The Study Of Tunneling Nanotubes Promoting Intercellular Mitochondria Transfer Followed By Increased Invasiveness In Bladder Cancer Cells

Objective: To investigate the existence of tunneling nanotubes(TNTs)and mitochondrial transfer between bladder cancer cells with different invasive abilities(RT4 and T24 bladder cancer cells),the direction of mitochondrial transfer and changes in biological behavior of bladder cancer cells caused by mitochondrial transfer.Methods: The bladder cancer cells(T24 cells and RT4 cells)were selected for experiment.The cells were labeled by the mitochondrial fluorescent probe Mito-Tracker Deep Red and the cell tracer probe CFSE Green respectively,and then co-cultured for 24 h in a 1:1 ratio.The presence of TNTs between co-cultured RT4 and T24 bladder cancer cells was observed using light microscopy,fluorescence imaging,and scanning electron microscopy(SEM).The cytoskeletal protein F-actin in the TNTs structure was labeled by Actin-Tracker Green.Mitochondrial transfer and the direction of mitochondrial transfer were determined using Laser scanning confocal microscopy(LSCM)and Fluorescence activated Cell Sorting(FACS).The cytological morphology and mitochondrial distribution of bladder cancer cells after mitochondrial transfer were observed using LSCM.The invasiveness of bladder cancer cells after mitochondrial transfer was detected in vitro by Wound-healing assay,Transwell assays,Western Blot and in vivo by xenograft tumor growth model.Results: The presence of TNTs between T24 cells and RT4 cells after co-culture was confirmed by light microscopy,fluorescence microscopy and SEM.TNTs had a diameter of 100-200 nm and a length of 20 ?m to 1 mm.TNTs showed continuous,membranous,microtubular structures with both ends continuing with the cell membranes of T24 cells and RT4 cells under SEM.TNTs were mainly composed of F-actin,were extended from T24 cells to RT4 cells,and in which fluorescently labeled mitochondria were observed.Mitochondria were observed spontaneously unidirectionally transfer from T24 cells to RT4 cells using LSCM and FACS assays.The distribution of mitochondria migrated from T24 cells was in good agreement with the original mitochondria in RT4 cells.New variety of cells,RT4-Mito-T24 cells,was sorted by FACS,and RT4-Mito-T24 cells were formed by RT4 cells receiving mitochondria from T24 cells.We detected cytoskeleton reconstruction in RT4-Mito-T24 cells by observing F-actin redistribution.Compared with RT4 cells,the invasive ability of RT4-Mito-T24 cells was improved and the expression of Akt(p=0.003),mTOR(p=0.001),p-mTOR(p<0.001),4EBP1(p=0.023)was increased.Conclusions: In the co-culture system of T24 cells and RT4 cells,TNTs formed from T24 cells to RT4 cells.The mitochondria of T24 cells were transferred into RT4 cells through TNTs and then RT4 cells became RT4-Mito-T24 cells.Compared with RT4 cells,Akt,mTOR,and their downstream mediators were activated and increased in RT4-Mito-T24 cells.The cytoskeleton reconstruction of RT4-Mito-T24 cells was detected.The resultant increase in the invasiveness of RT4-Mito-T24 cells was detected in vitro and in vivo.These data indicate that TNTs promote intercellular mitochondrial transfer between heterogeneous cells,followed by an increase in the invasiveness of bladder cancer cells.