(483e) Unraveling the Crosstalk between Neutrophils, Platelets, and Extracellular Vesicles in Breast-to-Lung Metastasis

Background & Rationale: Breast cancer (BC) is the most diagnosed cancer amongst women, and metastasis drives the vast majority of deaths in these patients. The lung is the most common metastatic site in highly aggressive basal-like triple negative BC and is also extremely common in all other subtypes; therefore, understanding how the pulmonary microenvironment fosters metastasis is of utmost importance. There is mounting evidence that crosstalk between neutrophils, platelets, neutrophil extracellular traps (NETs), and BC-associated extracellular vesicles (EVs) plays a role in the formation of the pre-metastatic niche; however, the exact cellular and molecular mechanisms mediating these interactions are not well understood.

NETs form when neutrophils extrude their nuclei as web-like structures decorated with proteolytic and pro-inflammatory granular proteins. In the context of cancer, NETs act as a scaffold allowing circulating tumor cells to escape the shear stresses of the vasculature and extravasate. Additionally, NETs enhance thrombosis, which itself may facilitate metastasis. Increased platelet activation may also lead to enhanced NET formation as a positive feedback loop forms in which NETs activate platelets which then activate neutrophils that form even more NETs. One recent study found tumor EVs to be able to generate NET release; however, it is limited by examining NETosis caused solely by interactions between neutrophils alone and cell-culture derived EVs.

We hypothesize that platelets will be driven towards a prothrombotic state in the presence of EVs isolated from mice with breast tumors, and that platelets and neutrophils will act synergistically to generate higher rates of NET formation through interaction with tumor EVs. Our preliminary data indicates that these BC-associated EVs not only modulate NET formation, but do so in a tumor progression-specific manner. BC-associated EVs isolated from tumorous mice with early metastases provoke the most NET formation, suggesting a correlation between the advent of metastasis and higher rates of NETosis. To investigate whether BC-associated EVs also modulate platelet phenotype, and if they can also indirectly modulate neutrophil behavior via platelet-dependent mechanisms, we will coculture isolated neutrophils and platelet rich plasma with or without BC-associated EVs.

Methods: In this study, EVs will be isolated via sequential centrifugation from the plasma of mice bearing orthotopic 4T1 murine mammary carcinomas 1, 2, 3, or 4 weeks post-tumor establishment. Platelets and neutrophils will be isolated from healthy mice and stimulated with BC-associated EVs ex vivo. Plasma EVs isolated from healthy mice will serve as controls. The potential of BC-associated EVs to induce NETosis and/or platelet activation will be compared to 4T1 EVs derived from conditioned cell culture medium. The stimulatory effects of BC-associated EVs on platelets and neutrophils will be compared to our preliminary data in which neutrophils alone were stimulated with BC-associated EVs (Fig. 1). Media alone or classical NET stimuli will serve as negative and positive controls, respectively. Confocal fluorescent microscopy will evaluate overall levels of NETosis and platelet/neutrophil association. Observed NETosis will be confirmed via ELISA assay detecting citrullinated histone 3 (a highly specific marker of NETosis) or via assays detecting DNA. Possible coagulation pathways by which EVs promote platelet activation will be revealed via platelet aggregometry. Finally, EV cargo and size characteristics will be determined using mass spectrometry and nanotracking analysis, respectively.

Results & Conclusions: Our preliminary results indicate that EVs isolated from week 3 of tumor progression enhance NET formation to a degree that is not statistically different than platelet activating factor (postive control) and is significantly higher than the rate of NETosis in unstimulated neutrophils (media alone) or healthy EV-stimulated neutrophils. All other groups had higher rates of NET formation than unstimulated neutrophils or those stimulated with healthy EVs; however, this was not significant. We expect to see similar trends when coculturing platelets and neutrophils ex vivo; however, significant differences may occur if platelets further enhance NETosis in the presence of BC-associated EVs in general. Unraveling the means by which tumor-associated EVs modulate disease progression through NET formation will be of great benefit to both breast cancer patients and cancer patients in general by fostering the development of new treatments targeting the means by which tumors communicate with the rest of the body.