Figure 4.
Preclinical targeting of MFF-VDAC1 complex for cancer therapy. A, Predicted structure of MFF peptide #8–11 with L-amino acids (top) or retro-inverso D-enantiomer (bottom). B, PC3 cells were treated with biotin-labeled MFF (D) 8–11 peptidomimetic (10 μM), incubated with streptavidin-FITC (TRITC) and intracellular peptidomimetic accumulation was analyzed after 20 min by fluorescence microscopy. Nuclei were stained with DAPI. Representative images (n=2). C and D, PC3 cells were incubated with the indicated concentrations (10–25 μM) of cell-permeable scrambled peptide (Scr), MFF peptide #8–11 (L) or MFF (D) 8–11 peptidomimetic and analyzed for mitochondrial membrane potential by TMRE labeling and flow cytometry (C) or cell death (D) at the indicated time intervals. Mean±SD (n=2). E, PC3 cells (5×106 in 50% Matrigel) were engrafted onto the flanks of immunocompromised athymic mice, and animals randomized in two groups were treated with cell-permeable scrambled peptide or MFF (D) 8–11 peptidomimetic (50 mg/kg, daily i.p.) with quantification of tumor growth. Mean±SD (n=8–10). **, p=0.008 by unpaired two-tailed t test (tumor measurements at d. 13). F, Patient-derived melanoma xenografts resistant to the combination of Dabrafenib (Dab) plus Trametinib (Tra) were treated with vehicle (Veh) or MFF (D) 8–11 peptidomimetic (50 mg/kg) and tumor growth was quantified at the indicated time intervals. Mean±SD (n=5) *, p=0.02 by unpaired two-tailed t test. G, Primary, patient-derived NSCLC 3D organoids were treated with cell-permeable scrambled peptide (Scramb, 100 μM) or MFF (D) 8–11 peptidomimetic (100 μM) and analyzed after 24 h by hematoxylin-eosin (H&E) staining or immunohistochemistry for Ki-67 or cleaved caspase-3 (Cl. Casp.3) expression. Scale bar, 100 μm. H and I, The conditions are as in (G) and the percentage of cells stained for Ki-67 or cleaved caspase-3 in patient-derived NSCLC (H) or breast adenocarcinoma (AdCa, I) 3D organoids was quantified. Mean±SD (average of 2–3 independent fields). *, p=0.02; ***, p=0.0004 for 100 μM MFF (D) 8–11 peptidomimetic compared to scrambled peptide, by unpaired two-tailed t test. J and K, Patient-derived human glioblastoma (GBM) neurospheres in culture were treated with cell-permeable scrambled peptide (50 μM) or the indicated increasing concentrations of MFF (D) 8–11 peptidomimetic (μM) for 1, 3 or 24 h (J, representative GBM neurospheres after 24-h treatment are shown), stained with calcein (live cells) or To-Pro (dead cells), and normalized fluorescence units (FU) were quantified (K). BF, bright field. Mean±SD, two individual patients analyzed. The statistical analysis for each time point is as follows: 1-h, Scrambled peptide (Scr) vs. MFF 10, p=0.002; Scr vs. MFF 25, p=0.03; Scr vs. MFF 50, ns; 3-h, Scr vs. MFF 10, p=0.001; Scr vs. MFF 25, p=0.01; Scr vs. MFF 50, p=0.01; 24-h, Scr vs. MFF 10, p=0.03; Scr vs. MFF 25, p=0.02; Scr vs. MFF 50, p=0.01, all by unpaired two-tailed t test.