Abstract Cellular heterogeneity is a prominent feature of the tumor microenvironment, and the organization of these cells has been linked to clinical outcomes such as disease progression and drug resistance. While retrospective analysis of patient tumors has produced a myriad of insights, samples are limited and highly complex making it challenging to validate causality. Here we present an engineered breast tumor microenvironment model, with single cell spatial resolution, to systematically identify which cell phenotypes and their spatial arrangements may be driving ductal carcinoma in situ disease progression. A microfluidic dispenser (Biopixlar, Fluicell) was optimized to enable the spatial patterning of single cells to replicate native histology. To demonstrate the high spatial precision of our method, we first replicated an annotated 2D section of a breast tumor biopsy region of interest (ROI) using MCF10A, MDA-MB-231, primary mammary fibroblasts, THP-1 derived macrophages, and primary mesenchymal stem cells. The XY coordinate of each cell was identified to create a print map matching the original biopsy. Deposited cells adhered to their intended target with an average print fidelity of 2.4 µm. Next, the stromal compartment of the ROI was altered to include phenotypes associated with tumor promoting microenvironments (i.e. cancer associated fibroblasts, M2 macrophages) and the core of the MCF10A ring filled with MDA-MB-231. Each deposited cell contained a fluorescent tag corresponding with its cell type to facilitate real time spatial tracking and annotation. The tumor microenvironments were live cell imaged for 24 hours at 5 minute intervals. MCF10A cells spread to form their junctions and create a cohesive ring, MDA-MB-231 cells proliferated and rapidly moved around within the confined space, and the cells of the stromal compartment spread to create a dense tissue like structure. The microenvironments were cultured for an additional 2-6 days, fixed, and stained for markers of proliferation, phenotype shifts, cell-cell junctions, and morphology. Unlocking the relative contributions of the specific cell types in ductal carcinoma in situ tumor microenvironment to the overall evolution of invasive breast cancer may hold the answer for many questions in early detection, treatment, and cancer avatar models. Citation Format: Haylie R. Helms, Kody A. Oyama, Alexander E. Davies, Ellen M. Langer, Luiz E. Bertassoni. An engineered breast tumor microenvironment model, with single-cell spatial resolution, to assess spatial dynamics of tumor evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4217.
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