N2-neutrophils Promote Invasion and Metastasis of Ovarian Cancer by Upregulating MAPK Signaling

Background: Metastasis is an important factor of high mortality in ovarian cancer. Neutrophils are involved in multiple pathologic mechanisms of cancer,including invasion and metastasis. However, the relationship of neutrophils and invasion and metastasis in ovarian cancer is unclear,as well as the exact mechanism. Methods:To verify the relationship of neutrophils and invasion and metastasis in ovarian cancer,we tested the expression of CD11b in 24 groups of benign and malignant ovarian tumor tissues.And then,we tested the expression of CD11b,CXCL8,and CXCR1 in 16 cases of ovarian cancer,including primary lesions, metastatic lesions and adjacent carcinoma tissues.We successfully build tumor associated neutrophils research model (N1 and N2) and prove that N2-neutrophils can promote the invasion and metastasis of ovarian cancer. Next,we screened the signicantly changed MAPK signaling pathway by high-throughput sequencing. And then conrmed this conclusion by molecular biology experiments. Results:The expression of CD11b was signicantly higher in malignant tumor than benign tumor tissues tested by western blot and Immunohistochemistry.The expression of CD11b,CXCL8 and CXCR1 is highest in ovarian cancer metastases ,followed by the primary lessions, and then the adjacent carcinoma tissues tested by PCR and WB methods.We proved that N2-neutrophils can promote the invasion and metastasis of ovarian cancer by transwell assay.Forthermore,we detected the related indicators of metastasis including MMP-2,MMP-9,E-Cadherin,N-cadherin and Vimentin by PCR and WB methods.Next,we screened the signicantly changed MAPK signaling pathway by High-throughput sequencing through comparing ovarian cancer cells before and after co-cultured with N2-neutrophils. At last,we found the key gene P38 of MAPK signaling pathway by molecular biology experiments. Conclusions: N2-neutrophils promote the invasion and metastasis of ovarian cancer by Upregulating MAPK signaling pathway, nd a key gene P38.


Introduction
Ovarian cancer is one of the common malignant tumors in the female reproductive system, and the mortality rate ranks rst among all kinds of gynecological tumors. More than 20,000 women worldwide are diagnosed with ovarian cancer each year 1 . In the past 30 years, the clinical outcome of ovarian cancer has not improved signi cantly, and the overall 5-year survival rate of patients is 25% to 30% 2 .
Ovarian cancer lacks typical clinical features and speci c diagnostic indicators in the early stage.It is often asymptomatic before metastasis.Most patients are already in advanced stage (FIGO III-IV stage) when diagnosed. Even accepted timely surgery and chemotherapy, only a small percentage of patients have achieved some relief, and most patients in advanced stage will relapse within 18 months. Many ovarian cancer patients experienced chemotherapy resistance after receiving multiple chemotherapy, which can not control local, distant metastasis effectively, leading to death 3 .
In condition of acute and suppurative infections, various poisoning, tissue damage, malignant tumors, etc., the number of neutrophils will increase signi cantly 4 . CD11b positive cells are granulocytes, mainly neutrophils, followed by macrophages. In mouse tissues, CD11b+ Ly6G+ cells are neutrophils, while CD11b+ Ly6G− cells are macrophages 5 . CXCL8,also known as IL-8, is a small molecule peptide secreted by macrophages,T cells, neutrophils and epithelial cells. CXCR1 and CXCR2 are co-ligands of CXCL8 and are mainly expressed on the surface of neutrophils 6 .
Neutrophils are highly mobile, and are rapidly recruited to the tumor microenvironment in the early stages of the tumor. We refer to neutrophils in ltrating in the tumor microenvironment as tumor-associated neutrophils (TANs). As early as 2009, Fridlender et al. demonstrated the existence of two different forms of tumor-associated neutrophils 5 . In the early stage of tumorigenesis, neutrophils chemotaxis to malignant sites, inhibiting the occurrence and development of tumors through cytotoxicity and immune activation, we de ned this group as N1-neutrophils. In the middle and late stages of the tumor, neutrophils accumulate in the tumor stroma.The phenotypic and functional characters changed under the action of factors produced by tumors and microenvironment cells. This type of cells can promote tumor cell proliferation, migration, invasion and angiogenesis, and inhibit the body's anti-tumor immunity, which was de ned as N2-neutrophils 7 . The plasticity of TAN is regulated by various cytokines in the tumor microenvironment. Studies have shown that N2-neutrophils can promote the development of tumor cells, participate in angiogenesis, and enhance the invasion and metastasis of tumor cells by secreting various cytokines and chemokines, biological agents such as NE, MMP-9 and ROS,presenting a tumor-promoting effect [8][9][10] .
TANs has been con rmed to be associated with the development and prognosis of various malignant tumors in humans, such as kidney cancer, liver cancer, gastric cancer, pancreatic cancer, colon cancer, malignant melanoma, cholangiocarcinoma, and uterine cancer [11][12][13][14][15][16] . However, there are few studies on neutrophils and ovarian cancer. The main focus of research in the past ve years on neutrophillymphocyte ratio (NLR) can be used to assess the prognosis of patients with ovarian cancer [17][18][19] . The relationship between neutrophils and invasion and metastasis of ovarian cancer has rarely been reported 20,21 , and the exact molecular mechanism is still unclear. This article intends to con rm the relationship between neutrophils and invasion and metastasis of ovarian cancer.Furtherly,we strive to seek for its possible molecular mechanism to explore new potential therapeutic targets for ovarian cancer.

Patients and specimens
All clinical pictures and tissue specimens were taken from the sample library of the Tenth People's Hospital a liated to Tongji University (2012-2016). The patients were enrolled with informed consent, and the specimens were collected and approved by the hospital ethics committee. All patients were con rmed by pathology. No treatment measures such as radiotherapy and chemotherapy were received before surgery, and other systemic neoplastic diseases were excluded at the same time.
Cell culture and plasmid transfection SKOV3 cells purchased from ATCC and cultivated in McCoys medium(Biochrom AG, Berlin, Germany) containing 10% fetal bovine serum at 37°C in a 5% CO2 humidi ed atmosphere. OVCAR3 cells purchased from ATCC (Manassas, Virginia, USA) were cultivated in RPMI medium (Invitrogen) containing 10% fetal bovine serum, .HL60 cells purchased from were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China)and cultivated in RPMI medium (Invitrogen) containing 10% fetal bovine serum which was induced by1.25%DMSO.TGF-b-RNAi lentiviral vectors were purchased from Shanghai GeneChem Company (Shanghai, China). and the shRNA control sequence was 5'-GGACTATCCACCTGCAAGA -3'.

RNA extraction and real-time PCR
Total RNA was extracted using Trizol(Invitrogen, 15596-018). First strand cDNA synthesis was performed using a reverse transcription kit (TransGen Biotech, Beijing, China) and the real-time PCR analyses were conducted on an iQ5 Multicolor Real-Time PCR Detection System(Bio-Rad, Hercules, CA, USA) using the SYBR Green dye (Roche Diagnostics, Mannheim, Germany). The GAPDH gene was used as an internal control, and the data are shown as the fold change. The experiment was performed in triplicate. Primers for MMP-2,MMP-9,E-Cadherin,N-cadherin,Vimentin and GAPDH are shown in Table 1.

Neutrophils in ltration in ovarian cancer
It is observed that ovarian cancer metastasis is common in the peritoneum, intestine, mesentery, and omentum surface. Late ovarian cancer often shows ascites, local necrosis of cancer, and similar purulent infection(Figure1A-D) It is well known that there is a large amount of neutrophils in the infection area when in ammation occurs. We examined the expression of neutrophil marker CD11b in 24 pairs of ovarian benign (serosal cystadenoma) and malignant tumors (serous cystadenocarcinoma) by western blot and immunohistochemistry. The western blot showed that the expression of CD11b in ovarian malignant tumor tissues was signi cantly higher than that in ovarian benign tumor tissues(Figure2A).
Immunohistochemical examination revealed that there were few neutrophils in benign ovarian tumor tissues; neutrophils were distributed in malignant tumor tissues, and the proportion of positive cells was more than 50%. Through statistical analysis, it is suggested that the condition of neutrophils in ltration were signi cant different between the two groups.(p≤ 0.001) (Figure2B).These results demonstrate that neutrophil in ltration is closely related to ovarian cancer.
We selected 16 pairs of ovarian serous carcinoma samples (including primary lesions, metastatic lesions, and adjacent tissues), and detected mRNA and protein expression of neutrophil CD11b, CXCL8 and CXCR1 by PCR and WB methods. The results showed that these indicators were the highest in ovarian cancer metastasis tissues, followed by primary tumor tissues, and the expression of adjacent tissues was the lowest (Figure2C).This result suggests that neutrophils may have a relationship with ovarian cancer metastasis.

N2 -neutrophils promote invasion and metastasis of ovarian cancer cells
We then applied the in vitro experiment to con rm the above human clinical data. Using HL60 cells as the initial cells, a TGF-b stable knockout cell line was constructed(HL60-si TGF-b). HL60-si TGF-b cells and HL60 cells were induced with 1.25% DMSO for 5 days to construct TAN cell models named N1-neutrophil and N2-neutrophil separately. Cell models was veri ed by western blot (Figure3A).First, We selected two ovarian cancer cell lines SKOV3 and OVCAR3 , co-cultured with N2-neutrophils for 48 hours, and then examined the changes of cell invasion and metastasis ability in the control and treatment groups. The results showed that the invasive and metastatic ability of ovarian cancer cells was signi cantly enhanced after co-culture with N2-neutrophils(Figure3B-C). Next,We selected a well-cultured ovarian cancer cell line OVCAR3 and OVCAR3-N2 (OVCAR3 co-cultured with N2-neutrophils for 48 hours) to extract mRNA and protein, and detected the expression of metastasis-related indicators.Results showed that the expression level of the these indicators signi cantly changed after co-cultured with N2-neutrophils(Figure3D-E).The above data prove that N2-neutrophils can promote the invasion and metastasis of ovarian cancer cells.

Mechanism of N2-neutrophils promoting the invasion and metastasis of ovarian cancer
In order to clarify the molecular mechanism by which N2-neutrophils promote invasion and metastasis of ovarian cancer, we performed high-throughput sequencing. The well-cultured cell line OVCAR3 (control group) and OVCAR3-N2 (treatment group) were selected to extract mRNA,and then perform highthroughput sequencing to analyse differentially expressed genes.Heatmap results are showed (Figure4A). Path analysis of the differentially expressed genes detected the signi cantly changed MAPK signaling pathway. The results are as follows (Figure4B).
MAPK is an important transmitter of signal transduction from the cell surface to the interior of the nucleus. MAPK subfamilies include ERK, p38, JNK and so on.We selected two ovarian cancer cell lines SKOV3 and OVCAR3 , co-cultured with N2-neutrophils for 48 hours, and then examined the changes of key factors of MAPK signaling pathway in the control and treatment groups by western blot method and p-P38 was detected(Figure5A). SB-202190 is a highly selective, potent and cell-permeable inhibitor of p38 MAP kinase. We added SB-202190 to the experimental system and then detect the expression of p-P38 by western blot methods.The results showed that p-P38 expression was signi cantly increased after co-culture with N2-neutrophils compared with the mono-inhibitor group(Figure5B).The above data con rmed that N2-neutrophils can promote ovarian cancer invasion and metastasis by upregulating the phosphorylation level of P38, a key factor in MAPK signaling pathway.

Discussion
In recent years, the mechanism of action of neutrophils in tumor metastasis has emerged in an endless stream. Neutrophils can promote tumor cell metastasis by degrading extracellular matrix and promoting tumor cell clonal formation 22

Conclusion
The expression of neutrophils in ovarian malignant tumors was signi cantly higher than that of ovarian benign tumor tissues tested by WB and immunohistochemistry. In vitro experiments show that the ability of ovarian cancer cells to invade and metastasize is signi cantly enhanced after co-culture with N2 neutrophils. Further, we analyzed the MAPK signaling pathway with signi cant changes through high sequencing analysis, and screened the key gene P38. From the neutrophil level, targeting P38, we hope to provide a new solution for the treatment of ovarian cancer.
Declarations Figure 1 Clinical features of ovarian cancer metastasis:ovarian cancer metastasis is common in the peritoneum A , intestine surface(B). Late ovarian cancer often shows ascites(C)and similar purulent infection(D)  after co-cultured with N2 neutrophils was assesed by PCR and WB. The expression of E-cadherin was signi cantly reduced after co-cultured with N2 neutrophils.While the expression of Ncadherin,Vimentin,MMP2 and MMP9 was signi cantly increased.

Figure 4
High-throughput sequencing seek for differentially expressed genes in the treatment group (OVCAR3-N2) and the control group (OVCAR3).(A)Gene heatmap was showed. Data analysis showed more than 700 differentially expressed genes, of which 73 were more than 3 times and 13 were more than 5 times. (B)Pathway analyses showed a signi cant up-regulation of MAPK signaling levels.