Evaluation of pluripotent stem cell-derived myeloid line-based chimeric antigen receptor macrophage-like cells identifies limited in vivo persistence.

Macrophages are of interest as candidates for adoptive cell therapy for solid tumors because they can infiltrate tumor tissue, engage target cells and influence the tumor microenvironment. However, macrophage products derived from primary monocytes remain difficult to standardize, expand and genetically modify. Pluripotent stem cell-derived myeloid cell lines (PSC-MLs) provide a renewable and engineerable myeloid platform, but their use as direct tumor-targeting effectors has remained incompletely defined. In this study, we generated macrophage-like cells from PSC-MLs carrying a doxycycline (DOX)-inducible anti-HER2 chimeric antigen receptor (CAR) and evaluated three separable features of the platform: in vitro tumor-cell control, contact-associated and CAR-modified transcriptional responses and postadministration persistence in vivo. Cells established from 2 human PSC backgrounds retained macrophage-associated phenotypes after differentiation. In coculture with HER2-expressing tumor cells, the cells showed measurable antitumor activity relative to untreated controls. The DOX-treated condition was compatible with a CAR-associated contribution in some comparisons, but uniform CAR-dependent enhancement was not established across tumor models. Live imaging documented phagocytic events but did not quantify their frequency. Transcriptomic profiling associated tumor-cell contact with inflammatory, trafficking and uptake-related programs, and the CAR-induced coculture condition with a relative emphasis on vesicular, lysosomal and proteostasis-related modules. In a subcutaneous coimplantation assay, in which tumor and effector cells were introduced together, treated groups showed short-term tumor-control signals relative to untreated controls at later observation points. However, an incremental benefit of the CAR-induced (DOX) condition was not clearly resolved. Luciferase-based tracking of ML-MPs showed marked loss by day 3 and no detectable signal by day 7. Repeated dosing was feasible, but its incremental benefit over single dosing could not be established in the small exploratory experiment.