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 we observed elongated, thin bridges with a length of 10 – 100μm between cancer cells and hpfs ( fig. 1c ), suggesting that the transfer of mitochondria occurs through tnts. consistently, we did not observe mitotracker - high hpfs in a transwell assay, which does not allow communication through tnts and gap junctions ( fig. 1d ). treatment of the cocultures with the gap junction inhibitor carbenoxolone even increased the transfer, as shown by flow cytometry analysis of mitotracker - high cells, and knockdown of connexin 26, a major connexin in skin cancer cells, had no effect ( fig. 1e, f ). these data further suggest that the transfer occurs through tnts. this was confirmed by real - time holotomographic imaging ( fig. 1g and supplementary video 1 ). phalloidin combined with mitotracker staining revealed actin - containing tnts transferring mitochondria from a431 cancer cells to hpfs ( fig. 1h ). because tnts include actin and, in some cases, also microtubules, we explored the requirement of these cytoskeletal components for mitochondrial transfer. treatment of the cocultures with the microtubule polymerization inhibitor nocodazole even enhanced the transfer, while the actin polymerization inhibitor dih ydrocytochalasin b had a strong inhibitory effect ( fig. 1i ). knockdown of the exocyst complex components sec3 ( exoc1 ) and sec5 ( exoc2 ), which have a documented role in the regulation of the actin cytoskeleton and in tnt formation, also reduced the transfer ( fig. 1j ). mitochondrial transfer was also observed in cocultures of hpfs with immortalized but nontumorigenic human keratinocytes ( hacat cells ). however, their transfer efficiency was significantly lower compared to a431 cancer cells ( fig. 1k ). in addition, mda - mb - 231 breast cancer and panc1 pancreatic cancer cells transferred mitochondria to fibroblasts, demonstrating that this process occurs in different types of cancer cells ( fig. 1l ). the selective effects of actin polymerization inhibitors and of sec3 – sec5 small interfering rnas ( sirnas ) strongly suggest that the bright signal observed in some fibroblasts adjacent to cancer cells resulted from mitochondrial transfer rather than from dye leakage. nevertheless, we performed additional controls to further confirm the specificity. fluorescence analysis of fluorescence - activated cell sorting ( facs ) - sorted hpfs showed that mitotracker was stably incorporated into their mitochondrial network after serial passages and mitotracker staining was not detectable after culture of hpfs in the conditioned medium ( cm ) of mitotracker - treated a431 cells ( extended data fig. 1a, b ). as an alternative, we used cocultures of human a431 cells and mitotracker green - positive mouse fibroblasts. after 24 h, we detected human mitochondrial dna in the fibroblasts by pcr ( fig. 2a ). they exclusively expressed murine fibronectin 1 ( fn1 ), confirming their murine origin ( fig. 2b ). therefore, human cancer cells also transfer their mitochondria into mouse fibroblasts, although the transfer efficiency was significantly lower than in the human – human cocultures ( fig. 2c ). we confirmed the transfer by making use of species - specific single-nucleotide polymorphisms ( snps ). unique sequence variants within the 16s ribosomal rna ( rrna ) gene region of a431 cell mitochondria were detected in the mitochondria of the recipient mouse fibroblasts ( fig. 2d ). fig. 2cancer cells transfer mitochondria to fibroblasts in vitro and in vivo. a, qpcr for the human mtdna encoding trna - leu ( uur ) relative to mouse nuclear dna encoding beta2 - microglobulin ( b2m ) using dna from mitotracker - positive and mitotracker - negative mouse fibroblasts sorted from n = 3 cocultures. total mtdna content was calculated on the basis of ct values. b, rt – qpcr for human and mouse fn1 and fn1 relative to rpl27 or rps29, respectively, using rna from mitotracker - high and mitotracker - low mouse fibroblasts sorted from n = 3 cocultures. c, transfer efficiency in human – human and human – mouse cocultures ( n = 3 cocultures per group ). d, comparison of snps within the 16s rrna gene region of a431 cell mitochondria with those of control and recipient mouse fibroblasts. snps from a431 cells in recipient fibroblasts are indicated with rectangles ( n > 300, 000 cells pooled from three independent cocultures ). e, experimental setup and fluorescence images of hpf su9 – rfp and a431 su9 – gfp cocultures. this image was created with biorender. com. bottom, ( 1 ) colocalization of a431 and hpf mitochondria ( orange ), ( 2 ) hpf periphery with own mitochondrial network ( red ) and ( 3 ) nonrecipient hpfs ( red ). f, section of a xenograft tumor formed by su9 – rfp a431 cells, showing su9 – rfp fluorescence in mitochondria of keratin 14 ( k14 ) - negative cells and cultured fibroblasts from these tumors showing su9 – rfp fluorescence ( red ) and vimentin expression. g, qpcr for the mtdna - encoded human trna - leu gene relative to the mouse b2m gene using total dna from cultured mouse fibroblasts isolated from noninjected ear skin ( ns ) or a431 xenograft tumors ( n = 3 normal skin samples and n = 3 tumor samples from different mice ). h, representative immunoflu orescence staining of a431 xenograft tumors for coli ( green ) and human mitochondria ( red ). costaining ( yellow ) of stromal cells adjacent to tumors was confirmed by colocalization analysis ( site indicated with an asterisk ). the white arrow indicates the line along which the intensity values of the different fluorescence signals were measured, starting from the initial position at the base of the arrow and ending at the arrowhead. separate channels of zoomed - in regions are displayed ( n = 3 sections from different tumors ). graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( a, c, g ) or two - sided one - way anova with bonferroni post hoc multiple comparison test ( b ) was used to determine statistical significance. one control value was set to 1. scale bars, 25 μm ( e, f, h ). source data a, qpcr for the human mtdna encoding trna - leu ( uur ) relative to mouse nuclear dna encoding beta2 - microglobulin ( b2m ) using dna from mitotracker - positive and mitotracker - negative mouse fibroblasts sorted from n = 3 cocultures. total mtdna content was calculated on the basis of ct values. b, rt – qpcr for human and mouse fn1 and fn1 relative to rpl27 or rps29, respectively, using rna from mitotracker - high and mitotracker - low mouse fibroblasts sorted from n = 3 cocultures. c, transfer efficiency in human – human and human – mouse cocultures ( n = 3 cocultures per group ). d, comparison of snps within the 16s rrna gene region of a431 cell mitochondria with those of control and recipient mouse fibroblasts. snps from a431 cells in recipient fibroblasts are indicated with rectangles ( n > 300, 000 cells pooled from three independent cocultures ). e, experimental setup and fluorescence images of hpf su9 – rfp and a431 su9 – gfp cocultures. this image was created with biorender. com. bottom, ( 1 ) colocalization of a431 and hpf mitochondria ( orange ), ( 2 ) hpf periphery with own mitochondrial network ( red ) and ( 3 ) nonrecipient hpfs ( red ).