Production of Uniform 3D Microtumors in Hydrogel Microwell Arrays for Measurement of Viability, Morphology, and Signaling Pathway Activation

Abstract

Despite significant investments in cancer research and drug discovery/development, the rate of new cancer drug approval is £5% and most cases of metastatic cancer remain incurable. Ninety-five percent of new cancer drugs fail in clinical development because of a lack of therapeutic efficacy and/or unacceptable toxicity. One of the major factors responsible for the low success rate of anticancer drug development is the failure of preclinical models to adequately recapitulate the complexity and heterogeneity of human cancer. For throughput and capacity reasons, high-throughput screening growth inhibition assays almost exclusively use two-dimensional (2D) monolayers of tumor cell lines cultured on tissue culture-treated plastic/glass surfaces in serum-containing medium. However, these 2D tumor cell line cultures fail to recapitulate the three-dimensional (3D) context of cells in solid tumors even though the tumor microenvironment has been shown to have a profound effect on anticancer drug responses. Tumor spheroids remain the best characterized and most widely used 3D models; however, spheroid sizes tend to be nonuniform, making them unsuitable for high-throughput drug testing. To circumvent this challenge, we have developed defined size microwell arrays using nonadhesive hydrogels that are applicable to a wide variety of cancer cell lines to fabricate sizecontrolled 3D microtumors. We demonstrate that the hydrogel microwell array platform can be applied successfully to generate hundreds of uniform microtumors within 3-6 days from many cervical and breast, as well as head and neck squamous cell carcinoma (HNSCC) cells. Moreover, controlling size of the microwells in the hydrogel array allows precise control over the size of the microtumors. Finally, we demonstrate the application of this platform technology to probe activation as well as inhibition of epidermal growth factor receptor (EGFR) signaling in 3D HNSCC microtumors in response to EGF and cetuximab treatments, respectively. We believe that the ability to generate large numbers of HNSCC microtumors of uniform size and 3D morphology using hydrogel arrays will provide more physiological in vitro 3D tumor models to investigate how tumor size influences signaling pathway activation and cancer drug efficacy.

Three-Dimensional Breast Cancer Models Mimic Hallmarks of Size-Induced Tumor Progression

“…Heterogeneity in spheroid sizes can affect non-cellular factors in the tumor microenvironment including nutrient/oxygen gradients, hypoxia and metabolic stress, which can further affect tumor biology and response to drug. To circumvent this challenge, we have microfabricated non-adhesive PEGDMA hydrogel microarrays with hundreds of defined diameter microwells using photolithography (29) that allows generation of uniform, defined size microtumors of various cell lines such as cervical, breast and head and neck cancer (24) (Fig. 1A).…”

“…1A). Microwell diameter, cell seeding density and cell lines determine the size of generated microtumors (24). Here, we have used this microfabricated hydrogel platform to develop and validate 3D breast tumor progression model.…”

“…Uniform size 3D microtumors of subtype specific breast cancer cell lines were fabricated using polyethylene glycol dimethacrylate (PEGDMA) hydrogel microwell arrays as described earlier (23,24) (Fig. 1A, Supplementary information, Methods).…”

Three-dimensional culture systems in cancer research: Focus on tumor spheroid model

“…Polydimethylsiloxane (PDMS) stamp with micropillars are photocrosslinked to PEGDMA to construct microwells. The TMS-PA pre-coating ensures covalent attachment of the hydrogel microwells onto the glass plate (Figure 3D) (Singh et al , 2015). In direct photo patterning, UV exposure produces reactive oxygen species (ROS), which in turn makes the protein-repellent part of a molecule that has been grafted on the substrate to detach, allowing ECM protein to further bind onto the substrate (Théry, 2010).…”

The production of 3D tumor spheroids for cancer drug discovery

“…Several methods to generate anchorage-independent 3D tumor spheroids have also been developed; spontaneous aggregation, liquid overlay on agarose, hanging drop cultures, spinner flask cultures, rotary cell culture systems, ultra-low attachment (ULA) plates, and encapsulation in biologically inert hydrogels lacking attachment cues (Table 1) [14, 16–18, 21, 24, 25, 32, 33, 38, 39]. However, not all tumor cell lines form 3D spheroids under these conditions [14, 33], and many of these methods generate irregular 3D cellular aggregates that have a wide range of morphologies and sizes [16, 17].…”