Signal Amplification for Fluorescent Staining of Single Particles in Liquid Biopsies: Circulating Tumour Cells and Extracellular Vesicles.
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| Názov: | Signal Amplification for Fluorescent Staining of Single Particles in Liquid Biopsies: Circulating Tumour Cells and Extracellular Vesicles. |
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| Autori: | Cavallaro S; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA.; Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Veiga SI; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA.; Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Ahmad R; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA.; Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.; Shriners Children's Boston, Boston, Massachusetts, USA., Aldikacti B; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA., Bienstock M; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA., Capen D; Program in Membrane Biology/Division of Nephrology, Massachusetts General Hospital, Boston, Massachusetts, USA., Rabe DC; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA.; Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA., Ho U; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA., Lee D; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA., Ruiz-Torres DA; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA.; Massachusetts Eye and Ear, Massachusetts General Hospital, Boston, Massachusetts, USA., Wakimoto H; Neurosurgery, Massachusetts General Hospital, Boston, Massachusetts, USA., Dietrich J; Brain Tumor Center, Massachusetts General Hospital, Massachusetts, USA., Nahed BV; Brain Tumor Center, Massachusetts General Hospital, Massachusetts, USA., Stott SL; Krantz-Family Center for Cancer Research, Massachusetts General Hospital, Boston, Massachusetts, USA.; Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.; Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. |
| Zdroj: | Journal of extracellular vesicles [J Extracell Vesicles] 2025 Oct; Vol. 14 (10), pp. e70167. |
| Spôsob vydávania: | Journal Article |
| Jazyk: | English |
| Informácie o časopise: | Publisher: Wiley Country of Publication: United States NLM ID: 101610479 Publication Model: Print Cited Medium: Internet ISSN: 2001-3078 (Electronic) Linking ISSN: 20013078 NLM ISO Abbreviation: J Extracell Vesicles Subsets: MEDLINE |
| Imprint Name(s): | Publication: 2020- : [Hoboken, NJ] : Wiley Original Publication: Järfälla : Co-Action Pub. |
| Výrazy zo slovníka MeSH: | Neoplastic Cells, Circulating*/pathology , Neoplastic Cells, Circulating*/metabolism , Extracellular Vesicles*/metabolism , Glioblastoma*/pathology , Glioblastoma*/metabolism , Fluorescent Antibody Technique*/methods, Humans ; Liquid Biopsy/methods ; Cell Line, Tumor ; Staining and Labeling/methods ; Fluorescent Dyes |
| Abstrakt: | Immunofluorescence (IF) staining represents a convenient and cost-effective approach to analysing single extracellular vesicles (EVs) and identifying subpopulations with specific roles or biological functions. However, the application of the method is challenged by the weak and unstable signals generated by the low abundant markers carried by the vesicles. In this study, we report the development of an IF strategy based on tyramide signal amplification (TSA) that employs tyramide probes for signal enhancement. The technique is first validated on glioblastoma circulating tumour cells (GBM CTCs) and systematically compared with conventional approaches using fluorescently labelled primary and secondary antibodies. Thereafter, the proposed method is adapted, tested and optimised for the multiplexed fluorescent staining of single EVs isolated from the parental GBM CTCs. The results demonstrate specific staining of single EVs by the developed TSA method, highlighting its advantages of amplified (>6×) signal intensities, more stable signals and broader (∼3×) signal dynamic ranges as compared to the conventional fluorescence methods. The developed protocol also supports multiplexing by incorporating a quenching buffer between the different staining colours. Finally, the protocol demonstrates its applicability to CTCs and EVs derived from plasma samples of GBM patients, with easy adaptation to other cancers or proteins of interest. (© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.) |
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| Grant Information: | 132030-RSG-18-108-01-TBG American Cancer Society; PF-23-1151433-01-CCB American Cancer Society; F32-CA236417 United States CA NCI NIH HHS; R01-CA226871 United States CA NCI NIH HHS; U18-TR003793 United States TR NCATS NIH HHS; DK043351 Inflammatory Bowel Disease Grant; d'Arbeloff MGH Research Scholar Award; DK135043 Boston Area Diabetes and Endocrinology Research Center (BADERC) Award; V Foundation for Cancer Research |
| Contributed Indexing: | Keywords: circulating tumour cells; extracellular vesicles; fluorescence microscopy; liquid biopsies; signal amplification; single particle; tyramide signal amplification |
| Substance Nomenclature: | 0 (Fluorescent Dyes) |
| Entry Date(s): | Date Created: 20251009 Date Completed: 20251009 Latest Revision: 20251012 |
| Update Code: | 20251012 |
| PubMed Central ID: | PMC12508258 |
| DOI: | 10.1002/jev2.70167 |
| PMID: | 41063443 |
| Databáza: | MEDLINE |
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Nájsť tento článok vo Web of Science | Abstrakt: | Immunofluorescence (IF) staining represents a convenient and cost-effective approach to analysing single extracellular vesicles (EVs) and identifying subpopulations with specific roles or biological functions. However, the application of the method is challenged by the weak and unstable signals generated by the low abundant markers carried by the vesicles. In this study, we report the development of an IF strategy based on tyramide signal amplification (TSA) that employs tyramide probes for signal enhancement. The technique is first validated on glioblastoma circulating tumour cells (GBM CTCs) and systematically compared with conventional approaches using fluorescently labelled primary and secondary antibodies. Thereafter, the proposed method is adapted, tested and optimised for the multiplexed fluorescent staining of single EVs isolated from the parental GBM CTCs. The results demonstrate specific staining of single EVs by the developed TSA method, highlighting its advantages of amplified (>6×) signal intensities, more stable signals and broader (∼3×) signal dynamic ranges as compared to the conventional fluorescence methods. The developed protocol also supports multiplexing by incorporating a quenching buffer between the different staining colours. Finally, the protocol demonstrates its applicability to CTCs and EVs derived from plasma samples of GBM patients, with easy adaptation to other cancers or proteins of interest.<br /> (© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.) |
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| ISSN: | 2001-3078 |
| DOI: | 10.1002/jev2.70167 |