Ovarian cancer (OC) is the most lethal gynecologic malignancy (1). The overall survival rate at 5-years follow-up is less than 50% and decreases to 5-21% within 10 years (2). This high mortality can be partly explained by the presence of metastases at the time of diagnosis, its high recurrence rate, and the acquisition of chemoresistance by the tumor (3).

High-grade serous ovarian cancer (HGSOC) is the most frequent and aggressive subtype of OC. It accounts for 70-80% of OC-related deaths, mainly due to its predominant diagnosis in advanced stages, when diffuse widespread peritoneal metastases are already present (1). Although OC metastases may occur via systemic or lymphatic routes, most HGSOC tumors spread across the peritoneal cavity following peritoneal fluid (PF) dynamics (4). Malignant accumulation of PF or ascites has been described in >90% HGSOC patients, contributing to chemoresistance, metastasis and poor prognosis (5). Since PF is considered a crucial component of the HGSOC tumor environment (6), an exhaustive characterization of its cellular and molecular components might open new avenues to improve patients’ management.

The gold standard therapy for advanced OC has remained mostly unchanged for decades, consisting of a primary debulking surgery followed by first-line chemotherapy based on platinum and taxanes (7). For patients who are medically unfit to undergo first line surgery or in the presence of unresectable disease, neoadjuvant chemotherapy followed by interval debulking surgery can be considered as an alternative approach (8). Nevertheless, despite advances in the field of surgery and chemotherapy, almost half of patients will develop disease recurrence within 18 months and most of them will die from the disease within 5 years (9). The fact that the 5-year overall survival of advanced OC is 29%, whereas the 5-year OS in early stages is 92% (10) underpins the need for a deeper understanding of the mechanism of disease progression and the evaluation of new therapeutic alternatives. In this context, intraperitoneal chemotherapy, either normo- or hyperthermic, is being considered to improve first line treatment outcomes (11–14).

Novel discoveries in the mechanisms of metastasis and disease progression in cancer have focused on neutrophils and their associated functions (15–17). Neutrophils are the most abundant circulating leukocytes, playing a predominant role in defense mechanisms through phagocytosis and degranulation (18). However, in response to different stimulus (19), neutrophils can release neutrophil extracellular traps (NETs), composed of DNA, histones and cytoplasmic and granular proteins such as calprotectin, myeloperoxidase (MPO) and elastase, in a process called NETosis (20). Since the decondensation of chromatin due to histone 3 citrullination has been described as a key step prior to the formation and release of NETs (21), citrullinated histone 3 (citH3) has been proposed as a specific marker of these structures (22–24). In cancer, NETs may contribute to tumor progression and metastasis (25). Thus, pharmacologically interfering in NET formation or destruction has been recently proposed as a promising therapeutic approach in oncology (26).

Regarding HSOC, recent evidence has proposed NETosis as one of the responsible mechanisms for initial establishment of omental metastases. Lee et al. (27) demonstrated in a murine model that early-stage OC cells can release several factors to recruit neutrophils into the omentum and induce NETosis. Subsequently, disseminated cells through the PF would get trapped into the formed NETs to conform to metastatic implants. However, to the best of our knowledge, the role of NETosis in advanced HGSOC has not been evaluated yet. Hence, our primary aim was to characterize a profile of NETosis biomarkers in biofluids (namely plasma and PF) from patients with advanced HGSOC and control women. Secondarily, we wished to assess its potential diagnostic value and to analyze the possible effect of neoadjuvant treatment (NT) on their levels.

HGSOC causes 70-80% of gynecological cancer-related deaths, mainly due to its asymptomatic nature, its diagnosis in advanced stages, the resistance to chemotherapy and the intraperitoneal recurrences (1). Difficulty in the management of affected women (38) highlights the need to better define the biological mechanisms that convert a localized potentially curable disease onto a disseminated fatal disease. In this sense, the contribution of NETosis in cancer is emerging as a hot topic in cancer research (26, 39, 40), although studies in OC in general and in advanced HGSOC, specifically, are scarce. In order to broaden the current knowledge in this field, our principal aim was to characterize a profile of NETosis biomarkers in plasma and PF from patients with advanced HGSOC and control women. Our results show that HGSOC patients have a higher concentration of cfDNA, calprotectin and citH3 in plasma and an increase in the 5 NETosis biomarkers scrutinized (i.e., cfDNA; nucleosomes, citH3, calprotectin and MPO) in PF compared to control women, which would suggest a possible contribution of NETosis in advanced HGSOC. Additionally, our results reveal plasma cfDNA and/or calprotectin as potential minimally invasive surrogate biomarkers for advanced HGSOC. Interestingly, we described that, after neoadjuvant treatment, a significant reduction of NETosis biomarkers occurs mainly systemically but not in the environment where the tumor develops, questioning the efficacy of systemic chemotherapy in the peritoneum, paving the way for alternative therapeutic approaches.

OC predominates in postmenopausal women over 50 years, in which PF accumulation or ascites forms mostly due to local inflammation (41). However, under begin conditions, this biofluid is generated as a mixture of plasma transudate and exudate from ovarian surface tissues under ovarian hormonal influence, predominantly in premenopausal stages (42, 43). Thus, the hormonal and/or inflammatory dependence on PF production has precluded us the recruitment of a control post-menopausal women cohort, resulting in differences in age and menopausal status between our study groups. High counts of blood neutrophils have been documented, as in our case, in OC patients (44), suggesting the key role that this cell type may play in the progression and prognosis of this disease (45–47). Nevertheless, the putative influence of age, menopausal status and neutrophil count as covariates has been excluded in our study using appropriate statistical treatments.

To the best of our knowledge, this is the first study to evaluate the differences in biomarkers of NETosis in biofluids of advanced HGSOC compared to control women. We observed a higher concentration of cfDNA, citH3 and calprotectin in plasma of HGSOC patients and an increase in the levels of the 5 NETosis biomarkers in their PF. Moreover, the subsequent analyses performed to assess the influence of the covariates age, menopausal status and neutrophil count allowed us to confirm that, with the only exception of citH3 in plasma, the differences in the levels of the remaining biomarkers were mainly attributable to the clinical group. As described, the greatest differences mainly occurred in the tumor environment (PF) and not at the systemic level (plasma), which could evidence the contribution of NETosis in the development of HGSOC in the pelvic-abdominal microenvironment.

In a recent work, Lee and coworkers (27) proposed that, under the influence of primary tumor, NETs released by neutrophils on the omentum’s surface serve as a trap for tumor cells migrated into the PF, crucially contributing to the metastasic process in early-stages of HGSOC. Accordingly, we have identified that NETs are present in advanced HGSOC patients’ PF, a biofluid considered a key element of the tumor environment, whose composition crucially conditions the development and progression of the disease (6). Therefore, our findings could suggest that the contribution of NETosis is maintained in advanced stages, converting omental neutrophils and NETs structures into an outstanding therapeutic target. In this regard, the lack of significant direct correlation in our results between NETosis levels and tumor burden (based on tumor size and PCI) might reflect that NETs would be essential for the initial establishment of metastasis at sites free of tumors. However, once the tumor cells have established a metastatic niche (potentially with an optimal blood supply, extracellular matrix invasion, etc.) the function of NETs would not be necessary to maintain the metastatic niche progression. Future studies in patients’ serial samples may shed light on the kinetics of NETs formation and its role in HGSOC progression. Although current literature describes NETs formation as a brief process that occurs in a short time interval (35, 48), analyses in patients’ serial samples might clarify whether NETosis activation in advanced HGSOC patients is a gradually increasing process or, on the contrary, presents an “all/nothing activation”. Besides, it has to be taken into account that PCI assessment depends on the visual acuity of the surgeon, as it evaluates the macroscopic disease, which could underestimate those initial metastatic niches areas with microscopic disease. Furthermore, the quantification of NETosis markers in tumor and omental samples from advanced HGSOC patients might favor a broader understanding of the contribution of NETosis in advanced HGSOC progression.

A challenge in NETosis research is represented by the selection of the best biomarker, since no standard marker or direct and simple method has been validated for the detection of NETs (49) and differences exist in terms of specificity, objectivity or quantification. Although in our work we have quantified the levels of citH3, which has been proposed as a specific marker of this process structures (22–24), the remaining molecules measured herein as markers of NETosis (cfDNA, nucleosomes, calprotectin and MPO) could have a cellular origin other than neutrophils. Specifically, calprotectin and MPO are also expressed, to a lesser extent, in monocytes, macrophages or eosinophils (50, 51); and cfDNA and nucleosomes can be released by apoptotic or necrotic cells present in cancer patients (52). In our hands, the levels of all 5 analyzed biomolecules correlated pairwise, mainly in PF’ HGSOC patients, reinforcing a potential common origin and their suitability as NETosis biomarkers. The different behavior noted for the molecules studied in both biofluids could suggest that the increased levels observed in HGSOC’ PF compared to control women might be attributed to a greater activation of NETosis process in the tumor environment. On the contrary, the differences observed in plasma could be attributable either to NETosis or other processes in which these molecules are involved.

Subsequently, we assessed the potential of the molecules studied as biomarkers of advanced HGSOC. ROC curve analyses showed a high accuracy for the 5 NETosis biomarkers in PF (AUC×0.87) and cfDNA, citH3 and calprotectin in plasma (AUC×0.67). Interestingly, the positive correlation of plasma cfDNA and calprotectin and the levels of the 5 NETosis biomarkers in PF, suggest the quantification of these makers in plasma as a minimal invasive determination potentially informative of the increased NETosis in the tumor environment.

A number of studies have proposed plasma cfDNA as a novel marker of OC (53). On the other hand, most of the studies focused on the potential role of calprotectin as biomarker are based on its fecal determination (54), without promising results in cancer in general (55, 56) and in OC in particular; and with limited studies in plasma calprotectin levels in cancer (57). Remarkably, Odegaard et al. (58) described an increase in circulating calprotectin in patients with OC, suggesting, in line with us, its possible use as a clinical tool, although further studies are required.

In line with our results, decreased DNaseI activity has been associated with increased levels of NETs in some disorders such as lupus erythematosus (59) and acute thrombotic microangiopathy (60). However, this is not responsible for the increase in NETosis markers in our HGSOC patients as similar DNaseI activity levels were detected than controls both in plasma and PF.

Regarding HGSOC therapeutic management, our results showed that biomarkers of NETosis decrease as a consequence of neoadjuvant chemotherapy, tending to those of the control group, predominantly in plasma but not in PF. This observation might imply that, at least for the NETosis process, the intravenous administration of neoadjuvant chemotherapy would produce more changes at the systemic level than in the peritoneal tumor environment, which could support the potential usefulness of intraperitoneal chemotherapy in OC. Besides, it has been reported that neutrophils reach the omentum via specialized high endothelial venules in inflammatory conditions of the peritoneum, from where they migrate to the surface area and extrude NETs that remain exposed into the peritoneal cavity (61). As far as OC is concerned, this location of NETs would mean an absence of close contact between tumor cells and the systemic circulation in early stages of metastasis. Therefore, systemic administration of chemotherapy may reach adequate cytotoxic effects in the well vascularized primary tumors, but might be insufficient for the recently migrated NETs-trapped ovarian cancer cells on the omental surface with an incipient vascularization via angiogenesis. Altogether, the clinical derivative of our findings could partially explain the intraperitoneal recurrences of HGSOC despite proper surgical and chemotherapy treatment and could envisage a benefit for the use of intraperitoneal chemotherapy for the microscopic disease (62, 63), which may be improved with the incorporation of NETs inhibitors or degradation components.

The difficulty in obtaining PF samples from healthy postmenopausal women is one of the main limitations of our study. However, devoted statistical analyses have been performed to rule out the possible effect of these covariates. The sample size studied is rather limited. Nonetheless, the selection of patients following the established exclusion criteria and the difficulty in recruiting paired samples of the biofluids of interest (maintly PF) hinders the recruitment and management of a high number of patients for this type of studies. In addition, the potential of plasma cfDNA and calprotectin as biomarkers of HGSOC ought to be tested in larger independent cohorts and for early-stages HGSOC patients, for which effective diagnostic approaches have not been established yet. Finally, other studies designed to characterize the NETosis process and/or identify its role in HGSOC would be necessary.

Our results provide a proof of concept of the activation of the NETosis process in the tumor environment of patients with advanced HGSOC. Nevertheless, the research of the contribution of this process in OC is still at its infancy and several factors are yet to be deeply studied, as the association between plasma and omental neutrophil count, half-life time of omental neutrophils and the stoichiometry between neutrophils, NETs and tumor cells to establish the lower number of neutrophils capable of promoting metastasis as well as the exhaustive characterization of such interaction, to mention a few.

In conclusion, the present study represents a proof of concept on the alteration of the NETosis biomarkers in patients with advanced HGSOC and their potential implications in patients’ management. The positive correlations obtained in both biofluids and clinical groups indicate that the analyzed biomarkers could be useful as NETosis biomarkers in advanced HGSOC. Our results suggest that an increased NETosis occurs in biofluids from HGSOC patients, mainly at the tumor environment (PF) in comparison to the systemic level (plasma), potentially contributing to the progression of HGSOC in advanced stages. The correlation between PF and plasma levels of cfDNA and calprotectin might postulate these molecules as potential low-invasive biomarkers of HGSOC that may improve current diagnostic markers. Finally, we observed that the systemic neoadjuvant treatment has a major influence on NETosis at the systemic levels but its effect is rather limited in the tumor environment, which might improve the therapeutic landscape of HGSOC.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee, Research Foundation of the General University Hospital of Valencia, protocol code 48/2021, 28/05/2021. The patients/participants provided their written informed consent to participate in this study.

JM-A, PM and JG-E contributed to the conception and design of the study. CA, AC-L, FC, LM-F, NS, RR, SC-M and AL achieved and organized a clinical data base. ST-P, JO, RH and EG-C performed the experiments. ST-P, JA, MC, and AH-P performed the statistical analysis. ST-P, JO, EG-C, JA and PM visualized and interpreted the results. ST-P and JM-A wrote the first draft of the manuscript. JO, BM, MC, AL, PM and JG-E reviewed and edited the manuscript. JM-A, PM and JG-E supervised and acquired resources and funding. All authors contributed to the article and approved the submitted version.