FN1 Drives The Trans-differentiation Of Peritoneal Mesothelial Cells To Promote The Progression Of Gastric Cancer

Objective: The presence of free cancer cells in the abdominal cavity in patients with early gastric cancer has caused specific clinical diagnosis and treatment difficulties.It was previously believed that gastric cancer had to infiltrate the serosa before there was a possibility of shedding and implanting into the peritoneal cavity.Still,recent studies have found that some early gastric cancer patients with infiltration not reaching the serosa have free cancer cells in the abdominal cavity.Peritoneal mesothelial cells(PMCs)are a thin layer covering the peritoneum’s surface,which can respond to changes in the microenvironment and differentiate into other cell types under various pathological and physiological conditions.The relationship between PMCs and the progression of gastric cancer is still unclear.Some studies have suggested that overcoming the mechanical barrier of PMCs is necessary for gastric cancer to break through the gastric wall and further spread in the peritoneal cavity,while others have suggested that PMCs can promote the local progression and peritoneal dissemination of gastric cancer in various ways.The present study aims to further explore the role of PMCs in the progression of gastric cancer and the corresponding molecular mechanisms.It will provide a new perspective to slow down local invasion of gastric cancer,block peritoneal implantation metastasis and optimize the clinical therapy process.Methods:(1)PMCs and gastric cancer cells NUGC4/MKN45 were co-cultured.CCK8,Transwell,and clone formation experiments were used to detect PMCs’ and gastric cancer cells proliferation and migration ability.Flow cytometry was used to detect NUGC4 apoptosis and cell cycle changes.(2)Gastric cancer specimens were collected,and immunohistochemical staining was used to observe whether PMCs can differentiate into cancer-associated fibroblasts(CAFs).Validation was performed at the molecular level through RT-PCR and Western blot.(3)PMCs were collected that underwent morphological changes after co-culture with NUGC4,and transcriptome detection was performed using second-generation sequencing technology.Proteome detection was performed using label-free technology,and key driver genes for PMCs to differentiate into CAFs were identified through comprehensive bioinformatics analysis.(4)CRISPR/Cas9 technology was used to establish PMCs cell line with FN1 gene knockout(PMCs＿KO).FN1 wild type(PMCs＿WT),or PMCs＿KO cells,were cocultured with NUGC4.CCK8,Transwell,and clone formation experiments were used to observe NUGC4 proliferation and migration ability changes.Flow cytometry was used to detect changes in the NUGC4 cell cycle.Western blot was used to verify the molecular mechanism of PMCs phenotype conversion and enhancement of NUGC4 function after differentiation into CAFs.Further validation was performed at the level of nude mice.(5)Gastric cancer tissue microarray chip with 94 cases was established for FN1 immunohistochemical staining,and the relationship between FN1 expression characteristics and clinical pathology was analyzed.Results:(1)PMCs promote apoptosis and inhibit the proliferation and migration of gastric cancer cells after 24 hours of co-culture,while after seven days of co-culture,PMCs promote the proliferation and migration of gastric cancer cells.(2)In gastric cancer tissues,a cell population co-expressing the mesothelial marker Calretinin and the CAFs marker α-SMA was observed in the tumor stroma.After co-culturing with NUGC4 for seven days,the expression of Calretinin was down-regulated,while the expression of the stromal marker vimentin and the CAFs marker α-SMA was upregulated in PMCs.(3)Through transcriptome and proteomic analysis,FN1 was identified as the key driving gene for transforming PMCs into CAFs.(4)After coculturing with NUGC4 for seven days,PMCs＿KO maintained the mesothelial phenotype and significantly inhibited gastric cancer cells proliferation and migration ability.PMCs＿KO or PMCs＿WT co-cultured with NUGC4,and each NUGC4 was inoculated subcutaneously into nude mice;the tumorigenic ability of the PMCs＿KO co-cultured group was significantly weakened.After transforming into CAFs,PMCs promote gastric cancer progression through the secretion of FN1 and reaction with the FAK/PI3K/AKT pathway.(5)The epithelial type of FN1 is associated with OS in gastric cancer,while the stromal type of FN1 is associated with tumor progression.Conclusions: PMCs promote gastric cancer progression after transforming into CAFs,while inhibiting cancer progression when maintaining their mesothelial cell phenotype.FN1 is the key driver gene for PMCs to transform into CAFs.The transdifferentiation of PMCs into CAFs promotes gastric cancer progression through FN1 via FAK/PI3K/AKT pathway.