Fig. 4.

Direct and indirect mitochondrial transfer from fibroblasts to epithelial cells. a Before co-cultivation, NOFs were stained with MTDR (red mitotracker), while NOKs and OSCCs were stained with MTG (green mitotracker). Confocal microscopy for live cells showing mitochondrial transfer from NOFs (red colour) to NOKs and OSCCs (green colour) at different time points (1 h, 3 h, 24 h). Already after 3 h of direct co-culture, mitochondrial transfer was observed from NOFs into epithelial cells (yellow colour), but predominantly in OSCCs. b Confocal microscopy for live cells showing mitochondrial transfer from NOFs to OSCCs through tunneling nanotubes (pointed by the arrow). Presence of mitochondria in TNTs protruding from NOFs into OSCCs and NOKs could be detected after 24 h. Again, mitochondria-containing TNTs were observed predominantly between NOFs and OSCCs (magnification × 40). c, d Quantification of the mitochondrial transfer after co-culture at distance (indirect transfer) by flow cytometry. NOFs were stained with MTDR and OSCC were stained with MTG, then co-cultured for 48 h in transwell. There was a significant increase of MTDR in OSCCs after co-culture with NOFs, suggesting presence of indirect mitochondrial transfer from NOFs to OSCCs (c). Less MTG signal could be found in NOFs after indirect co-cultured with MTG marked OSCCs (d). e Confocal microscopy showing mitochondrial transfer between NOF to NOK and OSCC after co-culture at distance, in transwells. Before seeding, NOFs were stained with MTDR and NOKs or OSCCs were stained with MTG. After 48 h of indirect co-cultivation, epithelial cells (mainly OSCCs) with both green and red mitochondria were distinguished (pointed by the arrow). Double-stained cells (i.e., with green and red fluorescent mitochondria) reflect mitochondrial exchange. Scale bar = 10 μm. The data points were presented from six different patient sets. All points represent mean values and SEM. Independent experiments were performed more than three times *p < 0.05; Wilcoxon signed-rank test was used