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Page 30 Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10

hypothesize that naturally secreted exosome (SEC-exosomes) vs. chelation-induced exosome (CI-exosomes)
treatment will lead to differential biophysical and molecular cellular responses to mediate more invasive
tumor microenvironment (TME).
Methods: OVCAR-3 EOC cells were cultured in exosome-free media for 48 hours. SEC-exosomes were
collected from media and harvested using standard ultracentrifugation methods. OVCAR-3 were washed
and treated with EDTA to harvest CI-exosomes. CI-exosomes were isolated using ultracentrifugation.
Exosome diameters and ubiquitous surface protein markers for CI- and SEC- exosomes were validated
using dynamic light scattering and immunoblots, respectively. Patient-derived EOC fibroblasts were treated
with either exosome population. High-throughput physical and molecular assays - single-cell migration,
immunocytochemistry, adhesion assays, and miRNA microarrays - were used to examine unique differences
between populations and their interactions with fibroblasts.
Results: Microarray data showed unique miRNA profiles in SEC- vs. CI- exosomes, suggesting heterogeneity in
cell-secreted exosomes. Specifically, there were 1,019 differentially expressed miRNAs between either exosome
population. Highly regulated miRNAs were linked to mechanosensitive pathways that impact cell motility and
cytoskeletal organization. Both populations altered actin fiber and focal adhesion protein organization to affect
fibroblast morphologies. Exosome populations further increased random and directional fibroblast migration;
however, fibroblast adhesion strength was only increased with CI-exosome treatment. For co-culture OVCAR-3
and fibroblast studies, CI-exosomes promoted adhesion and spreading of OVCAR-3 cells, while SEC-exosomes
led to dramatic elongation in a small percentage of OVCAR-3 cells.
Discussion/Conclusions: We showed that decreased extracellular calcium levels, which mirror various
gynecological conditions, led to the release of a unique exosome population (CI-exosomes) from a single
cancer cell line. This CI-exosome population contained unique miRNA content and led to different molecular/
physical fibroblast phenotypes compared to SEC-exosomes. This highlights that tumor cells can secrete
multiple exosome populations to mediate cancer progression.

12. Orchestration of human macrophage NLRP3 inflammasome activation by Staphylococcus
aureus extracellular vesicles

Authors: Jean C. Lee, Xiaogang Wang
E-mail: jclee@bwh.harvard.edu
Affiliations:
Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital and Harvard
Medical School, Boston, MA, USA.

Abstracts:
Staphylococcus aureus (S. aureus) is an opportunistic pathogen that causes a variety of diseases, including
bacteremia, endocarditis, skin and soft tissue infections, and food poisoning. The bacterium produces
a myriad of virulence factors, including toxins, enzymes, surface proteins, and multiple glycopolymers,
many of which are components of staphylococcal extracellular vesicles (EVs). We purified EVs from a
community-associated methicillin-resistant S. aureus strain and showed that EV-associated pore-forming
toxins, particularly alpha hemolysin and members of the leukocidin family, lysed a variety of cell types.
Staphylococcal alpha-type phenol-soluble modulins promoted EV biogenesis by disrupting the cytoplasmic
membrane, whereas peptidoglycan crosslinking and autolysin activity modulated EV production by altering
the permeability of the cell wall. Purified S. aureus EVs were internalized into human macrophages in vitro,
and this process was blocked by inhibition of the dynamin-dependent endocytic pathway. EVs triggered
NLRP3 inflammasome activation, resulting in the cellular release of IL-1b and IL-18 and induction of