Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays

Doulabi, Ehsan Manouchehri; Fredolini, Claudia; Gallini, R; Löf, Liza; Shen, Qiujin; Ikebuchi, Ryoyo; Dubois, Louise; Azimi, Alireza; Loudig, Olivier; Gabrielsson, Susanne; Landegren, Ulf; Larsson, Anders; Bergquist, Jonas; Kamali‐Moghaddam, Masood

doi:10.1038/s42003-022-04349-x

Cited by 8 publications

(3 citation statements)

References 84 publications

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“…Exosomes were isolated from the cell culture supernatant using ultracentrifugation as described in the literature [42,43]. Upon reaching 80% confluence, cells were washed with PBS to remove dead cells and residual media, then cultured in exosome-free complete media for 48 h. The cell culture supernatant was collected, and the collection process for exosomes was as follows: exosomes were isolated by differential centrifugation at 4 • C for 10 min (first at 300× g, subsequently at 2000× g, and next at 10,000× g).…”

Section: Exosome Isolation and Characterizationmentioning

confidence: 99%

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer

Li,

Qian,

Liu

et al. 2024

Biosensors

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Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an “APPROACH” strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL−1, 0.77 ng∙μL−1, and 1.1 ng∙μL−1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL−1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.

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Section: Exosome Isolation and Characterizationmentioning

confidence: 99%

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer

Li,

Qian,

Liu

et al. 2024

Biosensors

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“…These surface markers are crucial for cell recognition and cargo delivery, thereby influencing EV function and biological effects. Identification of surface markers associated with specific EV subtypes provides valuable insights into their physiological roles and pathophysiological implications [ 38 39 40 41 42 ].…”

Section: B Iomarkers Of Extracellular Vesiclesmentioning

confidence: 99%

Extracellular vesicles: Function, resilience, biomarker, bioengineering, and clinical implications

Sun,

Chang

2024

Tzu Chi Medical Journal

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Extracellular vesicles (EVs) have emerged as key players in intercellular communication, disease pathology, and therapeutic innovation. Initially overlooked as cellular debris, EVs are now recognized as vital mediators of cell-to-cell communication, ferrying a cargo of proteins, nucleic acids, and lipids, providing cellular resilience in response to stresses. This review provides a comprehensive overview of EVs, focusing on their role as biomarkers in disease diagnosis, their functional significance in physiological and pathological processes, and the potential of bioengineering for therapeutic applications. EVs offer a promising avenue for noninvasive disease diagnosis and monitoring, reflecting the physiological state of originating cells. Their diagnostic potential spans a spectrum of diseases, including cancer, cardiovascular disorders, neurodegenerative diseases, and infectious diseases. Moreover, their presence in bodily fluids such as blood, urine, and cerebrospinal fluid enhances their diagnostic utility, presenting advantages over traditional methods. Beyond diagnostics, EVs mediate crucial roles in intercellular communication, facilitating the transfer of bioactive molecules between cells. This communication modulates various physiological processes such as tissue regeneration, immune modulation, and neuronal communication. Dysregulation of EV-mediated communication is implicated in diseases such as cancer, immune disorders, and neurodegenerative diseases, highlighting their therapeutic potential. Bioengineering techniques offer avenues for manipulating EVs for therapeutic applications, from isolation and purification to engineering cargo and targeted delivery systems. These approaches hold promise for developing novel therapeutics tailored to specific diseases, revolutionizing personalized medicine. However, challenges such as standardization, scalability, and regulatory approval need addressing for successful clinical translation. Overall, EVs represent a dynamic frontier in biomedical research with vast potential for diagnostics, therapeutics, and personalized medicine.

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“…Our study is also limited in not knowing the immune cell origin of EVs released into peripheral blood circulation. Multiplex proximity extension assays and proteomic profiling of body-fluid derived EVs with matched cell lysates from human tumor biopsies or tumor derived cell lines could help trace EVs to their parental cells and further allow the characterization of cell type-specific signatures in highly heterogeneous tissue and/or permit the identification of biomarkers important for predicting tumor progression and metastasis (62)(63)(64)(65).…”

Section: Limitations Of This Studymentioning

confidence: 99%

Extracellular vesicles as biomarkers for AIDS-associated non-Hodgkin lymphoma risk

Martínez,

Magpantay,

Guo

et al. 2023

Front. Immunol.

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IntroductionExtracellular vesicles are membrane-bound structures secreted into the extracellular milieu by cells and can carry bioactive molecules. There is emerging evidence suggesting that EVs play a role in the diagnosis, treatment, and prognosis of certain cancers. In this study, we investigate the association of EVs bearing PD-L1 and molecules important in B-cell activation and differentiation with AIDS-NHL risk.MethodsEVs were isolated from archived serum collected prior to the diagnosis of AIDS-NHL in cases (N = 51) and matched HIV+ controls (N = 52) who were men enrolled in the Los Angeles site of the MACS/WIHS Combined Cohort Study (MWCCS). Serum specimens of AIDS-NHL cases were collected at a mean time of 1.25 years (range of 2 to 36 months) prior to an AIDS-NHL diagnosis. The expression of PD-L1 and other molecules on EVs (CD40, CD40L, TNF-RII, IL-6Rα, B7-H3, ICAM-1, and FasL) were quantified by Luminex multiplex assay.Results and discussionWe observed significantly higher levels of EVs bearing PD-L1, CD40, TNF-RII and/or IL-6Rα in AIDS-NHL cases compared with controls. Using multivariate conditional logistic regression models adjusted for age and CD4+ T-cell count, we found that EVs bearing PD-L1 (OR = 1.93; 95% CI: 1.10 – 3.38), CD40 (OR = 1.97, 95% CI: 1.09 – 3.58), TNF-RII (OR = 5.06; 95% CI: 1.99 – 12.85) and/or IL-6Rα (OR = 4.67; 95% CI: 1.40 – 15.53) were significantly and positively associated with AIDS-NHL risk. In addition, EVs bearing these molecules were significantly and positively associated with non-CNS lymphoma: PD-L1 (OR = 1.94; 95% CI: 1.01 – 3.72); CD40 (OR = 2.66; 95% CI: 1.12 – 6.35); TNF-RII (OR = 9.64; 95% CI: 2.52 – 36.86); IL-6Rα (OR = 8.34; 95% CI: 1.73 – 40.15). These findings suggest that EVs bearing PD-L1, CD40, TNF-RII and/or IL-6Rα could serve as biomarkers for the early detection of NHL in PLWH.

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Surface protein profiling of prostate-derived extracellular vesicles by mass spectrometry and proximity assays

Cited by 8 publications

References 84 publications

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer

APPROACH: Sensitive Detection of Exosomal Biomarkers by Aptamer-Mediated Proximity Ligation Assay and Time-Resolved Förster Resonance Energy Transfer

Extracellular vesicles: Function, resilience, biomarker, bioengineering, and clinical implications

Extracellular vesicles as biomarkers for AIDS-associated non-Hodgkin lymphoma risk

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