The production of 3D tumor spheroids for cancer drug discovery

Abstract

New cancer drug approval rates are ≤5% despite significant investments in cancer research, drug discovery and development. One strategy to improve the rate of success of new cancer drugs transitioning into the clinic would be to more closely align the cellular models used in the early lead discovery with pre-clinical animal models and patient tumors. For solid tumors, this would mandate the development and implementation of three dimensional (3D) in vitro tumor models that more accurately recapitulate human solid tumor architecture and biology. Recent advances in tissue engineering and regenerative medicine have provided new techniques for 3D spheroid generation and a variety of in vitro 3D cancer models are being explored for cancer drug discovery. Although homogeneous assay methods and high content imaging approaches to assess tumor spheroid morphology, growth and viability have been developed, the implementation of 3D models in HTS remains challenging due to reasons that we discuss in this review. Perhaps the biggest obstacle to achieve acceptable HTS assay performance metrics occurs in 3D tumor models that produce spheroids with highly variable morphologies and/or sizes. We highlight two methods that produce uniform size-controlled 3D multicellular tumor spheroids that are compatible with cancer drug research and HTS; tumor spheroids formed in ultra-low attachment microplates, or in polyethylene glycol dimethacrylate hydrogel microwell arrays.

Figure 1. Tumor cells cultured in 3D microenvironments experience different cellular cues that alter their responses and behaviors

Tumor cells cultured in spheroids exist in a highly interactive 3D microenvironment where cell-cell interactions, cell-ECM interactions and local gradients of nutrients, growth factors, secreted factors and oxygen regulate cell function and behavior. Cells in 3D cell cultures are exposed to different adhesive, topographical and mechanical forces than cells growing in 2D on treated surfaces, and the cell-cell and cell-ECM interactions of cells in multi-layer tumor spheroids constitute a permeability barrier through which therapeutic agents must penetrate. The 3D microenvironment alters numerous cellular and functional activities including; morphology, signal transduction, histone acetylation, gene expression, protein expression, drug metabolism, differential zones of proliferation, viability, hypoxia, pH, differentiation (epithelial to mesenchymal transition, EMT), migration, and drug sensitivity.

Figure 2. Production and analysis of head and neck cancer spheroids in 384-well ultra-low attachment plates

A. HNC tumor cell lines form Spheroids 24h after seeding in 384-well ULA-plates. We seeded 6 HNC (Cal33, Cal27, PE/CA-PJ-49, SCC9, BICR56 and FADU) and HET-1A cell lines at 5,000 cells per well into 384-well ULA-plates and after 24h in culture Calcein AM (live, green) and Ethidium homodimer (dead, red) reagents were added to the wells at 10 μM, plates were incubated for an additional 45 min before the cells were fixed in para-formaldehyde and stained with Hoechst, and single images per well were acquired with a 4× objective in each of 4 channels on the IXM automated HCS platform (Molecular Devices LLC, Sunnyvale, CA); transmitted light, Hoechst, FITC and Texas red. The scale bar in each of the transmitted light images represents 125 μm (). The diameters of the spheroids formed by the different HNC cell lines seeded into 384-well ULA-plates ranged between 400 and 600 μm. B. Transmitted light, Hoechst, CAM and EHD images of Cal33 spheroids exposed to PIK3CA inhibitors for 72h. Cal33 HNC cells were seeded at 5,000 cells per well into 384-well ULA-plates and after 24h in culture the indicated concentrations of the three PIK3CA inhibitors were added to the spheroids and the plates were returned to the incubator for an additional 72h of culture. Calcein AM (live, green) and Ethidium homodimer (dead, red) reagents were added to the wells at 10 μM, plates were incubated for an additional 45 min before the cells were fixed in para-formaldehyde and stained with Hoechst, and single images per well were acquired with a 4× objective in each of 4 channels on the IXM automated HCS platform; transmitted light, Hoechst, FITC and Texas red. The scale bar in each of the transmitted light images represents 125 μm (). The diameters of the Cal33 HNC spheroids exposed to DMSO or the three PIK3CA inhibitors ranged between 125 and 500 μm for 50 μM BMK-120 and DMSO controls respectively. C. Correlation between Cell Titer Glo® RLU Signal and Cal33 spheroid cell number and size. Cal33 HNC cells were seeded at seeding densities ranging from 625 to 20,000 cells per well into 384-well ULA-plates and after 24h in culture, the Cell Titer Glo™ (Promega, Madison, WI) reagent was added to the wells and the RLU signals were captured on the M5e microtiter plate reader (Molecular Devices LLC, Sunnyvale, CA). The mean ± SD (n=6) RLU signals from six wells for each seeding density are presented. Representative experimental data from multiple independent experiments are shown. D. Cytotoxicity towards Cal33 spheroids exposed to PIK3CA inhibitors for 72h. Cal33 HNC cells were seeded at 5,000 cells per well into 384-well ULA-plates and after 24h in culture the indicated concentrations of the three PIK3CA inhibitors were added to the spheroids and the plates were returned to the incubator for an additional 72h of culture. After 72h in culture, the CTG reagent was added to the wells and the RLU signals were captured on the M5e microtiter plate reader. For the HNC spheroid growth inhibition assays, we used DMSO control wells (Max controls, n=32) to represent uninhibited growth and 200μM Doxorubicin control wells (Min controls, n=32) to represent 100% inhibition of tumor cell growth respectively, and to normalize the data from the compound treated wells as % of DMSO controls. The mean ± SD (n=3) growth inhibition data from triplicate wells for each compound concentration are presented as the % of the DMSO plate controls. The Cal33 spheroid plate controls exhibited a 6.7-fold assay signal window (S:B ratio) and a Z-factor coefficient of 0.59, indicating that assay was robust, reproducible and suitable for HTS. Representative experimental data from three independent experiments are shown.

Figure 3. Size-controlled 3D uniform microtumors recapitulate physico-chemical changes in the tumor microenvironment

A. Fabrication of size-controlled microtumors. Schematic showing high throughput production of uniform microtumors on polyethylene glycol dimethacrylate-1000 (PEGDMA) hydrogel microarrays. Defined size (150–600 μm) hydrogel microwell arrays were fabricated using polydimethyl siloxane (PDMS) molds by photo-crosslinking 20% polyethylene glycol dimethacrylate (PEGDMA, 1000Da) solution containing 1% photoinitiator (Irgacure-1959) under OmniCure S2000 UV curing station. Hydrogel devices were seeded with cancer cells (0.5 – 1.0 × 10⁶/cm²) and cultured in 5% CO₂ at 37°C. Non-adhesive PEGDMA microwells facilitated cell-cell adhesion forming uniform microtumors where microwell size controlled the microtumor size. B. Hydrogel microwell devices containing arrays of uniform size Cal33 microtumors. Microarrays containing microtumors were stained with 2 μM Calcein-AM (live cells) and ethidium homodimer (dead cells) on day 3 and subsequently imaged using fluorescent microscope with 2.5× objective lens. C. Uniform size microtumors of various breast cancer cell lines. PEGDMA hydrogel microwell arrays (150 μm) were used to fabricate microtumors of subtype-specific breast cancer cell lines. Bright field images showed that hundreds of uniform size microtumors could be fabricated within short duration of 24–48 h. Hormone receptor positive cells (MCF7, T47D and BT474) formed compact microtumors as compared to triple negative breast cancer cell line (HCC1187). D. Spatial distribution of proliferating cells and hypoxia in 3D. Microtumors were harvested on day 6, fixed and immunostained with proliferation marker Ki-67 overnight followed by staining with respective Alexa flour 488 labeled secondary antibody. Images were obtained on Olympus FluoView confocal microscope using 488nm laser. Large (600 μm) microtumors showed Ki-67 positive proliferating cells only on the periphery unlike uniformly distributed Ki-67 positive cells in the 150 μm microtumor. Oxygen availability inside the microtumors was determined by staining microtumors harvested on day 6 with oxygen-sensitive fluorescent dye (Ru-dpp, 1 × 10⁻⁴ M) for 3 h and imaging under confocal microscope (Olympus Fluoview 1000). Images were captured by confocal microscope using 543-nm He-Ne laser for excitation and 604 LP emission filters. Large microtumors (600 μm) showed enhanced red fluorescence in the center as compared to small microtumors suggesting limited oxygen diffusion in large microtumors. E. Size-dependent expression of Hif-1α and vascular endothelial growth factor (Vegf) protein. Protein lysates were prepared using day 6 old microtumors along with 2D monolayer cells and western blot was performed using 40–50 μg protein on 8% polyacrylamide gel. Western blot results showed upregulation of Hif-1α and Vegf expression in 600 μm tumors as compared to 150 μm ones. F. Large microtumors show migratory phenotype. Microtumors were harvested on Day 6, fixed with 4% paraformaldehyde and subsequently with 95% methanol. They were immunostained with anti-E-cadherin primary and respective secondary antibody. Immunostained microtumors (green) were imaged under confocal microscope using 10× objective lens and 488nm laser. Star (*) represents the cells collectively migrated out of the wells from the microtumor.