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 next, we stably expressed mitochondria - targeted red fluorescent protein ( su9 – rfp ) in hpfs and green fluorescent protein ( su9 – gfp ) in a431 cells. after 24 h coculture, we detected hpfs with mitochondria that appeared orange in close proximity to the gfp - labeled cancer cells ( fig. 2e ), confirming the uptake of mitochondria from cancer cells and suggesting their fusion with mitochondria of recipient fibroblasts. a431 cells expressing su9 – rfp were then injected intradermally into the ears of immunocompromised nod scid mice. fluorescence microscopy analysis of the resulting tumors showed mitochondrial structures of stromal fibroblasts that were positive for the mitochondrial protein expressed by cancer cells. we also detected human mitochondrial dna ( mtdna ) in cultured primary mouse fibroblasts from the tumors ( fig. 2f, g ). lastly, we stained sections from skin cancer xenograft tumors formed by a431 cells with an antibod y specific for human mitochondria. in addition to the expected strong staining of the tumor cells, adjacent stromal cells showed clear staining, which overlapped with staining for the pan - fibroblast markers collagen type i ( coli ) or platelet - derived growth factor alpha ( pdgfrα ) ( fig. 2h and extended data fig. 1c ). the transfer of mitochondria into fibroblasts in vivo was verified with breast and pancreatic cancer cells by costaining of respective xenograft tumors with antibod ies to human mitochondria and coli ( extended data fig. 1d ). to assess the functional relevance of the mitochondrial transfer, we sorted viable hpfs with high and low mitotracker green fluorescence intensity ( extended data fig. 2a ) and analyzed them by rna sequencing ( rna - seq ). control fibroblasts were subjected to the sorting procedure but not maintained in cocultures. e - cadherin mrna was not detected in the sorted fibroblasts, confirming the efficient sorting. principal component analysis ( pca ) showed distinc t clustering ( extended data fig. 2b ). there were significant differences in gene expression between hpfs in the coculture ( mitotracker - high and mitotracker - low ) versus control hpfs in monoculture and also between mitotracker - high and mitotracker - low hpfs ( fig. 3a – c ), although the two latter populations were exposed to the same cancer cell secretome. this finding suggests a strong impact of cancer - cell - derived mitochondria on the recipient hpfs. genes significantly upregulated in the mitotracker - high versus mitotracker - low group were predominantly involved in pathways related to inflammation, immune response, cellular metabolism and stress responses ( extended data fig. 2c, d ). activation of the interferon pathway was reflected by the increased expression of several interferon response genes ( isgs ) ( extended data fig. 2e ). fig. 3transferred cancer cell mitochondria induce a caf phenotype. a – c, volcano plots displaying differentially expressed genes in mitotracker - low versus control ( a ), mitotracker - high versus control ( b ) and mitotracker - high versus mitotracker - low hpfs ( c ) sorted from n = 3 cocultures with a431 cells. d, e. rna - seq data from sorted hpfs depicting expression of inhba, il6, acta2 and col1a1 ( d ) or pdgfra, pdgfrb, s100a4, fap and cd74 ( e ) in mitotracker - high, mitotracker - low and control groups sorted from n = 3 cocultures. f – h, comparative analysis of gene signatures in mitotracker - high versus mitotracker - low hpfs with published caf datasets, showing similarities of mitotracker - high hpfs with mycafs and icafs. i, volcano plot displaying differentially abundant proteins in mitotracker - high versus mitotracker - low hpfs sorted from n = 4 cocultures with a431 cells. j, correlation analysis of gene and protein expression in mitotracker - high versus mitotracker - low hpfs. significantly regulated pathways ( q < 0. 1 ) are highlighted ( blue, hallmarks of cancer pathways; purple, wikipathways ). k, percentage of ki67 - positive hpfs and relative levels of intracellular atp and mitosox in sorted hpfs ( n = 3 cocultures for ki67 and 6 cocultures for atp and mitosox ). l, percentage of ki67 - positive a431 cells in spheroids cultured with cm from control hpfs and sorted mitotracker - high and mitotracker - low hpfs ( n = 3 spheroids per treatment group ). m, transwell migration of a431 cells in cm from sorted mitotracker - low and mitotracker - high and control hpfs ( n = 3 transwell cultures per treatment group ). n. relative colony size of a431 cells plated on decm from sorted hpfs ( n = 3 cultures ). graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( k ( right and middle ), n ) or two - sided one - way anova with bonferroni post hoc multiple comparison test ( d, e, k ( left ), l, m ) was used to determine statistical significance. one control value was set to 1. source data a – c, volcano plots displaying differentially expressed genes in mitotracker - low versus control ( a ), mitotracker - high versus control ( b ) and mitotracker - high versus mitotracker - low hpfs ( c ) sorted from n = 3 cocultures with a431 cells. d, e. rna - seq data from sorted hpfs depicting expression of inhba, il6, acta2 and col1a1 ( d ) or pdgfra, pdgfrb, s100a4, fap and cd74 ( e ) in mitotracker - high, mitotracker - low and control groups sorted from n = 3 cocultures. f – h, comparative analysis of gene signatures in mitotracker - high versus mitotracker - low hpfs with published caf datasets, showing similarities of mitotracker - high hpfs with mycafs and icafs. i, volcano plot displaying differentially abundant proteins in mitotracker - high versus mitotracker - low hpfs sorted from n = 4 cocultures with a431 cells. j, correlation analysis of gene and protein expression in mitotracker - high versus mitotracker - low hpfs. significantly regulated pathways ( q < 0. 1 ) are highlighted ( blue, hallmarks of cancer pathways; purple, wikipathways ). k, percentage of ki67 - positive hpfs and relative levels of intracellular atp and mitosox in sorted hpfs ( n = 3 cocultures for ki67 and 6 cocultures for atp and mitosox ). l, percentage of ki67 - positive a431 cells in spheroids cultured with cm from control hpfs and sorted mitotracker - high and mitotracker - low hpfs ( n = 3 spheroids per treatment group ). m, transwell migration of a431 cells in cm from sorted mitotracker - low and mitotracker - high and control hpfs ( n = 3 transwell cultures per treatment group ). n. relative colony size of a431 cells plated on decm from sorted hpfs ( n = 3 cultures ).