Cancer-Associated Fibroblasts Promote Migration And Invasion Via IL-17A/JAK2/STAT3 Signaling In Gastric Cancer Cells

Part1 Location and clinical significance of CAFs in gastric cancer Background Nowadays,gastric cancer is still a malignant tumor with a poor survival rate,ranking third in the mortality rate of all malignancies.Due to the lack of specific diagnostic biomarkers and effective anticancer treatment,the survival rate of GC patients is generally low,and the complexity of the tumor microenvironment(TME)may be an important cause of poor prognosis.There is a crosstalk between gastric cancer cells and the TME in which they promote each other and influence each other,which plays an extremely important role in the biological process of regulating the occurrence and development of gastric cancer.Cancer-associated fibroblasts(CAFs cancer-associated fibroblasts)are the most abundant cellular components in TME and are also the main source of various secretory molecules.CAFs play an important role in the occurrence,proliferation,invasion and metastasis of gastric cancer,pathological angiogenesis,immune escape and drug resistance.Existing studies have confirmed that the increased histological expression of CAFs in various types of cancer(colon,esophageal,breast and liver cancer)is significantly associated with poor patient prognosis,and the expression of CAFs in GC Its relationship with the prognosis of GC patients is rarely reported.CAFs play an important role in the occurrence and development of GC.Studying their distribution and role in GC helps to understand the mechanism of GC development and provide reference for the diagnosis and treatment of gastric cancer.Methods A total of 227 cases of GC were collected,and the localization andexpression of CAFs in GC tissue specimens were studied by immunohistochemistry and immunofluorescence methods.Then the SPSS19.0 software was used to analyze the expression level of specific marker ?-SMA and the corresponding GC The clinicopathological characteristics of patients and the relationship between the disease free survival(Disease Free Survival DFS)and disease specific survival(Disease Free Survival DSS).Results Immunohistochemistry and immunofluorescence of CAFs with positive?-SMA expression in gastric cancer tissues shows that CAFs are a type of polygonal or spindle cells of disorderly arrangement and different sizes,and are widely distributed in gastric tissues of gastric cancer patients,mainly in interstitial tissue.The number of CAFs in gastric cancer tissues was significantly related to the TNM stage(P = 0.001)and nerve invasion(P = 0.014)of gastric cancer patients,and was not associated with age,gender,ASA score,BMI ? tumor site,tumor size,vascular invasion,Lauren classification,pathological differentiation and postoperative adjuvant chemotherapy.Univariate analysis showed that: age,tumor diameter,tumor site,TNM stage,Lauren classification,high ?-SMA expression,and postoperative chemotherapy were significantly related to DFS in gastric cancer(P<0.05),while gender,ASA score,BMI?tissue type,Vascular invasion and nerve invasion were not related to DFS in gastric cancer(P> 0.05).Age,tumor site,tumor diameter,TNM staging,Lauren typing,vascular invasion and nerve invasion,high-expression of?-SMA and postoperative chemotherapy were all correlated with DSS of gastric cancer(P< 0.05),while gender,ASA score? BMI and tissue type were not correlated with DSS of gastric cancer(P>0.05).Multivariate analysis showed that: TNM staging(P<0.001),high ?-SMA expression(P = 0.009),and postoperative chemotherapy(P =0.001)were independent risk factors for DFS in GC patients.TNM staging(P<0.001),high ?-SMA expression(P = 0.008),and postoperative chemotherapy(P = 0.003)were independent risk factors affecting DSS in patients with GC.Conclusions1.CAFs are mainly distributed in the extracellular matrix of GC,and the number of CAFs is significantly related to TNM stage and nerve invasion in patients with GC.2.High ?-SMA expression,TNM staging,and postoperative adjuvant chemotherapy are all independent risk factors for DFS of patients with gastric cancer.3.High ?-SMA expression,TNM staging,and postoperative adjuvant chemotherapy are all independent risk factors for DSS of patients with gastric cancer.Methods A total of 227 cases of GC were collected,and the localization andPart2 CAFs activate the JAK2 / STAT3 signaling pathway by secreting IL-17 A to promote the migration and invasion of gastric cancer cell Background Tumor recurrence and metastasis are the main causes of malignant tumor death.Researchers have reported that CAFs are involved in the process of remodeling the tumor stroma and constitute a prerequisite for the later invasion and metastasis of tumor cells.CAFs in gastric cancer interact with a variety of Micro RNAs,activate a series of signaling pathways,regulate the expression of downstream target genes,and ultimately promote the invasion and metastasis of gastric cancer cells.At present,IL-17 A may have two sides in tumorigenesis and development.The related mechanism needs to be confirmed by further research.In gastric cancer,IL-17 expression levels in serum and tumor tissues of patients with gastric cancer were significantly increased compared with the normal control group.Studies have also found that IL-17 A and IL-17 F not only have higher expression levels in gastric cancer tissues than normal tissues,but also promote gastric cancer cell proliferation.However,the specific molecular mechanism of IL-17 A in the pathogenesis and development of gastric cancer cells remains unclear.CAFs can secrete multiple cytokines and activate multiple signal pathways to promote tumorigenesis and development.While,in gastric cancer,whether CAFs can secreted IL-17 A and the specific mechanism of action in gastric cancer progression have not been reported in relevant literatures.Methods The relationship between CAFs and IL-17 A positive cells in fresh gastric cancer tissues was analyzed by immunohistochemistry and immunofluorescence.ELISA was used to detect the expression levels of IL-17 A in gastric cells,gastric cancer tissue-isolated CAFs,CAFs and co-culture systems.QRT-PCR was used to detect the IL-17 a m RNA expression levels of gastric cancer cell lines,CAFs isolated from gastric cancer tissues,co-culture CAFs and gastric cancer cell lines.To explore the effect of CAFs on the migration and invasion ability of gastric cancer cells,IL-17 A neutralizing antibody and JAK2 blockers AG490 were used respectively to interfere with Transwell migration/invasion of the compartment.IL-17 A neutralizing antibody and neutralizing JAK2 blocker AG490 interfered with the Transwell cell co-culture compartment,respectively,to Explore the effects of CAFs and IL-17 A on the JAK2 /STAT3 signaling pathway in GC cells.Using Western blot to detect the expression of gastric cancer cell EMT-related markers(MMP-2,MMP-9,TIMP-1 and TIMP-2)and STAT3 signaling pathway marker proteins(p JAK2,JAK2,p STAT3 and STAT3).Results Immunohistochemistry and immunofluorescence staining of IL-17 A in gastric cancer tissues found that ?-SMA positively expressed CAFs and IL-17 A positive cells were mainly localized in gastric cancer interstitial tissue and co-occurred in gastric cancer interstitial.ELISA results showed that gastric cell lines(GES-1,AGS,and SGC7901)can secrete and produce IL-17 A,but the content is low,and the CAFs isolated from gastric cancer tissues have a significantly higher ability to produce IL-17 A than those from fibroblasts Cells,the difference is statistically significant(P<0.05).While,the CAFs co-cultured with gastric cancer cells AGS and SGC7901 were able to secrete and produce higher levels of IL-17A(P<0.05).QRT-PCR results showed that the m RNA expression level of IL-17 A in gastric cancer cell lines AGS and SGC7901 was slightly increased after co-culture,compared with the expression level of IL-17 A in gastric cancer cell lines cultured alone,and there was no statistical difference between the two group.Compared with CAFs cultured alone,the expression level of IL-17 A m RNA in CAFs isolated after co-culture was significantly increased,and there was a significant difference between the two group(P<0.001).After the CAFs were co-cultured with gastric cancer cells and the Transwell invasion test was performed.The migration and invasion capabilities of the two gastric cancer cell lines AGS and SGC7901 were significantly improved(P<0.05).After adding IL-17 A neutralizing antibody to the co-culture system,the migration and invasiveness of two kinds of gastric cancer cells were significantly reduced(P<0.05).After CAFs were co-cultured with gastric cancer cell lines,relevant cells were extracted and detected by Western blot.The results showed that: CAFs can increase the expression of p-STAT2 and p STAT3 related proteins of STAT3 signaling pathway in AGS and SGC7901(P<0.05 and P<0.05),and have no significant effect on the expression of JAK2 and STAT3.After adding the JAK2 inhibitor AG490 to the Transwell co-culture system of gastric cancer cells(AGS and SGC7901)and CAFs,the results showed that AG490 could also inhibit the expression of white-associated proteins p JAK2 and p STAT3 in the STAT3 signaling pathway(P<0.05 and P<0.05).),and had no significant effect on the expression of JAK2 and STAT3.Adding neutralizing antibodies to the cytokine IL-17 A to the Transwell co-culture system of gastric cancer cells AGS,SGC7901 and CAFs showed that the neutralizing antibodies of IL-17 A can inhibit the expression of the white-associated proteins p JAK2 and p STAT3 of the STAT3 signaling pathway(P<0.05 and P<0.05),but had no significant effect on the expression of JAK2 and STAT3.Western blot tests were performed on gastric cancer cells AGS and SGC7901 in a co-culture system of CAFs and gastric cancer cells.The results showed that the expressions of MMP-2 and MMP-9 were up-regulated(P<0.05,P<0.05),while TIMP-1 and TIMP-The expression of 2 decreased significantly(P<0.05,P<0.05).The addition of AG490,a JAK2 inhibitor,can significantly affect the expression of EMT-related markers in gastric cancer cells,and the expression of gastric cancer cells AGS and SGC7901 TIMP-1 and TIMP-2 is up-regulated(P<0.05,P<0.05),while the expression of MMP-2 and MMP-9decreased significantly(P<0.05,P<0.05).When IL-17 A neutralizing antibodies were added to CAFs and GC cell AGS and SGC7901 co-culture systems to neutralize IL-17 A,the expression levels of TIMP-1 and TIMP-2 in AGS and SGC7901 of GC cells were also increased(P <0.05,P <0.05),the expression levels of MMP-2 and MMP-9 decreased significantly(P <0.05,P <0.05).At the cellular level,the migration and invasion ability of gastric cancer cells AGS and SGC7901(P<0.001,P<0.05)were significantly reduced when AG490,an inhibitor of the transcription factor JAK2,was added to the Transwell invasion compartment.Conclusions1.CAFs may be the main source of IL-17 A secreted in gastric cancer tissues.2.CAFs can improve the migration and invasion of gastric cancer cells.3.CAFs may activate the JAK2 / STAT3 signaling pathway through IL-17 A,and promote gastric cancer cell migration and invasion.4.CAFs can promote the EMT of gastric cancer cells,and improve the invasion and migration ability of gastric cancer cells.