Embedded multicellular spheroids as a biomimetic 3D cancer model for evaluating drug and drug-device combinations
Introduction
Three dimensional (3D) in vitro cell culture models are being adopted as preclinical tools for studying tumor behavior and drug response [1]. This paradigm shift is in response to a growing body of evidence that 3D systems promote greater in vivo-like cell behavior than their two-dimensional (2D) counterparts due to recreating more of the characteristic traits of the native tumor environment [2], [3]. As such, these models are proving more predictive than monolayer based systems. The majority of these 3D tumor cell models are prepared by either: (a) growing cells on non-adherent surfaces or in suspension to induce cell clustering; (b) seeding cells within a preformed polymer scaffold [4], [5], [6], [7]; or (c) embedding cells within a hydrogel to promote cell cluster formation along with cell-matrix attachments [8], [9].
With regards to the latter technique, several polymer compositions including Matrigel™ [10], [11], collagen [12], and hyaluronic acid [13] are being used to create 3D scaffolds in an effort to recreate the native extracellular matrix (ECM)-like environments in vitro [14], [15], [16], [17]. These systems promote differential cell behavior when compared to 2D systems, but fail to reproduce the tumor macrostructure found in vivo [3], [18]. Clinical tumors usually consist of a singular structure with metabolically active cells at the surface and a necrotic core, while cell clusters in the 3D matrices are substantially smaller and numerous. Solid tumors also possess mass transport limitations stemming from decreased surface area-to-volume ratios and longer diffusion lengths, which are not present in single cells or small cell clusters [18], [19].
To address these challenges, several methods of creating large cell clusters (>350 μm) are reported in the literature [20], [21], [22]. These techniques eliminate or minimize the surface attachment sites for cells, forcing them to interact principally with each other, and include spinning flasks, hanging drops, and agarose-coated plates. The resulting clusters, or spheroids, are of a similar size to small tumors. Unlike clinical tumors, they exist in an attachment-free microenvironment with very different mechanical and biochemical properties than the native ECM [23]. This is an important caveat to their use, as matrix attachments via integrins and substrate mechanics play crucial roles in cell differentiation and survival [24]. The interplay between the ECM and the tumor drastically affects drug response, epigenetic state, and metastasis in cancer [2], [18]. Therefore, there is a need for additional methods to prepare stable and reproducible models which mimic the native tumor environment while being large enough for comparison to patient tumors.
In order to simultaneously study and model key cellular parameters that regulate form and function including cell adhesion, cell–ECM interaction, biochemical state, mechanical properties, and tumor macrostructure, we present a scalable and reproducible method for embedding and manipulating cancer cell spheroids inside a 3D collagen gel. It builds upon previous spheroid and spheroid-collagen models [25], [26], [27], [28], [29], [30], and enables individual spheroid manipulation along with quantitative and qualitative whole spheroid and single cell analyses. Specifically, we describe the formation of human osteosarcoma and breast adenocarcinoma multicellular spheroids and subsequent embedding within a collagen matrix (Fig. 1). We hypothesize that a multicellular spheroid contained in an ECM derived matrix will respond differently to the first-line chemotherapeutic agent paclitaxel based on its delivery route in contrast to that observed in a 2D monolayer system. Herein, we report the effects of matrix stiffness, cell seeding number, cell type, and chemotherapeutic treatment on a collagen embedded spheroid.
Section snippets
Cell culture
Experiments were performed on the pediatric osteosarcoma cell line U2OS and/or breast adenocarcinoma cell line MDA-MB-231 (ATCC, Manassas, VA). Both cell lines express high levels of E-Cadherin, readily form spheroids, and are well characterized, including their protein expression and secretion profiles as well as have been extensively studied in cancer research applications [12], [31], [32]. Cells were cultured in complete RPMI (U2OS) or DMEM (MDA-MB-231) media supplemented with 10% fetal calf
Spheroid size is linearly dependent on cell seeding number
To demonstrate the scalability and reproducibility of spheroid formation, we investigated the dependency of cell seeding number on subsequent spheroid diameter. Fig. 2 shows the resulting trend-line for both the U2OS and MDA-MB-231 cell lines. The results demonstrate the fidelity and reproducibility of our method. In both cases, the data was fit to a power-law (R2 > 0.98) with very small sample-to-sample error (<3% in U2OS cells and <5% in MDA-MB-231 cells). The horizontal error bars represent
Discussion
Over the last two decades, the identification of new anticancer agents for clinical evaluation has relied on the successful completion of in vitro cell toxicity assays, tumor bearing small animal models, and large animal biodistribution/toxicity studies. Animals do provide a physiological model for evaluating drug efficacy and toxicity, but are expensive, labor intensive, and low throughput. In contrast, the rapid and high throughput in vitro models used to analyze cellular and multicellular
Conclusion
While no tumor model fully recreates the in vivo system, the presented model combines the favorable attributes of a large spheroid structure with an extracellular matrix. It builds on the successes of previous 3D spheroid models and extends the methodology for preparation of single spheroids of controllable size incorporated into a collagen gel as a tumor model. The key features of this tumor model are: (1) a multicellular spheroid of controllable size; (2) a collagen based ECM structure
Acknowledgments
This work was supported in part by BU, BWH, BU Nanotheranostics ARC National Science Foundation (DMR-1006601 to MWG and DMR-1206635 to MHZ), the Boston University's Nanomedicine Program and Cross-Disciplinary Training in Nanotechnology for Cancer (NIH R25 CA153955), and the Boston University T32 Grant entitled Translational Research in Biomaterials (NIH T32EB006359).
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Contributed equally to the work.