Lab of Onco-Pharmacology and Experimental Therapeutics, Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
PLoS One. 2013 Sep 10;8(9):e73345. doi: 10.1371/journal.pone.0073345. eCollection 2013.
Intrinsic drug resistance of pancreatic ductal adenocarcinoma (PDAC) warrants studies using models that are more clinically relevant for identifying novel resistance mechanisms as well as for drug development. Tumor spheroids (TS) mimic in vivo tumor conditions associated with multicellular resistance and represent a promising model for efficient drug screening, however, pancreatic cancer cells often fail to form spheroids using conventional methods such as liquid overlay. This study describes the induction of TS of human pancreatic cancer cells (Panc-1, Aspc-1, Capan-2) in concave polydimethylsiloxane (PDMS) microwell plates and evaluation of their usefulness as an anticancer efficacy test model. All three cell lines showed TS formation with varying degree of necrosis inside TS. Among these, Panc-1 spheroid with spherical morphology, a rather rough surface, and unique adhesion structures were successfully produced with no notable necrosis in concave microwell plates. Panc-1 TS contained growth factors or enzymes such as TGF-β1, CTGF, and MT1-MMP, and extracellular matrix proteins such as collagen type I, fibronectin, and laminin. Panc-1 cells grown as TS showed changes in stem cell populations and in expression levels of miRNAs that may play roles in chemoresistance. Visualization of drug penetration and detection of viability indicators, such as Ki-67 and MitoSOX, were optimized for TS for quantitative analysis. Water-soluble tetrazolium (MTS) and acid phosphatase (APH) assays were also successfully optimized. Overall, we demonstrated that concave PDMS microwell plates are a novel platform for preparation of TS of weakly aggregating cells and that Panc-1 spheroids may represent a novel three-dimensional model for anti-pancreatic cancer drug screening.
胰腺导管腺癌 (PDAC) 的内在耐药性需要使用更符合临床实际的模型进行研究，以确定新的耐药机制并进行药物开发。肿瘤球体 (TS) 模拟与多细胞耐药相关的体内肿瘤条件，代表了一种用于高效药物筛选的有前途的模型，然而，使用传统方法（如液体覆盖）时，胰腺癌细胞往往无法形成球体。本研究描述了在凹形聚二甲基硅氧烷 (PDMS) 微井板中诱导人胰腺癌细胞 (Panc-1、Aspc-1、Capan-2) 形成 TS，并评估其作为抗癌功效测试模型的有用性。所有三种细胞系均显示出 TS 形成，TS 内部存在不同程度的坏死。在这些细胞系中，Panc-1 球体具有球形形态、相当粗糙的表面和独特的附着结构，在凹形微井板中成功形成，没有明显的坏死。Panc-1 TS 中含有生长因子或酶，如 TGF-β1、CTGF 和 MT1-MMP，以及细胞外基质蛋白，如 I 型胶原、纤连蛋白和层粘连蛋白。作为 TS 生长的 Panc-1 细胞显示出干细胞群体的变化以及可能在化疗耐药中发挥作用的 miRNA 的表达水平变化。优化了用于 TS 的药物渗透可视化和存活指标（如 Ki-67 和 MitoSOX）的检测，以便进行定量分析。水溶性四唑盐 (MTS) 和酸性磷酸酶 (APH) 测定也成功进行了优化。总之，我们证明凹形 PDMS 微井板是制备弱聚集细胞 TS 的新型平台，并且 Panc-1 球体可能代表用于抗胰腺癌药物筛选的新型三维模型。
