Extended Data Fig. 2: ENO2 and neuroendocrine differentiation are associated with resistance to antiangiogenic drugs in CRC.
a–d, Western blotting and qPCR assay were conducted to evaluate the protein and mRNA levels of ENO2 and neuroendocrine differentiation markers SYP and CHGA in (a, b) Bev-sensitive, Bev-resistant, (c, d) Rego-sensitive, and Rego-resistant CRC xenograft tumors. Bev, bevacizumab. Rego, regorafenib. (b, d) Relative mRNA expression in each group is shown. n = 3 mice per group. Representative blots are shown. e, The serum levels of ENO2 in mice bearing Rego-sensitive or -resistant CRC tumors was determined by ELISA. n = 5 mice per group. f, IHC staining and quantification of CHGA in tumors from the indicated groups. Scale bar, 50 μm. g, IHC staining and quantification of ENO2, SYP, and CHGA in tumors from the indicated groups. Scale bar, 50 μm. (f, g, n = 5 mice per group. Representative IHC images are shown). h, Diagram depicts the establishment of Rego-resistant HCT116 xenograft tumors in vivo. i, IF staining and quantification of CD31+ microvessel density in HCT116 xenograft tumors in each group. Scale bar, 20 μm. n = 6 mice per group. Representative IF images are shown. j, Western blot analysis of ENO2 in Rego-sensitive and -resistant HCT116 cells. Representative of n = 3 independent replicates. k, The viability of CRC cells treated with regorafenib for 24 h was evaluated by MTT assay. n = 3 biologically independent experiments. l, Disease free survival analysis of COAD and READ patients with high or low levels of ENO2 from TCGA (defined by RNA sequencing with group cutoff values of 50% and 50%). Data are presented as the mean ± s.e.m. ns, not significant. P values were determined by unpaired two-tailed Student’s t-test (b, d, e, f, g and k) or one-way ANOVA with Tukey’s multiple comparison test (i).
