Clonal lineage tracing of innate immune cells in human cancer

Innate immune cells constitute the majority of the tumor microenvironment (TME), where they mediate both natural anti-tumor immunity and immunotherapy responses. While single-cell T- and B-cell receptor sequencing has provided fundamental insights into the clonal dynamics of human adaptive immunity, the lack of appropriate tools has precluded similar analysis of innate immune cells. Here, we developed a method leveraging somatic mitochondrial DNA (mtDNA) mutations to reconstruct clonal lineage relationships between single cells across native human tissues. We jointly sequenced single-cell transposase-accessible chromatin and mtDNA to profile 124,958 cells from matched tumor, non-involved lung tissue (NILT) with peripheral blood of early-stage non-small cell lung cancer (NSCLC) patients, as well as 93,757 cells from matched tumor and peripheral blood of ovarian cancer patients. Our single-cell concomitant profiling of lineage and cell states of thousands of immune cells resolved clonality across cell types, tissue sites, and malignancies. By resolving clonal populations across innate immune subtypes, we demonstrated that TME-resident myeloid subsets, including macrophages and type 3 dendritic cells (DC3), are clonally linked to both circulating and tissue-infiltrating monocytes. Further, we identified distinct DC-biased and macrophage-biased myeloid clones, enriched in the tumor and NILT, respectively, and found that their circulating monocyte precursors exhibit distinct epigenetic profiles, suggesting that myeloid differentiation fate may be predetermined before TME infiltration. These results delineate the clonal pathways of intratumoral myeloid cell recruitment and differentiation in human cancer and suggest that remodeling of the tumor myeloid compartment may be peripherally programmed.