Journal Article

Antitumor Activity of Tumor‐Infiltrating Neutrophils Revealed by a Syngeneic Mouse Model of Cholangiocarcinoma

Sugiyama, Shigeaki, Nakayama, Keiichi I., Sugahara, Osamu, Koga, Daisuke, Higa, Tsunaki, Oka, Takeru, Wada, Reona

2025

Overview

The tumor immune microenvironment plays a key role in the regulation of cancer progression. Recent studies have suggested a relation between diverse tumor genotypes and tumor immune microenvironment phenotypes for cholangiocarcinoma (CCA). However, the contribution of tumor‐infiltrating immune cells to CCA progression has remained unclear, underscoring the need for genetically defined CCA models in immunocompetent mice. We here aimed to generate genetically engineered and transplantable CCA organoids from C57BL/6 mice and to investigate the role of tumor‐infiltrating immune cells in CCA progression with this model. CCA organoids were generated ex vivo with the use of the CRISPR/Cas9 system. Orthotopic transplantation of CCA organoids harboring mutations in Smad4, Trp53, and Kras into wild‐type C57BL/6 mice resulted in tumor formation accompanied by distant metastasis. Selective depletion of immune cell types in the tumor‐bearing mice revealed an antitumor action of tumor‐infiltrating neutrophils (TINs) that was mediated by direct killing of cancer cells through the production of reactive oxygen species. Furthermore, administration of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) increased the number and cytotoxicity of TINs, suppressed tumor growth, and prolonged the survival of tumor‐bearing mice. Finally, combination treatment with rhG‐CSF and standard chemotherapy resulted in a synergistic attenuation of tumor growth. Our study therefore provides a syngeneic and genetically defined mouse model of CCA and highlights the therapeutic potential of targeting TINs with rhG‐CSF. This study developed a genetically engineered and transplantable cholangiocarcinoma (CCA) organoid model in immunocompetent mice to investigate the role of tumor‐infiltrating immune cells in CCA progression. The findings revealed that tumor‐infiltrating neutrophils (TINs) exhibit antitumor activity by directly killing cancer cells through reactive oxygen species, and their effects were enhanced by recombinant human granulocyte colony‐stimulating factor (rhG‐CSF), which suppressed tumor growth and improved survival. Additionally, combining rhG‐CSF with standard chemotherapy showed synergistic tumor suppression, highlighting the therapeutic potential of targeting TINs in CCA.

• Publisher: John Wiley & Sons, Inc,Wiley
• Subject: Animal models / Animals / Antitumor activity / Apoptosis / Bile Duct Neoplasms - drug therapy / Bile Duct Neoplasms - genetics / Bile Duct Neoplasms - immunology / Bile Duct Neoplasms - pathology / Bioluminescence / Cancer / Cancer therapies / Cell cycle / Cell Line, Tumor / Chemotherapy / Cholangiocarcinoma / Cholangiocarcinoma - drug therapy / Cholangiocarcinoma - genetics / Cholangiocarcinoma - immunology / Cholangiocarcinoma - pathology / CRISPR / Cytokeratin / Cytotoxicity / Disease Models, Animal / Epidermal growth factor / Fibroblasts / Genes / Genetic aspects / Genetic engineering / Genetically modified organisms / Genotypes / Granulocyte Colony-Stimulating Factor - pharmacology / granulocyte colony‐stimulating factor (G‐CSF) / Granulocytes / Health aspects / Humans / Immunocompetence / Kinases / Leukocytes (granulocytic) / Leukocytes (neutrophilic) / Liver / Metastases / Metastasis / Mice / Mice, Inbred C57BL / Microenvironments / Mutagenesis / Mutation / neutrophil / Neutrophils / Neutrophils - drug effects / Neutrophils - immunology / Oncology, Experimental / organoid / Organoids / Organoids - immunology / Original / ORIGINAL ARTICLE / Phenotypes / Proteins / Reactive oxygen species / Reactive Oxygen Species - metabolism / Smad4 protein / Smooth muscle / Syngeneic grafts / tumor microenvironment / Tumor Microenvironment - immunology / Tumorigenesis / Tumors