g, rt – qpcr for inhba and il6 using rna from hpfs incubated with cm of sictrl or simiro2 a431 cells ( n = 3 cultures per treatment group ). h, rt – qpcr for inhba and il6 using rna from sorted hpfs cocultured with sictrl or simiro2 a431 cells ( n = 3 cocultures per group ). i, ocr of sictrl or simiro2 a431 cells. the time of drug injection is indicated ( n = 3 cultures per group ). j, rt – qpcr for miro2 using rna from a431 cells transfected with control or miro2 overexpression vectors ( oe - miro2 ) ( n = 3 cultures per group ). western blot of lysates from control or miro2 - overexpressing a431 cells for miro2 or gapdh ( n = 2 cultures per group ). k, percentage of mitotracker - high hpfs after coculture with mitotracker - stained control or miro2 - overexpressing a431 cells ( n = 3 cocultures per group ). l, percentage of ki67 hpfs after coculture with control or miro2 - overexpressing a431 cells ( n = 9 cocultures per group ). m, percentage of mitotracker - high hpfs after coculture with mitotracker - stained control or miro2 - overexpressing a431 cells, with or without treatment with dih ydrocytochalasin b ( n = 3 cocultures per group ). graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( a, c – e, g, h, l ) or two - sided one - way anova with bonferroni post hoc multiple comparison test ( m ) was used to determine statistical significance. scale bars, 100 μm ( b ) and 25 μm ( f ). source data a, rt – qpcr for miro2 using rna from sictrl or simiro2 a431 cells; western blot of total and mitochondrial lysates from these cells for miro2, vinculin or hsp60 ( loading controls ) ( n = 3 cultures per group ). b, fluorescence images of lifeact a431 cells ( red ) stained with mitotracker green and transfected with sictrl or simiro2 in coculture with hpfs, counterstained with hoechst. white arrowheads indicate a431 cells. c, percentage of mitotracker - high hpfs in cocultures with simiro2 or sictrl a431 cells, normalized to the number of cancer cells ( n = 3 cocultures per group ). d, qpcr for mtdna encoding trna - leu ( uur ) relative to nucdna encoding b2m using dna from a431 cells transfected with sictrl or simiro2. total mtdna content was calculated on the basis of ct values ( n = 3 cultures per group ). e, mitochondrial mass in sictrl and simiro2 a431 cells based on mitotracker green mean fluorescence intensity ( mfi ) ( n = 3 per group ). f, confocal images of sictrl or simiro2 a431 cells incubated with mitotracker green. the dashed line marks the outer edge of the cell ( n = 3 cultures per group ). g, rt – qpcr for inhba and il6 using rna from hpfs incubated with cm of sictrl or simiro2 a431 cells ( n = 3 cultures per treatment group ). h, rt – qpcr for inhba and il6 using rna from sorted hpfs cocultured with sictrl or simiro2 a431 cells ( n = 3 cocultures per group ). i, ocr of sictrl or simiro2 a431 cells. the time of drug injection is indicated ( n = 3 cultures per group ). j, rt – qpcr for miro2 using rna from a431 cells transfected with control or miro2 overexpression vectors ( oe - miro2 ) ( n = 3 cultures per group ). western blot of lysates from control or miro2 - overexpressing a431 cells for miro2 or gapdh ( n = 2 cultures per group ). k, percentage of mitotracker - high hpfs after coculture with mitotracker - stained control or miro2 - overexpressing a431 cells ( n = 3 cocultures per group ). l, percentage of ki67 hpfs after coculture with control or miro2 - overexpressing a431 cells ( n = 9 cocultures per group ). m, percentage of mitotracker - high hpfs after coculture with mitotracker - stained control or miro2 - overexpressing a431 cells, with or without treatment with dih ydrocytochalasin b ( n = 3 cocultures per group ). graphs show the mean ± s. e. m. an unpaired two - sided student ’ s t - test ( a, c – e, g, h, l ) or two - sided one - way anova with bonferroni post hoc multiple comparison test ( m ) was used to determine statistical significance. scale bars, 100 μm ( b ) and 25 μm ( f ). source data mitochondrial transfer was significantly reduced when hpfs were cocultured with simiro2 versus control a431 cells ( fig. 7c ), while knockdown of miro1, trak1 and trak2 even increased the transfer ( extended data fig. 9c, d ). miro2 knockdown did not lead to a decrease in mitochondrial dna copy number or mitochondrial mass ( fig. 7d, e ) and did not impede the activation of caf marker expression through mitoception ( extended data fig. 9e ), suggesting that miro2 is mainly responsible for the mitochondrial transfer rather than the effect on the recipient fibroblasts. the impaired mitochondrial transfer by miro2 - knockdown cells correlated with perinuclear clustering of mitochondria ( fig. 7f ). this is relevant, because proper distribution of mitochondria is important for protein secretion and cell migration. it is consistent with the role of miro family proteins in the regulation of mitochondrial distribution. the depletion of mitochondria at the periphery of cancer cells is likely to impact the mitochondrial transfer to fibroblasts. incubation of fibroblasts with the cm of simiro2 versus control a431 cells did not significantly affect caf marker gene expression, in contrast to the effect of miro2 knockdown in direct coculture ( fig. 7g, h ). these findings again demonstrate that direct contact between cancer cells and fibroblasts, which allows mitochondrial transfer through tnts, is necessary for the induction of a caf phenotype. depletion of miro2 did not significantly impact the metabolic activity of a431 cells ( fig. 7i ). therefore, the perinuclear clustering of mitochondria upon miro2 depletion is not attributed to major deficiencies in mitochondrial respiration or metabolism. instead, it is likely a consequence of altered mitochondrial motility and distribution within the cell. overexpression of miro2 promoted transfer activity of a431 and scc13 cells and a mild effect was also seen for hacat cells, as shown by flow cytometry analysis of mitotracker - high hpfs ( fig. 7j, k and extended data fig. 9f ). however, the increase in mitochondrial transfer from a431 cells to hpfs did not further promote hpf proliferation ( fig. 7l ). together with the results from mitoception studies with different amounts of mitochondria ( fig. 4k ), these data suggest a threshold for caf reprogramming, beyond which further mitochondrial transfer to fibroblasts has no additional effect. inhibition of actin polymerization nearly abolished the elevated mitochondrial transfer from miro2 - overexpressing a431 cells to fibroblasts ( fig. 7m ). the selective effect of miro2 knockdown or overexpression on the number of mitotracker - positive fibroblasts further confirms the suitability of mitotracker staining under our experimental conditions, as dye leakage would not be affected by these treatments. next, we tested the impact of miro2 depletion on the malignant features of a431 cells. a 24 - h knockdown of miro2 significantly reduced their proliferation without impacting their viability ( fig. 8a, b ). miro2 - knockdown cells also migrated more slowly than control cells in a transwell assay and formed smaller, less developed 3d spheroids ( fig. 8c, d ). similar results were obtained with scc13 cells ( fig. 8e – h ). lastly, nod scid mice injected with simiro2 a431 cells failed to develop tumors, whereas control cells rapidly formed large tumors ( fig. 8i, j ). microscopic examination of the ear tissue from the simiro2 a431 group revealed small, undeveloped cancer cell colonies with undetectable miro2 expression ( fig. 8j, inset ). therefore, even a transient reduction of miro2 levels during the early phase of tumor formation is sufficient to prevent tumorigenesis. fig. 8miro2 depletion in cancer cells reduces mitochondrial transfer and tumor growth. a, percentage of ki67 a431 cells 24 h after transfection with sictrl or simiro2 ( n = 3 cultures per group ).