Gastric Cancer Circulating Tumor Cells Phenotype And The Role Of IGF-1R Pathway In Gastric Cancer EMT

Part One Gastric Cancer Circulating Tumor Cells Phenotype and its Correlation with Clinicopathologic Features OBJECTIVES: To detect circulating tumor cells（CTCs）in peripheral blood of gastric cancer patients and classify CTCs into epithelial,mesenchymal and mixed（both epithelial and mesenchymal）phenotypes,and investigate the correlation between CTCs and clinicopathological characteristics and prognosis of gastric cancer patients.METHODS: A single-center prospective observational study was established using the REDCap（Research Electronic Data Capture）.Peripheral blood were collected from gastric cancer（GC）patients for CTCs detection before and 3months after treatment.The Can Patrol^TM assay was used to detect and classify CTCs into EMT phenotypes,and survival was obtained through follow-up.The relationship between the CTCs and clinical features and prognosis was analyzed.RESULTS: A total of 60 patients with GC were included in this study.CTCs were detected in 56 patients before treatment,and the detection rate was 93.3%.A total of 518 CTCs were detected,including 87 epithelial CTCs（E-CTCs）（16.8%）,139 mesenchymal CTCs（M-CTCs）（26.8%）,and 252epithelial-mesenchymal（EM-CTCs）CTCs（56.4%）.The percentage of EM-CTCs and M-CTCs were significantly higher than E-CTCs（P<0.05）.There were no significant differences in the detection rate of CTCs and its subtypes between subgroups of different age（≥60 vs <60）,gender（male vs female）,lymph node metastasis（positive vs negative）,Lauren classification（intestinal vs diffuse vs mixed）,and differentiation（well-moderately differentiated vs poorly-undifferentiated）（all P>0.05）.The total number of CTCs and M-CTCs in the distant metastasis groups were higher than those in the no-metastasis group,and the difference between the groups was statistically significant（P<0.05）.In patients with negative E-CTCs or EM-CTCs>7,the prognosis is poor.Univariate analysis suggested that the detection of E-CTCs and distant metastasis could significantly affect overall survival（all P<0.05）.And multivariate analysis found that positive detection of E-CTCs（HR=0.060,P=0.049）,lymph node metastasis（HR=13.693,P=0.040）and distant metastasis（HR=39.761,P=0.006）were independent factors for overall survival.CONCLUSIONS: Epithelial-mesenchymal transition is present in gastric cancer CTCs.There is more EM-CTCs and M-CTCs than E-CTCs in peripheral blood,suggesting that most CTCs have undergone EMT in GC patients.The total number of CTCs and the number of M-CTCs in gastric cancer patients can reflect the severity of the disease.Negative E-CTCs is closely related to poor prognosis of patients with GC.It is an independent prognostic indicator and a landmark event of EMT in CTCs.Part Two Biological Mechanisms of IGF-1R and Its Relationship with Clinicopathological Feature in Gastric Cancer by Bioinformatics Analysis OBJECTIVES: To investigate the expression and prognostic value of IGF-1R in gastric cancer,and to explore possible mechanisms of IGF-1R in regulating gastric cancer development.METHODS: The datasets of gastric cancer were downloaded from the The Cancer Genome Atlas（TCGA）.The relationship between IGF-1R expression and clinicopathological characteristics were analyzed online by using UCLCAN.We then used the c Bio Portal to determine the types and frequency of IGF-1R alterations in gastric cancer based on sequencing data.The survival analysis of IGF-1R was analyzed with Kaplan-Meier method and the log-rank test.Linked Omics was used to identify genes positively or negatively co-expressed with IGF-1R in gastric cancer.The STRING database was used to construct protein-protein networks（PPI）and GSEA（Gene Set Enrichment Analysis）was performed to explore possible signaling pathway of IGF-1R in gastric cancer development.RESULTS: The expression of IGF-1R was higher in gastric cancer than normal tissue.The expression of IGF-1R was correlated with stage,race,differentiation grade,N stage and TP53 status（P<0.05）.High expression of IGF-1R indicated poor prognosis（P=0.00018）。PPI and GSEA analysis of IGF-1R revealed that m TOR signaling pathway,calcium pathway,focal adhesion gene sets and other pathways were associated with gastric cancer invasion and metastasis.CONCLUSIONS: High expression of IGF-1R is a poor prognostic factor for gastric cancer and potential biomarker.High expression of IGF-1R was associated with focal adhesion pathway,which suggested that IGF-1R may promote gastric cancer invasion and metastasis through EMT by regulating focal adhesion.Part Three IGF-1R Pathway and EMT in Gastric Cancer and Its Significance OBJECTIVES: To detect expression of IGF-1R and EMT-associated markers in gastric cancer tissues,detect the expression of IGF-1R in gastric cancer CTCs,and explore the effect of IGF1 R and IGF1 on cell proliferation and migration.METHODS:RT-PCR and WB were used to detect the expression of IGF-1R and EMT-associated markers.The Can Patrol^TM assay was used to detect the expression of IGF-1R in CTCs,the relationship between IGF-1R and EMT-associated markers,CTCs and clinicopathological features was analyzed.IGF-1R knockdown cell line was constructed using lentivirus.CCK-8 and colony formation assays were used to detect the ability to proliferate and migrate in gastric cancer cell line after IGF-1R knockdown and IGF1 intervention.RESULTS: The expression of IGF-1R gene and protein was significantly higher in gastric cancer compared with paracancerous tissue.The m RNA expression of CK18,CK19,and Twist in gastric cancer tissue were significantly higher than those in paracancerous tissues（P<0.05）.The protein expression of Ep CAM and Vimentin was significantly higher than those in paracancerous tisssues,while Twist was significantly lower in cancer tissue than in paracancerous tissue（all P<0.05）.The expression of IGF-1R was not correlated with CTCs,gender,lymph node metastasis,differentiation,stage and distant metastasis,but significantly correlated with age and Lauren classification.IGF-1R expression in CTCs can be detected,the proportion of M-CTCs and EM-CTCs with high-medium expression of IGF-1R was significantly higher than those of E-CTCs（P<0.05）.Silence of IGF-1R was succeeding in MGC-803 cell lines.CCK-8 and colony formation assay demonstrated that IGF-1R silencing could inhibit gastric cancer cell proliferation and migration,while IGF1 could promote gastric cancer cell proliferation and migration.CONCLUSIONS: EMT phenomenon occurs in gastric cancer,with IGF-1R pathway playing an important role in gastric cancer EMT.IGF-1R was expressed in CTCs,the proportion of M-CTCs and EM-CTCs with high-medium expression of IGF-1R was significantly higher than those in E-CTCs.IGF-1R knockdown inhibits proliferation and migration,while IGF-1promotes proliferation and migration.