Single-Cell Transcriptomic Analysis Reveals Dynamic Changes in the Liver Microenvironment During Colorectal Cancer Metastatic Progression

Background: Metastasis is a leading cause of cancer-related deaths, with the liver being the most frequent site of metastasis in colorectal cancer. Previous studies have predominantly focused on the influence of the primary tumor itself on metastasis, with relatively limited research examining the changes within target organs. Methods: Using an orthotopic mouse model of colorectal cancer, single-cell sequencing was employed to profile the transcriptomic landscape of pre-metastatic and metastatic livers. The analysis focused on identifying cellular and molecular changes within the hepatic microenvironment, with particular emphasis on inflammatory pathways and immune cell populations. Results: A neutrophil subpopulation with high Prok2 expression was identified, showing elevated levels in the pre-metastatic and metastatic liver. Increased infiltration of Prok2? neutrophils correlated with poor prognosis in liver metastatic colorectal cancer patients. In the liver macro-metastatic niche (MMN), these neutrophils showed high App and Cd274 (PD-L1) expression, suppressing macrophage phagocytosis and promoting T-cell exhaustion. Conclusion: A Prok2? neutrophil subpopulation infiltrated both pre-metastatic and macro-metastatic liver environments, potentially driving immunosuppression through macrophage inhibition and T-cell exhaustion. Targeting Prok2? neutrophils could represent a novel therapeutic strategy for preventing liver metastasis in colorectal cancer patients. Overall design: Liver single cell suspension preparation was performed by the laboratory staff of NovelBio Biopharmaceutical Technologies Ltd. Liver lysis was performed as described previously, whole mouse livers were removed, cut into small pieces on ice, and then enzymatically lysed with 1 mg/mL Collagenase II (Worthington) for 30 min on a shaker at 37°C. 70-micron cell filters were sieved and then erythrocytes were lysed in erythrocyte lysis buffer (Miltenyi Biotec), rinsed twice with PBS, and then the MACS Dead Cell Removal Kit (Miltenyi Biotec) was used to further enrich the single cell suspension.