Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity
Cancer associated fibroblasts (CAFs) are one of the most abundant components of the breast tumor microenvironment (TME) and major contributors to immune modulation in the TME. CAFs are well known to regulate the activity of diverse types of immune cells including T cells, macrophages and dendritic cells, however little is known about their interaction with Natural killer (NK) cells. NK cells constitute an important arm of anti-tumor immunity, yet the regulation of NK cell activity by CAFs in solid tumors is poorly understood. Here we find, using mouse models of cancer and ex-vivo cocultures, that immunosuppressive CAF subsets severely inhibit NK cell cytotoxicity towards cancer cells. We unravel the mechanism by which this suppression occurs, through CAF-mediated downregulation of the NK-surface receptors, Natural Killer Group 2D (NKG2D) and DNAX Accessory Molecule-1 (DNAM-1). Ligands for these receptors are known to be expressed by cancer cells and minimally expressed in healthy tissue. Here we find that CAFs also upregulate ligands for NKG2D and DNAM-1. Ligand-receptor engagement between NK cells and CAFs leads to CAF cytolysis, which in turn diminishes the expression of NKG2D and DNAM-1 on NK cells via a negative feedback loop, and promotes cancer escape from NK cell surveillance. These results reveal a CAF-mediated immunosuppressive mechanism with implications for treatment of solid tumors. Overall design: To investigate the transcriptomic state of NK cells in the 4T1 TNBC model and assess how they may be influenced by CAFs, we sequenced 1) primary NK cells from the spleens of healthy BALB/C mice as controls (3 independent replicates) 2) primary NK cells from spleens of BALB/C mice harboring 4T1-GFP tumors as controls (4 independent replicates) and 3) 4T1 tumor infiltrating (TI) NK cells (4 independent replicates). NK cells were isolated using  magnetic seperation followed by FACS with the following markers: GFP-EPCAM-CD45+CD3-CD31-CD14-NKp46+. To investigate the genes in sCAFs that may influence NK cell migration and retention in stromal areas, we sequenced sCAFs (3 independent replicates) isolated from 4T1-GFP tumors. sCAFs were isoalted using magnetic seperation followed by FACS with the following markers: EPCAM-GFP-CD45-CD31-PDPN-