Using Three-dimensional Polystyrene (PS) ScaffoldsTM to Produce Tumor Spheroids

In vitro tumor spheroids are generated using scaffold-free technologies, e.g. hanging drop, agar gel sandwich, and low adhesive surfaces, etc, for drug screening applications. Some of these platforms are currently adapting towards reducing spheroid time formation and increasing highthroughput workflow. Here, we show the use of 3-dimensional (3D) polymer scaffolds to generate multicellular spheroids in an efficient manner. Using 3D Polystyrene (PS) InsertsTM, a 3D scaffold with 100% pore interconnectivity, several cancer cell lines were able to produce spheroids as early as 2 days in culture. Further characterization of spheroid formation showed that the number and size of the spheroids appear to be dependent on cell type and scaffold geometry. For example, endometrial cancer cells generated a large pool of small spheroids and osteosarcoma cultured in the scaffold produced a fewer pool of larger spheroids. In conclusion, the 3D PS InsertTM represents a novel platform to generate spheroids for standard drug screening and potential in vitro cancer and stroma co-culture models. Main Text Introduction Tumor spheroids are the closest organotypic structures mimicking in vivo cancer tumor morphology. Direct comparison between cancer cells grown as 3D spheroid and 2D monolayer showed that 3D spheroids closely resemble the morphology and biology of primary tumors. Recent evidence has shown that angiogenic suppression and extracellular matrix deposition in adenocarcinomas causes hypoxic/oxidative microenvironments, increasing aggressive and highly oncogenic cancer phenotypes. Thus, 3-dimensional (3D) tumor spheroids are intrinsically more resistant to most anticancer drugs than conventional monolayer in vitro models. The resistance encountered in cells cultured as spheroids seems to be analogous to cancer drug resistance observed in patients, so the use of three-dimensional cell culture may provide a model for studies on anti-cancer drug screening. There are some reported methods to generate tumor spheroids, e.g. embryoid body (EB) formation, reconstituted basement membrane, and soft-agar 4, . In these platforms, tumor spheroids form through anchorage-independent mechanisms, allowing cells to self-assemble in the absence of scaffolding. In this study, we report a novel tumor spheroid producing method that uses 3D polymer scaffolds. The 3D polymer scaffolds (3D InsertsTM -PS) is made from polystyrene, the same polymer use to produce tissue culture plates and flasks. The scaffolds are produced using microfabrication technology, creating scaffolds with controlled fiber diameter and open-pore geometry for facilitated exchange of waste/nutrients. Figure 1 is an illustration showing the 4layer structure of the 3D PS scaffold. Each layer was purposely colored for easy viewing of the 3D layering structure. The 3D scaffolds have been successfully used in Tissue and Cancer Engineering. For example, they have been used to amplify liquid tumors in the presence of 3D stroma and as a 3D platform for stem cell-based therapies . In this study, we found that 3D polymer scaffolds can be used to produce tumor spheroids in an efficient manner. (Figure 1 linked to “Introduction”) Anchorage-Dependent Spheroid Formation In this study, we used 3D InsertsTM -PS as a tool to generate tumor spheroids. Several cancer cells lines such as ECC-1 (Endometrial Adenocarcinoma), HepG2 (Hepatocacinoma), and U2OS-actin-RFP (Osteosarcoma) were seeded and cultured on 12-Well 3D InsertsTM -PS scaffolds at a seeding density of 25,000 cells/cm following manufacturer’s recommended protocols. After one day in culture, the scaffolds were transferred to 12-well non-treated culture plates and shaken for 6 days. As control, cells were grown on non-treated 12-well culture plates (2D Control) under the similar culture conditions. Spheroid formation was seen at day 2 for ECC-1 cells and at day 4 for both HepG2 and U2OS-β-actin-RFP when cultured in 3D InsertsTM -PS scaffolds. As expected, cellular aggregation was seen on 2D cell culture controls. However, aggregate compaction was different for spheroids formed in 3D culture. Thus, 3D scaffold geometry potentially plays a role in the formation of cell spheroids. Figure 2(a-c), shows a qualitative comparison between aggregates from 2D plates and spheroids from 3D scaffolds. HepG2 and U2OS-β-actin-RFP formed some type of spheroid-like aggregates under shaking conditions, but ECC-1 tumor cells did not produce any spheroidlike aggregates probably due to strong cellular adhesion to non-treated 2D plates on Fig. 2(a)-left. On the other hand, when using 3D scaffolds, spheroids formed easily and quickly, even for ECC-1 cells unable to produce unbound aggregates in 2D control plates on Fig. 2(a)-right. On figure 2(c), a close inspection of the spheroid morphology using fluorescent staining on ECC-1 fixed spheroids showed cortical localization of F-actin between cells (Green, F-acin phalloidin) and peripheral DAPI staining (Blue). While on figure 2(c)-right, U2OS-β-actin-RFP spheroids were live-imaged using fluorescence microscopy. These spheroids showed an even distribution of βactin-RFP fluorescent throughout the spheroid’s surface, showing evidence of cytoskeletal function. (Figure 2 linked to “Anchorage-Dependent Spheroid Formation”) Effect of 3D Scaffold on Spheroid Shape To further illustrate the differences in aggregation, we computed the shape factor (Length to Width Ratio) and spheroid diameter to show quantitative differences between 3D spheroids and 2D aggregates. The shape factor of a perfect spheroid is 1 and for non-spherical aggregates the shape factor is greater than 1. In table I all spheroids from 3D culture had shape factors close to 1, while the shape factor from 2D aggregates had mean values of 1.5. The shape factor data from ECC1 2D culture was not listed because 2D conditions did not yield detached tissue aggregates (N.A). Although cells were seeded at comparable seeding density, spheroids from different cell lines showed different number of spheroid and average diameters. Among the 3 cell lines, ECC-1 had the smallest average diameter of 110.44 ± 12.93-μm. In addition, spheroid diameter variation was more prominent for HepG2 3D cell culture with an average diameter of 179.49 ± 54.81-μm. (Table I linked to “Effect of 3D Scaffold on Spheroid Shape”) Effect of Scaffold Fiber Diameter on Spheroid Size To understand the mechanism of spheroid formation in 3D scaffolds, we tested two different scaffold geometries, i.e. scaffolds with fiber diameter 150-um and fiber-to-fiber space 200-um (PS(1520)), and scaffolds with fiber size 300-um and fiber –to fiber space 400-um (PS(3040)). ECC1 cells were seeded at 25,000 cells/cm and grown for 1 day. After one day, the scaffolds were transferred into non-treated wells and placed under shaking conditions. At day 6, the spheroids pool was characterized using spheroid size distribution. Figure 3 shows differences in distribution and spheroid diameter, indicating that scaffold fiber diameter does have an effect on spheroid size and size distribution. On figure 3(a), the number of spheroids from ECC1 cells on scaffolds with 150-um fiber diameter was 92 with an average diameter of 110 +/-10-um (STDEV). For scaffolds with 300-um fiber diameter, there were a total of 46 single spheroids formed with an average diameter of 151 +/-20-um (STDEV). (Figure 3 linked to “Effect of Scaffold Fiber Diameter on Spheroid Size”) Discussion As opposed to scaffold-free technology, we show multicellular spheroids can be easily produced using 3D porous scaffolds. The novel geometry of the scaffold such as homogeneous fiber/pore sizes and 100% pore-to-pore connectivity, allow the formation and easy retrieval of these multicellular spheroids. The mechanism of spheroid formation seemed to be related to the formation of 3D aggregates on the curved surface of polymer fiber and the amount of pore volume available. Once the initial small spheroids formed on the surface of the polymer fiber, the small spheroids will grow to certain sizes and then detach from the curved surfaces. The detachment of the spheroids from polymer fiber is very likely due to the dynamic momentum generated from the shaker used in culture. Since the surface seems to play a role, the scaffold can be coated with different extracellular matrix (ECM) molecules or stroma to study the rate of spheroid formation under normal and drug treatment conditions. This offers an integrative approach to more complex models in cancer drug screening.