YSCCC

RNF213 Knockdown by shRNA Induces Cell Migration and Invasion in CCA Cell Lines

Intraductal papillary neoplasm of the bile duct type (IPNB) is a rare subtype of intrahepatic cholangiocarcinoma (ICC) in which cancer cells originate in the bile duct and then invade surrounding liver tissue, and therefore, is a suitable model for studying local invasion, the initial step of metastasis.

As shown in Fig. 1A, RNA-Seq data from the Human Protein Atlas database reveal that CCA cell lines have high RNF213 expression. RNA-Seq of 81 normal cell types indicates that cholangiocytes have moderate expression of RNF213, in the top one-third of all cell types. To investigate the role of RNF213 loss-of-function in ICC carcinogenesis, they first examined RNF213 expression and protein levels in CCA cell lines (KKU-100, KKU-213A, HuCCT1, YSCCC) and normal cholangiocytes (MMNK-1). RNF213 was significantly upregulated in KKU-100, HuCCT1, and YSCCC compared to MMNK-1 (Fig. 1A). Stable HuCCT1, YSCCC, and KKU-100 cells with shRNF213#1, shRNF213#2, and Control were generated using lentiviral transduction. shRNF213#2, which showed higher knockdown efficiency, was chosen for subsequent experiments (Fig. 1A). shRNF213#2 knockdown did not affect cell proliferation (Fig. 1B). The cell migration and invasion capacity of HuCCT1 and YSCCC were significantly increased upon shRNF213#2 knockdown (≥ 3.2- and ≥ 1.5-fold, respectively; Fig. 1C). No significant differences in wound closure rates were observed (Fig. 1D), but 3D spheroids of the shRNF213#2 knockdown group invaded Matrigel more than that of the control group (Fig. 1E). Last, shRNF213#2 knockdown significantly reduced branch point formations of tube-like structures in HuCCT1 and YSCCC cells (Fig. 1F).

Cholangiocarcinoma Cell Line YSCCC Exhibits High MUC1 Surface Expression

The dense glycocalyx of cancer cells can hinder recognition by immune cells and effective immunotherapy. Park et al. profiled therapeutic agents that reduce the glycocalyx to aid anti-cancer immune responses, using Scanning Angle Interference Microscopy (SAIM). To this end, they developed a new membrane labeling strategy with leucine zipper pairs to define the glycocalyx boundary for its accurate measurement.

After establishing a method for membrane labeling, they surveyed various cancer cell lines to identify models for glycocalyx-mediated immune avoidance. Given that MUC1 levels are proportional to glycocalyx thickness, they queried DepMap data and determined that CCA has the highest MUC1 expression (Fig. 2a). Next, they measured MUC1 protein density on the cell surface using a MUC1-specific antibody and normalized the values to the SKBR3 cell line. Linear regression demonstrated high correlation between MUC1 protein and RNA expression (Fig. 2b). The YSCCC intrahepatic CCA cells had the highest MUC1 surface expression. The authors characterized the YSCCC cell line, which form both adherent and suspended cells in culture, which is indicative of a repulsive glycocalyx that prevents effective cell adhesion. To test this, they treated the YSCCC cells with low-dose mucin-digesting enzymes (StcE and SmE) overnight. Enzyme-treated cells had lower MUC1 levels (Fig. 2c) and increased adhesion and spreading compared to control cells (Fig. 2d-g), though SmE only showed a modest effect (Fig. 2e-g).