Figure 3. Targeting EphA2 suppresses tumor progression and enhances overall survival in MET/CAT-induced murine model of HCC.

(A) Western blot validating MET/CAT HCC model and analyzing for EphA2 signaling and expression. ACTIN as a loading control.

(B) Schematic of the experiment of targeted inhibition of EphA2 in MET/CAT induced murine HCC model. pX330 plasmids expressing Cas9 and sgRNA targeting EphA2 (sgEphA2) or empty vector (PX330) were hydrodynamically delivered to mice liver in conjunction with MET/CAT; mice were observed for development of HCC for 8–9 weeks. n = 6 mice in the PX330 group, and n = 9 mice in the sgEphA2 group.

(C) IHC validated the efficacy of 3 independent sgEphA2 constructs in MET/CAT model 10 days after injection. Scale bars, 100 μm.

(D) Western blot confirmed the efficacy of sgEphA2–2 in MET/CAT model 10 days after injection. GAPDH as a loading control.

(E) Immunofluorescence analyzing EphA2 knockout in the context of MET overexpression in MET/CAT model 10 days after injection. EphA2, green;, c-MET, red. Nuclei were stained with DAPI (blue). Scale bars, 30 μm.

(F) Top: a representative image of the mouse abdomen in MET/CAT mouse treated with PX330 or sgEphA2 45 days after injection. Bottom: gross pictures of livers extracted from mice from the upper panels.

(G) Liver-to-bodyweight ratios were calculated for MET/CAT mice injected with PX330 or sgEphA2 for 8 weeks (56 days). N = 3 mice per group.

(H) Livers of MET/CAT mice injected with PX330 or sgEphA2 were collected at day 45 for H&E and IHC for HCC and proliferative markers, AFP and Ki67. Scale bars, 100 μm.

(I) Kaplan-Meier plot for experiment illustrated in (B) represented the percent of survival (y axis) at days elapsed after injection (x axis).

Statistical significance was determined by the two-tailed Student’s t test ****p < 0.0001 (G) and log-rank test (I).