b, proton leak in the same cultures as in a. c, percentage of ki67 a431 cells and transwell migration of a431 cells cultured in cm of mitocepted or mock - treated hpfs ( n = 3 cultures per group ). d, rt – qpcr for inhba and il6 using rna from mitocepted ( mitochondria from mda - mb - 231 breast cancer cells ) or mock - treated hpfs ( n = 3 cultures per group ). e, clonogenicity of mda - mb - 231 cells cultured in cm from hpfs subjected to mitoception with mitotracker green - stained mitochondria from mda - mb - 231 cells or mock treatment ( n = 3 cultures per treatment group ). f, representative image of 3 - week - old ear xenograft tumors ( arrowheads ) following intradermal coinjection of a431 cells and mitocepted ( with a431 mitochondria ) or mock - treated hpfs and tumor volume at various time points ( n = 5 tumors per group from different mice ). g, representative immunoflu orescence stainings of tumors formed by a431 cells and mitocepted or mock - treated hpfs for e - cadherin and fn1 ( green ) and meca32 ( red ), counterstained with hoechst ( blue ) ( n = 5 tumors per group from different mice ). h, percentage of ki67 hpfs subjected to mitoception with a431 lmt mitochondria or mock treatment and rt – qpcr for inhba using rna from hpfs subjected to mitoception with a431 lmt mitochondria or mock treatment ( n = 9 ki67 or n = 3 rt – qpcr cultures per treatment group ). i, percentage of ki67 a431 cells ( left ) or transwell migration of a431 cells ( right ) cultured in cm from hpfs subjected to mitoception with a431 lmt mitochondria or mock treatment ( n = 9 ki67 or n = 3 transwell migration cultures per treatment group ). j, left, tumor volume at various time points during tumor development by a431 cancer cells coinjected with mitocepted hpfs, which received mitochondria from control or lmt a431 cells ( n = 4 tumors per group from different mice ). right, histological stainings of a tumor from each group. graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( c – e, h, i ) or two - sided one - way ( a, b ) or two - way ( f, j ) anova with bonferroni post hoc multiple comparison test was used to determine statistical significance. scale bars, 200 μm ( g ) and 1 mm ( j ). source data together, these results suggest that the transfer or transplantation of epithelial cancer cell - derived mitochondria alone is sufficient to reprogram fibroblasts. this is not limited to scc cells, as transfer of mitochondria from the breast and pancreatic cancer cell lines mda - mb - 231 and panc1, respectively, also increased the expression of caf markers and their cm promoted clonogenic growth of mda - mb - 231 and panc1 cells ( fig. 5d, e and extended data fig. 5a, b ). at day 5 after seeding, the proliferation rate of hpfs declined, particularly after mitoception ( extended data fig. 5c versus fig. 4d ). concomitantly, the number of β - galactosidase - positive fibroblasts increased among the mitocepted fibroblasts, indicating senescence ( extended data fig. 5d ). this is supported by their increased expression of the senescence markers cdkn1a and cdkn2b, while expression of most caf markers was no longer increased at this time point ( extended data fig. 5e, f ). the only exception was il6, which is also a senescence marker. nevertheless, the recipient hpfs may still exert protumorigenic effects because senescent cells often have a protumorigenic senescence - associated secretory phenotype. indeed, when we coinjected a431 - mitocepted or mock - treated hpfs with a431 cells into the ears of immunode ficient mice, the tumors that formed in the presence of fibroblasts containing a431 - derived mitochondria were significantly larger and showed increased deposition of fn1 and more blood vessels ( fig. 5f, g ). this was associated with the long - term presence of the mitocepted fibroblasts as determined in a separate experiment with hpfs, which received su9 – rfp - expressing a431 cancer cell mitochondria. then, 2 weeks after injection, these tagged fibroblasts were still detectable ( extended data fig. 5g ). to test whether disrup tion of mitochondrial function in cancer cells prevents caf differentiation upon mitochondrial transfer, we used fluidfm to extract mitochondria from a431 su9 – rfp cells, which were depolarized using carbonyl cyanide m - chlorophenylhydrazone ( cccp ). hpfs, which received cccp - treated mitochondria, had a mildly but significantly lower proliferation rate compared to hpfs, which received mock - treated mitochondria ( extended data fig. 6a ). in addition, hpfs mitocepted with mitochondria from a431 cells, which were pretreated with cccp, had a strongly reduced ocr compared to hpfs that received mitochondria from vehicle - treated cancer cells ( extended data fig. 6b ). we next generated a431 cancer cells with a 40 % reduction in the amount of mtdna ( termed low mtdna ( lmt ) cells ) using extended low-dose ethidium bromide treatment ( extended data fig. 6c ). mitochondria from lmt cells had a similar protein content to those from control cells and the viability and proliferation of lmt a431 cells were not reduced. however, their mitochondrial respiration was significantly impaired ( extended data fig. 6d – g ). upon transplantation of these mitochondria, the proliferation rate of the recipient cells was even reduced and expression of most caf markers was not significantly altered ( fig. 5h and extended data fig. 6h ). the cm of hpfs, which received mitochondria from lmt cancer cells, did not promote proliferation and migration of cancer cells ( fig. 5i ). in xenograft experiments, tumors formed by a431 cells coinjected with hpfs containing mitochondria from lmt a431 cells were significantly smaller compared to those formed with hpfs containing mitochondria from control a431 cells ( fig. 5j ). this finding underscores the critical role of mitochondrial dna, which encodes important components of the respiratory chain, in the induction of a protumorigenic caf phenotype in mitocepted fibroblasts. additional experiments using only lmt a431 cells showed that mice injected with these cells did not develop tumors within 15 days ( extended data fig. 6i, j ). to identify potential regulators of the mitochondrial transfer from skin cancer cells to fibroblasts, we used published single - cell rna ( scrna ) - seq data of tumors and site - matched normal skin from persons with cutaneous sccs. we used preprocessed annotations to identify keratinocyte populations and focused on genes with a documented function in mitochondrial trafficking, including miro1 ( rhot1 ), miro2 ( rhot2 ), trak1 and trak2 ( ref. ). we defined a gene as ‘ highly expressed ’ when its expression level exceeded the mean expression level observed across all cell populations examined. expression of miro2 was significantly elevated in malignant versus nonmalignant epithelial cells ( fig. 6a ). tumor - specific keratinocytes ( tsks ), a cluster exclusively present in tumor samples, exhibited particularly high miro2 expression ( fig. 6b ). this was confirmed with another scrna dataset from skin sccs ( extended data fig. 7a, b ). analysis of spatial transcriptomics data revealed elevated miro2 expression in invasively growing cells at tumor margins ( fig. 6c ). to delineate miro2 mrna localization relative to caf subtypes, we used coexpression analysis with pdgfra and specific markers for icafs ( mmp11 ), mycafs ( acta2 ) and adipose caf ( adicaf; cfd ). we detected substantial colocalization of miro2 mrna with these caf subtypes, particularly with mmp11 - positive icafs ( extended data fig. 7c ). we further applied cell2location spatial deconvolution using skin scc data as a single - cell ( ref. ) to estimate cell type distributions per spot. we computed caf scores using fibroblast - specific expression inferred by cell2location and identified miro2 spots on the basis of keratinocyte - specific expression.