Mechanism Of Rapamycin-induced Autophagy In Tumor-associated Macrophages To Regulate Epithelial-mesenchymal Transition Of Bladder Cancer Cells

Objective: To investigate the mechanism of epithelial-mesenchymal transition(E MT)in bladder cancer cells by rapamycin-induced autophagy of tumor-associated macr ophages(TAMs)and regulation of autophagy in TAMs.Methods: Immunohistochemical staining and immunofluorescence were used to d etect the expression levels of autophagy-related proteins LC3 and EMT-related regulato ry factors in bladder cancer tissues and adjacent tissues.The number of autophagosome s was observed by electron microscopy.Human monocyte(THP-1)cells were treated w ith PMA and IL-4+IL-10 for 72 h,induced cell polarization to TAMs(M2),cell morpho logy was observed,and M1 was detected by flow cytometry and immunofluorescenc e.And M2 surface CD86,CD206,F4/80 and other signature molecules,identify the pol arization efficiency of TAMs.The autophagy regulator rapamycin and 3-MA were use d to intervene TAMs for 24 h,and the autophagy-regulating drug was removed for 48 h.The expression of LC3 was detected by Western Blot and immunofluorescence,an d the number of autophagosomes was observed by electron microscopy.The level of aut ophagy in TAMs.The TAMs after rapamycin intervention were non-contacted with hu man bladder cancer cells(T24).Western blot was used to detect the expression of EMTrelated factors in bladder cancer cells,and the invasion and migration of bladder cance r cells were detected by transwell and scratch methods.ability.Results: Immunohistochemical staining showed that the expression levels of autoph agy-related proteins LC3 and EMT-related regulatory factors and the number of autopha gosomes in bladder cancer tissues were higher than those in adjacent tissues.Immunoflu orescence assay showed that LC3 was expressed in bladder cancer.Extracellularly,expr ession is also present in TAMs of bladder cancer tissues,and the expression level of LC3 in TAMs in MIBC tissues is higher than that in NMIBC tissues and adjacent tissues.Human monocytes(THP-1)were treated with PMA and IL-4+IL-10 for 72 h,and th e cell status changed from semi-suspended to adherent.The expression of CD206 on T AMs was detected by flow cytometry and immunofluorescence.Compared with the M1 group,the difference was statistically significant(P<0.05).Immunofluorescence,Wes tern blot and electron microscopy showed that the expression of LC3 was significantly i ncreased and the expression of LC3 was significantly increased in TAMs after 48 day s of rapamycin stimulation.The number of autophagosomes was increased by electro n microscopy.TAMs and bladder cancer cells(T24)were co-cultured for 48 hours.Scra tch and Transwell experiments showed that TAMs after rapamycin could significantly e nhance the migration and invasion of bladder cancer cells.The difference was statistical ly significant(P <0.05),Western Blot assay showed up-regulation of EMT-related gene s and proteins in bladder cancer cells.Conclusion: Autophagy of TAMs is associated with the occurrence of EMT in blad der cancer;rapamycin can regulate the occurrence of EMT in bladder cancer by inducin g autophagy of TAMs.