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

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Proteomic Profiling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2261))

Abstract

Cancer cells secrete membranous extracellular vesicles (EVs) which contain specific oncogenic molecular cargo (including oncoproteins, oncopeptides, and RNA) into their microenvironment and the circulation. As such, EVs including exosomes (small EVs) and microvesicles (large EVs) represent important circulating biomarkers for various diseases, including cancer and its progression. These circulating biomarkers offer a potentially minimally invasive and repeatable targets for analysis (liquid biopsy) that could aid in the diagnosis, risk stratification, and monitoring of cancer. Although their potential as cancer biomarkers has been promising, the identification and quantification of EVs in clinical samples remain challenging. Like EVs, other types of circulating biomarkers (including cell-free nucleic acids, cf-NAs; or circulating tumor cells, CTCs) may represent a complementary or alternative approach to cancer diagnosis. In the context of multiple myeloma (MM), a systemic cancer type that causes cancer cells to accumulate in the bone marrow, the specific role for EVs as biomarkers for diagnosis and monitoring remains undefined. Tumor heterogeneity along with the various subtypes of MM (such as non-secretory MM) that cannot be monitored using conventional testing (e.g. sequential serological testing and bone marrow biopsies) render liquid biopsy and circulating tumor-derived EVs a promising approach. In this protocol, we describe the isolation and purification of EVs from peripheral blood plasma (PBPL) collected from healthy donors and patients with MM for a biomarker discovery strategy. Our results demonstrate detection of circulating EVs from as little as 1 mL of MM patients’ PBPL. High-resolution mass spectrometry (MS)-based proteomics promises to provide new avenues in identifying novel markers for detection, monitoring, and therapeutic intervention of disease. We describe biophysical characterization and quantitative proteomic profiling of disease-specific circulating EVs which may provide important implications for the development of cancer diagnostics in MM.

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Acknowledgments

This work was supported by Monash University, Melbourne, Australia—Australian Government Training Program (RTP) scholarship and Monash Departmental Scholarship (AR). This work was also funded by NHMRC project grants (#1057741 and #1139489; DWG), Helen Amelia Hains Fellowship (DWG). The authors acknowledge Prof. Richard Simpson, Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia for access to equipment and expertise employed in this study. We also acknowledge AMREP Flow Cytometry Core Facility (85 Commercial Road, Melbourne, VIC, Australia), Malarmathy Ramachandran and Kawa Choi (Myeloma Research Group, Monash University/The Alfred Hospital, Melbourne, VIC, Australia), Haoyun Alexandra Fang, Molecular Proteomics (Baker Heart and Diabetes Institute, Melbourne, VIC, Australia), Dr. Julian Ratcliffe and Dr. Peter Lock (LIMS BioImaging Facility, La Trobe University, Melbourne, VIC, Australia) for technical assistance.

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Authors and Affiliations

  1. Myeloma Research Group, Australian Centre for Blood Diseases, Monash University/The Alfred Hospital, Melbourne, VIC, Australia

    Antonia Reale, Tiffany Khong, Maoshan Chen, Sridurga Mithraprabhu, Nicholas Bingham & Andrew Spencer

  2. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia

    Rong Xu & David W. Greening

  3. Malignant Haematology and Stem Cell Transplantation, The Alfred Hospital, Melbourne, VIC, Australia

    Andrew Spencer

  4. Department of Clinical Haematology, Monash University, Melbourne, VIC, Australia

    Andrew Spencer

  5. Molecular Proteomics Group, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia

    David W. Greening

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  1. Bio-Rad Laboratories GmbH, Feldkirchen, Germany

    Anton Posch

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Reale, A. et al. (2021). Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy in Multiple Myeloma. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 2261. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1186-9_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1186-9_10

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