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Tissue Microarrays for Analysis of Expression Patterns
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are essential building blocks in every living cell, and since the complete human genome was sequenced in 2004, researchers have attempted to map the human proteome, which is the functional representation of the genome. One such initiative is the Human Protein Atlas programme (HPA), which generates monospecific antibodies towards all human proteins and uses these for high-throughput tissue profiling on tissue microarrays (TMAs). The results are publically available at the website www.proteinatlas.org.

In this thesis, TMAs were used for analysis of expression patterns in various research areas. Different search queries in the HPA were tested and evaluated, and a number of potential biomarkers were identified, e.g. proteins exclusively expressed in islets of Langerhans, but not in exocrine glandular cells or other abdominal organs close to pancreas. The identified candidates were further analyzed on TMAs with pancreatic tissues from normal and diabetic individuals, and colocalization studies with insulin and glucagon revealed that several of the investigated proteins (DGCR2, GBF1, GPR44 and SerpinB10) appeared to be beta cell specific. Moreover, a set of proteins differentially expressed in lung cancer stroma was further analyzed on a clinical lung cancer cohort in the TMA format, and one protein (CD99) was significantly associated with survival. In addition, TMAs with tissue samples from different species were generated, e.g. for mapping of influenza virus attachment in various human and avian tissues. The results showed that the gull influenza virus H16N3 attached to human respiratory tract and eye, suggesting possible transmission of the virus between gull and human. TMAs were also used for analysis of protein expression differences between humans and other primates, and two proteins (TCF3 and SATB2) proved to be significantly differentially expressed on the human lineage at both the protein level and the RNA level.  

In conclusion, this thesis exemplifies the potential of the TMA technology, which can be used for analysis of expression patterns in a large variety of research fields, such as biomarker discovery, influenza virus research or further understanding of human evolution.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 846
Keyword [en]
Tissue microarrays, Antibody-based proteomics, Immunohistochemistry, Biomarker discovery, Diabetes, Lung cancer, Influenza virus, Evolution
National Category
Endocrinology and Diabetes Infectious Medicine Cancer and Oncology Medical Genetics
Research subject
Pathology
Identifiers
URN: urn:nbn:se:uu:diva-186272ISBN: 978-91-554-8551-1 (print)OAI: oai:DiVA.org:uu-186272DiVA: diva2:572818
Public defence
2013-01-25, Rudbeck hall, Rudbeck laboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-12-21 Created: 2012-11-28 Last updated: 2013-02-11Bibliographically approved
List of papers
1. A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues
Open this publication in new window or tab >>A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues
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2008 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 7, no 5, 825-844 p.Article in journal (Refereed) Published
Abstract [en]

Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-103798 (URN)10.1074/mcp.M700411-MCP200 (DOI)000255830200001 ()17913849 (PubMedID)
Available from: 2009-05-20 Created: 2009-05-20 Last updated: 2015-10-29
2. Antibody-based proteomics for discovery and exploration of proteins expressed in pancreatic islets
Open this publication in new window or tab >>Antibody-based proteomics for discovery and exploration of proteins expressed in pancreatic islets
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2010 (English)In: Discovery Medicine, ISSN 1539-6509, E-ISSN 1944-7930, Vol. 9, no 49, 565-578 p.Article in journal (Refereed) Published
Abstract [en]

Abnormal glucose tolerance and deviant blood glucose levels are late stage clinical parameters that signify diabetes mellitus. To be able to diagnose the disease at an earlier stage and develop new tools for beta cell imaging, new molecular markers are needed. In the present study, five proteins highly expressed in pancreatic islets with no expression in the surrounding exocrine glandular cells of pancreas, and one protein with the opposite expression pattern, were identified by searches in the Human Protein Atlas (www.proteinatlas.org). The proteins were analyzed immunohistochemically on a specially designed tissue microarray, containing isolated human islets and pancreatic tissues with different characteristics, and compared to the expression of previously known markers of endocrine and exocrine pancreatic cells. Of the five novel endocrine markers, tetraspanin-7 was identified as a membrane-bound protein with exclusive positivity in islet cells. Also beta-2-microglobulin and ubiquitin carboxyl-terminal hydrolase isozyme L1 were expressed in a majority of islet cells, whereas sad1/unc-84 domain-containing protein 1 and beta-1,3-glucuronyltransferase 1 were positive in a smaller subset of islet cells. The potential exocrine marker galectin-2 was expressed in both exocrine acinary cells and pancreatic ductal cells, with no or low positivity in islet cells. In conclusion, antibody-based proteomics and specially designed tissue microarrays enable identification and exploration of novel proteins with differential expression in pancreatic islets. Here we describe 5 candidate proteins for further investigation of their physiological role and potential involvement in the pathogenesis of diabetes. One of these proteins, tetraspanin-7, is expressed on the cell membrane and could thus be a potential candidate for future development of tracers for beta cell imaging.

National Category
Medical and Health Sciences
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-153099 (URN)000208638200011 ()20587347 (PubMedID)
Available from: 2011-05-05 Created: 2011-05-05 Last updated: 2017-12-11Bibliographically approved
3. Novel pancreatic beta cell-specific proteins: Antibody-based proteomics for identification of new biomarker candidates
Open this publication in new window or tab >>Novel pancreatic beta cell-specific proteins: Antibody-based proteomics for identification of new biomarker candidates
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2012 (English)In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 75, no 9, 2611-2620 p.Article in journal (Refereed) Published
Abstract [en]

Beta cell-specific surface targets are required for non-invasive monitoring of beta cell mass, which could be used for evaluation of new diabetes treatments as well as to help unravel pathogenic mechanisms underlying beta cell dysfunction. By antibody-based proteomics, we have identified and explored a set of islet cell-specific proteins. A search algorithm in the Human Protein Atlas was set up for identification of islet-specific proteins that yielded 27 hits, of which twelve showed a clear membranous expression pattern or had predicted transmembrane regions. The specificity of the identified proteins was investigated by immunohistochemical staining of pancreas sections from diabetic and non-diabetic subjects. No expression of these antigens could be detected in the exocrine pancreas. Colocalization with insulin and glucagon was further determined by confocal microscopy using isolated human islets. All antibodies specifically stained human islets and colocalization analysis revealed that four proteins were exclusively expressed in beta cells. Importantly, these antibodies were negative in sections from subjects with long-standing type 1 diabetes. In the present study, we present four proteins; DGCR2, GBF1, GPR44 and SerpinB10, the expression of which has not previously been described in beta cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-175948 (URN)10.1016/j.jprot.2012.03.008 (DOI)000304634000009 ()22465717 (PubMedID)
Available from: 2012-06-14 Created: 2012-06-14 Last updated: 2017-12-07Bibliographically approved
4. CD99 is a novel prognostic stromal marker in non-small cell lung cancer
Open this publication in new window or tab >>CD99 is a novel prognostic stromal marker in non-small cell lung cancer
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2012 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 131, no 10, 2264-2273 p.Article in journal (Refereed) Published
Abstract [en]

The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein composition underlying tumour-stroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non-small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC, VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p=0.037) and multivariate (p=0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells, and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p=0.008). Furthermore, double-staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.

National Category
Clinical Laboratory Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-170991 (URN)10.1002/ijc.27518 (DOI)000309185300007 ()22392539 (PubMedID)
Note

Karolina Edlund and Cecilia Lindskog are shared first authors.

Available from: 2012-03-14 Created: 2012-03-14 Last updated: 2017-12-07
5. H16 Gull Influenza Virus Attaches to the Human Respiratory Tract and Eye
Open this publication in new window or tab >>H16 Gull Influenza Virus Attaches to the Human Respiratory Tract and Eye
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(English)In: Emerging Infectious Diseases, ISSN 1080-6059Article in journal (Other academic) Submitted
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-186270 (URN)
Available from: 2012-11-28 Created: 2012-11-28 Last updated: 2015-10-29
6. Analysis of Candidate Genes for Lineage-Specific Expression Changes in Humans and Primates
Open this publication in new window or tab >>Analysis of Candidate Genes for Lineage-Specific Expression Changes in Humans and Primates
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2014 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 8, 3596-3606 p.Article in journal (Refereed) Published
Abstract [en]

RUNX2, a gene involved in skeletal development, has previously been shown to be potentially affected by positive selection during recent human evolution. Here we have used antibody-based proteomics to characterize potential differences in expression patterns of RUNX2 interacting partners during primate evolution. Tissue microarrays consisting of a large set of normal tissues from human and macaque were used for protein profiling of 50 RUNX2 partners with immunohistochemistry. Eleven proteins (AR, CREBBP, EP300, FGF2, HDAC3, JUN, PRKD3, RUNX1, SATB2, TCF3, and YAP1) showed differences in expression between humans and macaques. These proteins were further profiled in tissues from chimpanzee, gorilla, and orangutan, and the corresponding genes were analyzed with regard to genomic features. Moreover, protein expression data were compared with previously obtained RNA sequencing data from six different organs. One gene (TCF3) showed significant expression differences between human and macaque at both the protein and RNA level, with higher expression in a subset of germ cells in human testis compared with macaque. In conclusion, normal tissues from macaque and human showed differences in expression of some RUNX2 partners that could be mapped to various defined cell types. The applied strategy appears advantageous to characterize the consequences of altered genes selected during evolution.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-186271 (URN)10.1021/pr500045f (DOI)000339983600010 ()
Available from: 2012-11-28 Created: 2012-11-28 Last updated: 2017-12-07Bibliographically approved

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