Oral Presentation Australasian Extracellular Vesicles Conference 2020

Challenges in exosomes isolation from primary biological samples (#4)

Antonia Dr Reale 1 , Tiffany Khong 1 , Rong Xu 2 , Maoshan Chen 1 , Richard Prof Simpson 2 , Ioanna Dr Savvidou 1 , Malarmathy Ramachandran 1 , David Dr Greening 2 3 , Andrew Prof Spencer 1 4
  1. Monash University, Melbourne, VIC, Australia
  2. La Trobe University, Melbourne, VIC, Australia
  3. Baker Institute, Melbourne, VIC, Australia
  4. Alfred Hospital, Melbourne, VIC, Australia

Aim: Multiple myeloma (MM) remains incurable despite advances in its treatment. The crosstalk between myeloma cells and stromal cells within the bone marrow has long been known to protect the survival of malignant cells. Exosomes are important regulators of the cellular niche, and their importance for diagnostic and therapeutic applications has been proven in many diseases including cancer. In this context we hypothesized that a better understanding of the molecular role and features of myeloma-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-MM drugs already in use in clinical settings, given the optimisation and validity of their isolation/purification method.
Methods: Exosomes were isolated from human myeloma cell lines (HMCLs) supernatants and peripheral blood plasma (PBPL) isolated from healthy individuals, myeloma and MGUS (monoclonal gammopathy of undetermined significance) patients. Both fresh and frozen samples were tested. We evaluated 3 commercially available kits (Exoquick ULTRA, Exoeasy, Norgen exosomes purification kit), OptiPrep™ density-based separation and the conventional ultracentrifugation.
Results: The quality and quantity of exosomes for all methods were confirmed by western blotting, nanoparticle tracking analysis and electron microscopy. Enriched exosomes were found to increase the proliferation of the immortalized stromal cell line HS5 when compared to untreated cells. A higher increase in proliferation of HS5 treated with MM-exosomes when compared to exosomes derived from normal and MGUS PBPL samples was also observed.
Conclusion: The yield and purity of exosomes are critical for the evaluation of exosomes in MM. Our data demonstrated that OptiPrep™ density-based and resin-based isolation methods provided functional myeloma-derived exosomes with proliferative effects on stromal cells. In summary, our findings may serve as a guide to choose exosome isolation/purification methods for myeloma studies. Further optimisation steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications.


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