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Background Cancer is a systemic disease that results in broad immune alterations throughout malignant progression. Previous studies have shown that tumors exert immunosuppressive effects on dendritic cells (DCs) and T cells both within the tumor microenvironment and at distant sites. 1 Strategies aimed at rescuing cell functionality under these conditions and enhancing DC-mediated T cell priming could pave the way for novel therapeutic approaches. CRISPR-based genetic engineering tools have presented a unique opportunity to alter individual cell subtypes for therapeutic gain, but the study of single cell-cell interactions following genomic perturbations in vitro and ex vivo remains challenging with existing technologies.Methods To overcome these limitations, we leverage Cellanome’s R3200 platform, which can culture tens of thousands of single cell-cell interactions, longitudinally track their function via imaging, and link imaging data to genetic perturbations ( figure 1). We enclosed thousands of single, edited DCs with a distribution of T-cells in semi-permeable hydrogel compartments (CellCage™ enclosures - CCEs) with light-guided polymerization. Single DC-T-cell co-cultures were analyzed with time-lapse imaging over days to assess their interactions by evaluating morphology, proliferation, and surface receptor expression. Imaging and sequencing data are integrated, as cells are lysed within CCEs to generate barcoded libraries, enabling detection of sgRNAs and transcriptomics following perturbation.Results We optimized a DC-T cell priming assay within polymer cages using ex vivo-differentiated mouse bone marrow-derived dendritic cells (BMDCs) and OT-I T cells, which exhibited proliferation and upregulation of the early activation marker CD69 upon successful priming ( figure 2). To simulate the immunosuppressive conditions encountered by DCs during tumor progression, we exposed BMDCs to various immunosuppressive factors during the final stages of their differentiation. This conditioning resulted in downregulation of co-stimulatory molecules, changes in transcriptomic profile, and a reduced ability to prime OT-I T cells. Finally, we generated a pooled library of CRISPR-edited BMDCs targeting key positive and negative regulators of DC-mediated T cell priming and demonstrated that specific gene perturbations could be directly linked to changes in the priming capacity of BMDCs.Conclusions These findings demonstrate the potential of our approach to capture dynamic immune cell-cell interactions and link them to genetic perturbations. By establishing a pooled CRISPR screening workflow for cell-extrinsic phenotypes, we provide proof of concept for scalable discovery of regulators of DC priming in immunosuppressive tumor environments—offering a path toward identifying therapeutic targets to enhance tumor-specific T cell responses.Reference Allen BM, Hiam KJ, Burnett CE, Venida A, DeBarge R, Tenvooren I, Marquez DM, Cho NW, Carmi Y, Spitzer MH. Systemic dysfunction and plasticity of the immune macroenvironment in cancer models . Nat Med. 2020;26(7):1125–1134.Abstract 1029 Figure 1Cellanome’s technology enables the measurement of multiple phenotypic and functional assays from the same cellsAbstract 1029 Figure 2Time-lapse imaging of T cell priming following caging of a single BMDC with OT-I T cells