Fig. 9
Ex vivo treatment of human 3D tumor/CAF/macrophage spheroids with VitE:SM nanoemulsions. a Schematic of human 3D tumor/CAF/macrophage spheroid generation, illustrating the process of generating human spheroids, comprising three populations of human cells: pancreatic cancer cells (PANC1), cancer-associated fibroblasts (CAFs) and macrophages. The 3D model was created as follows: on day one, PANC1/GFP and CAFs/mCherry were seeded in wells of a non-adherent plate, and on day 3, macrophages were added. Spheroids were treated with VitE:SM nanoemulsions (0.5 mg/mL) on day 4, the medium was changed on day 6, and on day 7, the spheroids were disaggregated and implanted orthotopically in the NOD.SCID mice. Mice were monitored for 90 days. b Representative fluorescence microscopy image of a human spheroid co-culture. Scale = 1mm. c Schematic representation of the in vivo orthotopic human spheroid model in NOD.SCID mice. Mice were euthanized at 90 days post-implantation. d Representative macroscopic photos of the tumors at the experimental end point (90 days post-implantation). S = Spleen served as an anatomical reference for the pancreas. Scale = 1cm. e Bar graph ± SD of tumor weight from d. Data compiled from two independent experiments (n = 2) were normalized, and the control was set as 1.0. Unpaired t test. ∗  = p < 0.05. f Percentage of GFP + cells (PANC1/GFP) ± SD in the tumor. Data were normalized for two independent experiments (n = 2), and the control was set as 1.0. Unpaired t test. ∗  = p < 0.05. g Left: Representative images (40X) of H&E-stained tumor samples from d are shown. T  Tumor, P Pancreas. Scale = 400 µm. Right: Ratio of the % area of tumor/healthy pancreas ± SD compared to the total histologic pancreas/tumor area quantified in the H&E slides from the left panel. Unpaired t test. ns not-significant. h Percentage of GFP + cells (PANC1/GFP) infiltrated in the liver. Bars represent the mean fold change ± SD, with Control set as 1.0. Unpaired t test. ∗  ∗  ∗  = p < 0.001. .i qRT-PCR analysis of the ratio of the human GAPDH gene/murine beta actin (mActb). Bars represent the mean fold change ± SD, (n = 2 independent experiments), with Control set as 1.0. Unpaired t test. ∗  = p < 0.05