Background More than 50% of patients with colorectal cancer (CRC) present with liver metastases and there are few effective medications for treating CRC liver metastases (CRLM). Innate immunity plays an important role in CRLM. Neutrophils are major players in the innate immune system. Herein, we aimed to investigate the role of neutrophils and to identify a potential therapeutic target in CRLM.

Methods Tumor tissues, blood samples and tumor-associated neutrophils (TANs) from patients with CRLM were isolated for phenotypical and functional analyses by single-cell RNA sequencing, flow cytometry, and spatial mapping at the RNA and protein levels. The biological roles and mechanisms of BHLHE40+ TANs in CRLM were determined in vitro assays and in vivo models, including chromatin immunoprecipitation (ChIP) qPCR, patient-derived CRLM organoids and neutrophil-specific Bhlhe40 knockout (Bhlhe40^f/f; Mrp8-Cre) mice.

Results A comprehensive transcriptional landscape of tumor-associated neutrophils (TANs) was provided by profiling more than 55, 000 neutrophils from six CRLM patients using single-cell RNA sequencing. A terminally differentiated pro-tumor neutrophils subset (TAN-1), with glycolysis signature and senescent phenotype, was significantly enriched in liver metastases and associated with poor prognosis. TAN-1 was converged from other TAN subsets under conditions of hypoxia and glucose starvation, resulting from enhanced glucose metabolism of tumor cells in CRLM. Functionally, TAN-1 exhibited a pro-angiogenic activity via secretion of vascular endothelial growth factor A (VEGFA) and recruited pro-tumor macrophages via CCL3L1-CCR1 to foster immunosuppressive microenvironment in liver metastases. Further spatial transcriptomics analysis of CRLM tissues revealed the immunosuppressive niche contained TAN-1 and pro-tumor macrophages, which promotes CRLM progression. Mechanistic studies revealed that BHLHE40 was a key regulator in the polarization of neutrophils towards TAN-1 phenotype, and direct transcriptional regulation of BHLHE40 on TAN-1 marker genes was demonstrated by ChIP assay. Furthermore, neutrophil-knockout of BHLHE40 in liver metastases mice models significantly suppressed tumor metastasis to the liver.

Conclusions BHLHE40+ neutrophils play a critical role in the immunosuppressive nature of the CRLM microenvironment. Eliminating BHLHE40+ neutrophils represents a new strategy to inhibit CRLM progression.