A DLL1-culture system to propagate murine patrolling monocytes from bone marrow progenitors.

Patrolling monocytes (pMos) scavenge debris from vessel walls and mediate antibody-dependent cellular phagocytosis, making them attractive cell therapy candidates for cancer, atherosclerosis, and Alzheimer's disease. However, methods to generate pMos for cell therapy are not available. To address this, we developed a 2-step procedure to produce pMos in vitro from murine bone marrow (BM). First, myeloid progenitors were expanded and enriched from BM using cytokines for 4 d. Second, expanded progenitors were differentiated into pMos on delta-like ligand 1 (DLL1)-expressing monolayers for 8 d. We confirmed that in vitro grown pMos expressed the transcription factor Nr4a1 (Nur77) and other canonical pMos surface proteins, and depended on Notch signaling for their development. RNA-sequencing revealed that in vitro pMos expressed hallmark pMos genes, including Cx3cr1, Itgax (CD11c), CD43, Fcγr4, and Cd274 (PD-L1), and their gene signatures clustered closely with in vivo blood and BM pMos. Transcriptomic and phenotypic analyses further demonstrated that in vitro pMos were distinct from classical BM macrophages. Phagocytosis assays demonstrated the function of in vitro pMos in cancer cell uptake. Adoptive transfer studies demonstrated that in vitro pMos persisted within the circulation and lung vasculature during the early post-transfer period compared with BM-derived macrophages, consistent with the vascular-patrolling properties of pMos. Adoptive transfer of pMos reduced lung tumor burden in a metastatic model, supporting an anti-tumoral role for pMos and their ability to mediate immune surveillance in vivo. These findings demonstrate that the DLL1 culture system allows for propagation of functional pMos, enabling studies of pMos biology and their therapeutic potential.