Fig. 7

LARGE2/α-DG signaling is elevated in primary and liver metastatic CRC cells and affects CRC cell adhesion and migration. A)LARGE2 gene expression analysis on human TCGA-COAD dataset: comparison of normal human mucosa samples and tumor tissues. (**** p < 0.0001). B) qRT-PCR analysis of LARGE2 in PDOs and PDTOs: colonic organoids (PDOs, blue), primary tumor organoids (PDTO, dark red), and liver metastasis-derived tumor organoids (mPDTO, light red). Shown are mean values ± SD (n = 3 technical replicates). C) TaqMan™ qRT-PCR analysis of LARGE2 expression on primary FFPE tissue samples comparing M0 and M1 CRC cases. D) Immunoblot analysis of glycosylated α-DG on WGA-AE purified glycoproteins from primary and metastatic (m)PDTOs. β-DG and α-Tubulin were detected on input WCL. E) IHC analysis of glycosylated α-DG (IIh6 antibody) on FFPE CRC tissues matching to the indicated PDTOs. Black arrowheads: specific staining. Clear arrowhead: negative staining. Scale bars: 50 μm. F,G) Immunoblot analysis of glycosylated α-DG and laminin overlay (Laminin-OL) on WGA-AE purified glycoproteins from KM12c and KM12-L4a cells (F) and from KM12-L4a-NE or -E cells non-treated or after treatment with 400 nM 4-OHT for 72 h (G). β-DG and α-Tubulin were detected on WCL. H) Adhesion of KM12-L4a cells, stably transduced with a luciferase (luc)-encoding lentivirus and either wild-type or LARGE2 KO, was quantified at the indicated time points by measuring luc activity. T = 0 represents the baseline control and was also used for sample normalization. The results are shown as mean ± SD (n = 3), **** p < 0.0001. I,J) Transwell migration assays of CRC cells through laminin-111 coated membranes. LS174T cell pools harboring a LARGE2 knockout (I), and HT-29 cells overexpressing LARGE2 (J) were compared to their control cell pools (*: p < 0.05; **: p < 0.01; ***: p < 0.001)