Fig. 4: Targeting SLC25A15 robustly inhibits hepatic tropism of mut-ER BC cells in vitro, ex vivo and in vivo.

WT-ER (control) and Y537S-ER (control and SLC-KO) cells were seeded in U-shaped plates with A hepatocytes conditioned media (hCM), B astrocytes conditioned media (aCM), C and small airways epithelial cells (SAEC) conditioned media (saCM) for 3 days. Images were captured using the Incucyte live image device. Spheroids average area of triplicate was quantified using the Live-Cell Analysis Incucyte Software. D A scratch assay was conducted in control and SLC25A15 KO cells of WT-ER and Y537S-ER. The monolayer was scraped, then treated with either control conditions (maintenance media; MM) or hCM. Scratch-wound area was monitored for 48 hours. Cells were photographed at 6, 24, and 48 hours, and migratory lengths were quantified. E A schematic representation of a precision-cut liver slice. F mCherry-labeled MCF-7 cells expressing WT-ER and the Y537S-ER (both control and SLC25A15 KO clones) were seeded on top of fresh murine liver slices. 96 hours later, adherent viable cells were imaged for at least 3 fields per sample at 20x magnification. Cancer cells that occupied liver slices surface were quantified. The percentages of liver slice surface occupied by tissue-adhered fluorescent cancer cells were quantified using the ImageJ software. The figures depict a representative graph output of at least three independent experiments. G A quantitative analysis of mCherry fluorescence is depicted. H Luciferase-labeled MCF-7 cells expressing Y537S-ER with or without SLC25A15 knockout (SLC-KO (#12) or Con (#44), respectively) were injected to the spleen of athymic nude mice. Luciferase activity was measured every 10 days and 8 weeks after injection. The experiment was ended, liver was excised, and luciferase activity was imaged and measured. I Liver metastasis incidence as evidenced by luminescence. J Luciferase activity of liver metastasis measured by average radiance K Representative H&E staining confirming liver metastasis of mice injected with Y537S Con (#45). Photomicrographs were taken at 4x, 10x, and 20x objectives. Each bar represents the mean ± S.D. *P < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Con Control, SLC-KO SLC25A15 knockout.