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Publications (10 of 118) Show all publications
Gallant, C. J. & Landegren, U. (2019). A compendium on single-cell analysis for the curious. The FEBS Journal, 286(8), 1442-1444
Open this publication in new window or tab >>A compendium on single-cell analysis for the curious
2019 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 286, no 8, p. 1442-1444Article in journal, Editorial material (Other academic) Published
Place, publisher, year, edition, pages
John Wiley & Sons, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-384089 (URN)10.1111/febs.14820 (DOI)000466783900001 ()31012288 (PubMedID)
Available from: 2019-06-19 Created: 2019-06-19 Last updated: 2019-06-19Bibliographically approved
Herr, A. E., Kitamori, T., Landegren, U. & Kamali-Moghaddam, M. (2019). Next wave advances in single-cell analyses. The Analyst, 144(3), 735-737
Open this publication in new window or tab >>Next wave advances in single-cell analyses
2019 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 144, no 3, p. 735-737Article in journal, Editorial material (Other academic) Published
Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Analytical Chemistry Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-377491 (URN)10.1039/c9an90011j (DOI)000457394400001 ()30656308 (PubMedID)
Available from: 2019-02-20 Created: 2019-02-20 Last updated: 2019-02-20Bibliographically approved
Franzen, B., Alexeyenko, A., Kamali-Moghaddam, M., Hatschek, T., Kanter, L., Ramqvist, T., . . . Lewensohn, R. (2019). Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer. Molecular Oncology, 13(2), 376-391
Open this publication in new window or tab >>Protein profiling of fine-needle aspirates reveals subtype-associated immune signatures and involvement of chemokines in breast cancer
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2019 (English)In: Molecular Oncology, ISSN 1574-7891, E-ISSN 1878-0261, Vol. 13, no 2, p. 376-391Article in journal (Refereed) Published
Abstract [en]

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and for follow-up of personalized cancer therapy, including immunotherapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissue samples; however, the minute amounts of sample require sensitive multiplex molecular analysis to be of clinical biomarker utility. We have applied proximity extension assays (PEA) to analyze 167 proteins in FNA samples from patients with breast cancer (BC; n = 25) and benign lesions (n = 32). We demonstrate that the FNA BC samples could be divided into two main clusters, characterized by differences in expression levels of the estrogen receptor (ER) and the proliferation marker Ki67. This clustering corresponded to some extent to established BC subtypes. Our analysis also revealed several proteins whose expression levels differed between BC and benign lesions (e.g., CA9, GZMB, IL-6, VEGFA, CXCL11, PDL1, and PCD1), as well as several chemokines correlating with ER and Ki67 status (e.g., CCL4, CCL8, CCL20, CXCL8, CXCL9, and CXCL17). Finally, we also identified three signatures that could predict Ki67 status, ER status, and tumor grade, respectively, based on a small subset of proteins, which was dominated by chemokines. To our knowledge, expression profiles of CCL13 in benign lesions and BC have not previously been described but were shown herein to correlate with proliferation (P = 0.00095), suggesting a role in advanced BC. Given the broad functional range of the proteins analyzed, immune-related proteins were overrepresented among the observed alterations. Our pilot study supports the emerging role of chemokines in BC progression. Due to the minimally traumatic sampling and clinically important molecular information for therapeutic decisions, this methodology is promising for future immunoscoring and monitoring of treatment efficacy in BC.

Place, publisher, year, edition, pages
WILEY, 2019
Keywords
breast cancer subtypes, fibroadenomas, fine-needle aspiration, immune-related protein biomarker, proximity extension assay
National Category
Cancer and Oncology Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-377673 (URN)10.1002/1878-0261.12410 (DOI)000457747900016 ()30451357 (PubMedID)
Funder
VINNOVA, 2016-00595Swedish Foundation for Strategic Research Swedish Research CouncilEU, FP7, Seventh Framework Programme, 316929EU, FP7, Seventh Framework Programme, 294409Stockholm County Council, 20151043Stockholm County Council, 20160287Swedish Cancer Society, 170246Swedish Foundation for Strategic Research The Breast Cancer Foundation
Available from: 2019-02-25 Created: 2019-02-25 Last updated: 2019-02-25Bibliographically approved
Franzen, B., Kamali-Moghaddam, M., Alexeyenko, A., Hatschek, T., Becker, S., Wik, L., . . . Lewensohn, R. (2018). A fine-needle aspiration-based protein signature discriminates benign from malignant breast lesions. Molecular Oncology, 12(9), 1415-1428
Open this publication in new window or tab >>A fine-needle aspiration-based protein signature discriminates benign from malignant breast lesions
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2018 (English)In: Molecular Oncology, ISSN 1574-7891, E-ISSN 1878-0261, Vol. 12, no 9, p. 1415-1428Article in journal (Refereed) Published
Abstract [en]

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and to follow-up personalized cancer therapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissues; however, the minute sample amounts require sensitive multiplex molecular analysis to achieve clinical utility. We have applied proximity extension assays (PEA) and NanoString (NS) technology for analyses of proteins and of RNA, respectively, in FNA samples. Using samples from patients with breast cancer (BC, n=25) or benign lesions (n=33), we demonstrate that these FNA-based molecular analyses (a) can offer high sensitivity and reproducibility, (b) may provide correct diagnosis in shorter time and at a lower cost than current practice, (c) correlate with results from routine analysis (i.e., benchmarking against immunohistochemistry tests for ER, PR, HER2, and Ki67), and (d) may also help identify new markers related to immunotherapy. A specific 11-protein signature, including FGF binding protein 1, decorin, and furin, distinguished all cancer patient samples from all benign lesions in our main cohort and in smaller replication cohort. Due to the minimally traumatic sampling and rich molecular information, this combined proteomics and transcriptomic methodology is promising for diagnostics and evaluation of treatment efficacy in BC.

Keywords
breast cancer diagnosis, fine-needle aspiration, protein biomarker, proximity extension assay
National Category
Cancer and Oncology Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-364195 (URN)10.1002/1878-0261.12350 (DOI)000443402000002 ()30019538 (PubMedID)
Funder
VINNOVA, 201600595Swedish Research CouncilEU, FP7, Seventh Framework Programme, 316929 294409Stockholm County Council, 20151043The Breast Cancer FoundationSwedish Cancer SocietyThe Cancer Research Funds of Radiumhemmet
Available from: 2018-10-29 Created: 2018-10-29 Last updated: 2018-11-16Bibliographically approved
Landegren, U., Al-Amin, R. A. & Björkesten, J. (2018). A myopic perspective on the future of protein diagnostics. New Biotechnology, 45, 14-18
Open this publication in new window or tab >>A myopic perspective on the future of protein diagnostics
2018 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 45, p. 14-18Article, review/survey (Refereed) Published
Abstract [en]

Plasma proteome analyses of the future promise invaluable insights into states of health, not only by measuring proteins whose role it is to ensure blood homeostasis, but increasingly also as a window into the health of practically any tissue in the body via so-called leakage protein biomarkers. Realizing more of this vast potential will require progress along many lines. Here we discuss the main ones, such as optimal selection of target proteins, affinity reagents, immunoassay formats, samples, and applications, with a view from ongoing work in our laboratory.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2018
Keywords
Protein leakage markers, Liquid biopsy, Early detection of disease, Tissue-specific proteins, Exocrine secretion, Recombinant protein binders, Dual-recognition assays, Sandwich immune assay, Proximity ligation assay, Proximity extension assay, Dried blood spots, Consecutive patient samples, Wellness testing, Point of care
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-363863 (URN)10.1016/j.nbt.2018.01.002 (DOI)000441913800003 ()29309916 (PubMedID)
Funder
Swedish Research CouncilSwedish Foundation for Strategic Research EU, European Research Council, 313010
Available from: 2018-11-14 Created: 2018-11-14 Last updated: 2018-11-14Bibliographically approved
Klaesson, A., Grannas, K., Ebai, T., Heldin, J., Koos, B., Leino, M., . . . Landegren, U. (2018). Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes. Scientific Reports, 8, Article ID 5400.
Open this publication in new window or tab >>Improved efficiency of in situ protein analysis by proximity ligation using UnFold probes
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 5400Article in journal (Refereed) Published
Abstract [en]

We have redesigned probes for in situ proximity ligation assay (PLA), resulting in more efficient localized detection of target proteins. In situ PLA depends on recognition of target proteins by pairs of antibody-oligonucleotide conjugates (PLA probes), which jointly give rise to DNA circles that template localized rolling circle amplification reactions. The requirement for dual recognition of the target proteins improves selectivity by ignoring any cross-reactivity not shared by the antibodies, and it allows detection of protein-protein interactions and post-translational modifications. We herein describe an improved design of the PLA probes -UnFold probes - where all elements required for formation of circular DNA strands are incorporated in the probes. Premature interactions between the UnFold probes are prevented by including an enzymatic "unfolding" step in the detection reactions. This allows DNA circles to form by pairs of reagents only after excess reagents have been removed. We demonstrate the performance of UnFold probes for detection of protein-protein interactions and post-translational modifications in fixed cells and tissues, revealing considerably more efficient signal generation. We also apply the UnFold probes to detect IL-6 in solution phase after capture on solid supports, demonstrating increased sensitivity over both normal sandwich enzyme-linked immunosorbent assays and conventional PLA assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-340077 (URN)10.1038/s41598-018-23582-1 (DOI)000428618900043 ()29599435 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568 264737 294409Swedish Foundation for Strategic Research Swedish Research Council
Note

Ola Söderberg and Ulf Landegren jointly supervised this work

Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2018-08-06Bibliographically approved
Zieba, A., Ponten, F., Uhlen, M. & Landegren, U. (2018). In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation. Modern Pathology, 31(2), 253-263
Open this publication in new window or tab >>In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation
2018 (English)In: Modern Pathology, ISSN 0893-3952, E-ISSN 1530-0285, Vol. 31, no 2, p. 253-263Article in journal (Refereed) Published
Abstract [en]

Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Immunology in the medical area Other Industrial Biotechnology
Identifiers
urn:nbn:se:uu:diva-347648 (URN)10.1038/modpathol.2017.102 (DOI)000424761400003 ()28937142 (PubMedID)
Funder
Knut and Alice Wallenberg Foundation, 2008:0143EU, FP7, Seventh Framework Programme, 222635 241481Swedish Research CouncilVINNOVAEU, European Research Council, 294409
Available from: 2018-04-06 Created: 2018-04-06 Last updated: 2018-04-06Bibliographically approved
Lindblom, R. P., Shen, Q., Axén, S., Landegren, U., Kamali-Moghaddam, M. & Thelin, S. (2018). Protein Profiling in Serum and Cerebrospinal Fluid Following Complex Surgery on the Thoracic Aorta Identifies Biological Markers of Neurologic Injury.. Journal of Cardiovascular Translational Research, 11(6), 503-516
Open this publication in new window or tab >>Protein Profiling in Serum and Cerebrospinal Fluid Following Complex Surgery on the Thoracic Aorta Identifies Biological Markers of Neurologic Injury.
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2018 (English)In: Journal of Cardiovascular Translational Research, ISSN 1937-5387, E-ISSN 1937-5395, Vol. 11, no 6, p. 503-516Article in journal (Refereed) Published
Abstract [en]

Surgery on the arch or descending aorta is associated with significant risk of neurological complications. As a consequence of intubation and sedation, early neurologic injury may remain unnoticed. Biomarkers to aid in the initial diagnostics could prove of great value as immediate intervention is critical. Twenty-three patients operated in the thoracic aorta with significant risk of perioperative neurological injury were included. Cerebrospinal fluid (CSF) and serum were obtained preoperatively and in the first and second postoperative days and assessed with a panel of 92 neurological-related proteins. Three patients suffered spinal cord injury (SCI), eight delirium, and nine hallucinations. There were markers in both serum and CSF that differed between the affected and non-affected patients (SCI; IL6, GFAP, CSPG4, delirium; TR4, EZH2, hallucinations; NF1). The study identifies markers in serum and CSF that reflect the occurrence of neurologic insults following aortic surgery, which may aid in the care of these patients.

Keywords
Biomarkers, Cardiovascular surgery, Neurologic injury, Thoracic aortic disease
National Category
Cardiac and Cardiovascular Systems
Identifiers
urn:nbn:se:uu:diva-369702 (URN)10.1007/s12265-018-9835-8 (DOI)000453355000007 ()30367354 (PubMedID)
Available from: 2018-12-16 Created: 2018-12-16 Last updated: 2019-01-15Bibliographically approved
Shen, Q., Björkesten, J., Galli, J., Ekman, D., Broberg, J., Nordberg, N., . . . Landegren, U. (2018). Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays. Clinical Chemistry and Laboratory Medicine, 56(4), 582-594
Open this publication in new window or tab >>Strong impact on plasma protein profiles by precentrifugation delay but not by repeated freeze-thaw cycles, as analyzed using multiplex proximity extension assays
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2018 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 56, no 4, p. 582-594Article in journal (Refereed) Published
Abstract [en]

Background: A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles.

Methods: Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 degrees C or 22 degrees C for 1-36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at -80 degrees C and thawing at 22 degrees C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins.

Results: Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 degrees C or 22 degrees C, respectively. Some increases became noticeable after 8 h delay at 4 degrees C but already after 1 h at 22 degrees C. For samples stored at 4 degrees C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 degrees C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 degrees C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles.

Conclusions: Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Place, publisher, year, edition, pages
WALTER DE GRUYTER GMBH, 2018
Keywords
biobank, protein detection, proteome, proximity extension assay (PEA), sample collection and handling
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-350276 (URN)10.1515/cclm-2017-0648 (DOI)000426657400016 ()29040064 (PubMedID)
Funder
Swedish Research Council, 829-2009-6285EU, European Research Council, 313010, 294409
Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2018-05-14Bibliographically approved
Ebai, T., de Oliveira, F. M., Löf, L., Wik, L., Schweiger, C., Larsson, A., . . . Kamali-Moghaddam, M. (2017). Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification. Clinical Chemistry, 63(9), 1497-1505
Open this publication in new window or tab >>Analytically Sensitive Protein Detection in Microtiter Plates by Proximity Ligation with Rolling Circle Amplification
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2017 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 63, no 9, p. 1497-1505Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Detecting proteins at low concentrations in plasma is crucial for early diagnosis. Current techniques in clinical routine, such as sandwich ELISA, provide sensitive protein detection because of a dependence on target recognition by pairs of antibodies, but detection of still lower protein concentrations is often called for. Proximity ligation assay with rolling circle amplification (PLARCA) is a modified proximity ligation assay (PLA) for analytically specific and sensitive protein detection via binding of target proteins by 3 antibodies, and signal amplification via rolling circle amplification (RCA) in microtiter wells, easily adapted to instrumentation in use in hospitals.

METHODS: Proteins captured by immobilized antibodies were detected using a pair of oligonucleotide-conjugated antibodies. Upon target recognition, these PLA probes guided oligonucleotide ligation, followed by amplification via RCA of circular DNA strands that formed in the reaction. The RCA products were detected by horseradish peroxidase-labeled oligonucleotides to generate colorimetric reaction products with readout in an absorbance microplate reader.

RESULTS: We compared detection of interleukin (IL)-4, IL-6, IL-8, p53, and growth differentiation factor-15 by PLARCA and conventional sandwich ELISA or immuno RCA. PLARCA detected lower concentrations of proteins and exhibited a broader dynamic range compared ELISA and iRCA using the same antibodies. IL-4 and IL-6 were detected in clinical samples at femtomolar concentrations, considerably lower than for ELISA.

CONCLUSIONS: PLARCA offers detection of lower protein levels and increased dynamic ranges compared to ELISA. The PLARCA procedure may be adapted to routine instrumentation available in hospitals and research laboratories.

National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-326672 (URN)10.1373/clinchem.2017.271833 (DOI)000408421200013 ()28667186 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 294409 264737 313010
Available from: 2017-07-21 Created: 2017-07-21 Last updated: 2018-09-17Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-7820-1000

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