| Background and purposeGastric cancer is one of the most frequent gastrointestinal malignant tumors in China,and its morbidity and mortality rate take the 2nd and 3rd place among all malignant tumors,respectively,with the proportion of advanced stages as high as 80% and the 5-year survival rate less than 10%.Peritoneal metastasis of gastric cancer and its combined malignant ascites are one of the main causes of death of gastric cancer patients.At present,the clinical treatment effect of peritoneal metastasis of gastric cancer is poor,and the exact mechanism has not been fully elucidated,and further research on the molecular mechanism of peritoneal metastasis is urgently needed.Compared with gastric cancer,ovarian cancer ascites is often treated as a typical object of study of the ascitic microenvironment of malignant tumors,which may be related to the special metastatic propensity of ovarian cancer.Existing studies suggest that the malignant ascites microenvironment is an immunosuppressive and metabolically disturbed microenvironment where various different non-tumor cells and cytokines play different roles on the tumor cells themselves,which directly contributes to the shorter survival of gastric cancer with peritoneal metastasis than ovarian cancer with peritoneal metastasis,and therefore it is of great importance to study the effect of ascites on the biological behavior of gastric cancer cells.Meanwhile,cell migration is a key process for ascites free cancer cells to swim out from the primary foci and then generate metastases on the peritoneum,so we aimed to investigate the effect of gastric cancer ascites supernatant on the migration ability of gastric cancer cells and to find possible molecular targets and downstream mechanisms.Research Methodology1.Clinical ascites specimens were collected: Ascites specimens from gastric cancer seen in our hospital from January 1,2021 to February 1,2023 were collected,and all patients had focal pathology reports confirming gastric cancer,along with peritoneal effusion,and tumor cells were detected in ascites exfoliative cytology tests,excluding patients with other malignant tumors and abdominal infections.All ascites specimens were collected before the patients received intravenous chemotherapy or peritoneal perfusion therapy.The cirrhotic ascites admitted to the infection department of our hospital during the same period were also collected as control ascites.2.In vitro cell experiments: The specimens were processed promptly within 24 hours after specimen acquisition,and the ascites supernatant was isolated.After 72 hours of treatment with gradient ascites supernatant,cell migration ability was detected by cell scratching and Transwell assay,and the expression of cell migration markers was detected by Real-time PCR and protein blotting assay(Western blot,WB).3.Vivo experiment in nude mice: a subcutaneous tumor-bearing model was constructed in nude mice,and ascites injection was regularly performed under the tumor-bearing skin.Tumor tissues were collected after 14 days for H&E and immunohistochemical staining,and the expression of ascites-promoted gastric cancer cell migration markers was verified in vivo.4.Ascites 4D-label Free proteomics: ascites supernatant from 4 non-malignant patients were selected as the control group and ascites supernatant from 4 malignant patients were selected as the experimental group for proteomic analysis,and protein content difference analysis and signaling pathway enrichment analysis were performed after quality control to identify proteins with high content in malignant ascites supernatant.5.ELISA of FGF family: The collected supernatant of malignant ascites of gastric cancer and control supernatant were subjected to ELISA to detect the content of FGF family,and three representative cases of malignant ascites and peripheral blood serum of gastric cancer patients were taken to detect the content of FGF21,to explore the content of FGF21 in different body fluid environments,and to analyze the expression of FGF21 and its receptors in TCGA gastric cancer patients by bioinformatics.6.Transcriptome sequencing of ascites supernatant: transcriptome sequencing analyzed the differences in gene expression of gastric cancer cells before and after treatment of gastric cancer cells with gastric cancer ascites supernatant,and found that the expression of FGF21 differed significantly.The expression of FGF21 was verified by q PCR and WB at the gene transcription level and protein level,respectively,while the changes of FGF21 content in the supernatant of gastric cancer cells cultured in vitro were detected by ELISA.7.Bioinformatics analysis and in vitro validation: Biosign analyzed the relationship between FGF21 receptor and cell migration markers,added recombinant human-derived FGF21 protein into the in vitro culture system of gastric cancer cells,and verified the expression of cell migration-related markers using scratch assay,Transwell assay,q PCR and WB.8.Expression knockdown assay: use small interfering RNA(si RNA)technology to down-regulate FGF21 expression in gastric cancer cells,q PCR and WB to detect the silencing efficiency,use Transwell assay to verify the effect of FGF21 down-regulation on cell migration,and detect the expression of cell migration markers by q PCR and WB.9.FGFR1/AKT signaling pathway mechanism study: verify the activation effect of FGF21 on AKT pathway and FGFR1 receptor activation by WB,detect the changes of protein expression and phosphorylation after down-regulating AKT pathway using AKT inhibitor GSK690693,and then verify the changes of cell migration ability by Transwell assay.Results1.A total of 27 ascites specimens from patients with gastric adenocarcinoma and 4ascites specimens from patients with non-gastric cancer were included.All patients with gastric adenocarcinoma had pathology reports confirming gastric cancer,and patients with non-gastric cancer had no cancer cells after 3 or more ascites exfoliation cytology examinations and no other clinicopathological features suggestive of tumor.All patients’ specimens were collected and used in compliance with the Declaration of Helsinki and approved by the Ethics Committee of Changhai Hospital.2.RT-PCR and WB experiments showed that gastric cancer malignant ascites supernatant could promote the expression of cell migration marker Fibronectin and inhibit the expression of cell adhesion protein E-cadherin,and Transwell assay confirmed that gastric cancer malignant ascites supernatant could promote the in vitro migration invasion of gastric cancer cells.3.In a nude mouse subcutaneous load tumor model,the injection of gastric cancer ascites supernatant could also promote the expression of Fibronectin and inhibit the expression of cell-linked E-cadherin.4.Ascites proteomics showed that the protein species in malignant ascites supernatants were significantly different compared to non-malignant ascites supernatants,and the differential proteins were mainly enriched in pathways such as glucose metabolism,lipid response,protein response,calcium mucin linkage,antioxidant response and metal ion response.Moreover,FGF21,which regulates glucose and lipid metabolism,was higher in malignant ascites supernatant.5.ELISA results showed that the content of FGF21 was higher in the ascites supernatant of gastric cancer patients and FGF21 aggregated in the ascites of gastric cancer patients,while the content of FGF21 was lower in both peripheral blood serum and non-malignant ascites supernatant of non-gastric cancer patients.6.By transcriptome sequencing,gastric cancer ascites supernatant can also promote the secretion of FGF21 by gastric cancer cells,suggesting that tumor cells themselves are involved in the formation of high FGF21 in the malignant ascites environment of gastric cancer.7.Bioinformatics analysis showed that FGF21 receptor expression and migration gene expression were positively correlated,and gene expression of cell adhesion was negatively correlated,and the difference was statistically significant.And the promotion effect of FGF21 on cell migration ability was verified by scratch assay,Transwell assay,q PCR and WB on in vitro cell lines.8.When FGF21 expression in gastric cancer cells was down-regulated by using small interfering RNA,Transwell assay showed that migration of gastric cancer cells was attenuated,FGFR1 phosphorylation was decreased,E-cadherin expression was increased and Fibronectin expression was decreased.9.FGF21 can promote tumor cell migration through FGFR1/AKT signaling pathway.After adding AKT pathway inhibitor GSK690693 to the culture system,AKTSer473 site phosphorylation was inhibited,and the role of FGF21 in promoting gastric cancer cell migration through FGFR1/AKT pathway was partially inhibited.ConclusionGastric cancer ascites supernatant can promote migration of gastric cancer cells.Gastric cancer ascites supernatant can cause gastric cancer cells to secrete FGF21 by themselves,which promotes tumor cell migration through FGFR1/AKT pathway. |
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