Colorectal cancer is the third most frequent human malignancy characterized by high rates of hepatic metastasis and still lacks a curative approach. Patients with colorectal cancer liver metastases (CRLMs) have a 5-year overall survival between 20 and 45%, a recurrence of 60% and poor clinical benefits from immunotherapies. The liver is a preferential tissue for Natural Killer (NK) cells that are classified into two main subsets, namely CD56Bright/CD16- (CD56Bright) and CD56Dim/CD16+ (CD56Dim). Exploring the heterogeneity of intrahepatic NK cells beyond this dichotomy is key to identify specific immune checkpoints (ICs) and to unveil novel therapeutic targets.

We conducted in depth single cell RNA-sequencing combined with multiparametric flow cytometry analysis of NK cells and total CD45+ immune cells. In addition, functional studies were performed to assess the relevance of immune-surveillance by NK cells and to leverage their anti-tumor potential against CRLMs.

We demonstrated that tissue-resident CD56Bright NK cells in CRLMs are mature and potent anti-tumor effectors, showing minimal developmental relationship with intrahepatic CD56Dim and circulating NK cell subsets. In particular, unique transcriptional profile and peculiar expression of ICs were observed in CD56Bright and CD56Dim liver NK cell clusters, in line with their specific end-terminal differentiation programs in the metastatic niche. Among ICs, CXCR4 emerged as targetable inhibitory receptor involved in CXCL12-dependent mitigation of intrahepatic NK cells. CXCR4 negatively correlated with Interferon-y (IFNγ) and increased production of this anti-tumor molecule was reported after CXCR4 blockade with the antagonist plerixafor. In addition, we identified IL-1R8 as an IC ubiquitously expressed by NK cells in CRLMs, demonstrating that its pharmacological inhibition with an original monoclonal antibody significantly improved NK cell anti-tumor effector functions.

In summary, the present study discloses the origin, heterogeneity and effector potential of matched circulating and intrahepatic NK cells from CRLM patients by using unbiased analytic approaches. It sheds light on an abundant tissue-resident IFNγ+ CD56Bright NK cell population with an anti-tumor signature, and provides two possible therapeutic targets (i.e., IL-1R8 and CXCR4) to complement current CRLM immunotherapy.