b, relative viability of a431 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). c, transwell migration of a431 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). d, relative spheroid area of a single hanging drop formed by sictrl or simiro2 a431 cells and representative images of the spheroids ( n = 9 spheroids per group ). e, percentage of ki67 scc13 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). f, relative viability of scc13 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). g, transwell migration of scc13 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). h, relative spheroid area of a single hanging drop formed by sictrl or simiro2 scc13 cells and representative images of the spheroids ( n = 15 spheroids per group ). i, photo of 5 - week - old ear xenograft tumors ( indicated by arrowheads ) formed after injection of 200, 000 a431 cells transfected with simiro2 or sictrl and tumor volume at different time points ( n = 3 tumors per group from different mice ). j, representative photomicrographs of herovici - stained tumors ( left ) formed by simiro2 or sictrl a431 cells and immunoflu orescence staining of sections from these tumors for k14 ( green ) and miro2 ( red ), counterstained with hoechst ( blue ). inset, a tumor cell island with persistent miro2 knockdown ( n = 3 sections from independent tumors per group ). k, normalized cell count of a431 lifeact – rfp cells transfected with sictrl or simiro2, cocultured with or without hpfs in spheroids and analyzed by facs after 5 days ( n = 3 spheroids per group ). l, tumor volume during tumor development following coinjection of hpfs ( with or without a431 - derived mitochondria, introduced using mitoception ) and a431 cells transfected with either simiro2 or sictrl ( n = 5 tumors per group ). m, representative h & e stainings of tumors from each group ( n = 5 mice ). graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( a – h ) or two - sided one - way ( k ) or two - way ( i, l ) anova with bonferroni post hoc multiple comparison test ( i, k, l ) was used to determine statistical significance. scale bars, 100 μm ( d ), 200 μm ( j ) and 1 mm ( m ). source data a, percentage of ki67 a431 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). b, relative viability of a431 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). c, transwell migration of a431 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). d, relative spheroid area of a single hanging drop formed by sictrl or simiro2 a431 cells and representative images of the spheroids ( n = 9 spheroids per group ). e, percentage of ki67 scc13 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). f, relative viability of scc13 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). g, transwell migration of scc13 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ). h, relative spheroid area of a single hanging drop formed by sictrl or simiro2 scc13 cells and representative images of the spheroids ( n = 15 spheroids per group ). i, photo of 5 - week - old ear xenograft tumors ( indicated by arrowheads ) formed after injection of 200, 000 a431 cells transfected with simiro2 or sictrl and tumor volume at different time points ( n = 3 tumors per group from different mice ). j, representative photomicrographs of herovici - stained tumors ( left ) formed by simiro2 or sictrl a431 cells and immunoflu orescence staining of sections from these tumors for k14 ( green ) and miro2 ( red ), counterstained with hoechst ( blue ). inset, a tumor cell island with persistent miro2 knockdown ( n = 3 sections from independent tumors per group ). k, normalized cell count of a431 lifeact – rfp cells transfected with sictrl or simiro2, cocultured with or without hpfs in spheroids and analyzed by facs after 5 days ( n = 3 spheroids per group ). l, tumor volume during tumor development following coinjection of hpfs ( with or without a431 - derived mitochondria, introduced using mitoception ) and a431 cells transfected with either simiro2 or sictrl ( n = 5 tumors per group ). m, representative h & e stainings of tumors from each group ( n = 5 mice ). graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( a – h ) or two - sided one - way ( k ) or two - way ( i, l ) anova with bonferroni post hoc multiple comparison test ( i, k, l ) was used to determine statistical significance. scale bars, 100 μm ( d ), 200 μm ( j ) and 1 mm ( m ). source data to determine whether the poor spheroid growth of miro2 - knockdown cells and their failure to form tumors in mice is simply a consequence of their cell - autonomous defect in proliferation or migration or whether it involves non - cell - autonomous effects, such as impaired mitochondrial transfer to fibroblasts, we set up coculture spheroid assays. the difference in proliferation between sictrl and simiro2 cancer cells was even more pronounced in the presence of fibroblasts ( fig. 8k ), indicating an important non - cell - autonomous role of miro2. in xenograft experiments, coinjection of simiro2 a431 cells with control hpfs already caused a mild stimulation of tumor growth but the tumor - promoting effect was much stronger with mitocepted hpfs. this combination compensated for the deficiency of simiro2 cancer cells in tumor formation and the rate of tumor growth was almost comparable to that of the control group, in which sictrl a431 cells were coinjected with control fibroblasts ( fig. 8l, m ). these findings highlight the notable influence of fibroblasts with mitochondria from cancer cells on tumor formation and further suggest an important role of miro2 in this transfer. we identified mitochondrial transfer through tnts as a strategy of cancer cells to promote caf differentiation. because cancer cells and cafs often have direct contact in the tumor, particularly at its periphery, this transfer is likely to contribute to the increased invasiveness of cancer cells. we further show that caf differentiation through mitochondrial transfer from cancer cells is supported by two mechanisms. first, cancer cells at the invasive front overexpress miro2, which promotes the mitochondrial transfer. second, mitochondria from malignant cells but not from nontumorigenic epithelial cells can induce a caf phenotype. this could be explained by alterations in the proteome of mitochondria from cancer cells and the associated metabolic alterations. in support of this hypothesis, uptake of mitochondria from a431 cancer cells altered the expression of several metabolic proteins and promoted oxphos and atp production in the recipient fibroblasts. these features were shown to promote proliferation, matrix production and protein secretion by fibroblasts and cafs. consistent with an important role of oxphos in the fibroblast reprogramming by mitochondrial transfer, inhibition of this metabolic pathway in the recipient fibroblasts blocked the induction of important caf features. therefore, metabolic alterations induced by mitochondria from cancer cells contribute to the caf phenotype. the induction of a caf phenotype was associated with significant changes in the expression of genes and proteins associated with inflammation, immune response, cellular metabolism and stress response. in the cocultures, we found increased expression of many isgs. this is consistent with the sensing of genomic damage of cancer cells by fibroblasts, which resulted from transcytosis of cytoplasm from cancer cells into neighboring fibroblasts and activation of the stimulator of interferon genes – interferon regulatory factor 3 pathway.