Nature Zhang Ning/Zhang Zemin/Zhu Jiye collaborated to reveal liver cancer immune microenvironment subtypes and neutrophil heterogeneity

The heterogeneity of the immune microenvironment is one of
the important reasons for tumor resistance, recurrence and poor prognosis.
In recent years, immunotherapy and combination therapy have brought hope to patients with advanced tumors, and systematic exploration of the heterogeneity of tumor immune microenvironment has played an important guiding
role in treatment selection, efficacy prediction, protocol optimization and development of new immunotherapy targets.
In recent years, Zhang Ning's team at Peking University First Hospital has been focusing on the exploration of liver cancer heterogeneity, and revealed the genome, phenotype and single-cell copy number heterogeneity of liver cancer through a series of preliminary work (Gastroenterology, 2016; Cancer Cell, 2019; Gastroenterology, 2022）
。

However, we still lack systematic understanding
of the heterogeneity of the immune microenvironment of liver cancer.
Previous studies have been based on pathological analysis or Bulk transcriptome sequencing, and the results have not accurately resolved their cellular composition and tend to ignore the small number of key cell subsets
.
The development of single-cell sequencing technology has promoted the exploration of tumor immunity, and a number of studies have explored liver cancer at the single-cell level
.
However, most of these studies have focused on specific types of cells, and their results do not reflect the full profile of
the immune microenvironment.
Therefore, there is an urgent need for unbiased research strategies that include all cell subtypes to systematically reveal the heterogeneity of the immune microenvironment of liver cancer.

On November 9, 2022, Zhang Ning's team from the Cancer Translational Research Center of Peking University First Hospital, Zhang Zemin's team from Peking University Biomedical Frontier Innovation Center (BIOPIC) and Zhu Jiye's team from the Department of Hepatobiliary Surgery of Peking University People's Hospital published a report entitled "Liver tumor immune microenvironment subtypes and" in Nature Neutrophil Heterogeneity"
.
In the study, the researchers collected 160 samples from 124 cases and 29 fresh tissue samples from 8 mice to complete antibody-enriched, single-cell transcriptome sequencing for a total of 189 samples (Figure 1).

At the same time, the researchers performed exome sequencing on 84 supporting cases, collected 8 published single-cell transcriptome sequencing datasets, 453 tissue transcriptome sequencing and 10 spatial transcriptome data for comprehensive analysis, and comprehensively used tumor cell lines to construct the In vitro culture system, and built the Ex vivo experimental system based on clinical samples.
The In vivo experimental system based on mouse tumor model was comprehensively verified
.
This study defines the five immune microenvironment subtypes (TIMELASER) of liver cancer for the first time in single-cell precision, explores its cell composition, spatial distribution, genomic characteristics and chemokine receptor-ligand network, comprehensively reveals the heterogeneity of tumor-associated neutrophils (TAN) for the first time, and discovers and verifies CCL4⁺ and PD-L1 ⁺The pro-tumor mechanism of the two key subpopulations of TAN, which was finally constructed by constructing a mouse liver cancer model, from the three levels of In vitro, Ex vivo, and In vivo, It has been gradually proved that targeting tumor-associated neutrophils is expected to form a new immunotherapy regimen for liver cancer, and these achievements provide key information
for basic research and clinical diagnosis and treatment of liver cancer.

Figure 1 Experimental design of this study

1) Cell module analysis revealed five immune microenvironment subtypes of liver cancer

First, the researchers systematically resolved 89 cell subsets of the immune microenvironment of liver cancer, a discovery that far exceeds previous studies, including 11 previously unreported neutrophil subsets
for the first time.
The researchers found that these cell subsets were not evenly distributed
between different cases.
Through hierarchical clustering, the researchers successfully resolved 5 different immune microenvironment subtypes, including: (1) immune-activated TIME-IA (ImmuneActivation); (2) myeloid enrichment immunosuppressive TIME-ISM (ImmuneSuppressiveMyeloid); (3) stroma-enriched immunosuppressive TIME-ISS (Immune S uppressiveStromal); (4) Immunorejection ImmuneExclusion (TIME-IE); (5) Immune-resident (ImmuneResidence), combined with the TIMELASER typing system (Figure 2).

。 Subsequently, the researchers conducted multi-dimensional analysis of these five immune microenvironment subtypes, and verified the existence of the five subtypes through large-scale transcriptome data.
The spatial distribution of cells of the five subtypes was revealed by spatial transcriptome data and CODEX multicolor immunoassay; Through receptor ligand analysis, it was found that each isotype had different chemokine receptor-ligand networks, suggesting the formation mechanism of different isoforms.
Combined exon data analysis found that different subtypes were enriched with different driver gene mutations, such as TP53, CTNNB1, KRAS and IDH1, And found that different subtypes enriched different tumor cell gene modules
.
The discovery of these five immune microenvironments provides important reference information
for tumor immunotherapy.

FIG.
2 SCHEMATIC DIAGRAM OF 5 SUBTYPES (TIMELASER) OF TUMOR IMMUNE MICROENVIRONMENT

2) Systematically revealed 11 neutrophil subsets, identified 6 groups of tumor-associated neutrophils, and experimentally demonstrated the tumor-promoting mechanism of CCL4⁺TAN and PD-L1⁺TAN

Neutrophils are a very fragile class of cells that are generally considered to survive no more than a week after entering the body into the peripheral bloodstream and no more than 24 hours
in vitro.
Therefore, this population of cells
has not been captured in previous single-cell studies of liver cancer.
Thanks to the rapid experimental protocol and antibody-free enrichment strategy, more than 30,000 neutrophils
were successfully captured.
After cluster analysis, the researchers found a total of 11 subsets of neutrophils, which were enriched in peripheral blood, paracancer, and tumor tissues (Figure 3), and successfully identified 6 groups of tumor-associated neutrophils
.
Subsequently, the researchers analyzed the developmental trajectories and key transcription factors of these neutrophil subsets and found that two neutrophil subsets, CCL4⁺TAN and PD-L1⁺TAN, may promote tumor growth
through different mechanisms.
In order to further verify these findings, the researchers constructed the in vitro co-culture system of hepatoma cell line-neutrophils (In vitro) and the neutrophil ex vivo analysis system (Ex vivo) for liver cancer patients, and verified CCL4+ by transcriptome sequencing, ATAC-seq and multicolor immunofluorescence TAN promotes tumor growth by recruiting tumor-associated macrophages, while PD-L1+TAN promotes tumor growth
by inhibiting the killing function of CD8+ T cells.

Fig.
3 Tissue distribution, developmental trajectory, cancer enrichment and key transcription factors of neutrophil subsets

3) Construct a mouse liver cancer model, reveal that the subsets of neutrophils are highly conserved with humans, and confirm that removal of neutrophils can delay tumor growth

To further explore the tumor-promoting mechanism of neutrophils in vivo, the researchers based on Alb-cre/Trp53 fl^/fl Liver-specific knockout mice and liver cancer driver mutations were constructed as two mouse models of spontaneous tumors of liver cancer (Myc-90Ctnnb1, pTMC; Myc-Kras ^G12D, pTMK), and analyzed for single-cell sequencing
.
The researchers found that the neutrophil subsets of mice were highly conserved compared with humans, which indicates that experiments using mouse liver cancer models can provide important reference information
for the treatment of human liver cancer.
Subsequently, the researchers used Anti-Ly6G antibodies to perform neutrophil removal experiments in mouse models, and the results showed that liver cancer growth in mice was effectively inhibited after treatment (Figure 4).

Further, the researchers also revealed dynamic changes
in neutrophil subsets in mouse bone marrow, peripheral blood, paracancer, and tumor tissue during treatment.
These results suggest that the development of neutrophil-based immunotherapy targets is promising to lead to new therapeutic strategies
for liver cancer.

Fig.
4 Anti-Ly6G antibody can effectively alleviate the growth of liver cancer in mice by removing neutrophils

In summary, this study systematically revealed the immune microenvironment subtypes of liver cancer, and deeply analyzed the functional heterogeneity of tumor-associated neutrophils, and finally demonstrated that targeting tumor-associated neutrophils is expected to form a new immunotherapy strategy
for liver cancer through mouse liver cancer models.
The findings suggest that interventions targeting tumor-associated neutrophils are expected to significantly increase the number
of patients effectively treated by immune checkpoints.
These results provide key information
for basic research and clinical diagnosis and treatment of liver cancer and even solid tumors.

XUE RUIDONG, ASSOCIATE RESEARCHER OF PEKING UNIVERSITY FIRST HOSPITAL, DR.
ZHANG QIMING OF PEKING UNIVERSITY BIOPIC, CAO QI, DOCTORAL CANDIDATE OF PEKING UNIVERSITY FIRST HOSPITAL, ASSOCIATE RESEARCHER KONG RUIRUI OF PEKING UNIVERSITY FIRST HOSPITAL AND DR.
XIANG XIAO OF PEKING UNIVERSITY PEOPLE'S HOSPITAL ARE THE JOINT FIRST AUTHORS
OF THE PAPER.
Professor Zhang Ning, Cancer Transformation Center, Peking University First Hospital, Professor Zhang Zemin of Peking University BIOPIC and Professor Zhu Jiye of Peking University People's Hospital are co-corresponding authors
.
The research was supported and funded
by many national science foundations, such as the National Natural Science Foundation of China, the National Science and Technology Major Project of the 13th Five-Year Plan, the National Key Research and Development Program, and the Basic Science Center.

(Peking University First Hospital)