2.1. Patients and Specimens

We collected paraffin-embedded tissue sections and plasma samples from patients with pancreatic ductal adenocarcinoma (PDAC) diagnosed at the Zhongshan Hospital, Fudan University. The study protocol was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (protocol license number: B2020-062R2), and all procedures were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants before recruitment.

2.2. Cell Culture

BxPC-3, MIAPaCa-2, and PANC-1 cell lines were purchased from the American Type Culture Collection (ATCC). BxPC-3 was maintained in Roswell Park Memorial Institute (RPMI) 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). MIAPaCa-2 and PANC-1 were cultured in Dulbecco′s modified Eagle medium (DMEM) (Gibco) containing 10% FBS. All pancreatic cancer cell lines were cultured at 37°C in a humidified atmosphere with 5% CO₂ and 95% air.

2.3. Isolation of Neutrophils and NETs Production

Neutrophils were isolated from human blood according to the kit’s instructions (TBD Sciences). Briefly, the blood samples were layered over the neutrophil separation medium and centrifuged at 800 g for 30 min at room temperature. The lower leukocyte layer containing neutrophils was collected, followed by schistocyte and washing with phosphate buffered saline (PBS). The obtained cells were resuspended in different mediums depending on the subsequent experiments. For NETs production, isolated neutrophils were stimulated with 50 nM phorbol 12-myristate 13-acetate (PMA) (MKBio) for 4 h. After removing the supernatant, NETs adhered at the bottom were washed down by adding 2 mL of cell culture medium and were centrifuged to remove cell debris.

2.4. Immunohistochemistry (IHC) Assay

Paraffin-embedded tissue sections were deparaffinized, rehydrated, treated with 0.3% hydrogen peroxide, and processed for antigen retrieval with heat induction for approximately 10 min. Primary antibodies to the following proteins were used for IHC staining: myeloperoxidase (MPO) (1:50, R&D Systems, AF3667) and STING (1:100, Cell Signaling Technology, 13647S). Tissues were then incubated with peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibody. The slides were finally stained with 3,3 ^′-diaminobenzidine and counterstained with hematoxylin.

2.5. Immunofluorescence (IF) Assay

After cells were fixed and permeabilized, or paraffin-embedded tissue sections were deparaffinized, rehydrated, and processed for antigen retrieval, 1% bovine serum albumin (BSA) was used to block and then incubated with antibodies against citrullinated histone H3 (CitH3) (1:100, Abcam, ab5103), MPO (1:50, R&D Systems, AF3667), and STING (1:100, Cell Signaling Technology, 13647S) at 4°C for 12 h. Secondary antibodies with Alexa 488 or 594 (1:200, Abcam) were added for 60 min at 37°C. Finally, 4 ^′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei. The slices were visualized under a confocal microscope.

2.6. Cell Proliferation Assay

A Cell Counting Kit-8 (Dojindo Corp.) was used to test the relative cell proliferation rate for 24 h according to the manufacturer’s instructions. The absorbance (optical density, OD 450 nm) of each 96-well plate was measured with a Tecan Infinite F50 microplate reader.

2.7. Cell Migration and Invasion Assays

Pancreatic cancer cells were seeded into six-well plates and cultured for 12 h to achieve 100% confluence. Cell monolayers were manually wounded by scraping cells with a P200 pipette tip and followed by washing with PBS. Then, the cells were cultured in different mediums containing 1% serum. The wound width was measured at 0 h and 24 h after the scratch.

Cell migration and invasion assays were performed with chambers (Corning) containing 8-μm pores with or without Matrigel (BD Falcon), respectively. Cells were added to the upper chamber with different mediums in the lower chamber. After incubation for 24 h at 37°C, the noninvading cells were wiped off, and cells on the bottom side of the membrane were fixed with methanol and stained with crystal violet. The stained cells were counted under a light microscope. The migration ability of neutrophils was measured with chambers (Millipore) containing 3-μm pores.

2.8. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted with a TRIzol reagent (Sigma-Aldrich). The cDNA was synthesized with a PrimeScript RT reagent kit (RR036A, Takara), and RT-qPCR was performed with a TB Green PCR kit (RR820A, Takara). Quantification of gene expression was normalized to endogenous β-actin expression. The primer sequences used were as follows:

Zeb-1 forward: 5 ^′-GATGATGAATGCGAGTCAGATGC-3 ^′

Zeb-1 reverse: 5 ^′-ACAGCAGTGTCTTGTTGTTGT-3 ^′

Snail-2 forward: 5 ^′-CGAACTGGACACACATACAGTG-3 ^′

Snail-2 reverse: 5 ^′-CTGAGGATCTCTGGTTGTGGT-3 ^′

Vimentin forward: 5 ^′-GACGCCATCAACACCGAGTT-3 ^′

Vimentin reverse: 5 ^′-CTTTGTCGTTGGTTAGCTGGT-3 ^′

IL-8 forward: 5 ^′-TTTTGCCAAGGAGTGCTAAAGA-3 ^′

IL-8 reverse: 5 ^′-AACCCTCTGCACCCAGTTTTC-3 ^′

β-Actin forward: 5 ^′-CTCGCCTTTGCCGATCC-3 ^′

β-Actin reverse: 5 ^′-GAATCCTTCTGACCCATGCC-3 ^′

2.9. Western Blot

Cells with or without treatments were harvested and lysed with radio immunoprecipitation assay (RIPA) buffer (Solarbio) containing proteinase inhibitor phenylmethanesulfonyl fluoride (PMSF) (1 mM). Equal amounts of protein were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% milk for 1 h at room temperature and then was incubated with primary antibodies overnight at 4°C, including CitH3 (1:1000, Abcam, ab5103), MPO (1:1000, Abcam, ab208670), E-cadherin (1:1000, Cell Signaling Technology, 3195), N-cadherin (1:1000, Cell Signaling Technology, 13116), vimentin (1:2000, Cell Signaling Technology, 5741), alpha-smooth muscle actin (α-SMA) (1:1000, Cell Signaling Technology, 19245), STING (1:1000, Cell Signaling Technology, 13647), phospho-TBK1 (p-TBK1) (1:1000, Cell Signaling Technology, 5483), TBK1 (1:1000, Abcam, ab40676), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (1:1000, Abmart, TA5006), phospho-MEK (p-MEK) (1:1000, Cell Signaling Technology, 9154), MEK (1:1000, Cell Signaling Technology, 8727), phospho-ERK1/2 (p-ERK1/2) (1:1000, Cell Signaling Technology, 4370), ERK1/2 (1:1000, Cell Signaling Technology, 4695), phospho-Akt (p-Akt) (1:1000, Cell Signaling Technology, 4060), Akt (1:1000, Cell Signaling Technology, 4691), and β-actin (1:3000, Cell Signaling Technology, 3700). After being incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, ECL Western Blotting Substrate (Tanon) was used to detect the protein expression.

2.10. Enzyme-Linked Immunosorbent Assay (ELISA)

MPO (Abcam, ab119605) or IL-8 (Absin, abs5100004) ELISA kits were used to evaluate MPO-DNA and IL-8 levels according to the manufacturer’s instructions.

2.11. Statistical Analysis

The results were shown as means ± SEM. The difference between any two groups was analyzed using a two-tailed unpaired t-test. All statistical analyses were carried out using GraphPad Prism 8.0, and p < 0.05 was considered statistically significant ( ^∗p < 0.05,  ^∗∗p < 0.01,  ^∗∗∗p < 0.001, and  ^∗∗∗∗p < 0.0001).

3.1. NETs Formation Is Enhanced in PDAC Tumor Specimens

NETs have been considered an independent prognostic factor in PDAC [15]. To identify the existence of NETs in PDAC tumors, we performed IHC, IF, and Western blot analyses of NETs indicators, specifically MPO and CitH3, in tissue samples. We found a higher level of NETs in PDAC tissue sections compared with adjacent nontumor tissues (Figures 1(a), 1(b), and 1(c)). Neutrophils were isolated from the blood of PDAC patients or healthy controls (HCs) and subsequently treated with PMA to induce the formation of NETs in vitro. We observed that the neutrophils from PDAC patients exhibited an increased capacity for NETosis compared with those from HC individuals (Figure 1(d)). In addition, the serum level of MPO-DNA, a featured marker for systemic NETs formation, was also found to be significantly elevated in PDAC patients (n = 30) compared to HCs (n = 30, p < 0.0001) (Figure 1(e)). These data indicate an increased formation of NETs in PDAC tumor tissues, and neutrophils derived from PDAC patients possess a heightened potential to form NETs.

3.2. NETs Induce EMT to Promote Tumor Invasiveness

Having shown a higher level of NETs in clinical samples, we further investigated whether NETs promoted the proliferation and migration of pancreatic cancer cells. We treated pancreatic cancer cell lines BxPC-3, PANC-1, and MIAPaCa-2 with different mediums, and the results showed that NETs did not accelerate tumor proliferation in all cell lines (Figure 2(a)). Then, we performed experiments to identify the effects of NETs on the migration and invasion capabilities of tumor cells. Wound healing and Transwell assays demonstrated that NETs promoted both migration and invasion abilities of pancreatic cancer cells in vitro, which could be suppressed by the administration of DNase I (1000 U/mL) to degrade NETs (Figures 2(b), 2(c), and 2(d)).

To determine whether NETs influence EMT, we measured levels of several epithelial and mesenchymal markers. Western blot analysis showed that exposure to NETs resulted in the upregulation of mesenchymal markers including N-cadherin, vimentin, and α-SMA while downregulating the epithelial marker E-cadherin in PANC-1 and MIAPaCa-2 cells (Figure 2(e)). Additionally, IF analysis corroborated these findings by demonstrating an increased protein level of vimentin alongside a decreased protein level of E-cadherin in PANC-1 cells (Figure 2(f)). What is more, RT-PCR results indicated that NETs led to elevated mRNA levels of Zeb-1, Snail-2, and vimentin in PANC-1 cells (Figure 2(g)). Collectively, these results confirm that NETs can promote invasiveness in pancreatic cancer cells by inducing the EMT program.

3.3. NETs-Pretreated Tumor Cells Recruit More Neutrophils

Our previous study demonstrated that leukocytosis is associated with increased tumor-infiltrating NETs and is an independent prognostic factor for survival in esophageal cancer [16]. Infiltrating leukocytes are regarded as a hallmark of tumors, and the tumor microenvironment (TME) is considered to contribute to tumor initiation, progression, and patient prognosis [17, 18]. Furthermore, cytokines within the TME can recruit more immune cells such as neutrophils. To investigate this further, we evaluated the chemotaxis of neutrophils using a Transwell assay. Our findings revealed that the conditioned medium of PANC-1 cells pretreated with NETs significantly enhanced neutrophil chemotaxis compared with either the medium of PANC-1 cells alone or NETs alone (Figure 3(a)). Meanwhile, human umbilical vein endothelial cells (HUVECs) exposed to the conditioned medium of PANC-1 cells pretreated with NETs exhibited increased angiogenesis (Figure 3(b)). These results suggest that NETs may promote cytokine production in tumor cells.

IL-8 is known to be a classic neutrophil chemokine and has been shown to exert various tumor-promoting functions including angiogenesis [19]. Therefore, the concentration of IL-8 in different conditioned mediums was measured by ELISA, and the results showed that the level of IL-8 in the conditioned medium of PANC-1 cells pretreated with NETs was approximately 10 times higher than the others (Figure 3(c)). RT-PCR analysis also validated the upregulation of IL-8 expression in PANC-1 cells treated with NETs (Figure 3(d)). Taken together, our findings indicate that NETs enable pancreatic cancer cells to secrete IL-8, thereby recruiting more neutrophils into the TME.

3.4. NETs Activate the STING Signaling Pathway to Promote IL-8 Secretion

There is a strong correlation between persistent inflammation and tumor aggressiveness, with the STING and NF-κB pathways playing important roles in inflammation-driven tumor growth [20, 21]. In addition, NETs could induce activation of the STING pathway, which may subsequently activate NF-κB and thus boost IL-8 production [22]. As expected, data from the Gene Expression Profiling Interactive Analysis (GEPIA) dataset on pancreatic adenocarcinoma (PAAD) showed that patients with pancreatic cancer exhibited increased expression levels of STING, NF-κB, and IL-8 compared with normal controls (Figure 4(a)). IHC and IF analyses confirmed that the expression level of STING in tumor tissues was significantly higher than that in adjacent nontumor tissues (Figures 4(b) and 4(c)). Western blot analysis showed that NETs markedly elevated protein levels of the STING signaling pathway, including STING, p-TBK1, and NF-κB, in PANC-1 cells (Figure 4(d)).

To further explore the involvement of the STING/p-TBK1/NF-κB axis in IL-8 production, we used H-151 (10 μM, STING inhibitor, MedChemExpress) and BAY 11-7082 (25 μM, NF-κB inhibitor, Beyotime) to inhibit STING and NF-κB activation in PANC-1 cells, respectively. We found that the inhibition of the STING pathway markedly reduced protein levels of p-TBK1 and NF-κB (Figure 4(e)). Concurrently, both mRNA and protein levels of IL-8 were decreased upon suppression of the NF-κB pathway (Figures 4(f) and 4(g)). These results collectively demonstrate that NETs induced IL-8 production through the STING/p-TBK1/NF-κB axis in pancreatic cancer cells.

3.5. IL-8 Recruits Neutrophils and Mediates NETosis via the MEK/Mitogen-Activated Protein Kinase (MAPK)/Reactive Oxygen Species (ROS) Axis

To clarify whether IL-8 mediated the chemotaxis of neutrophils, Transwell assays were conducted. The addition of the anti-IL-8 antibody to the conditioned medium of PANC-1 cells pretreated with NETs resulted in a reduction of neutrophil chemotactic ability (Figure 5(a)). Furthermore, the chemotaxis of neutrophils elevated with the increase in IL-8 concentration (Figure 5(b)). We treated neutrophils with different conditioned mediums and found that levels of MEK and ERK1/2 phosphorylation increased in neutrophils exposed to the conditioned medium of PANC-1 cells pretreated with NETs, while AKT phosphorylation remained unchanged (Figure 5(c)). Consistent with these results, when IL-8 was added to the medium of neutrophils, enhanced phosphorylation of MEK and ERK1/2 was detected; this effect could be counteracted by the anti-IL-8 antibody (Figure 5(d)).

ROS are known to be a crucial mediator for NETs formation [13, 23], so we further investigated the impact of IL-8 on ROS production in neutrophils. The results indicated that IL-8 led to an increased level of ROS, which could be reversed by a MEK inhibitor (PD98059) or an anti-IL-8 antibody (Figure 5(e)). Both the conditioned medium of PANC-1 cells pretreated with NETs and medium containing IL-8 contributed to NETs formation, while the effect could be suppressed by inhibition of ROS (Figure 5(f)). Together, these findings suggest that IL-8 from the conditioned medium of PANC-1 cells pretreated with NETs promotes NETosis by regulating the MEK/MAPK/ROS axis of neutrophils.