In vitro patrolling monocytes in DLL cultures have similar gene expression profiles to their blood and bone marrow counterparts.

Patrolling monocytes (pMos) scavenge debris from vessel walls, mediate antibody dependent cellular-cytotoxicity (ADCC) and -phagocytosis (ADCP), and recruit natural killer cells to tumors. Pmos are therefore attractive cell therapy candidates to treat Alzheimer's disease, atherosclerosis and cancers. However, protocols to generate large numbers of pMos currently do not exist. To address this, we developed a two-step procedure to produce large numbers of pMos in vitro. We optimized cytokine combinations to facilitate (1) myeloid progenitor expansion from murine bone marrow (BM) over day 1-4, and (2) pMos development on a monolayer expressing DLL1 from day 5-12. In vitro pMos development depended on BM progenitors and Notch signaling, and the pMos expressed Nur77 and canonical pMos surface markers. Bulk RNA sequencing revealed that in vitro pMos expressed pMos genes, including Nr4a1 (Nur77), Cx3cr1, Itgax (CD11c), Fcgr4, and Cd274 (PD-L1), and their gene signatures clustered closely with blood and BM pMos. Further, in vitro pMos, but not cMos, performed ADCP on breast cancer cells, indicating that they reflect circulating cMos and pMos. Importantly, when cultured pMos were adoptively transferred into mice, they maintained a pMos phenotype and homed to blood and vascular organs such as the lungs and liver. The ability to propagate large numbers of functionally competent pMos, that can be transferred into mice will facilitate in vitro and in vivo studies of pMos biology and therapies. Overall design: RNA-seq study of mouse in vitro monocyte subsets (cMos, intMos, cMos) with 3 replicates per subset.