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Scalable In Situ Hybridization on Tissue Arrays for Validation of Novel Cancer and Tissue-Specific Biomarkers
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Pontén)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Pontén)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab. (Pontén)
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, e32927- p.Article in journal (Refereed) Published
Abstract [en]

Tissue localization of gene expression is increasingly important for accurate interpretation of large scale datasets from expression and mutational analyses. To this end, we have (1) developed a robust and scalable procedure for generation of mRNA hybridization probes, providing >95% first-pass success rate in probe generation to any human target gene and (2) adopted an automated staining procedure for analyses of formalin-fixed paraffin-embedded tissues and tissue microarrays. The in situ mRNA and protein expression patterns for genes with known as well as unknown tissue expression patterns were analyzed in normal and malignant tissues to assess procedure specificity and whether in situ hybridization can be used for validating novel antibodies. We demonstrate concordance between in situ transcript and protein expression patterns of the well-known pathology biomarkers KRT17, CHGA, MKI67, PECAM1 and VIL1, and provide independent validation for novel antibodies to the biomarkers BRD1, EZH2, JUP and SATB2. The present study provides a foundation for comprehensive in situ gene set or transcriptome analyses of human normal and tumor tissues.

Place, publisher, year, edition, pages
2012. Vol. 7, no 3, e32927- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-174583DOI: 10.1371/journal.pone.0032927ISI: 000303062000022OAI: oai:DiVA.org:uu-174583DiVA: diva2:529244
Available from: 2012-05-29 Created: 2012-05-22 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Expression and Mutation Analyses of Candidate Cancer Genes In Situ
Open this publication in new window or tab >>Expression and Mutation Analyses of Candidate Cancer Genes In Situ
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancers display heterogeneity in genetic profiles of the individual cancer cells and in the composition of different malignant and non-malignant cell populations. Such intra-tumor heterogeneity plays a role in treatment response and the emergence of resistance to cancer therapies. Approaches that address this complexity and improve stratification of patients for treatment are therefore highly warranted. Thus, the aims of this thesis were to further develop and apply in situ technologies for expression and mutation analyses of candidate cancer genes to gain a deeper understanding of cancer biology and to study intra-tumor heterogeneity.

In paper I, we established and validated a procedure for scalable in situ hybridization of large gene sets in human formalin-fixed paraffin-embedded tissues for analysis of gene expression. This method was used in paper II for large-scale expression analysis of the tyrosine kinome and phosphatome, two gene families whose members are frequently mutated in many forms of cancers. Systematic, compartment-specific expression mapping at cell type resolution enabled us to identify several novel vascular markers that have gone unnoticed in bulk transcriptomic analyses. In papers III and IV, we used padlock probes for in situ mutation detection in single cells for studies of genetic intra-tumor heterogeneity. In paper III, multiplex detection and genotyping of oncogenic point mutations was demonstrated in routinely processed tissue materials, whereas in paper IV we further the application by demonstrating multiplex detection of fusion gene transcripts.

Collectively, the work presented in this thesis employs in situ-based methods to obtain spatial resolution of gene expression and mutation patterns in normal and cancer tissues, thereby broadening our understanding of the cancer genome.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 50 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 842
Keyword
cancer, in situ, in situ hybridization, padlock probes, mRNA, tyrosine kinases, tyrosine phosphatases, fusion transcripts
National Category
Genetics Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-184510 (URN)978-91-554-8546-7 (ISBN)
Public defence
2012-12-21, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2012-11-30 Created: 2012-11-07 Last updated: 2013-02-11Bibliographically approved
2. Validation of antibodies for tissue based immunoassays
Open this publication in new window or tab >>Validation of antibodies for tissue based immunoassays
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In situ protein detection in human tissues using antibodies reveals the cellular protein localization, and affinity-based proteomic studies can help to discover proteins involved in the development of diseases. However, antibodies often suffer from cross-reactivity, and the lack of positive and negative tissue controls for uncharacterized proteins complicates the mapping of the proteome. The aim of this thesis is thus to improve the methodology for validating antibodies used for immunostaining on formalin-fixed paraffin-embedded tissues.

Two of the papers include comparisons between mRNA-expression and immunostaining of corresponding protein. In paper I, ISH and IHC staining patterns were compared on consecutive TMA-slides. The study of well-characterized genes showed that ISH could be used for validation of antibodies. ISH was further used for antibody evaluation, and could validate four out of nine antibodies showing potentially interesting staining patterns. In paper III, transcriptomic data generated by RNA-sequencing were used to identify tissue specific expression in lymphohematopoietic tissues. An increased expression in one or more of these tissues compared to other tissue types was seen for 693 genes, and these were further compared to the staining patterns of corresponding proteins in tissues.

Antibody labeling is necessary for many immunoassays. In paper II, two techniques for antibody-biotinylation were compared, aiming to find a stringent labeling method for antibodies used for immunostaining on TMAs. The ZBPA-method, binding specifically to Fc-part of antibodies, was found to be superior to the Lightning Link-biotinylation kit targeting amine groups, since labeling of amine groups on stabilizing proteins in the antibody buffer causes unspecific staining.

The localization of the estrogen receptor beta (ERβ) in human normal and cancer tissues was studied in paper IV. Thorough evaluation of 13 antibodies using positive and negative control cell lines showed that only one antibody, PPZ0506, is specific for ERβ in all three immunoassays used. Contradictory to previously published data, tissue profiling using PPZ0506 showed that ERβ is expressed in a limited number of normal and cancer tissues.

In conclusion, the present investigations present tools for validation of antibodies used for large-scale studies of protein expression in tissues.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 45 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1109
Keyword
Antibody validation, Conjugation, Estrogen receptor-beta, IHC, ISH, Lymphohematopoietic tissues, Proteomic, RNAseq, TMA, Transcriptomic
National Category
Medical and Health Sciences Biological Sciences
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-251344 (URN)978-91-554-9258-8 (ISBN)
Public defence
2015-06-13, Fåhreussalen, Rudbecklaboratoriet, hus C5, Dag Hammarskjölds väg 20, Uppsala, 12:30 (English)
Opponent
Supervisors
Available from: 2015-05-21 Created: 2015-04-15 Last updated: 2015-07-07

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Kiflemariam, SaraAndersson, SandraAsplund, AnnaPontén, FredrikSjöblom, Tobias
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