Application of Concave Microwells to Pancreatic Tumor Spheroids Enabling Anticancer Drug Evaluation in a Clinically Relevant Drug Resistance Model

Abstract

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.

Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique

“…It was previously reported that PDAC cells are difficult to cohere into spheroids capable of being used for high-throughput drug assays. 45 Data in Figure 2C and D show that the spheroids formed using the proposed method are highly reproducible. Light microscopy shows that these spheroids are uniform in shape, have smooth/defined boundaries, and are dense enough to be used for various assays (Fig.…”

“…We based this study on pancreatic adenocarcinoma cell lines (PDAC), since, in ours and others experience, PDAC cells are difficult to culture into cohesive and manageable spheres when any of the traditional spheroid forming methods are used. 45 The proposed technique enables simultaneous, cost-and time-effective generation of numerous spheroids. The tumor spheroids are extremely uniform in nature and do not easily dissemble when handled, allowing increased reproducibility and scalability.…”

Microfluidic 3D models of cancer

“…Microfabricated well arrays (Fig. 2a) were developed to increase the production of homogeneous tumor spheroids by using the geometrical confinement to trap similar numbers of cells in each well[30–34]. Other research groups have developed micro-chambers or micro-posts in microfluidic channels to trap clusters of cells to control spheroid size and simplify liquid handling procedures[35].…”

Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

“…Data obtained using these in vitro models may explain the involvement of interaction between cancer cells and the extracellular matrix (ECM) or between cancer cells and stromal cells in the induction of EMT. In contrast to these conventional 2-dimensional cell culture systems, 3-dimensional (3D) tumor spheroids (TSs) may be a more reliable model reflecting the characteristics of in vivo tumors more closely [12][13][14]. To incorporate tumor microenvironmental factors into TSs representing the architecture of in vivo tumors, a directmixed co-culture of TSs with fibroblasts using the hanging-drop method has been reported [15].…”