Effect of 3D mechanical microenvironment on breast tumor spheroid formation within microfluidics-generated microgels

Abstract

Introduction: Tumor spheroids have been considered valuable miniaturized three dimensional (3D) tissue models for fundamental biological investigation as well as drug screening applications. Most tumor spheroids are generated utilizing the inherent aggregate behavior of tumor cells, and the effect of microenvironmental factors such as extracellular matrix on tumor spheroid formation has not been extensively elucidated to date. Herein, a microfluidic flow-focusing device was adopted to create uniform-sized spherical micro-scale hydrogels (‘microgels’) encapsulated with spheroid-forming breast tumor cells, in order to investigate the effect of 3D microenvironment on the cellular behavior, leading to spheroid formation. Experimental methods: 1. Microfluidic fabrication of cell-laden microgels The flow-focusing microfluidic device was used to generate cell-laden droplets, which were photocrosslinked in situ to develop cell-laden microgels. Aqueous solution phases 1 and 2 (Aq1 and Aq2) both consisted of methacrylic gelatin (MGel) and 0.2 % Irgacure 2959® in phosphate buffered saline (PBS, pH 7.4). The channel geometry allowed Aq1 to enter Aq2 prior to droplet generation, with Aq1 becoming the core of a droplet (Fig. a). Oil phase (O) consisted of 20 % Span®80 as a surfactant in mineral oil. In Aq1, breast tumor cells, MDA-MB-231, at 1 × 107 cells mL-1, were dispersed. The flow rates of Aq and O were controlled to generate droplets having 100 μm in diameter, which were immediately photocrosslinked to develop cell-laden microgels. 2. In vitro evaluation of spheroids in microgels The viability of the cells encapsulated in the microgels were measured using LIVE/DEAD Cell Viability Assay kit. The proliferation rate (kP) of encapsulated cells was determined by counting the number of live cells at various time points up to 7 days, and the plot of the normalized number of viable cells (Nt/N0) vs. time (t) was fitted with the following power-law equation, Nt/N0 = 2kP t, where Nt was the number of viable cells at time, t, and N0 was the initial number of viable cells at t=0. To visualize the actin structure and nuclei of cells during spheroid formation, the cells were labeled with fluorescein-labeled phalloidin and DAPI, respectively. Results and discussions: To assess the effect of microenvironmental mechanics on the cell behavior and spheroid formation, the rigidity of the microgels was controlled by varying the MGel concentration. Five different pairs of concentrations were explored, denoted from ‘C1’ to ‘C5’. Their elastic moduli ranged from 1.2 to 40.3 kPa (Fig. b) MDA-MB-231 cells encapsulated in the microgels with varying rigidity maintained high viability (Fig. c and d). Interestingly, the cell proliferation increased substantially with microgel rigidity, with increased number of smaller spheroids within the microgels, suggesting the increased mechanotransduction signals imparted by higher microgel rigidity promoted the cellular proliferation (Fig. e and f). Several cells were shown to spread and display lamellipodial projections, suggesting the increase in migratory potential (Fig. g-i). In addition, the cells merged to form larger cells with multiple nuclei at earlier times at grew in size, while still demonstrating lamellipodial projections. This morphological changes suggested that the cells formed polyploid giant cancer cells (PGCC), a hallmark of aggressive tumor progression. Further cultured up to 21 days clearly demonstrated that at higher microgel rigidity of C4 and C5, the smaller cell aggregates merged and formed larger and more cohesive spheroids, occupying most of the microgels (Fig. j). It can be inferred that increased rigidity, coupled with restrictive environment elevating hypoxia, may have facilitated the spheroid formation. Conclusions: Taken together, the results of this study highlight the importance of both cellular and extracellular factors on the formation and pathophysiology of 3D tumor spheroids, and provide