Bioprinted spatially defined breast tumor microenvironment models of intratumoral heterogeneity and drug resistance

Cellular extracellular matrix (ECM) and spatial heterogeneity of tumor microenvironments (TME) regulate disease progression and treatment efficacy. Developing in vitro models that recapitulate the TME promises to accelerate studies of tumor biology and identify new targets for therapy. Here, we employed extrusion-based, multi-nozzle three-dimensional (3D) bioprinting to spatially pattern triple-negative MDA-MB-231 breast cancer cells, endothelial cells, and human mammary cancer-associated fibroblasts with biomimetic ECM inks. Bioprinted models captured key features of the spatial architecture of human breast tumors, including varying-sized dense regions of cancer cells and surrounding microvessel-rich stroma. Angiogenesis and ECM stiffening occurred in the stromal area but not the cancer cell rich regions, mimicking pathological changes in patient samples. Transcriptomic analyses revealed upregulation of angiogenesis-related and ECM remodeling-related signatures in the stroma region and identified potential ligand-receptor mediators of these processes. Breast cancer cells in distinct parts of the bioprinted TME showed differing sensitivities to chemotherapy, highlighting environmentally mediated drug resistance. In summary, our 3D bioprinted tumor model will act as a platform to discover integrated functions of the TME in cancer biology and therapy. we employed extrusion-based, multi-nozzle three-dimensional (3D) bioprinting to spatially pattern triple-negative MDA-MB-231 breast cancer cells, endothelial cells, and human mammary cancer-associated fibroblasts with biomimetic ECM inks.We identified spatial patterns based on morphology and staining of blood vessels. We mapped tumors into spatially distinct regions, including cancer cell rich (CCR) regions and adjacent stroma rich (SR) regions: (1) CCR regions were dominantly occupied by varying-sized cancer cell clusters with the occurrence of blood vessels and fibroblasts; (2) SR regions showed enrichment for blood vessels and fibroblasts. Based on the above spatial characteristics, we established vascularized breast cancer models with heterogeneous patterns of CCR and SR regions using an extrusion-based multi-nozzle 3D bioprinter. As a control, we fabricated unpatterned models with a generally uniform distribution of three cell types throughout the construct