Timing and location dictate monocyte fate and their transition to tumor-associated macrophages

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells whose phenotypes and functions are shaped by factors that are incompletely understood. Herein, we asked when and where TAMs arise from blood monocytes, and how they evolve during tumor development. We initiated pancreatic ductal adenocarcinoma (PDAC) in inducible monocyte fate-mapping mice and combined single-cell transcriptomics and high-dimensional flow cytometry to profile the monocyte-to-TAM transition. We revealed that monocytes differentiate first into a transient intermediate population of TAMs (IntTAM) that generates two longer-lived lineages of terminally differentiated TAMs with distinct gene expression profiles, phenotypes and intra-tumoral localization. Transcriptome datasets and tumor samples from patients with PDAC evidenced parallel TAM populations in humans and their prognostic associations. These insights will support the design of new therapeutic strategies targeting TAMs in PDAC. Overall design: tdTomato+/- cell populations were sorted from Ms4a3Cre x RosatdTomato and Ms4a3CreERT2 x RosatdTomato mice using the indicated gating strategies. The cells were processed using the Chromium Single Cell 3' (v3 Chemistry) platform (10X Genomics). tdTomato+ and tdTomato- cells were sequenced separately using the NovaSeq sequencer (Illumina). Briefly, cells were loaded onto a Chromium Next GEM Chip G, and single cell suspensions in gel beads-in-emulsion (GEMs) were generated using the chromium controller (10X genomics). Reverse transcription (RT) was performed to generate cDNA, which was amplified, cleaned, and fragmented. Finally, libraries were subjected to standard quality control (QC) steps before sequencing.