Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma

Reale, Antonia; Khong, Tiffany; Xu, Rong; Chen, Maoshan; Mithraprabhu, Sridurga; Bingham, Nicholas; Spencer, Andrew; Greening, David W.

doi:10.1007/978-1-0716-1186-9_10

Cited by 9 publications

(19 citation statements)

References 140 publications

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“…Importantly, sRNA tubes offer a significant advantage in a clinical setting based on extended processing times (up to 7 days at RT) [18, 119, 122] after blood collection in comparison to EDTA tubes which require immediate processing (within 2 h) [41]. Therefore, sRNA tubes were used for downstream psEV isolation and analysis and appropriate measures were employed to limit platelet activation and thus platelet‐derived EV presence in psEV analyses [58, 59].…”

Section: Resultsmentioning

confidence: 99%

“…All samples were acquired through collection of whole blood in ethylenediaminetetraacetic acid (EDTA) or Cell-Free RNA BCT ® -STRECK (sRNA) tubes. After blood collection, tubes were immediately inverted four to five times, transported vertically at room temperature (RT) without agitation and processed within 24 h. To obtain platelet-free plasma (PFP), PBPL was centrifuged at 1800 x g for 10 min, followed by 2000 x g, 15 min (4 • C) [58,59]. PFP was aliquoted (1 mL) and used immediately for isolation of EVs or stored at −80 • C.…”

Section: Human Blood Collection and Plasma Preparationmentioning

confidence: 99%

“…Therefore, sRNA tubes were used for downstream psEV isolation and analysis and appropriate measures were employed to limit platelet activation and thus platelet-derived EV presence in psEV analyses [58,59].…”

Section: Evaluation Of Plasma Small Evs: Srna Versus Edta Tube Typesmentioning

confidence: 99%

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Human myeloma cell‐ and plasma‐derived extracellular vesicles contribute to functional regulation of stromal cells

Reale

Carmichael

et al. 2021

Proteomics

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Circulating small extracellular vesicles (sEV) represent promising non-invasive biomarkers that may aid in the diagnosis and risk-stratification of multiple myeloma (MM), an incurable blood cancer. Here, we comprehensively isolated and characterized sEV from human MM cell lines (HMCL) and patient-derived plasma (psEV) by specific EV-marker enrichment and morphology. Importantly, we demonstrate that HMCL-sEV are readily internalised by stromal cells to functionally modulate proliferation. psEV were isolated using various commercial approaches and pre-analytical conditions (collection tube types, storage conditions) assessed for sEV yield and marker enrichment. Functionally, MM-psEV was shown to regulate stromal cell proliferation and migration. In turn, pre-educated stromal cells favour HMCL adhesion. psEV isolated from patients with both pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance [MGUS]; smouldering MM [SMM]) and MM have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre-malignant sEV in disease progression. Proteomic profiling of MM-psEV (305 proteins) revealed enrichment of oncogenic factors implicated in cell migration and adhesion, in comparison to non-disease psEV. This study describes a protocol to generate

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Section: Resultsmentioning

confidence: 99%

Section: Human Blood Collection and Plasma Preparationmentioning

confidence: 99%

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Human myeloma cell‐ and plasma‐derived extracellular vesicles contribute to functional regulation of stromal cells

Reale

Carmichael

et al. 2021

Proteomics

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“…The selection of a particular proteomic approach depends on the research objective. Complete identification of exosomal protein cargo can be achieved with a "global discovery" approach using multidimensional fractionation strategies followed by high-resolution mass spectrometric (MS) [326], which could include a quantitative cargo analysis if desired. This quantification can be performed with the Stable Isotope Labeling by Amino acids in Cell culture (SILAC) or Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) methods, involving the incorporation of a stable heavy isotope-labeled amino acid into the peptides of interests [327,328].…”

Section: Methods For the Study Of Exosomal Contentmentioning

confidence: 99%

Exosomes: Potential Disease Biomarkers and New Therapeutic Targets

et al. 2021

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Exosomes are extracellular vesicles released by cells, both constitutively and after cell activation, and are present in different types of biological fluid. Exosomes are involved in the pathogenesis of diseases, such as cancer, neurodegenerative diseases, pregnancy disorders and cardiovascular diseases, and have emerged as potential non-invasive biomarkers for the detection, prognosis and therapeutics of a myriad of diseases. In this review, we describe recent advances related to the regulatory mechanisms of exosome biogenesis, release and molecular composition, as well as their role in health and disease, and their potential use as disease biomarkers and therapeutic targets. In addition, the advantages and disadvantages of their main isolation methods, characterization and cargo analysis, as well as the experimental methods used for exosome-mediated drug delivery, are discussed. Finally, we present potential perspectives for the use of exosomes in future clinical practice.

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“…Recently, analyses of body fluids including blood (i.e. liquid biopsies) have generated significant interest due to their potential for the characterisation of tumours with a rapid and non-invasive procedure (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). In the context of MM, accumulating evidence suggest that liquid biopsy captures the genetic heterogeneity of the disease with important implications for circulating tumour DNA (ctDNA) and cell-free or extracellular RNA (exRNA) as valuable markers for tumour genome characterisation, prognostication and sequential monitoring of disease (6)(7)(8)(9)(10)(11)(12)(13).…”

Section: Introductionmentioning

confidence: 99%

Translational Potential of RNA Derived From Extracellular Vesicles in Multiple Myeloma

et al. 2021

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The cross-talk between tumour cells and stromal cells is a hallmark of multiple myeloma (MM), a blood cancer that still remains incurable despite increased knowledge of its biology and advances in its treatment. Extracellular vesicles (EVs) derived from both tumour and stromal cells have been shown to play an important role in mediating this cross-talk ultimately favouring MM progression and drug resistance. Furthermore, EVs and their content including RNA (EV-RNA) have been successfully isolated from blood and are being explored as liquid biomarkers in MM with the potential to improve diagnosis and monitoring modalities with a minimally-invasive and repeatable analysis, i.e. liquid biopsy. In this review, we describe both the role of EV-RNA in defining the biological features of MM and their potential translational relevance as liquid biomarkers, therapeutic targets and delivery systems. We also discuss the limitations and technical challenges related to the isolation and characterization of EVs and provide a perspective on the future of MM-derived EV-RNA in translational research.

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Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma

Cited by 9 publications

References 140 publications

Human myeloma cell‐ and plasma‐derived extracellular vesicles contribute to functional regulation of stromal cells

Human myeloma cell‐ and plasma‐derived extracellular vesicles contribute to functional regulation of stromal cells

Exosomes: Potential Disease Biomarkers and New Therapeutic Targets

Translational Potential of RNA Derived From Extracellular Vesicles in Multiple Myeloma

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