"Nature": Peking University scientists reveal the secret of liver cancer! Million-cell sequencing to identify five tumor microenvironments and clarify multiple mechanisms of cancer-promoting neutrophils

*For medical professionals only

Primary liver cancer (PLC) is the third leading cause of cancer-related death worldwide, with common types including hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and HCC-ICC mixed (CHC) [1].

However, at present, the treatment of liver cancer is limited, and a major stumbling block on the road of its mechanism research and precision treatment is the heterogeneity of the tumor immune microenvironment (TIME) [1,2].

This time, the team of Zhang Ning, Zhang Zemin and Zhu Jiye of Peking University cooperated to perform single-cell RNA sequencing analysis based on patient samples and mouse samples, define five immune microenvironment subtypes of liver cancer, and also reveal the significance of tumor-associated neutrophils in the progression of liver cancer, and found that neutrophil depletion can reduce tumor burden and inhibit the progression of liver cancer in mice [2].

The article was recently published in
the top issue of Nature.

Screenshot of the first page of the paper

Heterogeneity in TIME is determined by immune cells and stromal cells, and is one of the main factors of
tumor metastasis, recurrence and drug resistance.
If the TIME heterogeneity of primary liver cancer can be fully understood, patients can be better stratified by identifying biomarkers to achieve precision treatment [1,2].

In this study, Ning Zhang, Zemin Zhang, Jiye Zhu and their team first collected 160 samples from 124 patients with primary liver cancer who had been treated for single-cell RNA sequencing (scRNA-seq) to map a large-scale single-cell map of human liver cancer
.

Based on the results of the analysis, they identified a total of 89 liver cancer TIME cell subsets
from the 1092172 cells obtained.
It was also found that these cell subsets were specific between different patients and exhibited significant tissue and tumor type preferences
.

By performing hierarchical clustering analysis of these cell subpopulations, and considering their functional marker gene expression, prognostic correlation and other characteristics, the researchers defined five liver cancer TIME subtypes, namely:

(1) Immune-activated: TIME-IA (Immune Activation);

(2) Myeloid enrichment immunosuppressive type: TIME-ISM (Immune Suppressive Myeloid);

(3) Matrix-enriched immunosuppressive type: TIME-ISS (Immune Suppressive Stromal);

(4) Immune rejection: TIME-IE (Immune Exclusion);

(5) Immune residency: TIME-IR (Immune Residence).

THEY NAMED THIS SUBTYPE CLASSIFICATION SYSTEM TIMELASER
.

FIGURE 1: TIMELASER

Based on TIMELASER, the researchers stratified patients to observe the relationship
between different TIME subtypes and progression-free survival (PFS).

Figure 2: Association between five TIME subtypes and PFS in PLC patients

Not only that, the researchers also explored the expression patterns
of chemokines, cytokines and their receptors in TIME.
The results showed that these 5 different TIME subtypes exhibited different chemokine networks and correlated
with the transcriptome characteristics of tumor cells.

Since multiple neutrophil subsets have been enriched in myeloid enrichment immunosuppressive TIME, and myeloid enrichment immunosuppressive TIME is associated with poor prognosis in patients (see Figure 2), the association between neutrophils and liver cancer progression has attracted the attention of researchers
.

According to the analysis results, the researchers identified a total of 11 neutrophil subsets, of which 3 subsets were enriched in peripheral blood, 2 subsets were enriched in liver cancer paracancerous tissues, and 6 subsets were enriched in liver cancer tumor tissues, that is, tumor-associated neutrophils (TANs).

Specifically, these tumor-associated neutrophils promote liver cancer progression
by different mechanisms.
For example, CCL4⁺TANs (Neu_11_CCL4) can highly express the chemokines CCL3 and CCL4, and recruit tumor-infiltrating macrophages by secreting CCL3 and CCL4; On the other hand, PD-L1⁺TANs (Neu_09_IFIT1) highly express the immune checkpoint PD-L1 and inhibit the anti-tumor toxicity
function of peripheral blood CD8⁺ T cells through PD-L1.

Figure 3: PD-L1⁺TANs inhibit the proliferative capacity and toxic function of CD8⁺ T cells

So, can these tumor-associated neutrophils be used as a breakthrough to prevent the progression of liver cancer?

Therefore, the researchers further constructed two mouse models of spontaneous liver cancer (hepatocellular carcinoma and intrahepatic cholangiocarcinoma, respectively), and used antibody therapy targeting the neutrophil-specific marker Ly6G (anti-Ly6G antibody) to deplete neutrophils
of liver cancer mice.

The results showed that after receiving anti-Ly6G antibody treatment, 70% of tumor-associated neutrophils in mice were depleted
.
Compared with the control group, the tumor weight of mice treated with anti-Ly6G antibody was significantly reduced, and the inhibitory effect of neutrophils on T cytotoxic work and the recruitment of macrophages were weakened, the number of tumor-infiltrating macrophages was reduced by 46.
6%, and the expression of PD-L1 was also down-regulated
.

In addition, the researchers also found that the composition of tumor-associated neutrophils in liver cancer mice was also changing
during anti-Ly6G antibody treatment.
Although the number of most tumor-associated neutrophil subsets is decreasing, the number of Neu_09_Apoa2 (corresponding to human Neu_07_APOA2) in mouse tumors is relatively high
.

Figure 4: Neutrophil reduction using anti-Ly6G antibodies can inhibit liver cancer progression in mice

Overall, Zhang Ning, Zhang Zemin, and Zhu Jiye led their team to reveal five immune microenvironment subtypes of liver cancer, and found the relationship between neutrophils and liver cancer progression in the
process.
More importantly, the experimental results of liver cancer mice showed that the consumption of neutrophils could effectively inhibit tumor progression
.

In response to this research result, the researchers said that the discovery that tumor-associated neutrophils promote tumor progression by expressing CCL4 and PD-L1, which means that tumor-associated neutrophils are promising immunotherapy targets whether used alone or in combination with immune checkpoint inhibitors
.
In the future, we will further find new solutions for the treatment of liver cancer from neutrophils
.

References:

[1] Tang M, Zhao Y, et al.
Liver cancer heterogeneity modeled by in situ genome editing of hepatocytes.
Sci Adv.
2022 Jun 24; 8(25):eabn5683.
doi: 10.
1126/sciadv.
abn5683.
Epub 2022 Jun 22.

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The author of this article Eddy Zhang