High-Throughput Microfluidic Generation and Interrogation of 3D Co-Culture Cancer Spheroids

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions as 3D spheroids account for both cell-to-cell contacts and mass transfer limitations. Several promising breast cancer therapeutics with success in 2D systems failed in early clinical trials due to differences between 2D and 3D cell growth and behavior. Varied approaches exist for 3D cell culture; however, droplet microfluidics is a promising method to generate and interrogate uniform 3D breast cancer spheroids. One area in 3D cell culture with limited research is co-culturing two different cell types in a single spheroid. Paracrine signaling associated with tumor progression between cancer cells and stromal fibroblasts has been suggested as a key contributor to tumor proliferation and drug resistance. Moreover, monocultured cells cannot properly model the tumor microenvironment (TME) which can remodel in response to physical and chemical interactions between cancer cells and fibroblasts. The goal of this work utilizes a novel approach to grow, interrogate, and evaluate changes in the extracellular matrix (ECM) of 3D co-cultured spheroids. This is accomplished using a thiol-acrylate (TA) hydrogel scaffold supporting cell culture for 7 days in a droplet microfluidic trapping array that isolates single droplets in a 300-member trapping array with up to 99% trapping. Hydrogel gelation occurs rapidly (~30 min) allowing culture media that supports spheroid growth to replace the oil phase responsible for droplet generation. Spheroid size, circularity, and diameter were analyzed using custom MATLAB code that analyzes brightfield images of the circular traps. The program masks each trap and isolates the spheroid inside for data analysis.

Three different ratios (1:1, 2:1, and 4:1) of co-cultured primary fibroblasts:ER⁺ breast cancer MCF-7 cells were studied to determine how fibroblast density affects spheroid size. Increasing fibroblast concentration correlated to the formation and increase in size of a dark core inside each spheroid. Fibroblasts accumulation in the spheroid core was confirmed using CellTracker^TM, a live stain specific to fibroblasts. Fibroblast presence correlated to a significant increase in spheroid area over a 7-day incubation period compared to monocultured MCF-7 cells. To evaluate changes in the ECM, monocultured and co-cultured spheroids were immunostained for collagen I, an ECM protein thought to play a role drug resistance. Fluorescent imaging showed that monocultured MCF-7 cells secreted less collagen I than fibroblast/cancer co-cultured spheroids. Interestingly, a higher amount of collagen I was observed at the core of the spheroids suggesting fibroblast presence enhances collagen I production in the TME.