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Ronquist, Göran
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Publications (10 of 25) Show all publications
Wu, D., Yan, J., Shen, X., Sun, Y., Thulin, M., Cai, Y., . . . Kamali-Moghaddam, M. (2019). Profiling surface proteins on individual exosomes using a proximity barcoding assay. Nature Communications, 10, Article ID 3854.
Open this publication in new window or tab >>Profiling surface proteins on individual exosomes using a proximity barcoding assay
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2019 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 3854Article in journal (Refereed) Published
Abstract [en]

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2019
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-398851 (URN)10.1038/s41467-019-11486-1 (DOI)000482567200002 ()31451692 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationEU, FP7, Seventh Framework Programme, 294409
Available from: 2019-12-18 Created: 2019-12-18 Last updated: 2019-12-18Bibliographically approved
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
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
Ronquist, K. G., Sanchez, C., Dubois, L., Chioureas, D., Fonseca, P., Larsson, A., . . . Panaretakis, T. (2016). Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells. Journal of Extracellular Vesicles, 5, Article ID 29877.
Open this publication in new window or tab >>Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells
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2016 (English)In: Journal of Extracellular Vesicles, ISSN 2001-3078, E-ISSN 2001-3078, Vol. 5, article id 29877Article in journal (Refereed) Published
Abstract [en]

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process.

Keywords
extracellular ATP; prostate cancer; extracellular vesicles; exosomes; prostasomes; ATPase; glycolysis; energy metabolism; exosome uptake
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-281232 (URN)10.3402/jev.v5.29877 (DOI)000375921700001 ()26955882 (PubMedID)
External cooperation:
Available from: 2016-03-21 Created: 2016-03-21 Last updated: 2017-11-30Bibliographically approved
Carlsson, L., Ronquist, G., Elisasson, R., Dubois, L., Ronquist, K. G. & Larsson, A. (2016). High Concentrations of the Angiogenic Peptide VEGF-A in Seminal Fluid and its Association to Prostasomes. Clinical Laboratory, 62(8), 1515-1520
Open this publication in new window or tab >>High Concentrations of the Angiogenic Peptide VEGF-A in Seminal Fluid and its Association to Prostasomes
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2016 (English)In: Clinical Laboratory, ISSN 1433-6510, Vol. 62, no 8, p. 1515-1520Article in journal (Refereed) Published
Abstract [en]

Background: Angiogenesis is the formation of new blood vessels by capillary sprouting from pre-existing vessels. This process is associated with increased expression of angiogenic factors like vascular endothelial growth factor (VEGF). The VEGF family consists of five members denoted VEGF-A, B, C, D and placenta growth factor (PlGF). Prostasomes are exosome-like extracellular vesicles existing in seminal plasma. The present study aimed at investigating the possible relationship between VEGF-A in seminal fluid and blood plasma and the prostasomal association of VEGF-A.

Methods: Measurement of VEGF-A concentrations was carried out in seminal plasma from 40 males and in blood plasma from 40 male blood donors utilizing commercial ELISA kits. The prostasomal association of VEGF-A was investigated by flow cytometry.

Results: We found highly elevated concentrations of VEGF-A in seminal fluid (median value 150000 pg/mL) compared with those of blood plasma. Flow cytometric analysis showed that VEGF-A is bound to the surface of prostasomes.

Conclusions: Prostasomes and seminal plasma contain the angiogenic factor VEGF-A in high concentrations exceeding that of blood plasma by 1000 times.

Keywords
angiogenesis factor, ELISA, prostasomes, seminal fluid, vascular endothelial growth factor
National Category
Urology and Nephrology
Identifiers
urn:nbn:se:uu:diva-303148 (URN)10.7754/Clin.Lab.2016.151229 (DOI)000381716800017 ()28164613 (PubMedID)
Available from: 2016-09-15 Created: 2016-09-15 Last updated: 2017-11-21Bibliographically approved
Wikström, A.-K., Hagmar, M., Ronquist, G. & Larsson, A. (2015). Evaluation of a Plasma hCG Method for Point of Care Testing with the Aim of Shortening Test-Turnaround-Times. Open Journal of Obstetrics and Gynecology, 5(6), 341-343
Open this publication in new window or tab >>Evaluation of a Plasma hCG Method for Point of Care Testing with the Aim of Shortening Test-Turnaround-Times
2015 (English)In: Open Journal of Obstetrics and Gynecology, ISSN 2160-8792, E-ISSN 2160-8806, Vol. 5, no 6, p. 341-343Article in journal (Refereed) Published
Abstract [en]

Objective: To examine the correlation between plasma hCG results obtained with the new i-STAT® hCG point of care test with those concomitantly obtained from the central hospital laboratory utilizing the same patient samples.

Methods: Prospective cross-sectional laboratory test evaluation. We compared plasma hCG results obtained with the i-STAT® hCG test (Abbott Point of Care, Princeton, NJ, USA) with Architect Ci8200 (Abbott Laboratories, Abbott Park, IL, USA). We also calculated the total coefficient of variation (CV) for the i-STAT® method.

Results: The two methods showed a good linear correlation (R2 = 0.994; slope 1.03) and CV for the i-STAT® method was 2.1% - 5.2%.

Conclusion: We suggest that the i-STAT® hCG blood assay could be used as a complement to urine hCG assays in clinical situations when rapid test results are needed and urine is not available.

National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:uu:diva-259354 (URN)10.4236/ojog.2015.56049 (DOI)
Available from: 2015-07-31 Created: 2015-07-31 Last updated: 2017-12-04Bibliographically approved
Dubois, L., Stridsberg, M., Kharaziha, P., Chioureas, D., Meersman, N., Panaretakis, T. & Ronquist, G. (2015). Malignant Cell-Derived Extracellular Vesicles Express Different Chromogranin Epitopes Compared to Prostasomes. The Prostate, 75(10), 1063-1073
Open this publication in new window or tab >>Malignant Cell-Derived Extracellular Vesicles Express Different Chromogranin Epitopes Compared to Prostasomes
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2015 (English)In: The Prostate, ISSN 0270-4137, E-ISSN 1097-0045, Vol. 75, no 10, p. 1063-1073Article in journal (Refereed) Published
Abstract [en]

BACKGROUND. Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. METHODS. We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. RESULTS. A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. CONCLUSION. Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers.

Keywords
prostasomes, extracellular vesicles, PC3, LNCaP, DU145, chromogranins, synaptophysin, prostate cancer
National Category
Endocrinology and Diabetes Urology and Nephrology
Identifiers
urn:nbn:se:uu:diva-256204 (URN)10.1002/pros.22990 (DOI)000354203600006 ()25783430 (PubMedID)
Available from: 2015-07-06 Created: 2015-06-22 Last updated: 2017-12-04Bibliographically approved
Ronquist, G. (2015). Prostasomes: Their Characterisation: Implications for Human Reproduction. In: MALE ROLE IN PREGNANCY LOSS AND EMBRYO IMPLANTATION FAILURE: (pp. 191-209). Springer
Open this publication in new window or tab >>Prostasomes: Their Characterisation: Implications for Human Reproduction
2015 (English)In: MALE ROLE IN PREGNANCY LOSS AND EMBRYO IMPLANTATION FAILURE, Springer, 2015, p. 191-209Chapter in book (Refereed)
Abstract [en]

The prostate is a principal accessory genital gland that is vital for normal fertility. Epithelial cells lining the prostate acini release in a defined fashion (exocytosis) organellar nanosized structures named prostasomes. They are involved in the protection of sperm cells against immune response in the female reproductive tract by modulating the complement system and by inhibiting monocyte and neutrophil phagocytosis and lymphocyte proliferation. The immunomodulatory function most probably involves small non-coding RNAs present in prostasomes. Prostasomes have also been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, since they are rich in various transferable bioactive molecules (e.g. receptors and enzymes) that promote the fertilising ability of sperm cells. Antigenicity of sperm cells has been well documented and implicated in involuntary immunological infertility of human couples, and antisperm antibodies (ASA) occur in several body fluids. The propensity of sperm cells to carry attached prostasomes suggests that they are a new category of sperm antigens. Circulating human ASA recognise prostasomes, and among 12 identified prostasomal antigens, prolactin-inducible protein (95 %) and clusterin (85 %) were immunodominant at the expense of the other 10 that were sporadically occurring.

Place, publisher, year, edition, pages
Springer, 2015
Series
Advances in Experimental Medicine and Biology, ISSN 0065-2598 ; 868
Keywords
Prostasomes, Non-coding RNA, Seminal fluid, Spermatozoa, Antioxidants, Immunosuppression
National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:uu:diva-265648 (URN)10.1007/978-3-319-18881-2_9 (DOI)000361813200010 ()26178851 (PubMedID)978-3-319-18881-2 (ISBN)978-3-319-18880-5 (ISBN)
Available from: 2015-11-02 Created: 2015-11-02 Last updated: 2015-11-05Bibliographically approved
Dubois, L., Ronquist, K. G., Ek, B., Ronquist, G. & Larsson, A. (2015). Proteomic profiling of detergent resistant membranes (lipid rafts) of prostasomes. Molecular & Cellular Proteomics, 14(11), 3015-3022
Open this publication in new window or tab >>Proteomic profiling of detergent resistant membranes (lipid rafts) of prostasomes
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2015 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 14, no 11, p. 3015-3022Article in journal (Refereed) Published
Abstract [en]

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/mL were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10g/mL, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry and electron microscopy. The clearly visible band on top of 1.10g/mL sucrose in the Triton X-100 containing gradient was subjected to LC-MS/MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs and Ras-related proteins. This is the first comprehensive LC-MS/MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

National Category
Urology and Nephrology
Identifiers
urn:nbn:se:uu:diva-261543 (URN)10.1074/mcp.M114.047530 (DOI)000365636800014 ()26272980 (PubMedID)
Available from: 2015-09-01 Created: 2015-09-01 Last updated: 2018-12-11Bibliographically approved
Nickel, K. F., Ronquist, G., Langer, F., Labberton, L., Fuchs, T. A., Bokemeyer, C., . . . Renne, T. (2015). The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis. Blood, 126(11), 1379-1389
Open this publication in new window or tab >>The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis
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2015 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 126, no 11, p. 1379-1389Article in journal (Refereed) Published
Abstract [en]

Cancer is a leading cause of thrombosis. We identify a new procoagulant mechanism that contributes to thromboembolism in prostate cancer and allows for safe anticoagulation therapy development. Prostate cancer-mediated procoagulant activity was reduced in plasma in the absence of factor XII or its substrate of the intrinsic coagulation pathway factor XI. Prostate cancer cells and secreted prostasomes expose long chain polyphosphate on their surface that colocalized with active factor XII and initiated coagulation in a factor XII-dependent manner. Polyphosphate content correlated with the procoagulant activity of prostasomes. Inherited deficiency in factor XI or XII or high-molecular-weight kininogen, but not plasma kallikrein, protected mice from prostasome-induced lethal pulmonary embolism. Targeting polyphosphate or factor XII conferred resistance to prostate cancer-driven thrombosis in mice, without increasing bleeding. Inhibition of factor XII with recombinant 3F7 antibody reduced the increased prostasome-mediated procoagulant activity in patient plasma. The data illustrate a critical role for polyphosphate/factor XII-triggered coagulation in prostate cancer-associated thrombosis with implications for anticoagulation without therapy-associated bleeding in malignancies.

National Category
Cardiac and Cardiovascular Systems Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-267337 (URN)10.1182/blood-2015-01-622811 (DOI)000363479500019 ()26153520 (PubMedID)
Funder
Swedish Cancer Society, 100615Swedish Research Council, K2013-65X-21462-014-5Swedish Heart Lung Foundation, 20140741Stockholm County Council, 20140464German Research Foundation (DFG), SFB877German Research Foundation (DFG), SFB841EU, European Research Council, ERC-StG-2012-311575_F-12
Available from: 2015-11-24 Created: 2015-11-20 Last updated: 2017-12-01Bibliographically approved
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