Department of Molecular and Tumor Pathology, Graduate School of Medicine, Chiba University, Chiba, 260-8670, Japan.
Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8565, Japan.
Sci Rep. 2019 Aug 2;9(1):11247. doi: 10.1038/s41598-019-47101-y.
Tube-forming growth is an essential histological feature of pancreatic duct adenocarcinoma (PDAC) and of the pancreatic duct epithelium; nevertheless, the nature of the signals that start to form the tubular structures remains unknown. Here, we showed the clonal growth of PDAC cell lines in a three-dimensional (3D) culture experiment that modeled the clonal growth of PDAC. At the beginning of this study, we isolated the sphere- and tube-forming clones from established mouse pancreatic cancer cell lines via limiting dilution culture using collagen gel. Compared with cells in spherical structures, the cells in the formed tubes exhibited a lower CK19 expression in 3D culture and in the tumor that grew in the abdominal cavity of nude mice. Conversely, the expression of the transforming growth factor β (TGF-β)-signaling target mRNAs was higher in the formed tube vs the spherical structures, suggesting that TGF-β signaling is more active in the tube-forming process than the sphere-forming process. Treatment of sphere-forming clones with TGF-β1 induced tube-forming growth, upregulated the TGF-β-signaling target mRNAs, and yielded electron microscopic findings of a fading epithelial phenotype. In contrast, the elimination of TGF-β-signaling activation by treatment with inhibitors diminished the tube-forming growth and suppressed the expression of the TGF-β-signaling target mRNAs. Moreover, upregulation of the Fn1, Mmp2, and Snai1 mRNAs, which are hallmarks of tube-forming growth in PDAC, was demonstrated in a mouse model of carcinogenesis showing rapid progression because of the aggressive invasion of tube-forming cancer. Our study suggests that the tube-forming growth of PDAC relies on the activation of TGF-β signaling and highlights the importance of the formation of tube structures.
管状形成生长是胰腺导管腺癌（PDAC）和胰腺导管上皮的重要组织学特征；然而，启动管状结构形成的信号的性质仍然未知。在这里，我们通过使用胶原凝胶进行三维（3D）培养实验，模拟 PDAC 的克隆生长，显示了 PDAC 细胞系的克隆生长。在本研究开始时，我们通过有限稀释培养从已建立的小鼠胰腺癌细胞系中分离出球体和管状形成克隆。与球体结构中的细胞相比，在 3D 培养和在裸鼠腹腔中生长的肿瘤中，形成管腔的细胞 CK19 表达较低。相反，转化生长因子β（TGF-β）信号靶标 mRNA 的表达在形成的管腔中高于球体结构，表明 TGF-β 信号在管状形成过程中比球体形成过程更为活跃。用 TGF-β1 处理球体形成克隆诱导管状形成生长，上调 TGF-β 信号靶标 mRNA，并产生上皮表型逐渐消失的电子显微镜发现。相反，通过用抑制剂处理消除 TGF-β 信号激活，减少管状形成生长并抑制 TGF-β 信号靶标 mRNA 的表达。此外，在致癌模型中观察到 PDAC 管状形成生长的标志性基因 Fn1、Mmp2 和 Snai1 的 mRNA 上调，该模型显示出由于管状形成癌症的侵袭性快速进展而导致的快速进展。我们的研究表明，PDAC 的管状形成生长依赖于 TGF-β 信号的激活，并强调了管状结构形成的重要性。
