Neurotrophic receptor GFRalpha2 drives pro-metastatic programming of Kupffer cells in liver metastases

Gastrointestinal cancers such as colorectal, gastric and pancreatic cancers frequently metastasize to the liver, contributing significantly to cancer-related mortality and resistance to immune checkpoint inhibitors (ICIs). Recent multidisciplinary studies have highlighted a functional and mechanistic interplay between the nervous and immune systems in cancers, termed as neuroimmune axis. Given the rich hepatic innervation, we investigated whether neuroimmune signaling contributes to liver metastasis by analyzing the expression profiles of 217 known receptors involved in efferent signal transmission using single-cell omics datasets from colorectal cancer liver metastasis (CRLM) patients, among which glial cell line-derived neurotrophic factor (GDNF) family receptor alpha 2 (GFRalpha2) was identified due to its reproducibly and exclusively elevated expression on CRLM adjacent livers compared with healthy livers from transplantation donors. Using scRNA-seq, high-dimensional flow cytometry and multiplex-immunofluorescence analysis in mice and patients with CRLM, we further confirmed that GFRalpha2 was selectively expressed and upregulated on Kupffer cells (KCs). Notably, GFRalpha2 + KCs were found in close proximity to liver-infiltrating nerve fibers in CRLM, whereas chemical liver denervation led to reduced expression of GFRalpha2, suggesting a possible neural regulation on GFRalpha2 + KCs. Functionally, lentiviral shRNA-mediated Gfra2 knockdown significantly suppressed liver metastasis without affecting primary colorectal tumor growth, which could be abolished by KCs depletion using liposomal clodronate. Mechanistically, GFRalpha2 + KCs displayed impaired antigen presentation capacity, but enhanced pro-angiogenesis and neutrophil recruitment, suggesting a reprogrammed, metastasis-promoting phenotype. Notably, Gfra2 knockdown significantly reduced neutrophil accumulation and angiogenesis, but increased MHCII + KCs. Taken together, our findings pinpointed that GFRalpha2 upregulation reprograms KCs towards a pro-metastatic state, thereby facilitate liver metastasis.