Background: Fibroblast activation protein (FAP) is one of the surface markers of cancer-associated fibroblasts (CAFs) and is closely related to the malignant characterization of CAFs. SP13786 is a specific micromolecule inhibitor of FAP and this study is to investigate the effects and mechanism of SP13786 on the migration and invasion of A549 cells through regulating exosomes of CAFs.
Methods: CAFs and paracancerous fibroblasts (PTFs) were isolated and subcultured from freshly resected lung adenocarcinoma tissues and paracancerous normal tissues separately. MTT assay was used to detect the proliferation of CAFs incubated by different concentrations of SP13786; PTFs-exo, CAFs-exo and CAFs+SP13786-exo were extracted by polymer precipitation method. The A549 cells were divided into Ctrl group, PTFs group, CAFs group and SP13786 group and each group was incubated with DMEM, PTFs-exo, CAFs-exo and CAFs+SP13786-exo separately. Laser confocal microscope was used to observe the endocytoses of exosomes by A549 cells. The expression of alpha-smooth muscle actin (α-SMA) and FAP in PTFs and CAFs and the expression of E-cadherin, N-cadherin, Slug, Stat3 and P-Stat3 in A549 cells were detected by immunofluorescence, immunohistochemistry and Western blot. The migration and invasion ability of A549 cells were detected by cell scratch and transwell methods.
Results: α-SMA and FAP were expressed much higher in CAFs than that in PTFs which indicate that CAFs and PTFs were successfully obtained from lung adenocarcinoma and paracancerous tissues (P<0.05). MTT showed that the 50% inhibitory concentration (IC50) of SP13786 for CAFs was about 3.3 nmol/L. In addition, SP13786 can significantly decrease the expression of α-SMA and FAP in CAFs which means that targeted inhibition of FAP could reduce the malignant characteristics of CAFs (P<0.05). Laser confocal microscope found that exosomes from CAFs could be taken up by A549 cells and scratch and transwell tests showed that the endocytosed CAFs-exo could promote the migration and invasion of A549 cells (P<0.001), while FAP inhibitor SP13786 could inhibit the effects of CAFs-exo on A549 cells (P<0.05). Furthermore, Immunofluorescence and Western blot showed that CAFs-exo could promote EMT by decreasing E-cadherin expression and increasing N-cadherin, Slug expression in A549 cells while FAP inhibitor SP13786 could significantly supress CAFs-exo-induced epithelial-mesenchymal transition (EMT) of A549 cells (P<0.05). Moreover, the expression of P-Stat3 was obviously increased in A549 cells of CAFs group and significantly down-regulated in SP13786 group (P<0.05) whereas there was no significant difference in total Stat3 between CAFs and SP13786 groups (P>0.05). Finally, WP1066 (a specific inhibitor of Stat3) was used to comfirm whether SP13786 could influence EMT of A549 cells by inhibiting Stat3 phosphorylation via CAFs-Exo. The results showed that when the phosphorylation of Stat3 in CAFs group was inhibited by WP1066, SP13786 could not influence the P-Stat3 expression and EMT of A549 cells anymore (P>0.05).
Conclusions: As a specific micromolecule inhibitor of FAP, SP13786 indirectly inhibits the migration and invasion of A549 cells by affecting exosomes of CAFs. The possible mechanism is to inhibit the phosphorylation of Stat3 and thus affect the EMT of A549 cells.
【中文题目：SP13786通过CAFs外泌体抑制Stat3-EMT影响肺腺癌细胞A549的迁移侵袭】 【中文摘要：背景与目的 成纤维细胞活化蛋白（fibroblast activation protein, FAP）是肿瘤相关成纤维细胞（cancer-associated fibroblasts, CAFs）的表面标志物之一，与CAFs的恶性表征关系密切，SP13786是FAP的特异性小分子抑制剂。本研究探讨SP13786作用于CAFs后，CAFs外泌体（exosomes, exo）对A549细胞迁移、侵袭的影响与机制。方法 原代提取CAFs和癌旁成纤维细胞（peri-tumer fibroblasts, PTFs）；MTT实验检测不同浓度SP13786对CAFs增殖的影响；聚合物沉淀法提取PTFs-exo、CAFs-exo以及CAFs+SP13786-exo。将A549细胞设对照组、PTFs组、CAFs组及CAFs+SP13786组并分别以等体积的DMEM、PTFs-exo、CAFs-exo及CAFs+SP13786-exo孵育细胞。激光共聚焦实验检测A549细胞摄取外泌体的情况；免疫荧光、免疫组化和Western blot方法检测α平滑肌肌动蛋白（alpha-smooth muscle actin, α-SMA）、FAP在PTFs和CAFs中的表达及E-cadherin、N-cadherin、Slug、Stat3、P-Stat3在各组A549细胞中的表达；划痕实验和Transwell实验检测各组细胞的迁移和侵袭能力。结果 免疫荧光、免疫组化和Western blot结果均显示α-SMA、FAP在CAFs中高表达，在PTFs中低表达（P<0.05），表明从肺腺癌组织和癌旁组织中分别成功获得了CAFs 和PTFs。MTT实验测得SP13786对于CAFs细胞的半数抑制浓度（50% inhibitory concentration, IC50）约为3.3 nmol/L。免疫组化和Western blot结果显示与CAFs组相比，CAFs+SP13786组的α-SMA与FAP的表达显著降低（P<0.05），说明抑制FAP可以显著降低CAFs的恶性表征。激光共聚焦结果显示外泌体能够被A549细胞所摄取。划痕实验与Transwell实验显示SP13786可抑制CAFs-exo对A549细胞迁移和侵袭的促进作用（P<0.05）。与CAFs组比较，SP13786组A549细胞E-cadherin表达增多，N-cadherin与Slug表达降低（P<0.05）；免疫荧光与Western blot显示SP13786组A549细胞的P-Stat3较CAFs组明显降低（P<0.05），而总Stat3无显著差异。Stat3的特异性抑制剂WP1066明显抑制CAFs组A549细胞上皮间质转化（epithelial-mesenchymal transition, EMT），P-Stat3显著降低（P<0.05），而加入WP1066后再加入SP13786-exo，P-Stat3未见进一步减低，EMT的抑制亦未见显著变化（P>0.05）。结论 FAP的小分子特异性抑制剂SP13786通过影响CAFs外泌体间接抑制A549细胞的迁移、侵袭，其可能机制是抑制Stat3的磷酸化从而影响A549细胞的EMT。】 【中文关键词：肺腺癌；SP13786；肿瘤相关成纤维细胞；成纤维细胞活化蛋白；外泌体；EMT；Stat3】.
Keywords: Cancer-associated fibroblasts; EMT; Exosomes; Fibroblast activation protein; Lung Adenocarcinoma; SP13786; Stat3.