G-CSF PROMOTES ANTI-TUMOR RESPONSE OF NK CELLS BY ACTIVATING GRANULOPOIESIS IN HEPATOBLASTOMA

Background and Aims: Hepatoblastoma originates from abnormal differentiation of hepatocyte precursors during embryogenesis and is characterized by extramedullary hematopoiesis and enrichment of hematopoietic progenitor cells in tumor. Granulocyte-colony stimulating factor (G-CSF) is often used to manage neutropenia during chemotherapy, especially in HB patients. Recent studies have reported that G-CSF has a synergistic anti-tumor effect with other treatments in adults. However, the roles of G-CSF in HB, which exhibits exceptional extramedullary hematopoiesis properties, remain unknown.
Methods: We performed single-cell RNA sequencing (scRNA-seq) on tumors and matched liver samples from 18 treatment-naïve HB patients and 9 patients treated with neoadjuvant chemotherapy combined with G-CSF (including 6 pre- and post-treatment pairs). The impact of G-CSF on intratumoral granulocyte composition and other immunocytes was discussed. Pseudotime and cell-cell interaction analyses were conducted to unravel the differentiation procedure of granulocytes and their cellular crosstalk. Bulk RNA sequencing (RNAseq) on 110 HB patients who received the same regimen was performed for validation.
Results: We demonstrated that granulocyte-monocyte progenitors (GMPs) were significantly enriched in treatment-naïve HB tumors than in adjacent liver tissues. After chemotherapy and G-CSF treatment, GMPs displayed augmented development trajectory towards neutrophils and emergency-granulopoiesis-related transcriptomic signatures, including increased expression of transcription factor CEBPB and activation of JAK-STAT pathway. Further, MMP8⁺ neutrophils were the predominant neutrophil subset in the post-treatment HB tumors in comparison to non-tumor adjacent liver tissues. MMP8⁺ neutrophils showed immature and non-immunosuppressive phenotype with high expression of activation- and degranulation-associated genes. Granules encoded by MMP8⁺ neutrophils mainly included pro-inflammatory mediators RETN, CAMP and LTF. The MMP8⁺ neutrophils might enhance the anti-tumor cytotoxicity of natural killer (NK) cells through interact with them via RETN-CAP1 axis. Notably, based on bulk RNAseq, increased expression of MMP8⁺ neutrophil signature in tumor was significantly associated with better overall survival and recurrence-free survival for HB patients.
Conclusions: G-CSF triggers intratumoral granulopoiesis starting with GMP, leading to the enrichment of MMP8⁺ neutrophils and enhanced NK cell-mediated anti-tumor immune responses. Concurrent G-CSF administration with chemotherapy may provide novel treatment strategy for HB.