these results identify mitochondrial transfer from cancer cells to fibroblasts as a driver of tumorigenesis and provide a rationale for targeting miro2 and mitochondrial transfer in different malignancies. mitochondria are central to energy conversion and signaling events and engage in metabolism and cell fate decisions in health and disease. once an alphaproteobacterial species that evolved into an organelle, mitochondria persist as functionally specialized units, carrying the ability to move between cells. this phenomenon, known as mitochondrial transfer, has emerged as a powerful strategy for tissue revitalization and rejuvenation in injured or diseased organs. recent studies have identified important roles of mitochondrial dynamics in cancer, revealing how mitochondrial transfer contributes to metabolic heterogeneity among tumor cells and influences disease outcomes and treatment responses. mitochondrial transfer can occur through gap junctions, extracellular vesicles, direct mitochondrial release and uptake or tunneling nanotubes ( tnts ), which are thin membranous structures that form dynamic connections between cells. transfer of mitochondria from stromal or immune cells in the tumor microenvironment to cancer cells has also been reported, which promoted tumor growth. for example, mitochondrial transfer from cd8 t cells, mesenchymal stem cells or cancer - associated fibroblasts ( cafs ) into cancer cells has been described for different tumors, resulting in enhanced cancer cell proliferation, motility and lactate metabolism. however, the opposite process — mitochondrial transfer from cancer cells to stromal cells, including fibroblasts — has not been reported, although this may have important consequences for the fibroblast phenotype. here, we identify mitochondrial transfer from cancer cells to fibroblasts as a key regulator of caf differentiation. given the association of the caf phenotype with metabolic alterations, we tested whether cancer cells transfer their mitochondria to fibroblasts using cocultures of early - passage human primary skin fibroblasts ( hpfs ) with highly malignant a431 vulvar carcinoma cells. a431 cells stably expressing fluorescently labeled actin ( lifeact a431 ) were incubated with mitotracker green, which stains mitochondria in living cells ( fig. 1a ). this approach was chosen because of the strong fluorescence signal of mitotracker green. only some hpfs in close proximity to a431 cells became positive for mitotracker green after a 24 - h coculture, indicating that they received cancer cell mitochondria ( fig. 1b ). they were clearly discernible against the weak background fluorescence, which may have resulted from dye leakage — a previously reported limitation of mitotracker dyes that often produces false - positive results. fig. 1cancer cells transfer mitochondria to fibroblasts through tnts. a, coculture setup with lifeact a431 cells ( red ) stained with mitotracker green and unstained hpfs. this image was created with biorender. com. b, immunoflu orescence images of the cocultures, counterstained with hoechst. c, representative photomicrographs of cocultures of a431 cells prestained with mitotracker green and hpfs immunostained for coli ( white ) and counterstained with phalloidin ( red ) and hoechst ( blue ). tnt - like structures are indicated by white rectangles together with their length ( n = 3 a431 – hpf cocultures ). d, percentage of mitotracker - high hpfs in direct or transwell coculture with a431 cells ( n = 3 cocultures per setup ). e, percentage of mitotracker - high hpfs after coculture with a431 cells in the presence of carbenoxolone ( cbx ) or vehicle ( n = 3 cocultures per treatment group ). f, rt – qpcr for gjb2 ( encoding connexin 26 ) relative to rpl27 using rna from a431 cells transfected with control ( scrambled ) or connexin 26 ( cx26 ) sirna, and percentage of mitotracker - high hpfs after coculture of sictrl or sicx26 a431 cells ( n = 3 cultures per group ). g, holotomographic imaging showing mitochondrial transfer ( white arrows ) from a431 lifeact – mitotracker green cells to hpfs ( unstained ) ( supplementary video 1 ). h, representative image of a coculture of a431 cells stained with mitotracker green and hpfs, immunostained for coli and counterstained with phalloidin and hoechst ( n = 3 a431 – hpf cocultures ). white arrows point to tnt - like structures. i, percentage of mitotracker - high hpfs after coculture with a431 cells in the presence of nocodazole ( noc ), dih ydrocytochalasin b ( cyto b ) or vehicle ( n = 3 cocultures per treatment group ). j, western blot analysis for sec3 and sec5 using lysates from a431 cells transfected with sictrl, sisec3 or sisec5 ( n = 2 cultures per group ). graph shows the percentage of mitotracker - high hpfs after coculture with mitotracker - stained control or sec3 – sec5 - knockdown a431 cells ( n = 3 cocultures per group ). k, percentage of mitotracker - high hpfs after coculture with hacat or a431 cells ( n = 3 cocultures per cell line ). l, representative immunoflu orescence images depicting cocultures of mda - mb - 231 and panc1 cells prestained with mitotracker green and hpfs immunostained for coli ( white ) and counterstained with hoechst ( blue ) ( n = 3 cocultures per cell line ). graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( d – f, i, k ) or two - sided one - way anova with bonferroni post hoc multiple comparison test ( j ) was used to determine statistical significance. scale bars, 50 μm ( b, g ), 20 μm ( c ) or 25 μm ( h, l ). source data a, coculture setup with lifeact a431 cells ( red ) stained with mitotracker green and unstained hpfs. this image was created with biorender. com. b, immunoflu orescence images of the cocultures, counterstained with hoechst. c, representative photomicrographs of cocultures of a431 cells prestained with mitotracker green and hpfs immunostained for coli ( white ) and counterstained with phalloidin ( red ) and hoechst ( blue ). tnt - like structures are indicated by white rectangles together with their length ( n = 3 a431 – hpf cocultures ). d, percentage of mitotracker - high hpfs in direct or transwell coculture with a431 cells ( n = 3 cocultures per setup ). e, percentage of mitotracker - high hpfs after coculture with a431 cells in the presence of carbenoxolone ( cbx ) or vehicle ( n = 3 cocultures per treatment group ). f, rt – qpcr for gjb2 ( encoding connexin 26 ) relative to rpl27 using rna from a431 cells transfected with control ( scrambled ) or connexin 26 ( cx26 ) sirna, and percentage of mitotracker - high hpfs after coculture of sictrl or sicx26 a431 cells ( n = 3 cultures per group ). g, holotomographic imaging showing mitochondrial transfer ( white arrows ) from a431 lifeact – mitotracker green cells to hpfs ( unstained ) ( supplementary video 1 ). h, representative image of a coculture of a431 cells stained with mitotracker green and hpfs, immunostained for coli and counterstained with phalloidin and hoechst ( n = 3 a431 – hpf cocultures ). white arrows point to tnt - like structures. i, percentage of mitotracker - high hpfs after coculture with a431 cells in the presence of nocodazole ( noc ), dih ydrocytochalasin b ( cyto b ) or vehicle ( n = 3 cocultures per treatment group ). j, western blot analysis for sec3 and sec5 using lysates from a431 cells transfected with sictrl, sisec3 or sisec5 ( n = 2 cultures per group ). graph shows the percentage of mitotracker - high hpfs after coculture with mitotracker - stained control or sec3 – sec5 - knockdown a431 cells ( n = 3 cocultures per group ). k, percentage of mitotracker - high hpfs after coculture with hacat or a431 cells ( n = 3 cocultures per cell line ).