Neutrophil Extracellular Traps in Breast Cancer and Beyond: Current Perspectives on NET Stimuli, Thrombosis and Metastasis, and Clinical Utility for Diagnosis and Treatment

Abstract The formation of neutrophil extracellular traps (NETs), known as NETosis, was first observed as a novel immune response to bacterial infection, but has since been found to occur abnormally in a variety of other inflammatory disease states including cancer. Breast cancer is the most commonly diagnosed malignancy in women. In breast cancer, NETosis has been linked to increased disease progression, metastasis, and complications such as venous thromboembolism. NET-targeted therapies have shown success in preclinical cancer models and may prove valuable clinical targets in slowing or halting tumor progression in breast cancer patients. We will briefly outline the mechanisms by which NETs may form in the tumor microenvironment and circulation, including the crosstalk between neutrophils, tumor cells, endothelial cells, and platelets as well as the role of cancer-associated extracellular vesicles in modulating neutrophil behavior and NET extrusion. The prognostic implications of cancer-associated NETosis will be explored in addition to development of novel therapeutics aimed at targeting NET interactions to improve outcomes in patients with breast cancer

Impacts of diverse inflammatory stimuli on neutrophil behavior: extracellular vesicles, e-cigarettes, and nanoparticles

Neutrophils are the body’s front-line defenders against foreign insult and are key players in a variety of inflammatory conditions. This body of work examines the role of neutrophils in promoting pathology in three distinct inflammatory contexts. In the pro-inflammatory state provoked by breast cancer, neutrophils decondense their nuclei and release cytotoxic web-like structures known as neutrophil extracellular traps (NETs). NETs form most commonly via histone modifications facilitated by the enzyme PAD4. NETs are known to be a harbinger of disease progression and promote metastasis through capture of circulating tumor cells. It was hypothesized that breast tumors release small particles known as extracellular vesicles (EVs) into the circulation which interact with peripheral neutrophils to cause NET release. The first group of studies herein examined for the first time whether tumor-released EVs promote NET release in a PAD4- dependent manner, as well as whether PAD4 expression by the parent cell influences the capacity of EVs to promote NET formation. Altered neutrophil behavior is not unique to the inflammatory state of cancer. The second set of studies investigated the role of neutrophils in response to e-cigarette vapor (EC) exposure. Existing literature indicates that EC exposure dysregulates neutrophil function, promoting inflammation while compromising the ability to fight off infections. Neutrophil-platelet aggregates potentiate NET formation and can occlude small blood vessels, causing further inflammation and injury. Currently, no literature reports on the link between EC exposure and neutrophil interactions with platelets to form cellular aggregates, much less how the adhesion molecule P-selectin is involved in this process. It was observed that EC exposure potentiated neutrophil recruitment, neutrophil-platelet aggregates and vascular blockages in the pulmonary microcirculation following EC exposure and that these interactions were significantly abrogated through P-selectin blockade. The final set of studies explored NET formation at the intersection of toxicology and cancer. Often, neutrophils are ignored while designing novel nanoparticle (NP) contrast agents for cancer detection via magnetic resonance imaging (MRI). These studies investigated how NP metal oxide cargo and NP polymeric surface modification led to differential NET release, oxidative stress, and neutrophil cytokine release. Therefore, MRI contrast agents and more broadly, any NP intended for use in the circulation, should be designed with neutrophils in mind. These studies collectively provide a framework which illustrates the ubiquity of neutrophils in inflammation and offers novel insights into potential strategies for mitigating neutrophil-mediated pathology

PEGylation of Metal Oxide Nanoparticles Modulates Neutrophil Extracellular Trap Formation

Novel metal oxide nanoparticle (NP) contrast agents may offer safety and functionality advantages over conventional gadolinium-based contrast agents (GBCAs) for cancer diagnosis by magnetic resonance imaging. However, little is known about the behavior of metal oxide NPs, or of their effect, upon coming into contact with the innate immune system. As neutrophils are the body’s first line of defense, we sought to understand how manganese oxide and iron oxide NPs impact leukocyte functionality. Specifically, we evaluated whether contrast agents caused neutrophils to release web-like fibers of DNA known as neutrophil extracellular traps (NETs), which are known to enhance metastasis and thrombosis in cancer patients. Murine neutrophils were treated with GBCA, bare manganese oxide or iron oxide NPs, or poly(lactic-co-glycolic acid) (PLGA)-coated metal oxide NPs with different incorporated levels of poly(ethylene glycol) (PEG). Manganese oxide NPs elicited the highest NETosis rates and had enhanced neutrophil uptake properties compared to iron oxide NPs. Interestingly, NPs with low levels of PEGylation produced more NETs than those with higher PEGylation. Despite generating a low rate of NETosis, GBCA altered neutrophil cytokine expression more than NP treatments. This study is the first to investigate whether manganese oxide NPs and GBCAs modulate NETosis and reveals that contrast agents may have unintended off-target effects which warrant further investigation