Fig. 4: CAF reprogramming through transplantation of cancer cell mitochondria.
a, Representative fluorescence images of HPFs MitoCepted with MitoTracker green-stained A431 mitochondria (MitoCepted HPFs) or mock treatment, counterstained with Hoechst (blue) (n = 3 cultures per group). b, qPCR for the mtDNA encoding tRNA-Leu(UUR) relative to the nucDNA encoding B2M using DNA from MitoCepted (MC) or mock-treated (Ctrl) HPFs. Relative mtDNA content (based on Ct values) is indicated (n = 3 cultures per group). c, Representative confocal image in the xyz plane showing HPFs prestained with MitoTracker red and MitoCepted with MitoTracker green-labeled A431 mitochondria (left) and TOM20–GFP-expressing HPFs MitoCepted with mitochondria from A431 Su9–RFP cells (right). Yellow staining indicates mitochondrial fusion. d, Percentage of Ki67+ MitoCepted or control HPFs among all cells (n = 3 cultures per group). e, RT–qPCR for INHBA, IL6, ACTA2 and COL1A1 relative to RPL27 using RNA from MitoCepted or mock-treated HPFs (n = 3 cultures per group). f, FluidFM experimental setup, adapted from a previous study36. g, Image of FluidFM-mediated injection of mitochondria into HPFs. h, Percentage of Ki67+ fibroblasts in HPFs injected with A431-derived mitochondria using FluidFM or mock treatment (n = 3 cultures per group). i, RT–qPCR for INHBA using RNA from HPFs subjected to MitoCeption with mitochondria from HaCaT, HaCaT-Ras or A431 cell lines (n = 3 cultures per cell line). j, RT–qPCR for INHBA using RNA from (1) mock-treated HPFs or HPFs subjected to MitoCeption with mitochondria (2) from keratinocytes of a healthy individual, (3) from normal keratinocytes of a person with SCC or (4) malignant cancer cells of the same person with SCC. Right, representative FN1–COLI immunofluorescence stainings with quantification of staining intensity in the dECM produced by HPFs after MitoCeption with mitochondria from the different primary donor cells (n = 3 MitoCeptions per cell type). k, Percentage of Ki67+ HPFs subjected to MitoCeption with different amounts of mitochondria isolated from A431 cells. Numbers on the x axis show the ratio of donor A431 cells (used for mitochondrial isolation) and recipient HPFs (n = 3 cultures per group). Graphs show the mean ± s.e.m. An unpaired two-sided Student’s t-test (b,d,e,h) or two-sided one-way ANOVA with Bonferroni post hoc multiple comparison test (i–k) was used to determine statistical significance. Scale bars, 100 μm (a,j), 10 μm (c) and 25 μm (g).
