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. ### Competing Interest Statement J. L., X. S. and X. Y. are the employee of Roc Rock Biotechnology (Suzhou) Co., Ltd. And X. H., H.W.,Y. Z. and G. S. are the employee of Puheng Technology Co., Ltd. The other authors declare no competing interests.