Abstract LB161: Bioprinting 3D spatially resolved tumor avatars to mimic the native tumor microenvironment Free

Engineered tumor avatars provide opportunities to identify mechanisms of cancer initiation, progression, and treatment resistance. We previously reported the development of a single-cell bioprinting method, with subcellular resolution, to fabricate 2D tumor microenvironments (TME) replicating an annotated patient biopsy. Here we report the development of 3D tumor avatars that are cell dense, spatially resolved with 60 µm resolution, and heterogeneous containing up to 7 cell types. This approach overcomes current challenges in spheroid/organoid models that are limited in their ability to control the spatial arrangement of cells, geometry, and heterogeneity to dictate specific cell-cell interactions. A microfluidic dispenser (Biopixlar, Fluicell) was used to pattern cells of the ductal carcinoma in situ tumor microenvironment. The dispensed cells were supported by gelatin while they adopted tissue-like morphology and formed junctions to create an aggregated construct. The avatars contained a cancer cell line (MCF7, HCC1143, or MDA-MB-231) surrounded by an epithelial ring of either MCF10A or a combination of primary human mammary epithelial cells and myoepithelial cells. The stromal compartment included primary mammary fibroblasts along with combinations of primary human umbilical vein endothelial cells, mesenchymal stromal cells, cancer associated fibroblasts, and/or macrophages. Prints were cultured for up to two weeks, with or without secondary Cultrex matrix support, live cell imaged, fixed, and stained for markers of proliferation, cell-cell junctions, morphology, and phenotype states. We found that cells can be patterned with high precision, density, and heterogeneity while maintaining viability, proliferation, and migrational capacity. Cells spread to establish their expected morphology and cell-cell junctions with or without secondary Cultrex matrix support. By not including Cultrex, we demonstrate the epithelial cells can stabilize for 24 hours before introducing the cancer into the system. To demonstrate the high spatial precision of our method, a 1.5 mm x 1 mm region of interest was identified from an annotated breast cancer biopsy containing cancer filled mammary ducts, immune infiltrates, and surrounding stromal compartment of fibroblasts and vasculature. We extruded the 2D map 100 µm in the Z direction to create a cell dense tissue containing 7 cell types matching the size of the histological features. To assess function of the engineered avatars, we perturbed TMEs containing MCF7, MCF10A, and fibroblasts with 10 ng/mL transforming growth factor beta 1 (TGF-β1). Within 48 hours we observed greater fibroblast density, matrix remodeling, and MCF10A elongation in the treatment group relative to control. By controllably manipulating the cellular and extracellular environment in a spatially resolved manner we can begin to identify which conditions promote or inhibit cancer initiation and progression beyond what has been possible with conventional in-vivo and in-vitro models.