| Objective:The incidence of malignant tumors has been increasing globally,and lung cancer is the number one killer.Molecular regulation of immune response and remodeling of tumor microenvironment(TME)is a hot research topic in the treatment of lung cancer.TME is the internal environment in which tumors occur and develop.Carcinoma associated fibroblast(CAF)is an important component of TME.Fibroblast activation protein(FAP)is not only a molecular marker of CAF,but also an important factors in the progression of tumors.It is helpful to understand how the TME plays a role in the occurrence and progression of tumors,and provide a theoretical basis for molecular immune regulation and treatment of lung cancer to clarify the relationship and molecular mechanism between FAP,CAF and TME.Methods:1.The lung CAF1 and CAF2 were isolated from human lung tumor tissues and the expressions of FAP,?-SMA,Vimentin and FSP1 proteins were detecte d by Western Blot and immunofluorescence.The proliferation of lung cancer wa s detected by CCK8,using human normal fibroblast(NF)as a control.The abilit y of secretion of IL-6 of CAF was detected by ELISA.2.Lung adenocarcinoma cell line(SPC-A-1)and non-small cell lung cancer cell line(A549)were labeled with Luciferase.CAF1 and CAF2 were co-cultured with Luciferase-labeled SPC-A-1 and A549 in vitro at ratios of 4:1,2:1,1:1,1:2 and 1:4 one by one.NF+SPC-A-1 group was used as a control to detect the Luciferase reactivity at 24h,48h and 72h.3.Nude mice xenograft models were established with Luciferase-labeled SP C-A-1.The experimental group were injected subcutaneously with CAF+SPC-A-1+Metrigel,and the control group with NF+SPC-A-1+Metrigel and SPC-A-1+Met rigel.The tumor volume of nude mice were measured physically,and the proli feration of tumor cells in nude mice were observed by fluorescence imaging.T hese nude mice were sacrificed at day 28,and tumor volume and weight were measured.Then HE staining were performed.4.FAP overexpressing fibroblasts(FAP-BJ,FAP-HFF),FAP overexpressing lung adenocarcinoma cells(FAP-SPC-A-1)and negative control cells(NC-BJ,N C-HFF,NC-SPC-A-1)were established.The expression of FAP protein was det ected by WB,the levels of FAP and IL-6ST mRNA by real-time quantitative P CR,the proliferation ability by CCK8 and the ability of IL-6 secretion by ELIS A.5.FAP-BJ was co-cullured with Luciferase-labeled SPC-A-1 and A549 in v itro at ratios of 4:1,2:1,1:1,1:2 and 1:4 one by one,using SPC-A-1 Group a nd NC-BJ+SPC-A-1 group as controls to detect the Luciferase reactivity at 24h,48h and 72h.6.Co-culture experiments were performed to simulate the TME in vivo,an d the effect of FAP on IL-6 levels was studied.The co-culture systems were F AP-BJ+SPC-A-1,FAP-HFF+PBMC,FAP-SPC-A-1+PBMC and FAP-HFF+SPC-A-1+PBMC,and the culture supernatant was collected to detect IL-6 concentration.Results:1.CAF identification resultsWB detected Vimentin expression:NF>CAF1>CAF2;FSP1 expression:CAF 1>CAF2>NF;?-SMA expression:CAF2>CAF1>NF;FAP expression level in CA F1 and CAF2 is low.IF detected Vimentin was expressed in CAF1,CAF2 and NF;?-SMA was expressed in CAF1 and CAF2,but not in NF.Proliferation a bility:CAF2>CAF1>NF,the differences were statistically significant.IL-6 seer etion capacity:CAF2>CAF1>NF(P<0.0001).2.CAF2 promoted the proliferation of lung cancer cells in vitroLuciferase reactivity CAF2+SPC-A-1 group>NF+SPC-A-1 group?SPC-A-1 g roup(P<0.05).Luciferase reactivity CAF2+A549 group?NF+A549 group<A549 group,and the greater the proportion of fibroblasts,the greater the differenc e.3.CAF2 promoted the proliferation of lung cancer cells in vivoPhysical measurement of tumor volume(Day28):CAF2+SPC-A-1 group>NF+SPC-A-1 group(P<0.05);there was no statistical difference between CAF+SPC-A-1 group and SPC-A-1 group.Fluorescence imaging signals(Day28):CAF+SP C-A-1 group>NF+SPC-A-1 group(P<0.05);there was no statistical difference be tween CAF+SPC-A-1 group and SPC-A-1 group.These nude mice were Sacrific ed in day28.Physical measurement of tumor volume:CAF+SPC-A-1 group?SPC-A-1 group>NF+SPC-A-1group(P<0.05);there was no statistical difference in t umor weight between each group(P>0.05).4.FAP overexpressing cell lines identification resultsFAP protein expression level was detected by WB:FAP-BJ>NC-BJ,FAP-HF F>NC-HFF,FAP-SPC-A-1>NC-SPC-A-1.The level of FAP mRNA was detected b y PCR:FAP-HFF>NC-HFF(P<0.0001),FAP-SPC-A-1>NC-SPC-A-1(P<0.0001).T he level of IL-6ST mRNA was detected by PCR:FAP-HFF>NC-HFF(P<0.01),F AP-SPC-A-1>NC-SPC-A-1(P<0.05).Cell proliferation ability was detected by CCK8:FAP-BJ>NC-BJ(P<0.01).The secretion of IL-6 levels was detected b y ELISA:FAP-BJ>NC-BJ(P<0.0001).5.The results of co-culture FAP overexpression fibroblasts with lung cancer cells in vitroLuciferase reactivity FAP-BJ+SPC-A-1 group>NC-BJ+SPC-A-1 group?SP C-A-1 group(P<0.05).FAP-BJ+A549 group?NC-BJ+ A549 group<A549 g roup,and the greater the proportion of fibroblasts,the greater the difference.IL-6 secretion level:FAP-BJ+ SPC-A-1>NC-BJ+SPC-A-1,FAP-HF+PBMC>FAP-HF+PBMC,FAP-SPC-A-1+PBMC>NC-SPC-A-1+PBMC,FAP-HFF+SPC-A-1+PBM C>NC-HFF+SPC-A-1+PBMC.These differences were statistically significant.Conclusions:1.Isolated and identified two Lung cancer-associated fibroblasts CAF1 and CAF2.It was found that the proliferative capacity of CAF2 and the secretion o f IL-6 were stronger than CAF1 and NF.CAF2 can promote the proliferation o f SPC-A-1,but inhibit the proliferation of A549 in vitro.2.The proliferation of FAP overexpression fibroblast FAP-BJ was stronger t han NC-BJ.And FAP-BJ promoted the proliferation of SPC-A-1,but inhibited th e proliferation of A549 in vitro.3.FAP promotes the proliferation of SPC-A-1 by enhancing the expression of IL-6 in the TME. |
PDF Full Text Request