(616c) Dynamic 3D Co-Culture of Stromal and Immune Cells with ER+ Breast Cancer Using a Thiol-Acrylate Hydrogel Scaffold and Microfluidic Droplet Trapping Array

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions by mimicking cell-to-cell interactions and mass transfer limitations of metabolites, oxygen, and drugs. Recent drug studies have suggested that a high rate of pre-clinical and clinical failures results from mass transfer limitations associated with drug entry into solid tumors that 2D model systems cannot predict. Droplet microfluidic devices offer a promising alternative to grow 3D spheroids from a small number of cells to reduce intratumor heterogeneity which is lacking in other approaches. Many of the existing techniques for 3D cell culture focus on a single cell type; however, this does not adequately represent the tumor microenvironment (TME) which consists of numerous different cells types including stromal and immune cells. Moreover, recent co-culture studies have identified how cancer cells can manipulate nearby health cells to drive both tumor progression and drug resistance; however, many of the underlying mechanisms are still unclear. Our prior work demonstrated the feasibility of incorporating a thiol-acrylate (TA) hydrogel scaffold into a microfluidic droplet trapping array to generate uniform 3D spheroids of a model estrogen receptor-positive (ER⁺) cell line (MCF7). The TA hydrogel rapidly (~35 min) polymerized on-chip to provide an initial scaffold to support spheroid development followed by a time-dependent degradation The microfluidic device is coupled with a gravity-driven media infusion system to continually supply the 3D spheroids with fresh media to avoid growth stagnation or cell death. The goal of this work was to expand upon the utilities of our platform to allow for the dynamic 3D co-culture of stromal and immune cells with the MCF7 cells. A strength of the TA hydrogel is that it is transient and will degrade within ~24 hours allowing for the spatial rearrangement of the two different cell types in the spheroid which better recapitulates what occurs in the TME. Three different ratios (1:1, 2:1, and 4:1) of co-cultured primary fibroblasts and ER⁺ breast cancer MCF-7 cells showed that increasing the density of the fibroblasts increased the relative size of the spheroid. Spatial tracking of fluorescently-tagged fibroblasts showed that they localized in the center of the spheroid after ~3 days of culture. Terminal immunostaining for the extracellular matrix (ECM) protein Collagen I showed increased expression in the co-cultured spheroids when compared to the mono-cultured MCF-7 spheroid supporting the theory that cancer/fibroblast crosstalk can lead to remodeling of the TME. Co-culture of MCF-7 cells with a model monocyte cell line (THP-1) resulted in the chemical-free differentiation of the THP-1 cells into macrophages as determined by terminal CD11a staining. Unlike the fibroblasts, the macrophages had no spatial limitations within the spheroid and were even found to shed off of the spheroid onto the bottom of the well. Interestingly, the differentiated macrophages exhibited an M2 (or pro-cancer) phenotype when co-cultured with the MCF-7 cells as determined by terminal CD163 staining. These findings support the idea that 3D cultured ER⁺ breast cancer can manipulate healthy cells in the TME to adopt a pro-cancer phenotype.