Bioengineered Spatially Heterogeneous Tumor Microenvironment Model Mediated by Tumor-Stromal Interactions for Multi-Drug Synergy Evaluation

Abstract Stromal cell-involved heterogeneous tumor microenvironments (TMEs) critically regulate tumor metastasis, nutrient transport, metabolism, and proliferation. However, existing in vitro models are predominantly tumor cell-centric, lacking adequate cell-cell contact to recapitulate tumor-stromal crosstalk, and current 3D matrices fail to sustain multiple cell viability uniformly, restricting multicellular TME model utility. To address these limitations, this study presents a strategy for constructing a spatially heterogeneous TME model using two matrix materials with distinct properties: a modulus-tunable hybrid bioink (adEMA) with excellent printability for MCF-7 tumor cell culture, and high-bioactivity, low-modulus acellular adipose extracellular matrix (adECM) for stromal cells (MRC-5 fibroblasts and HUVECs), thus matching the growth demands of different cell types. Combining microfluidic chips and 3D bioprinting, a "chocolate sandwich ball"-mimetic spatially heterogeneous TME model mediated by three cell types was established. Herein, adECM acted as the primary matrix for HUVECs and MRC-5, while adEMA gel microspheres loaded with MCF-7 cells were uniformly distributed within the 3D model, maintaining an independent growth space from stromal cells. Results demonstrated that HUVEC- and MRC-5-mediated heterogeneous TME significantly upregulated the expression of malignant tumor markers, while these stromal cells enhanced tumor drug resistance and in vivo tumorigenicity. Using this model, the synergistic therapeutic effects of Cur, DOX, and OXA were confirmed, highlighting the utility of the three-cell-mediated spatially heterogeneous TME model for multi-drug screening.