2.2.7. Mitochondrial Activity

For mitochondrial assay, MitoTracker ™ Red (Molecular Probe, Eugene, OR, USA) cells were incubated for 72 h with CD-NHF and then subjected to 1 μg/mL MitoTracker and incubated for 30 min before the fluorescence of the resultant solutions was determined at 590 nm using a multiplate microplate reader.

### 2.2.8. 3D Matrigel Assays

The 3D Matrigel assays were conducted with 1000 cells seeded in Ibidi plates between 2 layers of Matrigel (BD Matrigel Matrix, Growth Factor Reduced (BD Biosciences)) and cultured for 14 days before microscopy analysis (TissueGnostic rig, Vienna, Austria, Europe). Twelve hours post-seeding, 3D embedded cells began to be treated with gels (CARB-F2, CMC-F3, AS-F5) alone and with gels containing 5% (50 μg/mL) CD-NHF (CARB-F4, CMC-F6, AS-F6) for 72 h. After 72 h, the treatments were removed and replaced with normal 3D Matrigel medium (medium corresponding to every cell type supplemented with 2% fetal bovine serum (FBS) and 1% Matrigel). The Live and Dead Cell Assay (Abcam) was used according to the manufacturer's instructions. Nuclei were counterstained with NucBlue Live Ready Probes Reagent (Thermo Fisher Scientific, Eugene, OR, USA). Fluorescence pictures were acquired at 20× magnification using a Zeiss Axio Observer Z1 Fluorescence Microscope from TissueGnostics rig. Single focal plane images were acquired using Tissue FAXS 4.2 software. The TissueQuest 6.0 software was used for total area segmentation analysis and to quantify the area and sum intensity of fluorescence signal for each spheroid (Figures S4 and S5).
