The Effect Of Substrate Stiffness On Immune Evasion Of Lung Cancer Cells

The changes of the mechanical microenvironment,especially the changes of substrate stiffness,play an important role in the occurrence of various diseases.Cells can sense the changes of mechanical signals caused by different stiffness substrates and convert them into biochemical signals to regulate multiple intracellular signaling pathways.The changes of these signaling pathways regulate the corresponding cell biological behavior and affect the development of the disease.Lung cancer is a primary malignant tumor with high morbidity and mortality and poor prognosis.Although in the research of the pathogenesis and treatment of lung cancer have major breakthroughs and progress.The rates of the prevalence,morbidity and death of lung cancer are still showing an upward trend and the research results are not very significant.There is an urgent need to develop more effective drugs and find new strategies for the treatment.Previous studies have found that immune evasion played an important role in the occurrence and development of lung cancer.Lung cancer cells can evade the anti-tumor response of the immune system through various mechanisms.However,how the changes of substrate stiffness affect the immune escape process and regulate the occurrence and development of lung cancer is still unclear.Based on this problem,this study intends to take human lung adenocarcinoma A549 cells as the experimental research object and use polydimethylsiloxane(PDMS)as the substrate material to study the effects of substrate stiffness on immune escape of A549 cells and related signaling pathways.Firstly,the differential expression and survival analysis of lung adenocarcinoma integrin β1(ITG-β1)were performed using the TCGA database.Secondly,the PDMS prepolymer and the curing agent were mixed uniformly according to the ratio of 10:1,30:1 and 50:1 to obtain substrates with different stiffness and the surface modification of PDMS substrates with different stiffness was carried out.A549 cells were seeded on different stiffness on PDMS substrate and the effect of substrate stiffness on the expression of ITG-β1 in A549 cells was detected.Next,in order to explore the underlying mechanism of substrate stiffness regulating immune escape,the effect of substrate stiffness on focal adhesion kinase(FAK)mediated immune escape-related signaling pathways in A549 cells was examined.Then,the method of stabilizing gene silencing by lentivirus was used to detect the effect of FAK silencing on the expression of immune escape molecules.Finally,A549 cells on different stiffness substrate were co-cultured with T cells,and the effects of substrate stiffness on the proliferation viability of A549 cells and T cell toxicity in the co-culture system were examined.The main experimental content and results of this article are as follows:(1)Using the TCGA database to analyzed the differential expression and survival of lung adenocarcinoma ITG-β1,the results showed that compared with normal tissues,lung adenocarcinoma tissues highly expressed ITG-β1,and the high expression of ITG-β1 is significantly associated with patient survival.A549 cells were seeded on PDMS substrates with different stiffness.Western blot was used to detect the effect of substrate stiffness on the expression of ITG-β1 protein in A549 cells.The experimental results showed that substrate stiffness regulated the expression of ITG-β1 protein.With the increase of substrate stiffness,the expression of ITG-β1 decreased.(2)Using Western blot,ELISA,QPCR technology to study the effect of substrate stiffness on FAK-mediated immune escape-related signaling pathways in A549 cells,the results showed that substrate stiffness had a very significant effect on the expression of FAK in A549 cells.As stiffness increased,FAK expression increased.Substrate stiffness regulated the expression of FAK.On the one hand,it affected the expression of inflammatory factors regulated by FAK.On the other hand,it affected the expression of immune escape-related molecules regulated by FAK/STAT1 signaling pathway.The study found that with the increase of substrate stiffness,the expression of inflammatory factor IL-33 and its downstream factors CCL5 and ST2 were up-regulated,and the expression of immunosuppressive factors TGF-β1 and IDO and surface molecules PD-L1 and Fas L were also up-regulated.(3)We used lentivirus to stably silence the FAK gene,and then used Western blot technology to detect the effect of silencing the FAK on the expression of immune evasion related molecules.Studies have shown that the FAK gene was closely related to the immune escape process.FAK gene silencing reduced the expression of IL-33,TGF-β1,PD-L1 and Fas L.It indicated FAK-related signaling pathways played an important role in the immune escape process.(4)MTT and CCK8 were used to detect the effect of substrate stiffness on the proliferation viability of A549 cells and T cell viability in the co-culture system.A549 cells on PDMS substrate with different stiffness were co-cultured with T cells.Substrate stiffness significantly affected the proliferation of A549 cells and T cell viability.With the increase of substrate stiffness,the proliferation of A549 cells increased,and T cell viability decreased.This experimental study confirmed that substrate stiffness can regulate the immune escape process of A549 cells.The results of the study are helpful to deepen our understanding of the mechanical microenvironment regulating the development of lung cancer.Prevention and treatment provide corresponding targets and improve the response rate of immunotherapy.