isotype control while the blue line is staining for CD10. The number in the top left is the cell positivity. There is a clear shift from a small shoulder population in SW480 to complete binding in SW620 cells. <b>C</b>) Immunoblotting for CD10 confirms the strong change in CD10 expression.</p

    Diagram of experimental methods used for multiplexed barcoded antibody array.

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    <p>The three cell lines were labeled with or without intracellular dye prior to admixing the cells into a single pool. The cells were then aliquoted into each well for antibody labeling. The contents of each well were then processed on a flow cytometer. The identity of each cell line was determined based on fluorescence intensity. The appropriate gates were drawn allowing for simultaneous analysis for each antibody. Histograms for mouse IgM isotype control are shown.</p

    Oncomine analysis.

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    <p>Oncomine heatmap analysis in 4 published datasets for expression of tumor antigens described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053015#pone-0053015-t001" target="_blank">Table 1</a>. Only those genes that were consistently upregulated across datasets with p<0.05 are shown. The numbers in parentheses indicate the number of samples analyzed. Abbreviations: normal colon, NC; ascending colon, AC; descending colon, DC; sigmoid colon, SC; transverse colon, TC (n = 1).</p

    Antigens downregulated in metastasis.

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    <p>Antibody array results showing surface antigens that were at least two-fold decreased in cell positivity in SW620 (metastatic) as compared to SW480 (primary). Abbreviations: epidermal growth factor receptor, EGFR; neural cell adhesion molecule, NCAM; C-X-C chemokine receptor 4, CXCR4; selectin P ligand, SELPLG; urokinase receptor, UPAR; FAS receptor, FasR; dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin, DC-SIGN; low density lipoprotein receptor related protein 1, LRP1; glycoprotein 130, gp130; natural cytotoxicity triggering receptor 3, NCR3; semaphorin-4D, SEMA4D; C-C chemokine receptor 3, CCR3; low affinity nerve growth factor receptor; LNGFR; interleukin 8 receptor alpha, IL8RA; P-glycoprotein, P-gp; intercellular adhesion molecule 1, ICAM1.</p>*<p>, Antigen was not two-fold decreased by comparison of mean fluorescence intensities (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053015#pone.0053015.s011" target="_blank">Table S4</a>).</p

    Validation of integrin α6 expression in colon cancer by immunohistochemistry.

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    <p><b>A</b>) H&E (top left) and integrin α6 IHC (top right) from clinical colon cancer specimens at low magnification. Areas of normal mucosa (N) and adjacent primary colon cancer (P) are indicated. Lower panels provide higher magnification fields of integrin α6 in normal (left) and tumor (right). <b>B</b>) Representative examples of liver and lymph node metastases. The regions of colon cancer metastases (M) are visible by H&E (left) and corresponding staining with integrin α6 (right). An area of normal liver (L) is indicated. All lymph node samples contained a high degree of fibrosis around the lesion that displaced normal lymphoid tissue from the field of view. Scale bar: 50 µm.</p

    Broadly expressed tumor antigens.

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    <p>Antibody array results showing surface antigens that were expressed on at least 50% of cells in all three colon cancer cell lines analyzed. Arranged in alphanumeric order.</p>*<p>, Antigens common to all nucleated human cells.</p><p>Abbreviations: lymphocyte function-associated antigen 3, LFA-3; membrane inhibitor of reactive lysis, MIRL; transferrin receptor protein 1, TFRC; large neutral amino acid transporter 1; LAT1; melanoma cell adhesion molecule, MCAM; basigin, BSG; L1 cell adhesion molecule, L1CAM; common leukocyte antigen, CLA.</p
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