Dmsi4000 b

Tissue samples were fixed in 4% formaldehyde, processed, embedded in paraffin and sectioned with Leica Microtome. Sections were stained using Ki67 antibody (Abcam) and CD31 antibody (Abcam). For each sample three randomly chosen fields were imaged by Leica camera and analyzed by ImageJ software. Cryo-sections (5 μm thick) were incubated with LAMP-1 primary antibody (Santa Cruz Biotechnology) followed by the Anti-Mouse IgG1 FITC (eBioscience) secondary antibody. Verteporfin auto-fluorescence was observed in the far-red spectrum. Coverslips were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, H-1500) and fluorescence images were taken using an automated inverted microscope (Leica DMSI4000 B).

HCC surgical resections were obtained from consented patients at University Hospital of Bern. Enzymatic digestion of minced tissue and 3D tumoroids culture were performed as previously described^{34 (link)}. For the drug-sensitivity assay, tumoroids were plated in Costar® Ultra-Low Attachment 96-well plate (Corning) with expansion medium supplemented with VP, SF and the respective vehicles. Tumoroid-viability assay was conducted as previously described^{34 (link)}. Briefly, HCC patient-derived tumoroids (PDT) viability was assessed by CellTiter-Glo® 3D Cell Viability Assay (Promega) after 2 days of drugs incubation, according to the manufacturer’s guidelines. To assess the tumoroid 3D structure and morphology, bright-field images were taken using an automated inverted microscope (Leica DMSI4000 B).

For live-cell imaging, HCC cell lines and primary human hepatocytes were seeded (1.5 × 10⁴ cells per well) in a chambered coverglass (Thermo Scientific) and treated as indicated before. After 24 h cells were washed twice in cold DPBS and stained with LysoTracker DNS-99 (Thermo Fisher). Nuclei were counterstained with DAPI (1:5000). Verteporfin auto-fluorescence was observed in the far-red spectrum (Cy5 channel). Fluorescence images of live-cells were obtained using an automated inverted microscope (Leica DMSI4000 B). For the Lysotracker fluorescence assay, HCC cells were seeded (1.5 × 10⁴ cells per well) on a 96-well assay plate and treated as described. After 24 h, medium was aspirated and discarded from all the wells followed by Lysotracker incubation as previously reported^{30 (link)}. LysoTracker fluorescence intensity was then measured by spectrophotometry (Ex = 577 nm, Em = 590 nm) on a fluorescence plate reader (Tecan Infinite® 200).