Show Author's information Hide Author's Information Mei Feng1,2,*,Fangyanni Wang3,*,Xinyu Liu1,Tingting Hao1,Ning Zhang1,3,4( 
),Mi Deng3,Yisheng Pan2 
( ),Ruirui Kong1 ( )
*These authors contributed equally to this work.
Abstract
Liver cancer is a deadly malignancy associated with high mortality and morbidity. Less than 20% of patients with advanced liver cancer respond to a single anti-PD-1 treatment. The high heterogeneity of neutrophils in the tumor immune microenvironment in liver cancer may contribute to resistance to immune checkpoint blockade (ICB). However, the underlying mechanism remains largely unknown.
We established an orthotopic liver cancer model by using transposable elements to integrate the oncogenes Myc and KrasG12D into the genome in liver cells from conditional Trp53 null/null mice (pTMK/Trp53−/−). Flow cytometry and immunohistochemistry were used to assess the changes in immune cells in the tumor microenvironment. An ex vivo coculture assay was performed to test the inhibitory effects of tumor-associated neutrophils (TANs) on CD8+ T cells. The roles of neutrophils, T cells, and NK cells were validated through antibody-mediated depletion. The efficacy of the combination of neutrophil depletion and ICB was evaluated.
Orthotropic pTMK/Trp53−/− mouse liver tumors displayed a moderate response to anti-Ly6G treatment but not PD-1 blockade. Depletion of neutrophils increased the infiltration of CD8+ T cells and decreased the number of exhausted T cells in the tumor microenvironment. Furthermore, depletion of either CD8+ T or NK cells abrogated the antitumor efficacy of anti-Ly6G treatment. Moreover, the combination of anti-Ly6G with anti-PD-L1 enhanced the infiltration of cytotoxic CD8+ T cells and thereafter resulted in a significantly greater decrease in tumor burden.
Our data suggest that TANs may contribute to the resistance of liver cancer to ICB, and combining TAN depletion with T cell immunotherapy synergistically increases antitumor efficacy.
Keywords: Liver cancer, neutrophil, PD-1, CD8+ T cell, exhaustion
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