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  1. Article ; Online: Fluorescent multiplexing of 3D spheroids: Analysis of biomarkers using automated immunohistochemistry staining platform and multispectral imaging.

    Bhaumik, Srabani / Boyer, Jean / Banerjee, Chaitali / Clark, Samantha / Sebastiao, Noemi / Vela, Elizabeth / Towne, Penny

    Journal of cellular biochemistry

    2020  Volume 121, Issue 12, Page(s) 4974–4990

    Abstract: In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate ... ...

    Abstract In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non-small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells.
    Language English
    Publishing date 2020-07-21
    Publishing country United States
    Document type Journal Article
    ZDB-ID 392402-6
    ISSN 1097-4644 ; 0730-2312
    ISSN (online) 1097-4644
    ISSN 0730-2312
    DOI 10.1002/jcb.29827
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1114: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
    ab Jg. 2022: Lesesaal (EG)

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  2. Article ; Online: A novel 5x multiplex immunohistochemical staining reveals PSMA as a helpful marker in prostate cancer with low p504s expression.

    Rüschoff, Jan H / Stratton, Steven / Roberts, Esteban / Clark, Samantha / Sebastiao, Noemi / Fankhauser, Christian D / Eberli, Daniel / Moch, Holger / Wild, Peter J / Rupp, Niels J

    Pathology, research and practice

    2021  Volume 228, Page(s) 153667

    Abstract: The ability to combine multiple immunohistochemical (IHC) markers within a single tissue section facilitates the evaluation and detection of co-expressions, while saving tissue. A newly developed 5x multiplex (MPX) IHC staining of five different IHC ... ...

    Abstract The ability to combine multiple immunohistochemical (IHC) markers within a single tissue section facilitates the evaluation and detection of co-expressions, while saving tissue. A newly developed 5x multiplex (MPX) IHC staining of five different IHC markers (Basal cell cocktail (34βE12 + p63), p504s (SP116), ERG (EPR3864), Ki-67 (30-9), PSMA (EP192)) was applied on whole sections of n = 37 radical prostatectomies (RPE) including normal and cancerous tissue. Four different colors including brown, magenta, yellow and teal coded for different stainings, whereas magenta was used twice for nuclear Ki-67 and cytosolic / membranous PSMA. The staining of multiplex IHC was compared to single stains of ERG, PSMA and p504s. The proper staining of the basal cell cocktail and Ki-67 could be assessed by internal positive controls in the multiplex staining. The proportion of PSMA and p504s expression revealed a significant correlation between multiplex and single stains (p < 0.01) as well as a concordant staining pattern for ERG (n = 14 prostate cancers were identified ERG positive with both methods). Our proof of concept study demonstrates a robust staining pattern of all five different antibodies with this newly developed 5x MPX IHC. This approach facilitates the recognition of prostate cancer, in particular by adding PSMA in cases with low p504s expression.
    MeSH term(s) Adenocarcinoma/diagnosis ; Biomarkers, Tumor/analysis ; Humans ; Immunohistochemistry/methods ; Male ; Proof of Concept Study ; Prostatic Neoplasms/diagnosis ; Racemases and Epimerases/analysis ; Staining and Labeling/methods
    Chemical Substances Biomarkers, Tumor ; Racemases and Epimerases (EC 5.1.-) ; alpha-methylacyl-CoA racemase (EC 5.1.99.4)
    Language English
    Publishing date 2021-10-21
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 391889-0
    ISSN 1618-0631 ; 0344-0338
    ISSN (online) 1618-0631
    ISSN 0344-0338
    DOI 10.1016/j.prp.2021.153667
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Ud III Zs.20: Show issues Location:
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    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)

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  3. Article ; Online: Gamma-interferon-inducible lysosomal thiol reductase is upregulated in human melanoma.

    Nguyen, Jennifer / Bernert, Richard / In, Kevin / Kang, Paul / Sebastiao, Noemi / Hu, Chengcheng / Hastings, K Taraszka

    Melanoma research

    2016  Volume 26, Issue 2, Page(s) 125–137

    Abstract: T-cell-mediated immunity has the ability to produce durable antimelanoma responses, resulting in improved survival of patients with advanced melanoma. Antigen presentation is a key determinant of T-cell responses. Gamma-interferon-inducible lysosomal ... ...

    Abstract T-cell-mediated immunity has the ability to produce durable antimelanoma responses, resulting in improved survival of patients with advanced melanoma. Antigen presentation is a key determinant of T-cell responses. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens to CD4+ T cells. However, GILT expression in melanoma has not been defined. We evaluated GILT and MHC class II expression in human primary and metastatic melanomas and nevi using immunohistochemical analysis. GILT staining in melanocytes was observed in 70% of primary and 58% of metastatic melanomas versus 0% of nevi. When present, the GILT staining intensity in melanocytes was typically faint. Both GILT and MHC class II expression were increased in melanocytes of primary and metastatic melanomas compared with nevi. GILT staining in antigen-presenting cells (APCs) was detected in 100% of primary and metastatic melanomas versus 31% of nevi, and it was typically intense. GILT expression was increased in APCs of primary and metastatic melanomas compared with nevi, whereas MHC class II had equivalent high expression in APCs of all melanocytic lesions. GILT staining in keratinocytes was detected in 67% of primary melanomas versus 14% of nevi and 6% of metastatic melanomas. GILT, but not MHC class II, expression was increased in keratinocytes of primary melanomas compared with nevi and metastases. GILT expression is anticipated to result in improved presentation of melanoma antigens and more effective antimelanoma T-cell responses. GILT expression may be a biomarker of immune recognition of melanoma.
    MeSH term(s) Adult ; Aged ; Aged, 80 and over ; Cell Line, Tumor ; Female ; Humans ; Male ; Melanoma/enzymology ; Melanoma/genetics ; Melanoma/pathology ; Middle Aged ; Oxidoreductases Acting on Sulfur Group Donors/genetics ; Oxidoreductases Acting on Sulfur Group Donors/metabolism ; Skin Neoplasms/enzymology ; Skin Neoplasms/genetics ; Skin Neoplasms/pathology ; Up-Regulation
    Chemical Substances IFI30 protein, human (EC 1.8.-) ; Oxidoreductases Acting on Sulfur Group Donors (EC 1.8.-)
    Language English
    Publishing date 2016-04
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 1095779-0
    ISSN 1473-5636 ; 0960-8931
    ISSN (online) 1473-5636
    ISSN 0960-8931
    DOI 10.1097/CMR.0000000000000230
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 3378: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (2.OG)
    ab Jg. 2022: Lesesaal (EG)

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  4. Article: Low GILT Expression is Associated with Poor Patient Survival in Diffuse Large B-Cell Lymphoma.

    Phipps-Yonas, Hannah / Cui, Haiyan / Sebastiao, Noemi / Brunhoeber, Patrick S / Haddock, Ellen / Deymier, Martin J / Klapper, Wolfram / Lybarger, Lonnie / Roe, Denise J / Hastings, Karen Taraszka

    Frontiers in immunology

    2013  Volume 4, Page(s) 425

    Abstract: The major histocompatibility complex (MHC) class II-restricted antigen processing pathway presents antigenic peptides acquired in the endocytic route for the activation of CD4(+) T cells. Multiple cancers express MHC class II, which may influence the ... ...

    Abstract The major histocompatibility complex (MHC) class II-restricted antigen processing pathway presents antigenic peptides acquired in the endocytic route for the activation of CD4(+) T cells. Multiple cancers express MHC class II, which may influence the anti-tumor immune response and patient outcome. Low MHC class II expression is associated with poor survival in diffuse large B-cell lymphoma (DLBCL), the most common form of aggressive non-Hodgkin lymphoma. Therefore, we investigated whether gamma-interferon-inducible lysosomal thiol reductase (GILT), an upstream component of the MHC class II-restricted antigen processing pathway that is not regulated by the transcription factor class II transactivator, may be important in DLBCL biology. GILT reduces protein disulfide bonds in the endocytic compartment, exposing additional epitopes for binding to MHC class II and facilitating antigen presentation. In each of four independent gene expression profiling cohorts with a total of 585 DLBCL patients, low GILT expression was significantly associated with poor overall survival. In contrast, low expression of a classical MHC class II gene, HLA-DRA, was associated with poor survival in one of four cohorts. The association of low GILT expression with poor survival was independent of established clinical and molecular prognostic factors, the International Prognostic Index and the cell of origin classification, respectively. Immunohistochemical analysis of GILT expression in 96 DLBCL cases demonstrated variation in GILT protein expression within tumor cells which correlated strongly with GILT mRNA expression. These studies identify a novel association between GILT expression and clinical outcome in lymphoma. Our findings underscore the role of antigen processing in DLBCL and suggest that molecules targeting this pathway warrant investigation as potential therapeutics.
    Language English
    Publishing date 2013-12-04
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2606827-8
    ISSN 1664-3224
    ISSN 1664-3224
    DOI 10.3389/fimmu.2013.00425
    Database MEDical Literature Analysis and Retrieval System OnLINE

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