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Löf, Liza
Publications (10 of 13) Show all publications
Dubois, L., Löf, L., Larsson, A., Hultenby, K., Waldenström, A., Kamali-Moghaddam, M., . . . Ronquist, K. G. (2018). Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents. Cancer Research Frontiers, 4(1), 13-26
Open this publication in new window or tab >>Human erythrocyte-derived nanovesicles can readily be loaded with doxorubicin and act as anticancer agents
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2018 (English)In: Cancer Research Frontiers, ISSN 2328-5249, Vol. 4, no 1, p. 13-26Article in journal (Refereed) Published
Abstract [en]

Purpose: In future therapeutics new formulas are needed that assure lower doses, fewer side effects, targeted administration and protection of the drug from degradation. In a first step to fulfil the requirements defined above, we carried out an in vitro study by developing a new procedure to encapsulate drugs using native vesicles first from prostasomes and then from erythrocyte membranes known to be well tolerated. The new method for production of drug delivery vesicles utilized osmotic loading of detergent resistant membranes (DRMs).

Materials and methods: DRMs of prostasomes and prepared human erythrocyte membranes were extracted and separated in a sucrose gradient at a density of 1.10 g/mL containing 1% Triton X-100. These DRMs were characterized by electron microscopy (transmission and scanning EM) and loaded with low and high molecular compounds. PC3 prostate cancer cells were treated with doxorubicin loaded DRMs in triplicate. DAPI (nuclear fluorescent stain) was included and fluorescence microscopic pictures were taken before the cells were trypsinized and counted after 48h.

Results: The content of the well separated band was observed ultrastructurally as small spherical, double layered membrane vesicles, (DRM vesicles) which harbored hyperosmolar sucrose of the gradient. Encapsulated hyperosmolar sucrose induced a transient osmotic lysis of the DRM vesicles when suspended in isotonic buffer containing loading molecules allowing vesicular inclusion. After this proof of concept, the method was finally employed for doxorubicin loading of DRM vesicles from human erythrocytes. When incubating such vesicles with PC3 cells a complete arrest of growth was observed in sharp contrast to PC3 cells incubated with plain doxorubicin in similar conditions.

Conclusion: The present results open up new possibilities for using DRM vesicles as drug delivery vesicles.

National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-364780 (URN)10.17980/2018.13 (DOI)
Note

Louise Dubois and Liza Löf contributed equally to this work.

Available from: 2018-11-02 Created: 2018-11-02 Last updated: 2019-01-31Bibliographically 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
Löf, L., Arngården, L., Olsson-Strömberg, U., Siart, B., Jansson, M., Dahlin, J. S., . . . Kamali-Moghaddam, M. (2017). Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia. Scientific Reports, 7, 1-9, Article ID 623.
Open this publication in new window or tab >>Flow Cytometric Measurement of Blood Cells with BCR-ABL1 Fusion Protein in Chronic Myeloid Leukemia
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-9, article id 623Article in journal (Refereed) Published
Abstract [en]

Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow). By targeting of the BCR and ABL1 parts of the fusion protein with one antibody each, and creating strong fluorescent signals through rolling circle amplification, PLA-flow allowed sensitive detection of cells positive for the BCR-ABL1 fusion at frequencies as low as one in 10,000. Importantly, the flow cytometric results correlated strongly to those of RQ-PCR, both in diagnostic testing and for MRD measurements over time. In summary, we believe this flow cytometry-based method can serve as an attractive approach for routine measurement of cells harboring BCR-ABL1 fusions, also allowing simultaneously assessment of other cell surface markers as well as sensitive longitudinal follow-up.

National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-319726 (URN)10.1038/s41598-017-00755-y (DOI)000398162400034 ()28377570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 294409Swedish Cancer SocietySwedish Research Council
Available from: 2017-04-07 Created: 2017-04-07 Last updated: 2017-05-15Bibliographically approved
Dahlin, J. S., Ekoff, M., Grootens, J., Löf, L., Amini, R.-M., Hagberg, H., . . . Nilsson, G. (2017). KIT signaling is dispensable for human mast cell progenitor development. Blood, 130(16), 1785-1794
Open this publication in new window or tab >>KIT signaling is dispensable for human mast cell progenitor development
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2017 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 130, no 16, p. 1785-1794Article in journal (Refereed) Published
Abstract [en]

Human hematopoietic progenitors are generally assumed to require stem cell factor (SCF) and KIT signaling during differentiation for the formation of mast cells. Imatinib treatment, which inhibits KIT signaling, depletes mast cells in vivo. Furthermore, the absence of SCF or imatinib treatment prevents progenitors from developing into mast cells in vitro. However, these observations do not mean that mast cell progenitors require SCF and KIT signaling throughout differentiation. Here, we demonstrate that circulating mast cell progenitors are present in patients undergoing imatinib treatment. In addition, we show that mast cell progenitors from peripheral blood survive, mature, and proliferate without SCF and KIT signaling in vitro. Contrary to the prevailing consensus, our results show that SCF and KIT signaling are dispensable for early mast cell development.

National Category
Hematology Clinical Laboratory Medicine
Research subject
Pathology
Identifiers
urn:nbn:se:uu:diva-339750 (URN)10.1182/blood-2017-03-773374 (DOI)000413246200005 ()28790106 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer SocietyCancer and Allergy FoundationThe Cancer Society in StockholmThe Cancer Research Funds of RadiumhemmetThe Karolinska Institutet's Research Foundation
Available from: 2018-01-25 Created: 2018-01-25 Last updated: 2019-03-29Bibliographically approved
Larssen, P., Wik, L., Czarnewski, P., Eldh, M., Löf, L., Ronquist, G., . . . Kamali-Moghaddam, M. (2017). Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay. Molecular & cellular proteomics (online), 16(3), 502-511
Open this publication in new window or tab >>Tracing Cellular Origin of Human Exosomes Using Multiplex Proximity Extension Assay
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2017 (English)In: Molecular & cellular proteomics (online), ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 3, p. 502-511Article in journal (Refereed) Published
Abstract [en]

Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA and KLK6, while prostasomes carried NKX31, GSTP1 and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-314142 (URN)10.1074/mcp.M116.064725 (DOI)000395670900013 ()28111361 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme, 259796 294409Swedish Cancer Society, 2013/867Stockholm County Council, 20140405Swedish Heart Lung Foundation, 20140497The Karolinska Institutet's Research FoundationCancer and Allergy Foundation
Available from: 2017-01-28 Created: 2017-01-28 Last updated: 2017-04-20Bibliographically approved
Löf, L. (2016). Applications of in situ proximity ligation assays for cancer research and diagnostics. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Applications of in situ proximity ligation assays for cancer research and diagnostics
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the field of cancer research and diagnostics it is crucial to have reliable methods for detecting molecules involved in the disease. New and better methods for diagnostics, prognostics and drug delivery therefore remain a permanent aim. In this thesis applications of the in situ proximity ligation assay (in situ PLA) were developed for diagnostics and research. Two new methods were developed, one more cost effective proximity assay without the use of enzymes and one method for loading pharmaceuticals in lipid rafts made from detergent resistant membranes (DRMs) to be used as a drug delivery platform.

In Paper I the aim was to develop a flow cytometric detection method of the fusion protein BCR-ABL that is the hallmark of chronic myeloid leukemia (CML). By using in situ PLA the malignant cells carrying the fusion protein could be detected in patients in a convenient workflow.

Paper II describes an application of multiplex in situ PLA, where extracellular vesicles (EVs) are detected and identified using flow cytometry. Up to five different antigens are targeted on the EVs, reflected in three different colors during detection in the flow cytometer. By using antibodies targeting proteins specific for prostasomes a population of prostasomes could be identified in human blood plasma.

In Paper III a new method is described for using lipid raft for drug delivery. In this method, lipid rafts, derived from prostasomes or erythrocytes, are loaded with pharmaceuticals. The vehicles were loaded with doxorubicin, added to cells and counted. Cells that received the vehicle with doxorubicin stopped proliferating and died, while controls that received the lipid raft vehicle without doxorubicin were not affected, suggesting that the vehicles are effectively loaded with the drug and that they are safe. This lipid raft vehicle could provide a safe drug delivery system.     

Paper IV investigates the crosstalk between the two major signal pathways Hippo and Wnt, and how these are affected in gastric cancer. When looking at different colon cancer tumor stages, we found that the cellular localization of TAZ/β-catenin interactions were different. We also found that protein complexes involved in the crosstalk increased in sparsely growing cells compared to more densely growing cells. On the basis of these results the protein E-cadherin, involved in maintenance of the epithelial integrity, was investigated and was found to have a probable role in regulating the crosstalk between Hippo and Wnt.    

A new method for localized protein detection is described in paper V. Here a proximity assay, based on the hybridization chain reaction (HCR), was developed. This assay, proxHCR, is more cost effective than in situ PLA because no enzymes are required. ProxHCR successfully detects protein interactions and can be used together with both fluorescence microscopy and flow cytometry. 

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. p. 56
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1241
Keywords
Cancer, Diagnostics, Research, Prognostics, Method development, Flow cytometry, Drug delivery
National Category
Medical and Health Sciences
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-300191 (URN)978-91-554-9638-8 (ISBN)
External cooperation:
Public defence
2016-09-23, B/B22, Husargatan 4, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2016-09-01 Created: 2016-08-04 Last updated: 2016-09-05
Löf, L., Olsson-Strömberg, U., Landegren, U. & Kamali-Moghaddam, M. (2016). Pla Flow; A Flow Cytometry-Based Assay For Detection Of Bcr-Abl Fusion Protein In Blood Cells From Cml Patients. Paper presented at 21st Congress of the European-Hematology-Association, JUN 09-12, 2016, Copenhagen, DENMARK. Haematologica, 101, 457-458
Open this publication in new window or tab >>Pla Flow; A Flow Cytometry-Based Assay For Detection Of Bcr-Abl Fusion Protein In Blood Cells From Cml Patients
2016 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 101, p. 457-458Article in journal, Meeting abstract (Other academic) Published
National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-301455 (URN)000379484601487 ()
Conference
21st Congress of the European-Hematology-Association, JUN 09-12, 2016, Copenhagen, DENMARK
Available from: 2016-08-23 Created: 2016-08-23 Last updated: 2017-11-28Bibliographically approved
Löf, L., Olsson-Strömberg, U., Landegren, U. & Kamali-Moghaddam, M. (2015). FLOW CYTOMETRY-BASED ASSAY FOR DETECTION OF BCR-ABL FUSION PROTEIN IN BLOOD CELLS FROM CML PATIENTS. Paper presented at 20th Congress of European-Hematology-Association, JUN 11-14, 2015, Vienna, AUSTRIA. Haematologica, 100, 694-695
Open this publication in new window or tab >>FLOW CYTOMETRY-BASED ASSAY FOR DETECTION OF BCR-ABL FUSION PROTEIN IN BLOOD CELLS FROM CML PATIENTS
2015 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 100, p. 694-695Article in journal, Meeting abstract (Other academic) Published
National Category
Hematology
Identifiers
urn:nbn:se:uu:diva-266179 (URN)000361204904294 ()
Conference
20th Congress of European-Hematology-Association, JUN 11-14, 2015, Vienna, AUSTRIA
Available from: 2015-11-06 Created: 2015-11-05 Last updated: 2017-12-01Bibliographically approved
Koos, B., Cane, G., Grannas, K., Löf, L., Arngården, L., Heldin, J., . . . Söderberg, O. (2015). Proximity-dependent initiation of hybridization chain reaction. Nature Communications, 6, Article ID 7294.
Open this publication in new window or tab >>Proximity-dependent initiation of hybridization chain reaction
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2015 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7294Article in journal (Refereed) Published
Abstract [en]

Sensitive detection of protein interactions and post-translational modifications of native proteins is a challenge for research and diagnostic purposes. A method for this, which could be used in point-of-care devices and high-throughput screening, should be reliable, cost effective and robust. To achieve this, here we design a method (proxHCR) that combines the need for proximal binding with hybridization chain reaction (HCR) for signal amplification. When two oligonucleotide hairpins conjugated to antibodies bind in close proximity, they can be activated to reveal an initiator sequence. This starts a chain reaction of hybridization events between a pair of fluorophore-labelled oligonucleotide hairpins, generating a fluorescent product. In conclusion, we show the applicability of the proxHCR method for the detection of protein interactions and posttranslational modifications in microscopy and flow cytometry. As no enzymes are needed, proxHCR may be an inexpensive and robust alternative to proximity ligation assays.

National Category
Medical and Health Sciences Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-255596 (URN)10.1038/ncomms8294 (DOI)000357171100008 ()26065580 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568, 316929, 259796Swedish Foundation for Strategic Research Swedish Research Council
Available from: 2015-06-17 Created: 2015-06-17 Last updated: 2018-01-25Bibliographically approved
Kamali-Moghaddam, M., Löf, L., Oliveir, F., Wik, L., Wu, D. & Yan, J. (2014). Advanced Molecular Tools for Proteomic Analyses of Microvesicles. Paper presented at 28th Annual Symposium of the Protein-Society, JUL 27-30, 2014, San Diego, CA. Protein Science, 23, 102-102
Open this publication in new window or tab >>Advanced Molecular Tools for Proteomic Analyses of Microvesicles
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2014 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 23, p. 102-102Article in journal, Meeting abstract (Other academic) Published
National Category
Other Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-231086 (URN)000339545700099 ()
Conference
28th Annual Symposium of the Protein-Society, JUL 27-30, 2014, San Diego, CA
Note

Special issue, DOI: 10.1002/pro.2504

Available from: 2014-09-04 Created: 2014-09-03 Last updated: 2017-12-05Bibliographically approved
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