Fig 2: Overexpression of mDia2 and MIRO1 in CAFs promotes tumorigenesis. A, Expression of MIRO1 in liver, breast, and ovarian cancer stroma (CS) vs. stroma of respective normal tissues (NS) based on data sets GSE45001 (N = 10 per group), GSE9014 (N = 6 and 53), and GSE40595 (N = 8 and 36). B, Single-cell expression profiles of mDia2/DIAPH3 and MIRO1 in immune cells, endothelial cells, and CAFs of melanomas based on data from Single-Cell Portal (Broad Institute). C, Correlation analysis between MIRO1 and mDia2, INHBA, and TGFB1 gene expression across different cancers using TIMER2. UVM, uveal melanoma; LUAD, lung adenocarcinoma; PAAD, pancreatic ductal adenocarcinoma; SKCM, skin cancer cutaneous melanoma; BRCA-Lum, luminal breast cancer; LIHC, liver hepatocellular carcinoma. Red or blue indicates a statistically significant positive/negative correlation (Spearman, P < 0.05), respectively, and gray denotes a nonsignificant result. D, Kaplan–Meier survival curves based on TCGA and GTEx data. High expression of mDia2/DIAPH3 and MIRO1 in patients with liver or lung cancer correlates with poor survival. E, Representative sections from normal human skin, cSCC, and BCC stained for MIRO1. Scale bars, 100 µm. T, tumor cells. F, qRT-PCR analysis for MIRO1 using RNA from CAFs or NFs from two SCC patients. n = 3. G, Representative Western blot of total lysates of NFs and CAFs from patient No. 1 for MIRO1, CAF markers, and GAPDH. H, Quantification of SCC13 tumor spheroid area in hanging drop including CM from NF and CAFs. n = 10–11. I, Mitochondrial distribution in NFs or CAFs from patient No.1. n = 15–19. J, Representative images and bar graph showing fluorescence intensity distribution of ATP biosensor (cyto-Ruby3-iATPSnFR1.0) relative to the highest intensity value from the perinuclear region to the plasma membrane of NFs and CAFs (patient No. 1). n = 19. K, Mitochondrial branch length of NFs or CAFs (patient No.1). n = 15–19. L, Representative immunofluorescence images of decellularized matrices obtained from NFs and CAFs (patient No. 1) stained for FN1 and COL1A1 and quantification of the stained area. n = 3. M, Quantification of SCC13 tumor spheroid area in hanging drop including CM from NFs transfected with an expression vector encoding human MIRO1 (pGEM-MIRO1 “+”) or empty vector (pGEM-EV “-”). The spheroid area in one control sample was set to 1. n = 10–12. N, SCC13 cells plated on either decellularized matrix from NFs transfected with pGEM-MIRO1 or pGEM-EV. After 3 days, the number of Ki67-positive SCC13 cells was quantified. n = 3. O, Representative FN1 and COL1A1 immunofluorescence stainings and quantification of the stained area in the decellularized matrix derived from NFs transfected with pGEM-MIRO1 or pGEM-EV. n = 3. P and Q, sh-EV and sh-mDia2 fibroblasts were transfected with pGEM-MIRO1 (“+”) or pGEM-EV (“-”). Expression of MIRO1 was analyzed by immunofluorescence 24 hours after transfection (P). n = 3. Bar graph shows the fluorescence intensity distribution of MitoTracker relative to the highest intensity value from the perinuclear region to the plasma membrane (Q). n = 6–8. Bar graphs show mean ± SEM. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [one-way ANOVA with Bonferroni post hoc test (P), two-way ANOVA with Bonferroni post hoc test (I, J, and Q), unpaired Student t test (A, F, H, and K–O)]. Scale bars, 50 µm.

Fig 3: mDia2 binds and stabilizes MIRO1 in primary human fibroblasts. A, Representative Western blot of total lysate of sh-EV or sh-mDia2 fibroblasts for mDia2, MIRO1, GAPDH, and a-tubulin. B, qRT-PCR analysis for MIRO1 using RNA from sh-EV and sh-mDia2 HDFs. n = 3. C, Representative Western blot of mitochondrial fractions from sh-EV and sh-mDia2 fibroblasts probed for mDia2, MIRO1, and HSP60, and quantification of mitochondrial mDia2/HSP60 or MIRO1/HSP60 ratios. n = 3. D, Representative images of sh-EV and sh-mDia2 HDFs immunostained for MIRO1 (red), counterstained with MitoTracker (green), and Hoechst (blue). Dotted lines outline the cell border. E, Western blot of total lysate from sh-EV or sh-mDia2 fibroblasts transfected with pMX-WT, pMX-IA, or pMX-control for MIRO1 and GAPDH, and quantification of the MIRO1/GAPDH ratio. n = 3. F, Representative Western blots for MIRO1 and vinculin using total lysates of sh-EV and sh-mDia2 fibroblasts treated for 2 hours with epoxomicin (Epox; 10 µmol/L) and quantification of the MIRO1/VINCULIN ratio. n = 3 biological replicates. G, Representative images of sh-mDia2 fibroblasts treated with epoxomicin (10 µmol/L) or vehicle and stained with MitoTracker (green) and rhodamine-coupled phalloidin (red). Bar graphs show the fluorescence intensity distribution of MitoTracker relative to the highest intensity value from the perinuclear region to the plasma membrane and mean network branch quantified using the MiNA toolset. n = 22–29. H and I, Representative images showing PLA signals (red) in HDFs stained with mDia2 and MIRO1 antibodies and in the negative control (-Ctrl) without primary antibodies (H), counterstained with Hoechst (blue), either alone (H) or together with MitoTracker (green; I). Graphs show mean ± SEM. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [one-way ANOVA with Bonferroni post hoc test (E–G for network analysis), two-way ANOVA with Bonferroni post hoc test (G for MitoTracker intensity), or unpaired Student t test (B and C)]. Scale bars, 50 µm.

Fig 4: Depletion of MIRO1 in CAFs suppresses skin tumorigenesis. A and B, sh-EV or -mDia2 HDFs cocultured for 7 days with SCC13 cells immunostained for E-cadherin (red) and counterstained with MitoTracker (green) and Hoechst (blue; A). The mitochondrial distribution relative to the highest intensity value from the perinuclear region of fibroblasts to the interface with SCC13 cells was quantified (B). Relative distances range from 1 to 10. n = 4–6. C, Representative Western blots for MIRO1, mDia2, and GAPDH using total lysates of NFs and CAFs (patient No. 1) cultured for 24 hours with SMIFH2 (25 µmol/L) or vehicle. The dotted line indicates cutting of the membrane; all samples were loaded on the same gel. D, Representative images of NFs and CAFs cultured with SMIFH2 (25 µmol/L) or vehicle and stained with rhodamine-coupled phalloidin (red), MitoTracker (green), and Hoechst (blue). E, Two-dimensional cocultures of SCC13 cells with si-Ctrl or si-MIRO1 CAFs at day 7. SCC13 cells were identified by E-cadherin (red) staining and counterstained with MitoTracker (green) and Hoechst (blue). The mitochondrial distribution relative to the highest intensity value from the perinuclear region of fibroblasts to the interface with SCC13 cells was quantified. Relative distances range from 1 to 10. n = 12–14. F, Chemotactic transwell migration of SCC13 cells using CM from si-Ctrl or si-MIRO1 CAFs. n = 3. G, Quantification of the SCC13 spheroid area in a single hanging drop including CM from si-Ctrl or si-MIRO1 CAFs. The spheroid area in one si-Ctrl sample was set to 1. N = 6. H, Tumor volume at different time points 2 weeks after injection (n = 4) and representative photos of 2-week-old tumors formed upon intradermal coinjection of SCC13 cells with si-MIRO1 or si-Ctrl CAFs. Inset, area where tumor cells invade into the cartilage. Asterisk, cartilage; arrow, site of invasion. I, Representative images of COL1A1 immunofluorescence and Herovici staining of tumors formed by SCC13 cells transfected with si-MIRO1 or si-Ctrl CAFs at day 15. J, Quantification of the percentage of the COL1A1-positive area and curvature of fibers per field of view. n = 3–4. K, Increase in SCC13 (coinjected with CAFs) tumor volume (mm3) upon injection of the tumors with MIRO1-Reducer (2.5 µmol/L per injection) or vehicle (DMSO) once every 3 to 4 days for 18 days. Red arrows, treatment time points. n = 5. Bar graphs show mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 [two-way ANOVA with Bonferroni post hoc test (B, E, H, and K), unpaired Student t test (F, G, and J)]. Scale bars, 25 µm (A, D, and E) and 50 µm (I).

Fig 5: Loss of Miro1 results in severe mitochondrial disorganization in early parthenotes. (A) Representative images of 1-cell parthenotes were imaged after the activation. Fire: TMRM. Dashed line: Oocyte membrane. White line: ROI (35 µm long) for analysis. Scale bar: 15 µm (B) Normalized mitochondrial intensity from the center to the cell periphery (35 µm) in a circle (360°). Data presented as mean ± SD. Paired t-test, ****p < 0.0001, ***p < 0.001, n.s., not significant. Data are collated from 3 independent experimental replicates (C) Mitochondrial distribution during the first mitotic cell division in control and Miro1 KO parthenotes. Fire: TMRM. Time: min. Scale bar: 15 µm. (D) Representative images of mitochondrial distribution in 2-cell parthenotes. Fire: TMRM. Scale bar: 15 µm. (E) Quantification data of mitochondrial inheritance in each blastomere at 2-cells. Normalized mitochondrial intensity was measured in z-projection images. (F) Ratio of mitochondrial intensity in B1 and B2. Unpaired t-test, n.s., not significant. Data are collated from 3 independent experimental replicates.