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Fasterius, E., Raso, C., Kennedy, S., Rauch, N., Lundin, P., Kolch, W., . . . Al-Khalili Szigyarto, C. (2017). A novel RNA sequencing data analysis method for cell line authentication. PLoS ONE, 12(2), Article ID e0171435.
Open this publication in new window or tab >>A novel RNA sequencing data analysis method for cell line authentication
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2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 2, e0171435Article in journal (Refereed) Published
Abstract [en]

We have developed a novel analysis method that can interrogate the authenticity of biological samples used for generation of transcriptome profiles in public data repositories. The method uses RNA sequencing information to reveal mutations in expressed transcripts and subsequently confirms the identity of analysed cells by comparison with publicly available cell-specific mutational profiles. Cell lines constitute key model systems widely used within cancer research, but their identity needs to be confirmed in order to minimise the influence of cell contaminations and genetic drift on the analysis. Using both public and novel data, we demonstrate the use of RNA-sequencing data analysis for cell line authentication by examining the validity of COLO205, DLD1, HCT15, HCT116, HKE3, HT29 and RKO colorectal cancer cell lines. We successfully authenticate the studied cell lines and validate previous reports indicating that DLD1 and HCT15 are synonymous. We also show that the analysed HKE3 cells harbour an unexpected KRAS-G13D mutation and confirm that this cell line is a genuine KRAS dosage mutant, rather than a true isogenic derivative of HCT116 expressing only the wild type KRAS. This authentication method could be used to revisit the numerous cell line based RNA sequencing experiments available in public data repositories, analyse new experiments where whole genome sequencing is not available, as well as facilitate comparisons of data from different experiments, platforms and laboratories.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-204084 (URN)10.1371/journal.pone.0171435 (DOI)000394423800024 ()28192450 (PubMedID)2-s2.0-85012231859 (Scopus ID)
Note

QC 20170329

Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2017-11-29Bibliographically approved
Uhlén, M., Zhang, C., Lee, S., Sjöstedt, E., Fagerberg, L., Bidkhori, G., . . . Ponten, F. (2017). A pathology atlas of the human cancer transcriptome. Science, 357(6352), 660-+.
Open this publication in new window or tab >>A pathology atlas of the human cancer transcriptome
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, no 6352, 660-+ p.Article in journal (Refereed) Published
Abstract [en]

Cancer is one of the leading causes of death, and there is great interest in understanding the underlying molecular mechanisms involved in the pathogenesis and progression of individual tumors. We used systems-level approaches to analyze the genome-wide transcriptome of the protein-coding genes of 17 major cancer types with respect to clinical outcome. A general pattern emerged: Shorter patient survival was associated with up-regulation of genes involved in cell growth and with down-regulation of genes involved in cellular differentiation. Using genome-scale metabolic models, we show that cancer patients have widespread metabolic heterogeneity, highlighting the need for precise and personalized medicine for cancer treatment. All data are presented in an interactive open-access database (www.proteinatlas.org/pathology) to allow genome-wide exploration of the impact of individual proteins on clinical outcomes.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE, 2017
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-214334 (URN)10.1126/science.aan2507 (DOI)000407793600028 ()2-s2.0-85028362951 (Scopus ID)
Note

QC 20170913

Available from: 2017-09-13 Created: 2017-09-13 Last updated: 2017-11-07Bibliographically approved
Thul, P. J., Åkesson, L., Wiking, M., Mahdessian, D., Geladaki, A., Ait Blal, H., . . . Lundberg, E. (2017). A subcellular map of the human proteome. Science, 356(6340), Article ID 820.
Open this publication in new window or tab >>A subcellular map of the human proteome
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2017 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 356, no 6340, 820Article in journal (Refereed) Published
Abstract [en]

Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

Place, publisher, year, edition, pages
American Association for the Advancement of Science, 2017
Keyword
antibody, proteome, biology, cells and cell components, disease incidence, image analysis, physiological response, protein, proteomics, spatial distribution, Article, cell organelle, cellular distribution, human, human cell, immunofluorescence microscopy, mass spectrometry, priority journal, protein analysis, protein localization, protein protein interaction, single cell analysis, transcriptomics
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-216588 (URN)10.1126/science.aal3321 (DOI)000401957900032 ()2-s2.0-85019201137 (Scopus ID)
Note

QC 20171208

Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2017-12-08Bibliographically approved
Gremel, G., Djureinovic, D., Niinivirta, M., Laird, A., Ljungqvist, O., Johannesson, H., . . . Ponten, F. (2017). A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma. BMC Cancer, 17, Article ID 9.
Open this publication in new window or tab >>A systematic search strategy identifies cubilin as independent prognostic marker for renal cell carcinoma
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2017 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 17, 9Article in journal (Refereed) Published
Abstract [en]

Background: There is an unmet clinical need for better prognostic and diagnostic tools for renal cell carcinoma (RCC). Methods: Human Protein Atlas data resources, including the transcriptomes and proteomes of normal and malignant human tissues, were searched for RCC-specific proteins and cubilin (CUBN) identified as a candidate. Patient tissue representing various cancer types was constructed into a tissue microarray (n = 940) and immunohistochemistry used to investigate the specificity of CUBN expression in RCC as compared to other cancers. Two independent RCC cohorts (n = 181; n = 114) were analyzed to further establish the sensitivity of CUBN as RCC-specific marker and to explore if the fraction of RCCs lacking CUBN expression could predict differences in patient survival. Results: CUBN was identified as highly RCC-specific protein with 58% of all primary RCCs staining positive for CUBN using immunohistochemistry. In venous tumor thrombi and metastatic lesions, the frequency of CUBN expression was increasingly lost. Clear cell RCC (ccRCC) patients with CUBN positive tumors had a significantly better prognosis compared to patients with CUBN negative tumors, independent of T-stage, Fuhrman grade and nodal status (HR 0.382, CI 0.203-0.719, P = 0.003). Conclusions: CUBN expression is highly specific to RCC and loss of the protein is significantly and independently associated with poor prognosis. CUBN expression in ccRCC provides a promising positive prognostic indicator for patients with ccRCC. The high specificity of CUBN expression in RCC also suggests a role as a new diagnostic marker in clinical cancer differential diagnostics to confirm or rule out RCC.

Place, publisher, year, edition, pages
BioMed Central, 2017
Keyword
Cubilin, Renal cell carcinoma, Independent prognostic biomarker, Immunohistochemistry
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:kth:diva-200752 (URN)10.1186/s12885-016-3030-6 (DOI)000391341500003 ()2-s2.0-85008151958 (Scopus ID)
Note

QC 20170210

Available from: 2017-02-10 Created: 2017-02-10 Last updated: 2017-11-29Bibliographically approved
Skogs, M., Stadler, C., Schutten, R., Hjelmare, M., Gnann, C., Björk, L., . . . Lundberg, E. (2017). Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins. Journal of Proteome Research, 16(1), 147-155.
Open this publication in new window or tab >>Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins
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2017 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, no 1, 147-155 p.Article in journal (Refereed) Published
Abstract [en]

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keyword
antibody validation, spatial proteomics, GFP, Human Protein Atlas, Cell Atlas, subcellular localization, immunofluorescence, confocal microscopy
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-201243 (URN)10.1021/acs.jproteome.6b00821 (DOI)000391782100014 ()2-s2.0-85017638855 (Scopus ID)
Note

QC 20170216

Available from: 2017-02-16 Created: 2017-02-16 Last updated: 2017-05-30Bibliographically approved
Hu, F. J., Volk, A.-L., Persson, H., Säll, A., Borrebaeck, C., Uhlén, M. & Rockberg, J. (2017). Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders. New Biotechnology.
Open this publication in new window or tab >>Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders
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2017 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347Article in journal (Refereed) Epub ahead of print
Abstract [en]

Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats.

Place, publisher, year, edition, pages
Elsevier, 2017
Keyword
Affinity maturation, Antibody, Cell-surface display, Flow cytometry, HER2, Phage display, S. carnosus, Binders, Bins, Cell membranes, Display devices, Genes, Libraries, Mammals, Cell surface displays, Antibodies
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-213019 (URN)10.1016/j.nbt.2017.07.011 (DOI)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20170828

Available from: 2017-08-28 Created: 2017-08-28 Last updated: 2017-08-28Bibliographically approved
Lindberg, H., Sandersjöö, L., Meister, S. W., Uhlén, M., Löfblom, J. & Ståhl, S. (2017). Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method. Biotechnology Journal, 12(1), Article ID 1600364.
Open this publication in new window or tab >>Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method
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2017 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 12, no 1, 1600364Article in journal (Refereed) Published
Abstract [en]

Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keyword
Affibody molecules, Aggregation-inhibitor, Amyloid beta, Combinatorial protein engineering, Intracellular selection system, Cells, Cytology, Diagnosis, Fluorescence, Glycoproteins, Molecules, Neurodegenerative diseases, Peptides, Aggregation inhibitors, Amyloid betas, Protein engineering, Selection systems, Proteins, alpha synuclein, amyloid beta protein, green fluorescent protein, recombinant protein, chemistry, Escherichia coli, flow cytometry, gene vector, genetics, metabolism, preclinical study, procedures, alpha-Synuclein, Amyloid beta-Peptides, Drug Evaluation, Preclinical, Genetic Vectors, Green Fluorescent Proteins, Recombinant Proteins
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-202248 (URN)10.1002/biot.201600364 (DOI)000395638400009 ()2-s2.0-85006293410 (Scopus ID)
Note

Funding text: We thank Affibody AB for providing the genetic construct of ZHER2-mCherry. The Wallenberg Center for Protein Research and the Swedish Brain Foundation (grant FO2015-0174) are acknowledged for funding. Conceived and designed the experiments: HL LS JL SS. Performed the experiments: HL LS SM. Contributed reagents/materials/analysis tools and analyzed the data: HL LS SM MU JL SS. Wrote the paper: HL LS JL SS. The authors declare no conflicts of interest. QC 20170313

Available from: 2017-03-13 Created: 2017-03-13 Last updated: 2017-11-29Bibliographically approved
Pin, E., Henjes, F., Hong, M.-G., Wiklund, F., Magnusson, P., Bjartell, A., . . . Schwenk, J. M. (2017). Identification of a Novel Autoimmune Peptide Epitope of Prostein in Prostate Cancer. Journal of Proteome Research, 16(1), 204-216.
Open this publication in new window or tab >>Identification of a Novel Autoimmune Peptide Epitope of Prostein in Prostate Cancer
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2017 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 16, no 1, 204-216 p.Article in journal (Refereed) Published
Abstract [en]

There is a demand for novel targets and approaches to diagnose and treat prostate cancer (PCA). In this context, serum and plasma samples from a total of 609 individuals from two independent patient cohorts were screened for IgG reactivity against a sum of 3833 human protein fragments. Starting from planar protein arrays with 3786 protein fragments to screen 80 patients with and without PCA diagnosis, 161 fragments (4%) were chosen for further analysis based on their reactivity profiles. Adding 71 antigens from literature, the selection of antigens was corroborated for their reactivity in a set of 550 samples using suspension bead arrays. The antigens prostein (SLC45A3), TATA-box binding protein (TBP), and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) showed higher reactivity in PCA patients with late disease compared with early disease. Because of its prostate tissue specificity, we focused on prostein and continued with mapping epitopes of the 66-mer protein fragment using patient samples. Using bead-based assays and 15-mer peptides, a minimal peptide epitope was identified and refined by alanine scanning to the KPxAPFP. Further sequence alignment of this motif revealed homology to transmembrane protein 79 (TMEM79) and TGF-beta-induced factor 2 (TGIF2), thus providing a reasoning for cross-reactivity found in females. A comprehensive workflow to discover and validate IgG reactivity against prostein and homologous targets in human serum and plasma was applied. This study provides useful information when searching for novel biomarkers or drug targets that are guided by the reactivity of the immune system against autoantigens.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keyword
autoimmunity, prostate cancer, prostein, planar microarray, suspension bead array, profiling epitope mapping, Human Protein Atlas, antigen, peptide
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-201244 (URN)10.1021/acs.jproteome.6b00620 (DOI)000391782100019 ()2-s2.0-85017653060 (Scopus ID)
Note

QC 20170216

Available from: 2017-02-16 Created: 2017-02-16 Last updated: 2017-11-29Bibliographically approved
Bosley, J., Borén, C., Lee, S., Grotli, M., Nielsen, J., Uhlén, M., . . . Mardinoglu, A. (2017). Improving the economics of NASH/NAFLD treatment through the use of systems biology. Drug Discovery Today, 22(10), 1532-1538.
Open this publication in new window or tab >>Improving the economics of NASH/NAFLD treatment through the use of systems biology
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2017 (English)In: Drug Discovery Today, ISSN 1359-6446, E-ISSN 1878-5832, Vol. 22, no 10, 1532-1538 p.Article, review/survey (Refereed) Published
Abstract [en]

Nonalcoholic steatohepatitis (NASH) is a severe form of nonalcoholic fatty liver disease (NAFLD). We surveyed NASH therapies currently in development, and found a significant variety of targets and approaches. Evaluation and clinical testing of these targets is an expensive and time-consuming process. Systems biology approaches could enable the quantitative evaluation of the likely efficacy and safety of different targets. This motivated our review of recent systems biology studies that focus on the identification of targets and development of effective treatments for NASH. We discuss the potential broader use of genome-scale metabolic models and integrated networks in the validation of drug targets, which could facilitate more productive and efficient drug development decisions for the treatment of NASH.

Place, publisher, year, edition, pages
ELSEVIER SCI LTD, 2017
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:kth:diva-217446 (URN)10.1016/j.drudis.2017.07.005 (DOI)000413799500008 ()28736156 (PubMedID)2-s2.0-85026322326 (Scopus ID)
Note

QC 20171117

Available from: 2017-11-17 Created: 2017-11-17 Last updated: 2018-01-13Bibliographically approved
Benfeitas, R., Uhlén, M., Nielsen, J. & Mardinoglu, A. (2017). New challenges to study heterogeneity in cancer redox metabolism. Frontiers in Cell and Developmental Biology, 5(JUL), Article ID 65.
Open this publication in new window or tab >>New challenges to study heterogeneity in cancer redox metabolism
2017 (English)In: Frontiers in Cell and Developmental Biology, ISSN 2296-634X, Vol. 5, no JUL, 65Article in journal (Refereed) Published
Abstract [en]

Reactive oxygen species (ROS) are important pathophysiological molecules involved in vital cellular processes. They are extremely harmful at high concentrations because they promote the generation of radicals and the oxidation of lipids, proteins, and nucleic acids, which can result in apoptosis. An imbalance of ROS and a disturbance of redox homeostasis are now recognized as a hallmark of complex diseases. Considering that ROS levels are significantly increased in cancer cells due to mitochondrial dysfunction, ROS metabolism has been targeted for the development of efficient treatment strategies, and antioxidants are used as potential chemotherapeutic drugs. However, initial ROS-focused clinical trials in which antioxidants were supplemented to patients provided inconsistent results, i.e., improved treatment or increased malignancy. These different outcomes may result from the highly heterogeneous redox responses of tumors in different patients. Hence, population-based treatment strategies are unsuitable and patient-tailored therapeutic approaches are required for the effective treatment of patients. Moreover, due to the crosstalk between ROS, reducing equivalents [e.g., NAD(P)H] and central metabolism, which is heterogeneous in cancer, finding the best therapeutic target requires the consideration of system-wide approaches that are capable of capturing the complex alterations observed in all of the associated pathways. Systems biology and engineering approaches may be employed to overcome these challenges, together with tools developed in personalized medicine. However, ROS- and redox-based therapies have yet to be addressed by these methodologies in the context of disease treatment. Here, we review the role of ROS and their coupled redox partners in tumorigenesis. Specifically, we highlight some of the challenges in understanding the role of hydrogen peroxide (H2O2), one of the most important ROS in pathophysiology in the progression of cancer. We also discuss its interplay with antioxidant defenses, such as the coupled peroxiredoxin/thioredoxin and glutathione/glutathione peroxidase systems, and its reducing equivalent metabolism. Finally, we highlight the need for system-level and patient-tailored approaches to clarify the roles of these systems and identify therapeutic targets through the use of the tools developed in personalized medicine. © 2017 Benfeitas, Uhlen, Nielsen and Mardinoglu.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2017
Keyword
Cancer heterogeneity, Personalized medicine, Reactive oxygen species, Redox biology, Systems biology
National Category
Cancer and Oncology Medical and Health Sciences
Identifiers
urn:nbn:se:kth:diva-216288 (URN)10.3389/fcell.2017.00065 (DOI)2-s2.0-85026835225 (Scopus ID)
Note

QC 20171211

Available from: 2017-12-11 Created: 2017-12-11 Last updated: 2017-12-11Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-8993-048X

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