Plasma Redox Imbalance Caused by Albumin Oxidation Promotes Lung-Predominant NETosis and Metastasis in Patients Treated with Definitive Radiation Therapy

Abstract

Neutrophils are associated with cancer progression and poor prognosis, but the mechanism is poorly understood. In preclinical models, neutrophil extracellular traps (NETs) entrap circulating tumor cells (CTCs) and promote the initiation of metastasis. In these models, NETosis is triggered by exogenous massive inflammatory stimuli, and thus it remains unknown whether cancer hosts under physiologic inflammation-free conditions experience NETosis and consequent cancer metastasis. The objective of this study was to uncover endogenous inflammatory-independent regulators of NETs and cancer metastasis. We hypothesized that a reservoir of free thiol in plasma provided by non-oxidized albumin counteracts the formation of NETs and decreases cancer metastasis. Human neutrophils were cultured with variable albumin and free thiol content. NETs were detected by immunocytochemistry and immunoblotting. Albumin thiols were depleted or chemically oxidized. NET deposition within organs of Albwt/wt and Alb-/- mice was detected by two-photon microscopy. NETs were blocked or degraded by PAD inhibition or DNase I, respectively. To model lung metastasis, Cal33 human head and neck squamous cell carcinoma (HNSCC) cells were injected via tail vein or 4T1 murine breast carcinoma cells were injected into mammary fat pads. Free thiol levels, albumin concentration/oxidation, NET marker proteins, and inflammatory cytokines were measured in plasma from HNSCC patients treated with definitive radiotherapy (RT). Albumin oxidation led to accumulation of intracellular reactive oxygen species within cultured neutrophils, causing NETosis. In mouse models, loss of albumin thiols resulted in enhanced NETosis. NETs were deposited specifically within pulmonary capillary beds where they contributed to the colonization of CTCs and initiation of lung metastases. These effects were abrogated by pharmacologic inhibition of NET formation. Within a cohort of 22 HNSCC patients, 8 developed subsequent lung metastasis. Decreased levels of plasma free thiol (hazard ratio [HR]=5.3, p=0.023) and non-oxidized albumin (HR=9.8, p=0.009) during RT were associated with elevated risk of lung metastasis. Elevated levels of NET markers were observed in plasma from patients with low levels of non-oxidized albumin (p = 0.008), whereas no difference was seen in the plasma concentration of inflammatory cytokines. Plasma albumin thiol content is an endogenous, physiologic, and inflammation-independent regulator of NETosis and pulmonary cancer metastasis. Patients undergoing definitive RT may develop depleted reservoirs of free thiol in plasma that could promote lung metastasis. Strategies that target albumin thiols and NETs could provide new therapeutic and diagnostic opportunities in combating cancer metastasis.