DNA origami assembled spheroid for evaluating cytotoxicity and infiltration of chimeric antigen receptor macrophage (CAR-M)

Chimeric antigen receptor (CAR) T-cell therapies have shown remarkable results in patients with hematological malignancies. However, their success in treating solid tumors has been limited. As an alternative candidate for the CAR approach, CAR-macrophages (CAR-M) have demonstrated activation and phagocytosis directed by tumor antigens, showing promise in the treatment of solid tumors. Nevertheless, the mechanisms by which CARs direct tumor chemotaxis and invasion of CAR-M remain poorly understood. In this study, we aim to investigate the role of CARs in CAR-M attachment and infiltration using 3D tumor micro-spheroids, which were created by utilizing a novel nucleic acid nanostructures decorated living cells (NACs) based origami assembly technique. First, the effectiveness of phagocytosis and killing conducted by CAR-M was validated in the conventional 2D well/plate-surface culture models. Then,peripheral blood mononuclear cell (PBMC) invasion assay confirmed that the 3D tumor micro-spheroids were feasible for cell invasion. Finally, our results demonstrated that CAR-M exhibited higher invasion and killing capacity in 3D tumor micro-spheroids. In summary, the 3D NACs-origami assembled tumor spheroid model provides a suitable platform for target screening and pharmacodynamic evaluation of CAR-M.