Fig. 4

Cav1.2 maintained Wnt/β-catenin signaling activity in AM tumoroids. A–C Bright field images of an AM tumoroid generated from a single cell. D The organoid forming efficiency was evaluated as a percentage of the number of tumoroids in each well. The CACNA1C overexpression effectively retained the organoid forming efficiency compared to vehicle and VPM treatment group. E–G Hematoxylin and eosin staining of paraffin sections of AM tumoroids. Spherical shaped AM tumoroids were observed in vehicle and VPM treatment group. Budding-like structures expended from CACNA1C-overexpressed AM tumoroids. H Quantification of the size of 14 days and 21 days cultured AM tumoroids. I–K Immunohistochemistry staining of β-catenin in an AM tumoroid. β-catenin was primary found in the plasma membrane. The nuclear accumulation of β-catenin was dominantly observed in the overexpression group compared to the vehicle and VPM treatment groups. L–M Immunohistochemistry staining of CK10 and CACNA1C. The white rectangle indicates the CK10 positive differentiated AM cells in the vehicle and VPM treatment groups. Nuclei were counterstained by TO-PRO-3 (TP3). O Western blot assay of nucleus and cytoplasm fractionated AM tumoroids with β-catenin antibody. P The relative mRNA expression of WNT3A, AXIN2, CTNNB1, and LGR5 in CACNA1C-overexpressed and VPM treated tumoroids. Scale bar: A–C, 300 μm; E–G, I–K, L–M, 100 μm. Data are presented as the mean ± SD of triplicate experiments. *p < 0.05; **p < 0.01. Veh: Vehicle, OE: CACNA1C overexpression