(533b) Neutrophil Extracellular Traps and Extracellular Vesicles: Mediators of Metastasis in Breast Cancer?

One in eight women will be diagnosed with breast cancer at some point in their lives. In the United States, the incidence of breast cancer approached 268,600 women in 2019 with almost 42,000 associated deaths. Approximately 90% of breast cancer mortality is caused from metastases to other vital organs including lungs, liver, brain and bone marrow. Once breast cancer has spread, the 5 year survival rate is only 25%. Thus, understanding the mechanisms by which metastasis occurs is critically important to improving patient outcome. Metastasis has been noted to be facilitated by processes involving the interaction of neutrophils, neutrophil extracellular traps (NETs), and extracellular vesicles (EVs). Indeed, higher concentrations of EVs in breast cancer patient serum as well as increased NET markers are both correlated with poor outcome and increased mortality. NETs (which form via a process called NETosis) occur when neutrophils unravel and extrude their nuclei into webs of DNA that are decorated with granular proteins; NETs have antimicrobial and proinflammatory properties. In the context of cancer, NETs have been shown to aid in arresting circulating tumor cells to promote metastasis, reactivating dormant metastatic tumor cells, and supporting the hypercoagulable state leading to other cancer-associated complications such as thromboembolism. EVs form when submicron membrane-bound particles are released from cells, and function as a mode of intercellular communication; however, their role in promoting NETosis and other metastasis-supporting phenomena remains unclear. In this study, 4T1 murine mammary carcinoma cells were orthotopically injected into n=32 mice, with IVIS performed weekly for 4 weeks to monitor tumor growth. Possible metastases were observed via IVIS in week 2, with gross metastases apparent upon lung collection after 3- and 4-weeks post-tumor injection. Plasma, lung, liver, and primary tumor were collected from groups on a weekly basis, with EVs subsequently isolated from plasma. EVs from each tumor group will be used to stimulate isolated neutrophils from healthy mice in vitro. The potential of tumor plasma-derived EVs to induce NETosis will be compared to 4T1 EVs derived from conditioned cell culture medium. Confocal microscopy will evaluate if EVs are fully internalized by neutrophils or bound to their surface. Electron microscopy and NanoSight Tracking Analysis (NTA) will be utilized to validate morphological differences in EV populations. Finally, western blots will be performed to validate the presence of EV-associated markers (CD9 and ALIX), tumor-specific EVs (EpCAM), and tissue factor, which can lead to hypercoagulability and NETosis downstream of platelet activation. We hypothesize that EVs from timepoints correlating with the onset of common metastases to lung and liver (week 2 and 3) will display enhanced potential to cause NETosis compared to EVs from timepoints closer to primary tumor establishment (week 1) or EVs in mice with later-stage metastases (week 4 and beyond).