Cancer is a systemic disease that drives immune dysregulation throughout malignant progression. Dendritic cells (DCs) are essential for initiating anti-tumor T-cell responses, yet tumors exert immunosuppressive effects on both DCs and T cells, limiting their ability to coordinate effective anti-tumor immunity. CRISPR-based genetic screens provide a powerful approach to identify regulators of immune cell functions for therapeutic gain, but existing technologies are limited in their ability to measure dynamic cell–cell interactions following genetic perturbation.

To overcome these limitations, we leverage Cellanome's R3200 platform, which uses light-guided polymerization to generate semi-permeable hydrogel compartments that capture thousands of single live cell–cell interactions. These interactions are tracked longitudinally by time-lapse imaging and followed by in-cage cell lysis to generate barcoded cDNA libraries, enabling imaging data to be linked to transcriptomic profiles and CRISPR perturbations. 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. Individual co-cultures were monitored by time-lapse imaging over several days to assess proliferation and surface receptor expression.

We then generated a pooled CRISPR library targeting key positive and negative regulators of DC biology and applied it to BMDCs in our system. Using longitudinal imaging together with sequencing-based identification of perturbations, our data demonstrate that this approach enables specific gene knockouts in DCs to be directly linked to enhanced or impaired T-cell priming at the level of individual DC–T-cell interactions.

Together, these findings demonstrate the potential of our platform to capture dynamic immune cell–cell interactions and connect them to defined genetic perturbations. By establishing a pooled CRISPR screening workflow for cell-extrinsic immune phenotypes, this work provides proof of concept for scalable discovery of regulators of DC priming in immunosuppressive tumor environments, with implications for identifying therapeutic targets that enhance tumor-specific T-cell responses.