| Objective:Lung cancer is the malignant tumor with the highest mortality rate in China,and one of the important reasons hindering effective clinical treatment is that tumor microenvironment(TME)leads to local immunosuppression of tumors.TME is the internal environment in which tumor stromal cells interact with tumor cells.Cancer-associated fibroblasts(CAFs)are important components of TME,and fibroblast activation protein(FAP)is an important molecular marker of CAFs,which participates in the tumor process by promoting tumor cell proliferation,TME matrix degradation and reconstruction,tumor vasculature establishment and mediating tumor immunosuppression.Studying the interaction mode and molecular mechanism between FAP-positive CAFs and tumor cells is conducive to explore the relationship among FAP,CAFs and TME,and laying a foundation for elucidating the mechanism of local immunosuppression in tumor microenvironment.Methods:1.Two CAFs were isolated and cultured from human lung cancer tissues.Theexpression of FAP,alpha-SMA and Vimentin was detected by WB and IF.Our group has successfully constructed and validated FAP-overexpressing fibroblast cell lines FAP-BJ,FAP-HFF and negative control cell lines NC-BJ,NC-HFF,WB and IF to detect the expression of alpha-SMA and Vimentin.2.In vitro direct co-culture,CAF3,CAF4 and Luciferase-labeled SPC-A-1 at 4:1,2:1,1:1,1:2,1:4 ratios respectively;CAF3,CAF4,FAP-HFF and Luciferase-labeled A549 at 1:8,1:16,1:32 ratios respectively,to detect SPC-A-1/A549 24h,48h,72h biofluorescein intensity.3.In vitro indirect co-culture,the ratio of CAF2,FAP-BJ and SPC-A-1 was 4:1;the ratio of CAF4,FAP-HFF and A549 was 1:8.The absorbance of SPC-A-1/A54924h,48h and 72h was detected by CCK8 method.4.In nude mice transplantation experiment,FAP-BJ,NC-BJ and SPC-A-1 were mixed with Matrigel at a ratio of 4:1 to inoculate nude mice.The tumor volume was measured physically,and the proliferation of tumor cells in vivo was observed by an in vivo fluorescence imaging system.The nude mice were sacrificed on the 28th day,the tumor volume was measured,and HE staining was performed.5.In vitro HUVECs tubule formation experiment,CAF2,CAF3,FAP-BJ,FAP-HFF and SPC-A-1 were directly co-cultured at a ratio of 4:1 for 48 h to collect the supernatant as the pretreatment medium and stimulate HUVECs cells to take pictures under light microscope for 6 h.ImageJ software quantified the total length of tubule formation.6.In vivo Matrigel-Plug experiment,nude mice were inoculated with CAF2,FAP-BJ,FAP-HFF and SPC-A-1 at a ratio of 4:1.FITC-dextran was injected into the tail vein of Day9.After 20 minutes,the animals were sacrificed and the tumor tissues were taken out to observe the vascular morphology and fluorescence intensity.ImageJ software quantified the vascular fluorescence intensity,and IHC detected the expression of CD31.7.FAP-BJ and NC-BJ were directly co-cultured with SPC-A-1 and A549 at a ratio of 4:1 for 48 h and then flow sorted cells.WB was used to detect FAP expression in SPC-A-1 and A549 after co-culture and SCUBE3 expression in FAP-BJ before and after co-culture with SPC-A-1.8.FAP-BJ,FAP-HFF,CAFN,CAF2,CAF3 and SPC-A-1 were directly co-cultured at a ratio of 4:1 for 48 h,and the supernatant was collected,SCUBE3 secretion level in the supernatant was detected by ELISA.Results:1.Identification of CAFs and FAP-overexpressing fibroblast cell linesWB and IF results showed that the expression of FAP was CAF4>CAF3>NF;Vimentin and alpha-SMA were expressed in CAF3,CAF4,NF,FAP-BJ,NC-BJ,FAP-HFF,NC-HFF,but the expression was not obvious in CAF4.2.In vitro direct co-culture of CAF3,CAF4 and FAP-HFF promotes lung cancer cell proliferationBiofluorescein intensity was measured,CAF3+SPC-A-1 group,CAF4+SPC-A-1 group>NF+SPC-A-1 group,SPC-A-1 group(P<0.05),and CAF4 promoted the proliferation of SPC-A-1 stronger than CAF3;CAF3+A549 group,CAF4+A549 group>NF+A549 group,A549 group(P<0.05),FAP-HFF+A549 group>NC-HFF+A549 group,A549 group(P<0.05).Within a certain range,the larger the proportion of fibroblasts,the more obvious the difference.3.In vitro indirect co-culture of CAF2,CAF4,FAP-BJ and FAP-HFF does not promote the proliferation of lung cancer cellsAbsorbance was measured.There was no statistical difference between CAF2+SPC-A-1 group and NF+SPC-A-1 group(P>0.05).CAF4 stimulated A549 to proliferate more slowly than NF group stimulated A549,and there was no significant proliferation effect compared with direct co-culture in vitro.There was no statistical difference between FAP-B J+SPC-A-1 group and NC-BJ+SPC-A-1 group,FAP-HFF+A549 group and NC-HFF+A549 group(P>0.05).4.In vivo FAP-BJ promotes tumor proliferationThe tumor volume was measured physically at 28 days,FAP-BJ+SPC-A-1 group>NC-BJ+SPC-A-1 group,SPC-A-1 group(P<0.05).In vivo fluorescence imaging signals were detected,FAP-BJ+SPC-A-1 group>NC-BJ+SPC-A-1 group,SPC-A-1 group(P<0.05).The nude mice were sacrificed and the tumor volume was measured physically,FAP-BJ+SPC-A-1 group>NC-BJ+SPC-A-1 group and SPC-A-1 group(P<0.05).5.In vitro angiogenesis of CAF2,CAF3,FAP-BJ and FAP-HFFTotal length of tubule formation,CAF2+SPC-A-1 group,CAF3+SPC-A-1 group>NF+SPC-A-1 group,SPC-A-1 group(P<0.05);FAP-B J+SPC-A-1 group>NC-BJ+SPC-A-1 group,SPC-A-1 group(P<0.05),FAP-HFF+SPC-A-1 group>NC-HFF+SPC-A-1 group,SPC-A-1 group(P<0.05).6.In vivo angiogenesis of CAF2,FAP-BJ and FAP-HFFImageJ quantified the vascular fluorescence intensity,CAF2+SPC-A-1 group>NF+SPC-A-1 group,SPC-A-1 group(P<0.05);FAP-BJ+SPC-A-1 group>NC-B J+SPC-A-1 group,SPC-A-1 group(P<0.05),FAP-HFF+SPC-A-1 group>NC-HFF+SPC-A-1 group,SPC-A-1 group(P<0.05).Immunohistochemical results showed that the expression of CD31 was stronger in the experimental group than in the control group(P<0.05).7.Expression of FAP in SPC-A-1 and A549 after co-cultureUpregulation of FAP expression was detected by WB in SPC-A-1 and A549 cells co-cultured with FAP-BJ.8.Expression of SCUBE3 in FAP-BJ before and after co-cultureThe expression of SCUBE3 was up-regulated by WB in FAP-BJ cells and was higher in FAP-BJ cells after co-culture with SPC-A-1,the protein expression rate is:FAP-BJ cells after co-culture>FAP-BJ cells>NC-BJ cells.9.SCUBE3 secretion level in supernatant of fibroblasts co-cultureThe levels of SCUBE3 secretion were up-regulated by ELISA in the supernatants of FAP-BJ,FAP-HFF,CAF2,CAF3 and SPC-A-1.Conclusions:1.Isolation and identification of CAF3 and CAF4,both naturally highly expressed FAP,and CAF4 was stronger than CAF3.2.In the study of lung cancer CAFs,the direction of proliferation was that lung cancer CAFs directly co-cultured in vitro to promote lung cancer cell proliferation,while in vitro co-cultured in vitro did not significantly promote lung cancer cell proliferation.The direction of angiogenesis,lung cancer CAFs in vivo and in vitro promote lung cancer angiogenesis.3.In the study of FAP overexpressing fibroblasts,the direction of proliferation is that FAP overexpressing fibroblasts directly co-culture in vitro promote lung cancer cell proliferation,and indirect co-culture in vitro does not promote lung cancer cell proliferation,and promotes lung adenocarcinoma growth in vivo.In the direction of angiogenesis,FAP overexpressing fibroblasts in vivo and in vitro all promote angiogenesis.4.The up-regulation of SCEBE3 protein after co-culture suggested that the interaction of lung cancer CAFs with FAP positive expression in the tumor microenvironment and lung cancer cells may promote lung cancer growth by enhancing the expression of SCEBE3. |
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