Neutrophil extracellular traps promote peritoneal metastasis of colon cancer cells
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Amr A. Al-Haidari1,*, Nader Algethami1,*, Mattias Lepsenyi1, Milladur Rahman1, Ingvar Syk1 and Henrik Thorlacius1
1Department of Clinical Sciences, Malmö, Section for Surgery, Lund University, 20502 Malmö, Sweden
*These authors contributed equally to this work
Henrik Thorlacius, email: email@example.com
Keywords: carcinomatosis; chemokines; metastases; neutrophils; peritoneum
Received: December 12, 2018 Accepted: January 31, 2019 Published: February 08, 2019
Cytoreductive surgery is the only curative option for patients with peritoneal carcinomatosis, however, intraperitoneal recurrence rate is high making new ways to prevent cancer recurrence an urgent need. Recent evidence suggests that neutrophils are involved in cancer progression. The purpose of our study was to examine the role of neutrophils in the spread of colon cancer cells in the peritoneal cavity.
The number of metastatic noduli in the peritoneal cavity was quantified in mice injected with murine colon cancer cells (CT-26) intraperitoneally after surgical laparotomy and treated with a neutrophil depleting antibody or DNase I. In addition, peritoneal metastases were harvested from patients with peritoneal carcinomatosis. Scanning and transmission electron microscopy showed extensive neutrophil extracellular trap (NET) formation in peritoneal colon cancer metastases in mice and patients. Neutrophil depletion markedly reduced the number of metastases in laparotomised animals. Administration of DNase I decreased the number of metastatic nodules by 88% in laparotomised animals as well as NET-induced chemokine-dependent colon cancer cell migration and adhesion in vitro. Finally, CT-26 cancer cells were found to express the αvβ3 integrin and inhibition of αv integrin abolished NET-induced adhesion of colon cancer cells to vitronectin. Taken together, our data show that NETs play an important role in colon cancer cell metastasis in the peritoneal cavity and regulate colon cancer cell migration and adhesion to extracellular matrix proteins. These novel findings suggest that targeting NETs might be an effective strategy to antagonize intrabdominal recurrences of colon cancer after cytoreductive surgery in patients with peritoneal carcinomatosis.
Metastasis is the main cause of mortality in patients with colorectal cancer . Peritoneal metastasis is an insidious feature and occurs in 10–30% of all cases of colorectal cancer [2, 3]. Dissemination of malignant cells can result from intestinal perforation of the primary tumor or exfoliation of carcinoma cells due to surgical division of lymphatics or blood vessels. Cytoreductive surgery with peritonectomy combined with hyperthermic intraperitoneal chemotherapy is the only treatment having documented curative benefits on colorectal peritoneal carcinomatosis achieving five-year survival rates above 50% in selected patients . However, intraperitoneal cancer recurrence rate is high (40–60%), which makes new ways to prevent cancer recurrence in the peritoneal cavity after surgery an urgent need . Thus, increased knowledge of the mechanisms promoting peritoneal spread of colon cancer cells could help to improve the efficacy of cytoreductive surgery in patients with peritoneal carcinomatosis. It is generally held that cancer cells have a preferential ability to accumulate at surgical wounds as a basis for tumor recurrence [6–8]. One explanation is that the acute inflammatory reaction generates numerous growth factors and chemoattractants in the wound that not only stimulate wound healing but also promote tumor cell adhesion and proliferation [5, 9].
Neutrophils are the most abundant of all leukocytes and play a key role in inflammatory reactions. Accumulating data suggest that neutrophils possess various functions that regulate cancer progression and metastasis. For example, neutrophils can promote proteolytic release of growth factors, including EGF, TGFβ and PDGF from the extracellular matrix (ECM), which stimulate tumor cell proliferation . Neutrophils also express high levels of metalloproteinases, mainly metalloproteinase-9, which can reconstruct the ECM facilitating tumor cell dissemination . Notably, elevated numbers of circulating neutrophils and a high neutrophil:lymphocyte ratio in the peripheral blood constitute independent markers of poor prognosis in patients with different tumors, such as liver, pancreas and colon cancers , suggesting an important role of neutrophils in cancer progression. Moreover, the observation that neutrophils accumulate in large numbers in pre-metastatic organs [13, 14] has led to the hypothesis that neutrophils play a role in metastatic niche formation. Activated neutrophils can eliminate invading microorganisms by expelling nuclear DNA forming extracellular web-like structures decorated with nuclear histones as well as granular and cytoplasmic proteins . These DNA structures are referred to as neutrophil extracellular traps (NETs). Interestingly, accumulating evidence indicate that NETs may play an important role in cancer development [16, 17]. For example, NETs induced within the vasculature by systemic bacterial infection or surgical stress have been shown to facilitate cancer metastasis to the liver and the lung . Intravascular NETs have also been documented to increase vascular permeability, promoting cancer cell extravasation from the vasculature . Data have shown that gastric, intestinal, hepatic and pancreatic cancers are physically associated with NETs [19, 20]. In fact, tissue specimens from human patients with colon cancer revealed the presence of NETs in the tumor as well as in associated metastatic lymph nodes [20, 21]. One study found extracellular DNA present on the surface of pancreatic cancer cells, which increased CXCL8 production, facilitating tumor cell invasion and angiogenesis of cancers . The authors of that study showed that DNase I inhibited pancreatic cancer metastasis by reducing NET levels and CXCL8 expression, suggesting that disruption of NETs can reduce cancer cell metastasis. Together, these findings implicate NETs in the metastatic process of malignant cells although the role of NETs in peritoneal metastasis of colon cancer cells is not known.
Based on the above, the aim of this study was to define the role of NETs in colon cancer spread and accumulation in the peritoneal cavity. For this purpose, we used a mouse model with intraperitoneal administration of colon cancer cells after laparotomy causing a midline incisional wound.
Neutrophils regulate peritoneal metastasis of colon cancer cells
In order to define the role of neutrophils in peritoneal spread of colon cancer cells, mice were made neutropenic by use of a specific antibody directed against Ly-6G expressed on neutrophils. It was observed that administration of the anti-Ly6G antibody reduced neutrophil systemic count by 95%, a reduction that was maintained for at least 3 days after each injection (Figure 1A). Intraabdominal administration of murine colon cancer cells (CT-26) resulted in clear-cut metastases in the peritoneal cavity (Figure 1B–1E). Notably, we found that neutrophil depletion significantly reduced the number of peritoneal metastasis by 70% (Figure 1B–1E), indicating a key role of neutrophils in the spread of colon cancer cells in the peritoneal cavity.
Figure 1: Neutrophils and NETs mediate colon cancer spread in the peritoneal cavity. (A) Circulating neutrophils (Ly-6G+/CD45+) at day 0, 3 and 6 after administration of the anti-Ly6G antibody. (B–D, F–H) shows representative images of the peritoneal wall and (E, I) illustrates the number of peritoneal metastases. CT-26 cells were injected intraperitoneally in laparotomised animals and mice received daily treatment with vehicle, control antibody (Ctrl Ab), anti-Ly6g Ab or DNase I. After 10 days the peritoneum wall was photographed, and the number of macroscopic tumor nodules was quantified in the peritoneum cavity. Data are mean ± SEM and n = 5. *P < 0.05 vs. Ctrl Ab or Vehicle.
NETs control colon cancer spread in the peritoneal cavity
Administration of DNase I is known to be an effective way to degrade NETs in vivo [23, 24]. It was found that treatment with DNase I decreased the number of peritoneal metastases by 88% (Figure 1F–1I), suggesting that NETs play an important role in the dissemination of colon cancer cells in the peritoneal cavity. NETs are composed of extracellular DNA, histones and neutrophil-derived granule proteins. Using scanning electron microscopy, we observed that peritoneal metastasis of colon cancer cells was associated with formation of extracellular fibrillar and web-like structures in the tumors compatible with NETs (Figure 2A). Importantly, it was found using transmission immunoelectron microscopy that the neutrophil-derived granule protein elastase and citrullinated histone 3 co-localized with the extracellular DNA in these extracellular fibrillar and web-like structures (Figure 2B), showing that NETs are formed in peritoneal metastases. Moreover, administration of DNase I abolished NET formation in peritoneal metastasis of colon cancer cells (Figure 2A–2B). We used correlative light and electron microscopy to examine co-localization of colon cancer cells and NETs in peritoneal metastasis. Figure 3A and 3D show a fluorescence microscopy image of a selected part of a section with visible colon cancer cells (green indicating CT-26-GFP cells). This selected region was examined by scanning electron microscopy showing extracellular fibrillar and web-like structures (Figure 3B and 3E). Figure 3C and 3F shows an overlay of the ROI shared by fluorescence and electron microscopy, revealing that NETs co-localize with CT-26-GFP cells and that DNase I reduced NETs formation in peritoneal metastases.
Figure 2: NET formation in peritoneal colon cancer metastasis in mice. (A) Scanning electron microscopy (SEM) showing extracellular web-like structures in metastases from animals injected with CT-26 cells. (B) Transmission electron microscopy (TEM) of the indicated area of interest from Figure 2A incubated with gold-labeled anti-citrullinated histone 3 (large gold particles, arrow) and anti-elastase (small gold particles, arrowhead) antibodies. CT-26 cells were injected intraperitoneally in laparotomised animals and mice received daily treatment with vehicle or DNase I (50 μg) and 10 days later, the metastases were harvested for electron microscopy.
Figure 3: CLEM images indicating that NETs co-localized with murine colon cancer metastasis tissue. (A, D) selected region of mouse GFP labeled-tumor tissue (green) containing citrullinated histone 3 (H3Cit-red) from vehicle-treated group and DNase1 treated group (B, E) Scanning electron microscope of tumor tissue shows web-like NET structure and (C, F) overlay of region of interest with SEM. ROI; Region of Interest, SEM; Scanning Electron Microscope.
NETs are generated in human colon cancer peritoneal metastases
We next wanted to examine if tumor cell metastasis in the peritoneal cavity in humans is also associated with NET formation. Similar to peritoneal metastases in mice, we observed that colon cancer metastases in the peritoneal cavity of patients with peritoneal carcinomatosis contained numerous extracellular fibrillar and web-like structures (Figure 4A) expressing elastase as well as citrullinated histone 3 (Figure 4B). In contrast, we did not find any extracellular fibrillar and web-like structures nor any expression of elastase or citrullinated histone 3 in pseudomyxoma tumors, which is a non-malignant tumor, in the peritoneal cavity of humans (Figure 4A–4B).
Figure 4: NET formation in peritoneal colon cancer metastasis in humans. (A) Scanning electron microscopy (SEM) showing extracellular web-like structures in peritoneal metastases. (B) Transmission electron microscopy (TEM) of the indicated area of interest from (A) incubated with gold-labeled anti-citrullinated histone 3 (large gold particles, arrow) and anti-elastase (small gold particles, arrowhead) antibodies. Metastases were harvested from patients undergoing cytoreductive surgery due to peritoneal carcinomatosis. Representative examples are shown from patients with spread colon cancer and pseudomyxoma.
NETs stimulate colon cancer migration and adhesion
We next wanted to investigate the impact of NETs on colon cancer cell migration and adhesion. The potent neutrophil chemoattractant CXCL2 was used to stimulate neutrophil migration. It was found that CXCL2 stimulation of neutrophils provoked expulsion of DNA containing citrullinated histone 3 and MPO (Figure 5), suggesting that CXCL2 is an effective stimulus of NET formation. Next, we stimulated neutrophils with CXCL2 and harvested conditioned media from the cells. It was found that co-incubation with this conditioned media potentiated CXCL2-induced colon cancer cell migration (Figure 6A–6B). Importantly, addition of DNase I to this conditioned media markedly reduced CXCL2-triggered migration (Figure 6A–6B). By use of flow cytometry, we found that CT-26 cells express the αv (Figure 6C) and β3 integrin (Figure 6D) subunits. Vitronectin is one of the most important ligands of αv integrins and we therefore evaluated CT-26 cell adhesion to wells coated with vitronectin. It was found that conditioned media from CXCL2-stimulated neutrophils increased CT-26 cell adhesion to vitronectin (Figure 6E). This increased colon cancer cell adhesion was abolished by immunoneutralization of CD51 (αv integrin) (Figure 6E). Moreover, addition of DNase I abolished colon cancer cell adhesion to vitronectin provoked by conditioned media from CXCL2-stimulated neutrophils (Figure 6E). Finally, co-incubation of neutrophils with the CXCR2 antagonist SB225002 markedly reduced the effect of CXCL2 conditioned media on colon cancer adhesion to vitronectin (Figure 6E).
Figure 5: NET regulates colon cancer cell migration and adhesion. Representative confocal imaging showing NETs formation in isolated neutrophils. NETs were generated by CXCL2-stimulation (3 h), co-incubated with or without DNase I (50 μg). Neutrophils were immune-stained with antibodies to citrullinated histone 3 (H3Cit-green), myeloperoxidase (MPO-red), and DAPI-blue to counterstain DNA. Non-stimulated neutrophils served as a control. One representative image from four independent experiments; Scale bar, 25 μm.
Figure 6: NET regulates colon cancer cell migration and adhesion. (A) Conditioned media from neutrophils stimulated with CXCL-2 and co-incubated with vehicle or DNase I (50 μg) was used to study colon cancer cell migration as described in Materials and Methods. Migration was quantified by counting cells in 10 High Power Fields in 5 different fields. Migration index was calculated as the ratio of the number of migrated cells divided by the number of cells in the insert wells. (B) Representative images showing colon cancer cell migration. (C, D) CT-26 cell surface expression of αv and β3 integrins. (E) Conditioned media from neutrophils stimulated with CXCL-2 and co-incubated with vehicle, the CXCR2 antagonist (SB225022), anti-CD51 antibody or DNase I (50 μg) was used to study colon cancer cell adhesion to vitronectin as described in Materials and Methods. Data represents mean ± SEM and n = 5.
Intraabdominal recurrences constitute a major challenge in the treatment of patients with peritoneal carcinomatosis. This study demonstrates that neutrophils play an important role in peritoneal dissemination of colon cancer cells. Our findings show that peritoneal metastasis is associated with extensive NET formation. In fact, disintegration of NETs markedly decreased peritoneal spread of colon cancer cells. Moreover, we could show that NETs promote chemokine-dependent colon cancer cell migration and αv integrin-mediated adhesion to the extracellular matrix protein vitronectin. Thus, targeting NETs could be an effective way to antagonize peritoneal dissemination of colon cancer cells.
Accumulating data suggest that neutrophils exert numerous functions facilitating metastasis of malignant cells to the liver and lung . For example, neutrophils have been shown to remodel extracellular matrix, promote angiogenesis and cause T-cell dysfunction, which help to explain parts of the pro-metastatic effects of neutrophils [25, 26]. Herein, we found that immuno-depletion of neutrophils greatly attenuated the number of metastasis in the peritoneal cavity, suggesting that neutrophils play an important role in peritoneal metastasis of colon cancer cells. This notion is also supported by a previous study reporting that reducing postoperative influx of neutrophils decreases peritoneal colon cancer recurrence . One explanation might be related to findings showing that neutrophil adhesion to mesothelial cells provokes retraction, gap formation, and detachment, resulting in substantial mesothelial cell injury and exposure of extracellular matrix components and thereby create a preferential site for adhesion of free-floating carcinoma cells . Nonetheless, considered together, neutrophils appear to be involved in the dissemination of colon cancer to many of the most critical sites in the body, including liver, lung and peritoneal cavity. NETs are normally generated in response to infections with the purpose to eradicate invading pathogens. However, a growing body of evidence in the literature suggest that NET formation is elevated in patients with different forms of cancer . Notably, recent reports suggest that NETs are involved in the spread of breast and colon cancer cells to the liver and lung [18, 30]. In the present study, we provide evidence showing that peritoneal spread of colon cancer cells is associated with widespread DNA deposition in the metastatic nodules, which co-localized with the neutrophil-derived granule protein elastase and citrullinated histone 3. Thus, our data indicate that colon cancer cell metastasis is associated with extensive NET generation in the peritoneal cavity. Several studies have documented that pharmacologic inhibition by use of DNase I is an effective method to disintegrate NETs in vivo . In the present study, we found that treatment with DNase I markedly decreased NET formation in colon cancer metastases. Moreover, administration of DNase I reduced the number of colon cancer metastases by 88%. Thus, these findings suggest that NET formation plays an important role in the pathophysiology in the peritoneal spread of colon cancer. This conclusion is in line with a recent study reporting that NETs enhance peritoneal spread of gastric cancer cells . Indeed, NETs contain numerous proteins, such as elastase, MMP-9, HMGB1 and cathepsin G, with pro-metastatic properties. For example, both elastase and cathepsin G have been shown to enhance tumor cell proliferation and migration . In addition, convincing data has documented that MMP-9 supports tumor angiogenesis and invasion . Interestingly, HMGB1 can cause immunosuppression via stimulation of myeloid derived suppressor cells, and thereby promote peritoneal spread of colon cancer cells after surgical resection . Although the detailed mechanisms remain to be elucidated, our study propose that targeting NET induction or function might be a useful way to attenuate intraabdominal recurrences in patients undergoing surgery due to peritoneal carcinomatosis. This notion is supported by our patient data showing that colon cancer metastases in the peritoneal cavity of patients contains clear-cut formation of NETs while benign conditions such as pseudomyxoma showed absence of NETs. Considered together, these findings provide incitement for future studies to address whether targeting NETs is also effective in reducing intrabdominal recurrences in patients with peritoneal carcinomatosis undergoing cytoreductive surgery.
It is generally held that cancer cells have a preferential ability to accumulate at surgical wounds causing tumor recurrence [6–8]. One reason is that numerous growth factors and chemoattractants are formed in the wound that not only stimulate wound healing but also promote tumor cell adhesion and growth [5, 9]. Previous work has shown that damaged mesothelium can secrete IL-8, the human analogue of murine CXCL2  and that CXCL2 is a potent stimulator of colon cancer cell migration , which was also confirmed in the present study. As previously shown, CXCL2 was a potent stimulator of NET formation . Moreover, when conditioned media from CXCL2-stimulated neutrophils were co-incubated with colon cancer cells CXCL2-induced migration was markedly increased. Notably, this enhanced migration was dose-dependently decreased by addition of DNase I, indicating that NETs can potentiate chemokine-dependent migration of colon cancer cells. At the site of peritoneal wounds, the extracellular matrix containing numerous different proteins with adhesive capacity is exposed to floating cancer cells. Herein, we found that colon cancer cells express the αv and β3 integrin subunits, indicating that colon cancer cells express the αvβ3 integrin, which is also known as the vitronectin receptor . This finding is in line with a previous study showing that human colon cancer cells also express the αvβ3 integrin . We therefore tested the role of NETs in supporting adhesion of colon cancer cells to vitronectin. Notably, co-incubation with conditioned media from CXCL2-stimulated neutrophils markedly increased colon cancer cell adhesion to vitronectin. Interestingly, addition of DNase I to the conditioned media from CXCL2-stimulated neutrophils abolished colon cancer cell adhesion to vitronectin, suggesting that NETs are potent stimulators of colon cancer cell binding to extracellular matrix proteins. Pretreatment of colon cancer cells with the CXCR2 antagonist markedly decreased the stimulatory effect of CXCL2 on tumor cell adhesion to vitronectin, indicating a functional role of CXCR2 on colon cancer cells in CXCL2-induced adhesion. Moreover, inhibition of CD51 (αv integrin subunit) greatly reduced colon cancer cell adhesion to vitronectin induced by conditioned media from CXCL2-stimulated neutrophils, showing that αvβ3 integrin expressed on colon cancer cells mediate binding to vitronectin. In this context, it is important to note that inhibition of adhesion to peritoneal surfaces and ECM results in cancer cell death . In addition, the majority of free tumor cells in the peritoneal cavity are rapidly destroyed by immune cells . This concept is in line by the observation that the presence of free-floating carcinoma cells does not necessarily result in metastases in the peritoneal cavity [43, 44].
Taken together, our findings show that neutrophils play an important role in peritoneal dissemination of colon cancer cells via release of NETs. Moreover, these results demonstrate that NETs increase chemokine-mediated migration and firm adhesion to vitronectin. In conclusion, formation of NETs constitutes a key component in peritoneal spread of colon cancer cells and targeting NETs might be a useful strategy to attenuate intraabdominal recurrences in patients undergoing cytoreductive surgery due to peritoneal carcinomatosis.
MATERIALS AND METHODS
Cells and reagents
The murine colon adenocarcinoma cell line, CT-26, transfected with green fluorescent protein (GFP) was a kind gift from Dr Otto Kollmar (University of Saarland, Germany). Cells were cultured in Roswell Park Memorial Institute medium 1640 (RPMI 1640, Sigma-Aldrich, Stockholm, Sweden) supplemented with 10% fetal bovine serum (FBS) containing 2 mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin at 37°C and 5% CO2. Calcein AM, CXCR2 antagonist; N-(2-Hydroxy-4-nitrophenyl)-N′-(2-bromophenyl) urea (SB225002), and accutase were obtained from Sigma-Aldrich. Recombinant mouse CXCL2 was purchased from Peprotech (Rocky Hill, NJ, USA). APC-labeled anti-mouse β3 integrin and PE-labeled αv integrin antibodies were purchased from R&D systems Europe (Abingdon, UK). Blocking anti-CD51 antibody was purchased from Affymetrix (eBioscience, San Diego, CA, USA). Pulmozyme (DNase I) was from Roche (Copenhagen, Denmark).
Mice were injected intraperitoneally (I.P) with 300 μl solution containing 500 μg of the mAb anti-ly6G, clone 1A8 (BioX Cell, West Lebanon, NH, USA) every three days. Control mice were injected with equivalent amount of an isotype control IgG antibody. A blood sample was taken every three days for quantification of the number of neutrophils in the circulation using flow cytometry. No distress feature or infection was observed in mice during the neutrophil depletion period.
Flow cytometry analysis of cell surface receptors
Flow cytometry was used to evaluate the efficacy of the neutrophil depletion antibody. Antibodies were administered intraperitoneally at day 0, 3 and 6. Blood was collected through a small puncture of facial vein. To reduce nonspecific antibody binding via Fc receptors, cells (2 × 106) were incubated with 1 μg of mouse Fc R inhibitor (eBioscience, San Diego, CA, USA) for 15 min. Cells were incubated with 0.2 μg of APC-labeled anti-CD45 antibody (eBioscience, San Diego, CA, USA) and 0.2 μg of PE-labeled anti-Ly6G antibody (BD Bioscience, NJ, USA) for 90 min at room temperature in the dark. Subsequently, cells were washed twice, resuspended in 0.4 ml final volume FACS buffer and analyzed in duplicates at least three times using cytoflex (Beckman Coulter, Mountain View, CA, USA). Unstained cells were served as negative control. Histograms were made using CytExpert software with assessment of 10 000 events per sample. In other set of experiments, αV and ß3 integrin expression in CT-26 cancer cells were assessed as above using APC-labeled anti-β3 and PE-labeled anti-αv antibodies.
Mice were euthanized and femurs and tibia were collected by careful dislocation of the epiphyses. Bone marrow was flushed from both ends of the bones with a 25-gauge needle and a 1 ml syringe containing RPMI 1640, supplemented with 10% FBS and 2 mM EDTA (Sigma-Aldrich, Stockholm, Sweden). Following hypotonic lysis (5 ml ice-cold 0.2% NaCl, added for 45 s followed by addition of 5 ml 1.6% NaCl), neutrophils were separated from mononuclear cells by density gradient centrifugation using a Ficoll-Paque gradient (GE Healthcare, Uppsala, Sweden). The neutrophil layer was harvested and washed with RPMI 1640 and cells were resuspended at (5 × 106 cells/ml). Isolated neutrophils were then used for subsequent experiments including chemotaxis, adhesion studies and NET formation.
Chemotaxis of CT-26 cancer cells in response to neutrophil co-culture was evaluated using 24-well cell migration chambers with 8 μm pore size inserts (Corning Coster, Corning, NY, USA). Colon cancer cells were serum starved overnight and resuspended in serum-free DMEM with 0.5% BSA and co-cultured with neutrophils in 2:1 neutrophils/cancer ratio and incubated with different concentrations of DNase I and loaded in the inserts. DMEM with or without 200 ng CXCL2 (Peprotech, Rocky Hill, NJ, USA) was added in the lower chambers and incubated for 24 h at 37°C (5% CO2). Non-migrated cells were removed by cotton swabs from the upper surface of the insert and cells on the lower surface of the insert membrane were fixed in ice-cold 100% methanol and stained with 1% crystal violet. All migrated cells were counted microscopically in at least 5 different fields. Migration index was then calculated as the ratio of the number of migrated cells divided by the number of cells in the control wells.
Cancer cell adhesion capacity to vitronectin was assessed using 96-well plate coated with vitronectin. Briefly, 96-well plate was coated with 10 μg/ml vitronectin (Sigma-Aldrich) in a final volume of 75 μl/well and blocked using 1% BSA for 2 h and then washed 3 times in sterile HBSS buffer and stored at 4°C until use. CT-26 cells were plated and stimulated with either PMA-induced NET conditioned media or CXCL2-induced NET conditioned media and co-incubated with the CXCR2 antagonist SB225002, anti-CD51 antibody, and DNase I. Cancer cells were then lysed in lysis buffer and stained with the fluorescent CyQuant® GR Dye (Cell Biolabs, San Diego, CA). Cancer cell adhesion was measured by fluorometer (Tecan’s Infinite M200, Mannedorf, Switzerland) at 480 nm/520 nm and at least three times of experiments were performed. The adhesion index was then calculated as the ratio of the number of adhered cells in all groups divided by the number of adhered cells in the control group and expressed as a percentage of adhesion.
Experimental model of peritoneal cancer cell metastasis
All experimental procedures were performed in accordance to legislation on protection of animals and were approved by the Regional Ethical Committee for Animal Experimentation at Lund University, Sweden. Male Balb/c mice weighing 20 to 25 g were housed on an animal facility with 12-12 h light dark cycle at 22°C and fed with a laboratory diet and water ad libitum. Mice were anesthetized with 7.5 mg ketamine hydrochloride (Hoffman-La Rhoche, Basel, Switzerland) and 2.5 mg of xylazine (Janssen Pharmaceutics, Beerse, Belgium) per 100 g body weight intraperitoneally. Mice were anesthetized, and a 1 cm long median incision was performed. After laparotomy, 1 ml buprenorphine analgesia (0.05 mg/kg, Temgesic, Schering-Plough, NJ, USA) was administered subcutaneously to alleviate postoperative pain. Mice were then randomly assigned into five different groups; sham, vehicle (laparotomy + CT-26 cancer cells), DNase I, anti-Lys6G, and isotype control antibody. CT-26 cells were detached from cell cultures using accutase and 98% viable cells were prepared for injection. 7 × 105 viable CT-26 cells in 0.4 ml phosphate-buffered saline (PBS) were injected I.P directly after the surgery. Animals received I.P injection according to their assigned groups. Vehicle (PBS) and DNase I (2 mg/kg) groups were treated on a daily basis while neutrophil depletion and isotype control antibodies were given every three days. Mice were examined daily for 10 days with no signs of distress, such as changes in appearance, respiration and physical activity. Upon experimental termination, mice were euthanized, and tumor growth was evaluated by counting macroscopic tumor nodules in the peritoneum cavity.
Confocal fluorescence microscopy
For immunofluorescence detection of NETs, neutrophils were isolated from mice bone marrow and preincubated with or without DNase I and then stimulated with 200 ng CXCL2. Neutrophils were fixed in 4% paraformaldehyde and permeabilized by 0.1% TritonX-100/PBS. Nonspecific binding sites were blocked with 1% BSA. NETs were stained with a rabbit anti-citrullinated histone H3 polyclonal antibody (Abcam, UK; ab5103) and mouse anti-myeloperoxidase monoclonal FITC antibody (Abcam, UK; 90812). A goat anti-rabbit APC antibody (Thermofisher scientific, Waltham, MA, USA) was utilized as a secondary antibody for CitH3 detection. Neutrophils were then counterstained with Hoechst stain and mounted using ProLong Diamond Antifade mountant (Life Technologies, Carlsbad, CA, USA) and visualized in a confocal microscopy LSM 800 confocal (Carl Zeiss, Jena, Germany) by a × 63 oil immersion objective (numeric aperture = 1.25). The pinhole was ~ 1 airy unit and the scanning frame was 1024 × 1024 pixels. Images were later processed using ZEN2012 software.
Electron microscopy (EM) and correlative light and electron microscopy (CLEM)
Deparaffinized peritoneal carcinomatosis tissue samples were fixed in 2.5% glutaraldehyde in 0.15 mol/L sodium cacodylate, pH7.4 (cacodylate buffer), for 30 min at room temperature. Specimens were washed with cacodylate buffer and dehydrated with an ascending ethanol series from 50% (vol/vol) to absolute ethanol (10 min/step). The specimens then were subjected to critical-point drying in carbon dioxide, with absolute ethanol as intermediate solvent, mounted on aluminum holders, and finally sputtered with 20 nm palladium/gold. Specimens were examined in a Jeol/FEI XL 30 FEG scanning electron microscope at the Core Facility for Integrated Microscopy at Panum Institute (University of Copenhagen, Denmark). The location of individual target molecules was analyzed at high resolution by ultrathin sectioning and transmission immunoelectron microscopy. Specimens on coverslips were embedded in Epon 812 and sectioned into ultrathin (50 nm) sections with a diamond knife in an ultramicrotome. For immunohistochemistry, sections were incubated overnight at 4°C with primary antibodies against elastase and histone H3. Controls without primary antibodies were included. The grids were incubated with species-specific, gold-conjugated secondary antibodies (Electron Microscopy Sciences, Fort Washington, MD). Finally, sections were post-fixed in 2% glutaraldehyde and post-stained with 2% uranyl acetate and lead citrate. Specimens were observed in a Jeol/FEI CM100 transmission electron microscope operated at 80-kV accelerating voltage at the Core Facility for Integrated Microscopy at Panum Institute. To confirm that the extracellular meshes observed by scanning electron microscopy were NETs, we performed correlative light immunofluorescence-electron microscopy (CLEM) to detect the expelled histone3-DNA complex in the meshes using tumor tissue sections labelled with anti-GFP and stained with immunogold labeled anti-DNA and anti-H3 antibodies.
Samples from two patients with colon cancer and one with pseudomyxoma were included in this study. All human peritoneal metastases samples were collected at the time of surgery after informed consent was obtained from patients or their legal guardians prior to the surgery. This study was approved by the ethics committee at Lund University, Lund, Sweden (permit no. 2017/626). All methods involving human tumor samples were performed in accordance with the relevant guidelines and regulations. Samples were directly collected in formalin and processed for subsequent electron microscopy.
All statistical analyses were performed using SigmaPlot® 10 software. For multiple comparisons, Kruskal–Wallis One Way Analysis of variance on ranks followed by the Dunnett’s post hoc test was used. P- value < 0.05 was considered significant. Mann Whitney test was used for comparison of two groups.
CONFLICTS OF INTEREST
Authors have no financial conflicts of interests.
This study was supported by the Swedish Medical Research Council (2017-01621), Einar och Inga Nilssons stiftelse, Malmö University Hospital Cancer Foundation and Lund University.
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