## 3 Results  
### Evaluation of MUC1 Expression in Pancreatic Duct Lesions by Immunohistochemical Staining  
The 26 PanIN/IPMN tissues had absent or weak intensities of MUC1 protein expression (Figure 1A). Similarly, in the non-invasive adenocarcinoma tissue samples, the majority (10 of 12) showed negative or weak intensities (Figure 1A). In contrast, 27 PDAC samples showed intermediate or strong intensities (Figure 1A). This indicates that MUC1 tends to be highly expressed in invasive carcinomas. However, the ANOVA test did not yield significant differences among the three groups evaluated. The suppression of MUC1 expression significantly decreased cell proliferation 72 h after transfection with MUC1 siRNA in Panc0203 and Panc1 (Figure 1B). The cell proliferation rate of PL45 was low, and its MUC1 inhibitory effect could not be fully evaluated (Figure 1B). Therefore, Panc0203 and Panc1 were used in the subsequent experiments. In the Matrigel assay, both Panc0203 and Panc1 showed decreased invasive capacity due to the suppression of MUC1 expression (Figure 1C). These results indicate that MUC1 has the potential to contribute to cell proliferation and invasion in PDAC cells. Figure 1: Expression of MUC1 in pancreatic ductal lesion tissues and its involvement in cell proliferation and invasion. (**A**) MUC1 expression in pancreatic intraepithelial neoplasia/intraductal papillary mucinous neoplasm (PanIN/IPMN); adenocarcinoma in situ; and PDAC samples are shown. The left panel is hematoxylin-eosin stained and the right panel anti-MUC1 immuno-stained. The degree of MUC1 expression at the cell membrane was assessed semi-quantitatively as 0 (negative), 1 (weak intensity), 2 (intermediate intensity), and 3 (strong intensity). (\(\times\)100; scale bar: 250 μm) A one-way ANOVA comparison of the three groups showed no statistically significant differences (\(p\) = 0.69). (**B**) The MTS assay under transfection with MUC1 siRNA at 24, 48, and 72 h in PDAC cell lines (\({}^{\prime}p\) < 0.05). (**C**) Matrigel invasion assay showing the promotion of invasive capability in Panc0203 and Panc1 cells transfected with MUC1 siRNA. (\(\times\)100; scale bar: 100 μm). Characterization of Panc0203 and Panc1 and Analysis of the Mechanism of Regulation of Cell Proliferation by MUC1 in PDAC Cells  
In order to use the two types of cells for subsequent experimentation, we confirmed the immunophenotype of Panc0203 and Panc1 by immunocytochemical staining with mucin molecular markers and epithelial markers. Panc0203 was positive for MUC4, CAM5.2, CK7, CK19, and MUC1/EMA, while Panc1 was weakly positive for MUC1/EMA and positive for CAM5.2 and CK19 (Figure 2A). This suggests that Panc1 may be a poorly differentiated adenocarcinoma compared to Panc0203. Evaluation of MUC1 mRNA in the two cell types by Ct value using quantitative RT-PCR showed that the Ct value of MUC1 was above 20 in both cases, and even higher in Panc1, suggesting that MUC1 mRNA expression is also low (Figure 2A). Therefore, since evaluation at the protein level is not sensitive enough for the following experiments using these two cells, mRNA was used to evaluate MUC1 expression levels. Furthermore, PL45, Panc0203, and Panc1 were genetically characterized by NGS analysis using a panel of 50 cancer-related genes. Although each cell line had different genetic variants, the KRAS mutation (G12V), a characteristic of PDA, was observed in all cell lines (Figure S1). This confirmed that the cells were PDACs, although they differed in origin and degree of differentiation. Figure 2: Characterization and function analysis under MUC1 suppression in Panc0203 and Panc1. (**A**) (**Left**) Immunocytochemical staining of various epithelial markers. Panc0203 and Panc1 cell blocks were prepared and immunohistochemistry stained. (\(\times\)100; scale bar; 100 μm) (**Right**) Evaluation of Ct value of MUC1 mRNA by quantitative RT-PCR method. (**B**) Annexin V analysis for the detection of apoptosis in Panc0203 and Panc1 cells, (”_p_ < 0.05).