Abstract 4878: Ablation of neutrophil-specific CXCR2 and PD-L1 alters neutrophil phenotype and can reduce disease burden in a murine model of advanced colorectal cancer liver metastases

Introduction: We have previously shown that as colorectal liver metastases (CRCLM) develop, there is a pressure that drives phenotypic development of neutrophils within the metastatic niche. Through transcriptional and protein analysis, different neutrophil transcriptional states can be delineated. Combination therapy with CXCR2 and PD-1/PD-L1 checkpoint inhibition has been shown to be a strategy to reduce disease burden in mouse models of hepatocellular carcinoma. Through loss of neutrophil-specific target proteins CXCR2 and PD-L1 in mouse models of CRCLM, we show modification of neutrophil phenotype and changes in disease burden. Methods: KrasG12D/+, Trp53fl/fl, Rosa26N1CD/+ (KPN) tumour cells modelling CMS4 pMMR colorectal cancer generated from murine CRCLM organoids were injected orthotopically into the spleen of male and female C57BL/6 mice with loss of neutrophil-specific proteins using the Cre-loxP system. Mrp8CreCxcr2fl/fl and Mrp8CrePd-l1fl/fl genetically engineered mouse models with neutrophil-specific loss of CXCR2 and PD-L1 respectively were sampled at 14 days and 35 days, and neutrophils isolated using MACS prior to bulk sequencing to assess the impact on neutrophil transcriptional phenotype. Neutrophil protein expression, phenotype and functional role was assessed using flow cytometry and ELISA. Results: Loss of neutrophil-specific CXCR2 does not alter CRCLM burden at a late timepoint following intrasplenic injection but leads to an altered neutrophil phenotype with reduced PD-L1 checkpoint inhibitor expression, and increased Lactoferrin expression, a neutrophil granule protein with known anti-cancer properties. These neutrophils are strongly suppressive of T cell proliferation when co-cultured ex vivo. Loss of neutrophil-specific PD-L1 leads to a reduction in CRCLM tumour size but not number, suggesting an important role for neutrophil PD-L1 in growth of CRCLM without affecting tumour seeding. Ex vivo assays will examine the effect of loss of neutrophil-specific CXCR2 on NETosis and ROS generation, and the effect of lactoferrin upon these neutrophil functions in neutrophils with and without CXCR2 expression. Conclusions: We speculate that inhibition of CXCR2 and PD-L1 and loss of these neutrophil proteins alters neutrophil phenotype to be more “anti-tumour”. This may be a strategy to alter the immunosuppressive microenvironment of CRCLM and allow for downstaging of CRCLM allowing for resection and preventing recurrence. Citation Format: Alistair S. McLaren, John Falconer, Sarah Ressel, Leo M. Carlin, Owen J. Sansom, Colin W. Steele. Ablation of neutrophil-specific CXCR2 and PD-L1 alters neutrophil phenotype and can reduce disease burden in a murine model of advanced colorectal cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4878.