Panel A: Immunofluorescence images showing EPHA2 and phosphorylated EPHA2 in tumor and adjacent normal tissue. The tumor tissue shows strong expression of EPHA2 and phosphorylated EPHA2, while the adjacent tissue shows little to no expression. Panel B: Box plots quantifying EPHA2 and phosphorylated EPHA2 mean intensity in tumor and adjacent normal tissue, showing significantly higher expression in tumor tissue. Panel C: Immunofluorescence images showing EPHA2 and phosphorylated EPHA2 in mouse PDAC tissue. Panel D: Immunofluorescence images of KPC spheroids treated with a Kras-targeting cET-ASO, showing the ability of the ASO to reduce spheroid size. Panel E: High-resolution micrographs of KPC cells treated with Kras-targeting cET-ASO, showing EPHA2 and ASO staining in intracellular vesicles. Panel F: Box plot showing the number of nuclei per spheroid after treatment with Kras-targeting cET-ASO, indicating a reduction in spheroid size. Panel G: Box plot showing the volume of spheroids after treatment with Kras-targeting cET-ASO, indicating a reduction in spheroid volume. Panel H: Line graph showing Kras mRNA expression in wild-type and Epha2 knockout KPC cells after treatment with Kras-targetingcET-ASO, indicating a reduction in Kras mRNA expression. Panel I: Western blot images showing MEK and ERK activation status in KPC tumor-derived cell lines from Epha2 wild-type and knockout mice after treatment with Kras-targetingcET-ASO. Panel J: Bar graph showing the proliferation of KPC Epha2 wild-type and knockout cells after treatment with Kras-targeting ASO, indicating a reduction in cell proliferation.
EPHA2 is required for cET-ASO–mediated suppression of KRAS in PDAC. (A and B) EPHA2 (green) and Ser897 phosphorylated EPHA2 (red) in PDAC patient tumor (top and right), and non-transformed adjacent tissue (bottom and left), and (B) their quantification in patient samples. Tumor n = 6; normal n = 8, unpaired t test. (C) EPHA2 (green) and Ser897 phosphorylated EPHA2 (red) in mouse PDAC (KPC). (D) EPHA2 (green) in KPC spheroids treated with cET-ASOKras (orange). (E) High-resolution micrograph of KPC cells either untreated (control) or treated with cET-ASOKras for 72 h. EPHA2 (green) and cET-ASOKras (red) staining are shown overlapping in intracellular vesicles. Side panels correspond to an image amplification of the area inside the white dotted line frames in the adjacent panels. (F and G) High-content image analysis of spheroid volume (G) and number of nuclei per spheroid (F) after 72-h treatment with Kras-targeting cET-ASO (ASO; 1 μM) of vehicle control (control). n = 3 independent experiments, one-way ANOVA (OWA), Dunnett. (H)Kras mRNA expression in WT (Epha2+/+, blue) or Epha2 knockout (Epha2−/−, magenta) KPC cells after 72-h treatment with cET-ASOKras. Data are mean ± SEM, n = 5 independent experiments. (I) MEK and ERK activation status in KPC tumor-derived cell lines from either Epha2+/+ or Epha2−/− mice after 72 h cET-ASOKras treatment. (J) KPC Epha2+/+ (blue) or Epha2−/− (magenta) cell proliferation after treatment with Kras-targeting ASO for 96 h. Proliferation index expressed as the fold change of cell number relative to untreated cells. Data are mean ± SEM, n = 9 independent experiments. (H and J) Two-way ANOVA (TWA), Sidak. Source data are available for this figure: SourceData F1.