- Open Access
Tumor-derived exosomes induce N2 polarization of neutrophils to promote gastric cancer cell migration
- Xu Zhang†1, 2, 3Email authorView ORCID ID profile,
- Hui Shi†1,
- Xiao Yuan1,
- Pengcheng Jiang2, 3,
- Hui Qian1, 2, 3 and
- Wenrong Xu1, 2, 3Email author
© The Author(s). 2018
- Received: 6 May 2018
- Accepted: 27 September 2018
- Published: 6 October 2018
Exosomes are extracellular vesicles that mediate cellular communication in health and diseases. Neutrophils could be polarized to a pro-tumor phenotype by tumor. The function of tumor-derived exosomes in neutrophil regulation remains unclear.
We investigated the effects of gastric cancer cell-derived exosomes (GC-Ex) on the pro-tumor activation of neutrophils and elucidated the underlying mechanisms.
GC-Ex prolonged neutrophil survival and induced expression of inflammatory factors in neutrophils. GC-Ex-activated neutrophils, in turn, promoted gastric cancer cell migration. GC-Ex transported high mobility group box-1 (HMGB1) that activated NF-κB pathway through interaction with TLR4, resulting in an increased autophagic response in neutrophils. Blocking HMGB1/TLR4 interaction, NF-κB pathway, and autophagy reversed GC-Ex-induced neutrophil activation. Silencing HMGB1 in gastric cancer cells confirmed HMGB1 as a key factor for GC-Ex-mediated neutrophil activation. Furthermore, HMGB1 expression was upregulated in gastric cancer tissues. Increased HMGB1 expression was associated with poor prognosis in patients with gastric cancer. Finally, gastric cancer tissue-derived exosomes acted similarly as exosomes derived from gastric cancer cell lines in neutrophil activation.
We demonstrate that gastric cancer cell-derived exosomes induce autophagy and pro-tumor activation of neutrophils via HMGB1/TLR4/NF-κB signaling, which provides new insights into mechanisms for neutrophil regulation in cancer and sheds lights on the multifaceted role of exosomes in reshaping tumor microenvironment.
- Gastric cancer
Neutrophils are important players in cancer development and progression [1–3]. In various cancers, neutrophils have been shown to promote carcinogenesis, growth and metastasis, angiogenesis, and immunosuppression. Neutrophils produce genotoxic substances such as reactive oxygen species (ROS) that can damage DNA in epithelial cells and initiate carcinogenesis. Neutrophils can also generate a wide spectrum of factors such as neutrophil elastase (NE) and prostaglandin E2 (PGE2) to promote tumor cell proliferation [4, 5]. In addition, neutrophils can promote tumor metastasis by enhancing tumor cell migration and invasion, degrading extracellular matrix, and promoting tumor cell colonization [6–8]. Meanwhile, neutrophils impair immunity to help tumor growth and metastasis [9–12]. Moreover, neutrophils produce a number of molecules such as matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) to induce angiogenesis . Increased neutrophil infiltration and elevated neutrophil/lymphocyte ratio (NLR) have been linked to disease progression and poor prognosis in cancer patients . Targeting neutrophils to inhibit their pro-tumor function has shown therapeutic potential in mouse models . Thus, better understanding of neutrophil regulation in cancer will provide new approaches for cancer diagnosis and therapy.
Accumulating studies suggest that tumor can induce a pro-tumor phenotype in neutrophils that, in turn, help tumor progression. The previous studies have shown that tumor cells produce oxysterol , C-X-C motif chemokine ligand 5 (CXCL5) , hyaluronan fragments (HA) , granulocyte-macrophage colony stimulating factor (GM-CSF) , and macrophage migration inhibitory factor (MIF) . These factors induce pro-tumor activation of neutrophils, leading to increased tumor growth and metastasis. We have reported that IL-6 derived from tumor-resident mesenchymal stem cells induces neutrophil activation, resulting in enhanced angiogenesis and tumor metastasis . Recently, Coffelt et al. demonstrated that IL-17 produced by tumor infiltrating γδ T cells could recruit, expand, and activate neutrophils to promote lung metastasis of breast cancer . Nonetheless, mechanisms for the modulation of neutrophil phenotype and function in tumor milieu remain not fully characterized.
Exosomes are small lipid bilayer membrane vesicles of endocytic origin. Exosomes, as a novel mechanism of intercellular communication, can shuttle bioactive molecules from one cell to another, leading to the exchange of genetic information and reprogramming of recipient cells. Increasing evidence suggests that tumor cells release excessive amount of exosomes that promote tumor growth . In addition, tumor-derived exosomes signal immune cells in tumor microenvironment, helping tumor cells escape immune surveillance and form pre-metastatic niche [24, 25]. We have recently shown that tumor cells interact with mesenchymal stem cells via exosomes to promote tumor growth, metastasis, and drug resistance [26–28]. However, the function of tumor-derived exosomes in neutrophil activation has not been well characterized.
In this study, we demonstrated that gastric cancer cells induced pro-tumor activation of neutrophils via exosomes. Gastric cancer cell-derived exosomes carried high mobility group box-1 (HMGB1) that interacted with toll-like receptor 4 (TLR4) to activate NF-κB and induce autophagy in neutrophils, which in turn promoted gastric cancer cell migration. Collectively, our findings indicate that exosomes represent a new regulator of neutrophil activation in gastric cancer.
The conditioned medium from gastric cancer cells induces autophagy and pro-tumor activation of neutrophils
The promoting role of neutrophils in cancer progression has been linked to autophagic activation. Thus, we collected GC-CM-treated neutrophils and analyzed for autophagosomes. The results of transmission electron microscope (TEM) analyses showed that there were more autophagosomes in GC-CM-treated neutrophils than control cells (Fig. 1d). Immunofluorescent staining results confirmed the increase in the number of LC3-positive puncta in GC-CM-treated neutrophils compared to control cells (Fig. 1e). Moreover, western blot results showed an increase in LC3-II expression, an autophagosomal marker, in GC-CM-treated neutrophils (Fig. 1f). We then examined the expression of autophagy related 7 (ATG7) and Beclin 1 (BECN1) genes, important regulators of autophagosome formation, for cells treated with GC-CM. As expected, the increased expression of ATG7 and BECN1 was observed in GC-CM-treated neutrophils compared to controls (Fig. 1g). We next treated neutrophils with autophagy inhibitors and evaluated their phenotypic and functional changes in response to GC-CM treatment. As shown in Fig. 1h and i, the pre-treatment with autophagosome formation inhibitor 3-MA, but not autophagosome degradation inhibitor CQ, remarkably reversed the effects of GC-CM on neutrophil survival and CD11b expression. More importantly, the pre-treatment with 3-MA inhibited gastric cancer cell migration induced by GC-CM-primed neutrophils. This effect, however, was not seen for CQ (Fig. 1j). Taken together, these results demonstrate that gastric cancer cell-derived conditioned medium upregulates autophagy-related gene expression and induces autophagy in neutrophils, leading to pro-tumor activation of neutrophils.
GC-CM activates NF-κB pathway in neutrophils
Gastric cancer cell-derived exosomes induce autophagy and pro-tumor activation of neutrophils
GC-Ex induces neutrophil activation through NF-κB pathway
GC-Ex induces neutrophil activation through the interaction between high mobility group box-1 and Toll-like receptor 4
Exosomes can induce signaling pathway activation in recipient cells by delivering bioactive molecules. To investigate the molecules in GC-Ex that induce neutrophil activation, we treated GC-Ex with proteinase and evaluated the effects of digested exosomes on neutrophil activation. Compared to undigested GC-Ex, the treatment of proteinase-digested GC-Ex decreased LC3-II expression as well as ATG7 and BECN1 expression in neutrophils (Additional file 5: Figure S5A and B). In similar, GC-Ex-induced activation of NF-κB, STAT3, and ERK pathways in neutrophils was also impaired by proteinase treatment (Additional file 5: Figure S5C). Moreover, the treatment with proteinase decreased GC-Ex-induced expression of inflammatory factors in neutrophils compared to undigested GC-Ex (Additional file 5: Figure S5D). Thus, we focused on exosomal proteins in the subsequent studies.
We next investigated the role of exosomal HMGB1 in neutrophil activation. Neutrophils were treated with HMGB1 antagonist and then exposed to GC-Ex. HMGB1 antagonist reversed GC-Ex-induced increases in LC3-II expression and ATG7 and BECN1 expression in neutrophils compared to GC-Ex alone (Fig. 5c and d). In addition, GC-Ex-induced apoptotic inhibition as well as increases of CD11b expression in neutrophils were inhibited by HMGB1 antagonist (Fig. 5e). Moreover, HMGB1 antagonist inhibited GC-Ex-induced activation of NF-κB pathway and expression of inflammatory factors in neutrophils (Fig. 5f and g). Finally, the promoting effect of supernatant from GC-Ex-treated neutrophils on gastric cancer cell migration was impaired by HMGB1 antagonist (Fig. 5h).
HMGB1 activates NF-κB pathway by interacting with toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE). We thus examined the effects of TLR2, TLR4, and RAGE inhibitors on the observed roles of GC-Ex in neutrophils. As expected, TLR4 inhibitor blocked GC-Ex-induced autophagy and NF-κB pathway activation in neutrophils (Fig. 5c, d and f). TLR4 inhibitor decreased the expression of inflammatory factors in GC-Ex-treated neutrophils and impaired its gastric cancer cell migration-promoting effect (Fig. 5g and h; Additional file 5: Figure S5E). However, the inhibitors for TLR2 and RAGE showed minimal effects (data not shown). Taken together, these results suggest that GC-Ex delivers HMGB1 to neutrophils and induces pro-tumor activation through TLR4/NF-κB signaling.
Gastric cancer tissue-derived exosomes induce autophagy and pro-tumor activation of neutrophils
To confirm our findings from gastric cancer cell lines, we tested neutrophil activation by using exosomes from matched gastric cancer and non-cancerous tissues. Similar to GC-Ex, exosomes from gastric cancer tissues prevented the apoptosis of neutrophils and induced CD11b expression in neutrophils (Fig. 6d and e). Gastric cancer tissue-derived exosomes also induced higher ATG7 and BECN1 expression (Fig. 6f) and NF-κB activation (Fig. 6g) in neutrophils than non-cancerous tissue-derived exosomes. Neutrophils treated with exosomes from gastric cancer tissues promoted gastric cancer cell migration more efficiently (Fig. 6h). In addition, HMGB1 antagonist reversed gastric cancer tissue-derived exosomes-induced increases of ATG7 and BECN1 expression in neutrophils (Fig. 6i). Moreover, gastric cancer tissue-derived exosomes-induced expression of inflammatory factors in neutrophils was inhibited by HMGB1 antagonist (Fig. 6j). Finally, the promoting effect of supernatant from gastric cancer tissue-derived exosomes-treated neutrophils on gastric cancer cell migration was impaired by HMGB1 antagonist (Fig. 6k). Taken together, these findings indicate that gastric cancer tissue-derived exosomes could induce autophagy and pro-tumor activation of neutrophils.
HMGB1 knockdown impairs GC-Ex-induced neutrophil activation
In this study, we reported that gastric cancer cells induced a pro-tumor phenotype in neutrophils via exosomes. Gastric cancer cell-derived exosomes induced autophagy in neutrophils by activating NF-κB pathway through HMGB1/TLR4 interaction. Activated neutrophils, in turn, promoted gastric cancer cell migration in vitro. Our findings reveal a novel mechanism for neutrophil modulation in the tumor milieu and provide new evidence for the important roles of exosomes in tumor microenvironment.
Inflammation in tumor microenvironment is a hallmark of cancer. Immune cells can be modulated by tumor signals to acquire tumor-promoting phenotype. The previous studies have shown that neutrophils could be redirected to a pro-tumor phenotype at the late stage of tumor progression . The presence of neutrophils in tumors is considered as an independent and unfavorable factor for the prognosis of gastric cancer patients . Wu et al. demonstrated that the number of neutrophils infiltrated in gastric cancer tissues was positively associated with lymph node metastasis. Furthermore, the supernatant from gastric cancer cells induced IL-1β and TNFα expression in neutrophils and prolonged the half-life of neutrophils . In consistent with this report, we found that gastric cancer cell-derived conditioned medium protected neutrophils from spontaneous apoptosis and induced IL-1β and TNFα expression, among other inflammatory factors. Although tumor-derived HA (hyaluronan) fragments can mediate neutrophil activation [18, 31], we showed in the current study that gastric cancer cell-derived exosomes could induce neutrophil activation, suggesting that both soluble factors and non-soluble extracellular vehicles produced by tumor cells could induce neutrophil activation. Moreover, neutrophils activated by gastric cancer cell-derived exosomes highly express several inflammatory factors (such as IL-1β and OSM) that have been previously shown to promote cancer cell migration and invasion [18, 32], suggesting that the activated neutrophils may promote gastric cancer metastasis by releasing these factors.
Autophagy, a mechanism for intracellular degradation and energy recycling, is emerging as an important regulator of immune responses. Autophagy has been linked to the generation, expansion, and function of neutrophils. Autophagy deficiency reduces degranulation in neutrophils, suggesting the requirement of autophagy for neutrophil-mediated inflammation . In addition, autophagy is essential for intracellular bacterial killing by human neutrophils . Kimmey et al. suggest that stimulating autophagy in neutrophils increases bacterial killing but inhibiting autophagy increases bacterial survival , indicating that autophagy is essential for the function of neutrophils. It has been reported that granulocyte-colony stimulating factor (G-CSF) activates autophagy in neutrophils and G-CSF-induced neutrophil expansion is impaired in the absence of autophagy . More recently, Li et al. suggest that increased autophagy sustains the pro-tumor effects of neutrophils in human hepatocellular carcinoma . Interestingly, Dutta et al. demonstrate that exosomes from breast cancer cells induce autophagy in primary mammary epithelial cells, which in turn, produce factors to promote breast cancer cell growth . We found that exosomes from gastric cancer cells induced autophagy in neutrophils, leading to the release of factors that promoted the migration ability of gastric cancer cells. Thus, our results, together with findings from the others, indicate that tumor-derived exosomes may regulate neutrophil phenotype and function by inducing autophagic activation.
Interaction of HMGB1 and TLR4 is involved in infection, tissue injury, and cancer . The previous studies have shown that UV irradiation-damaged skin cells produce HMGB1 that recruits and activates neutrophils, promoting angiogenesis and melanoma metastasis . In addition, tumor cell-derived HMGB1 mediates tumor cell-platelet interaction to promote metastasis . HMGB1 is shown to be generated in a vesicle form in monocytes and tumor cells [40, 41]. Activated platelets present HMGB1 to neutrophils, inducing autophagy and promoting the formation of neutrophil extracellular traps . In this study, we found that HMGB1 was present in GC-Ex. Inhibition of HMGB1/TLR4 interaction suppressed GC-Ex-induced pro-tumor activation of neutrophils, supporting that HMGB1 is a key factor for the roles of GC-Ex. However, this does not necessarily exclude other factors in GC-Ex that may contribute to neutrophil activation. Finally, overexpression of HMGB1 is reported to be associated with adverse prognosis in cancer patients . Indeed, we also found that increased HMGB1 expression in gastric cancer patients was associated with poor outcomes. Nonetheless, large cohort studies are warranted to determine the potential of exosomal HMGB1 as a biomarker for gastric cancer diagnosis and prognosis.
Tumor-derived exosomes can promote tumor growth by regulating immune cell phenotype and function . For instance, tumor-derived exosomes act on T cells [45, 46], NK cells [12, 47], macrophages [48, 49], dendritic cells , and myeloid-derived suppressor cells (MDSCs)  to induce an immunosuppressive microenvironment. Exosomes from ovarian cancer cells polarize macrophages to an M2 phenotype that, in turn, promotes ovarian cancer growth and metastasis . Similarly, neutrophils can be polarized to an N2 phenotype by tumor-derived factors in murine tumor models . The N2-polarized neutrophils display pro-metastatic and immunosuppressive activities [53–55]. Although polarization of neutrophils in human cancers has not been well characterized, we showed in this study that exosomes from human gastric cancer cells induced neutrophils to represent an N2-like phenotype, supporting a role of tumor-derived exosomes in neutrophil polarization.
Neutrophils in the circulation can be divided into high density neutrophils (HDN) and low density neutrophils (LDN). In various diseases including cancer, the frequency of LDN increased . Sagiv et al. suggest that LDN can be induced from HDN by TGF-β stimulation to promote cancer progression . It should be noted that in this study, HDN were isolated whereas low density neutrophils (LDN) were excluded due to the density gradient preparation method we used. The effects of tumor derived exosomes on the phenotype and function of LDN will be tested in future studies.
In conclusion, we demonstrate in this study that gastric cancer cell-derived exosomes induce autophagy and pro-tumor activation in neutrophils through the HMGB1/TLR4/NF-кB signaling pathway, which finally promotes the proliferation and migration of gastric cancer cells (Fig. 7i). These findings provide new insight into the mechanisms by which neutrophils are regulated in tumor microenvironment and contribute to tumor growth and metastasis, and will permit the development of new strategies for gastric cancer diagnosis, prognosis, and therapy.
Patients and biopsy specimens
Fresh tissue specimens were obtained from patients with GC who underwent surgical resection at the Affiliated People’s Hospital of Jiangsu University between April 2016 and December 2016. None of these patients had received chemotherapy or radiotherapy before surgery. Patients with infectious diseases, autoimmune disease or multi-primary cancers were excluded. The study was approved by the ethics committee of Jiangsu University and informed consent was obtained from all patients.
Human gastric cancer cell lines BGC-823, MGC80-3, SGC-7901, and HGC-27 were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) at 37 oC in humidified air with 5% CO2. When cells reached 80% confluence, medium was replaced with serum-free medium. Following a 24-hour incubation, medium was collected, centrifuged to remove cell debris, and stored at -80 oC in aliquots as conditioned medium (CM).
Neutrophil isolation and treatment
Peripheral blood was collected from healthy volunteers after obtaining written informed consent. The study was approved by the ethics committee of Jiangsu University. Neutrophils were isolated by using Polymorphprep (Axis-Shield PoC AS, Norway) as previously described . RBCs were lysed using hypotonic lysing procedure. The purity of neutrophils was 98% after this procedure. Neutrophils were seeded in RPMI 1640 (Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin at a density of 1×106 per well and treated with or without CM or exosomes from gastric cancer cells for 12 h. For experiments that use inhibitors, cells were pretreated with inhibitors for 1 h prior to adding CM or exosomes. The inhibitors used in this study include autophagy initiation inhibitor 3-methyladenine (3-MA, 5 mM), autophagosome degradation inhibitor chloroquine (CQ, 20 μM), HMGB1 inhibitor Glycyrrhizin (Gly, 10 μM), TLR4 antagonist TAK-242 (TAK, 10 μM), ERK inhibitor U0126 (10 μM), NF-κB inhibitor Bay11-7082 (10 μM), and STAT3 inhibitor WP1066 (10 μM). For HMGB1 treatment, cells were treated with recombinant human HMGB1 (10 μg/ml; Biovision, Shanghai, China) for 12 hours.
Exosomes were isolated from the conditioned medium of gastric cancer cells as previously described . In brief, cells were cultured in exosome-depleted medium and the supernatants were collected after 48 h. The conditioned medium was centrifuged at 1,000 g for 10 min to remove cell debris followed by 30 min at 10,000 g using 100 KDa MWCO before the concentrated solutions were filtrated through a 0.22-μm pore filter (Millipore, Shanghai, China). Exosomes were precipitated by adding the exosome quick extraction solution (System Biosciences, Palo Alto, CA, USA) at a ratio of 1:5 at 4 oC for 12 h. Exosomes were dissolved with PBS and stored at -80 oC. Protein concentration was determined by BCA protein assay kit (ThermoFisher Scientific, Shanghai, China). The size and concentration of exosomes were measured by Nanoparticle Tracking Analysis (NTA). The morphology of purified exosomes was identified by transmission electron microscopy (Tecnai 12; Philips) and the expression of exosomal markers CD9 and CD81 by western blot.
Exosome samples (250 μg) were lysed in STD buffer (4% SDS, 100 mM Tris/Hcl, and 1 mM dithiothreitol, pH 7.6) and centrifugated at 1000 g for 10 min to collect the supernatants. Proteins were identified using Q Exactive Orbitrap LC-MS/MS system (ThermoFisher Scientific).
Tissue array was purchased from Shines Pharmaceuticals (Shanghai, China). A total of 76 pairs of tumor tissues and non-tumor tissues were included in the tissue array. Tissue array was incubated with antibody against HMGB1 (Cell Signaling Technology). Immunohistochemical staining was performed as described elsewhere. IHC scoring was assessed by two pathologists in a double-blinded manner.
The supernatants from gastric cancer tissues and adjacent non-cancerous tissues were collected for ELISA. The concentrations of HMGB1 in tissue supernatants were determined by using ELISA kit according to the manufacturer’s instructions (Chondrex, Redmond, WA, USA).
ROS detection assay
Neutrophils treated with or without CM or exosome from gastric cancer cells for 12 h were collected and resuspended in serum-free medium. Cells were stained with DCFH-DA (10 μM; Beyotime Biotechnology, Shanghai, China) at 37 oC for 30 min and subjected to analyses of florescence intensity by flow cytometry.
Real-time quantitative PCR
Total RNA was extracted from cells using Trizol reagent (Thermo Fisher Scientific) and 1 μg of RNA was reverse transcribed to cDNA by using reverse transcriptase (Vazyme). Real-time quantitative PCR was performed by using the SYBR Green I real-time detection kit (Cwbio, Beijing, China) on a Bio-Rad CFX96 Detection System. The relative gene expression was normalized to β-actin. The primers for target genes were listed in Additional file 10: Table S1.
The siRNA against HMGB1 was produced by Genepharrm (Suzhou, Jiangsu, China). The sequences of HMGB1 siRNA and the scramble control were shown in Additional file 11: Table S2. BGC-823 cells (1×105 cells/well) were grown in 6-well plates and transfected with siRNAs by using LipoFiter transfection reagent (Hanbio, Shanghai, China) for 36 hours.
Cells were lysed in RIPA buffer containing proteinase inhibitors. Equal amount of proteins was separated by a 12% SDS-PAGE gel. Following electrophoresis, proteins were transferred to a PVDF membrane, blocked in 5% non-fat milk, and incubated with primary antibodies at 4 oC overnight. Antibodies for ERK1/2, p-ERK1/2, NF-кB p65, p-p65, STAT3, p-STAT3, p-p38, p38, p-Akt, Akt, LC3, CD9, CD63, Alix, and TSG101 were purchased from Cell Signaling Technology (Louis Park, MN, USA). After washing with TBST for three times, membrane was incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Bioworld Technology) at room temperature for two hours. The protein bands were visualized by enhanced chemiluminescence. GAPDH served as the loading control.
Cell apoptosis assay
Neutrophil apoptosis was analyzed by using an Annexin V apoptosis detection kit according to the manufacturer’s instructions (Invitrogen). The binding of Annexin V-FITC and PI to the cells was analyzed on FACS Calibur (BD Biosciences, NJ, USA) by using Cell Quest software.
For transmission electron microscopic analysis, neutrophils treated with or without CM or exosome from gastric cancer cells were washed and fixed in 4% glutaraldehyde, followed by post-fixation in 2% osmium tetroxide. Thereafter, cells were dehydrated, treated with propylene oxide, and embedded. The sections were subsequently stained with uranyl acetate and lead citrate and examined in a Tecnai 12 transmission electron microscopy. For immunofluorescent staining, neutrophils treated with or without CM or exosomes were stained with an autophagy detection kit (Enzo Lifesciences, NY, USA) and analyzed by Cytasion 3 cell imaging multi-mode reader (BioTek, Shanghai, China).
Transwell migration assay
Cell migration assay was performed in a 24-well Boyden chamber with an 8-μm pore size polycarbonate membrane (Corning, Union City, CA, USA). Cancer cells (2×104 in 100 μl of serum-free medium) were added into the upper chamber with 600 μl of supernatant from neutrophils in the lower chamber. After a 12-h incubation, cancer cells on the upper surface of the membrane were removed. The migrated cells on the lower surface of the membrane were fixed by paraformaldehyde, stained with crystal violet, and counted under a microscope.
Tube formation assay
Neutrophils were treated with or without CM or exosome from gastric cancer cells for 12 h, followed by washing with PBS once and culturing in fresh medium for additional 12 h. The supernatant from activated neutrophils were collected and filtered through a 0.22 μm filter. Human umbilical vein endothelial cells (HUVEC) were seeded at 2×104 cells/well and incubated with or without conditioned media from neutrophils at 37 oC for 12 h. The formation of tube-like structure by HUVECs were observed under a phase-contrast microscope and photographed at 100× magnification. The number of tubules from five random fields in each well was counted. The experiments were repeated for three times.
Gastric cancer cells were seeded at 4×103 cells/well and incubated with or without supernatant from the activated neutrophils. CCK8 reagent (10 μl; Vazyme, Nanjing, China) was added at 3 hours before the end of the experiments. The absorption was measured at 450nm in a microplate reader. The experiments were repeated for three times.
Tumor tissue-derived conditioned medium and exosomes
Fresh surgically removed gastric cancer tissues and adjacent non-cancerous tissues (at least 5 cm distant from the tumor site) were cut into 1 cm3 and placed in 1 ml serum-free RPMI 1640 medium for 24 h. The supernatants were centrifuged at 300 g for 10 min and filtered through a 0.22 μm filter and stored at -80 oC until use. Neutrophils were cultured in 50% T-CM or N-CM for 12 h. Tumor-derived exosomes were extracted from the conditioned medium as described in previous section.
Data were expressed as means ± SD from at least three independent experiments. The statistical significance of differences between groups was determined by two-tailed Student’s t test. Survival time was analyzed by Kaplan–Meier method and log-rank test. P<0.05 was considered statistically significant.
We thank the members of Zhang lab for excellent technical assistance and helpful discussion.
This work was supported by the National Natural Science Foundation of China (81672416, 81572075, 81670502), the Natural Science Foundation of the Jiangsu Province (BK20141303), Jiangsu Province’s Major Project in Research and Development (BE2015667, BE2017694), the Key Research and Development Project of Zhenjiang (SH2015034), Major Natural Science Reasearch Project for Universities in Jiangsu Province (18KJA320001), Jiangsu Province’s 333 project, Funds for Young Academic Leader of Jiangsu University, Startup Funds for Senior Talents of Jiangsu University (13JDG086).
Availability of data and materials
All of the data and material in this paper are available when requested.
XZ and WX: conception and design. XZ, HS and XY: development of methodology. XZ, HS and XY: acquisition of data. XZ, HS, PJ, HQ and WX: analysis and interpretation of data. XZ, HS and WX: writing, review, and revision of the manuscript. XZ, PJ, HQ, and WX: administrative, technical, or material support. XZ and WX: study supervision. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The use of clinical samples was approved by the ethics committee of Jiangsu University and informed consent was obtained from all patients.
Consent for publication
All of the authors are aware of and agree to the content of the paper and their being listed as a co-author of the paper.
The authors declare that they have no competing interests.
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