Pei Wang,1,2,* Ming-Hao Xu,3,* Wen-Xin Xu,3,* Zi-Ying Dong,4 Ying-Hao Shen,3 Wen-Zheng Qin1 

1Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China; 2Department of Digestive Medicine, Wuwei People’s Hospital, Wuwei City, Gansu Province, 733000, People’s Republic of China; 3Department of Liver Surgery and Transplantation, Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China; 4Department of CT/MRI Center, Wuwei People’s Hospital, Wuwei City, Gansu Province, 733000, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Wen-Zheng Qin, Endoscopy Center and Endoscopy Research Institute, Zhongshan Hospital, Fudan University, Shanghai, 200032, People’s Republic of China, Tel/Fax +86-21-64041990, Email qin.wenzheng@zs-hospital.sh.cn Ying-Hao Shen, Department of Liver Surgery and Transplantation, Liver Cancer Institute and Zhongshan Hospital, Fudan University, No. 180, Fenglin Road, Shanghai, 200032, People’s Republic of China, Tel/Fax +86-21-64041990, Email syh12268@163.com

Background: Anti-programmed death-1 (PD1) antibodies have changed the treatment landscape for hepatocellular carcinoma (HCC) and exhibit promising treatment efficacy. However, the majority of HCCs still do not respond to anti-PD-1 therapy.
Methods: We analyzed the expression of CXCL9 in blood samples from patients who received anti-PD-1 therapy and evaluated its correlation with clinicopathological characteristics and treatment outcomes. Based on the results of Cox regression analysis, a nomogram was established for predicting HCC response to anti-PD-1 therapy. qRT‒PCR and multiple immunofluorescence assays were utilized to analyze the proportions of N1-type neutrophils in vitro and in tumor samples, respectively.
Results: The nomogram showed good predictive efficacy in the training and validation cohorts and may be useful for guiding clinical treatment of HCC patients. We also found that HCC cell-derived CXCL9 promoted N1 polarization of neutrophils in vitro and that AMG487, a specific CXCR3 inhibitor, significantly blocked this process. Moreover, multiple immunofluorescence (mIF) showed that patients with higher serum CXCL9 levels had greater infiltration of the N1 phenotype of tumor-associated neutrophils (TANs).
Conclusion: Our study highlights the critical role of CXCL9 as an effective biomarker of immunotherapy efficacy and in promoting the polarization of N1-type neutrophils; thus, targeting the CXCL9-CXCR3 axis could represent a novel pharmaceutical strategy to enhance immunotherapy for HCC.

Keywords: CXCL9, anti-PD-1 antibody, N1 polarization of neutrophils, nomogram