N2-neutrophils promote invasion and metastasis of ovarian cancer by upregulating MAPK signaling

Abstract

Background Neutrophils is an important immune cell in microenvironment of various cancers. Previous studies reported that neutrophils were associated with the development and prognosis of various malignant tumors, but how different phenotypic and functional heterogeneity of neutrophils exert in regulating invasion and metastasis of ovarian cancer were rare reported. Objective This study aimed to verify functions and mechanisms of different phenotypes of neutrophils in ovarian cancer invasion and metastasis. Methods The expression of CD11b, one of the most important biomarkers of N2-neutrophils, was assessed by immunohistochemistry and western blot (WB) in 24 benign and malignant clinical ovarian cancer tissues respectively. 48 samples from 16 ovarian serous carcinoma patients were averagely divide into adjacent tissues, primary lesions and metastatic lesions groups, detecting the mRNA and protein expression levels of neutrophil biomarkers CD11b, CXCL8 and CXCR1 by reverse-transcription quantitative PCR (qRT–PCR) and WB. Tumor associated N1 and N2 neutrophils phenotypes research models were constructed. Invasion and metastasis changes of ovarian cancer cells were assessed by transwell assay and wound-healing test with or without N1/N2 neutrophils co-culturing. The roles and mechanisms of N2-neutrophils in promoting ovarian cancer progression were probed by high-throughput RNA sequencing and gain- and loss-of-function analysis. Results The expression of CD11b was significantly increased in malignant tissue groups compared with benign groups. The expression levels of CD11b, CXCL8 and CXCR1 were the highest in groups of metastatic lesions, followed by the primary lesions, and the lowest in the adjacent tissues. N2-neutrophils co-cultured with ovarian cancer cell lines (SKOV3 and OVCAR3) could promote the invasion and metastasis of ovarian cancer cell lines, but N1-neutrophils played the opposite role. MAPK signaling pathway was significantly enhanced in ovarian cancer cells by phosphorylating of P38 after co-culturing with N2-neutrophils and the p-P38 mono-inhibitor was able to reverse this function. Conclusions N2-neutrophils promoted the invasion and metastasis of ovarian cancer by upregulating MAPK signaling through phosphorylating the P38.