Deciphering the tumour immune microenvironment of hepatocellular carcinoma

Current treatments for hepatocellular carcinoma (HCC) are less effective and prone to recurrence after surgery, so it's needed to seek new ideas for its therapy. Tumour immune microenvironment (TME) is crucial for the pathogenesis, development and metastasis of HCC. Interactions between immune cells and tumour cells significantly impact responses to immunotherapies and patient prognosis. In recent years, immunotherapies for HCC have shown promising potential, but the response rate is still unsatisfactory. Understanding their cross‐talks is helpful for selecting potential therapeutic targets, predicting immunotherapy responses, determining immunotherapy efficacy, identifying prognostic markers and selecting individualized treatment options. In this paper, we reviewed the research advances on the roles of immune cells and multi‐omic research associated with HCC pathogenesis and therapy, and future perspectives on TME.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is a common primary liver cancer, accounting for nearly 85% of global cases, and is a major global health concern.Surgical resection and liver transplantation are still the most effective treatments for HCC.Because of the limited availability of donor organs and the high recurrence rates following resection, these treatments are not suitable for all patients.In contrast, non-surgical therapies such as radiofrequency ablation and transarterial chemoembolization are less invasive and can be administered on an outpatient basis. 1 However, the 5year survival rate of HCC patients remains less than 20%. 2 Therefore, new and effective therapies are urgently needed to improve the prognosis of HCC patients.One promising approach to treating HCC is immune checkpoint blockade therapies, including the usage of PD-(L)1 antibody or CTLA-4 antibody.However, more HCC patients easily acquire resistance to immunotherapies.To this end, targeting the tumour microenvironment (TME), a complex network of stromal cells, communication molecules and structural components (ECM), will offer a novel avenue for HCC therapy. 3Immune evasion is impacted by interactions between stromal cells and tumour cells, which promote HCC cell proliferation and metastasis.We will address the advances in studies on the TME of HCC in this review (Figure 1).

| REPRESENTATIVE CELL TYPES IN THE HCC TUMOUR MICRO ENV IRO NMENT
The tumour microenvironment of HCC plays an important role in tumorigenesis, progression and the development of drug resistance.Except for tumour cells, the immune microenvironment of HCC is mainly composed of stromal cells such as myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs), tumour-associated neutrophils (TANs), tumour infiltrating lymphocytes (TILs), hepatic stellate cells (HSCs), endothelial cells (ECs) and dendritic cells (DCs).

| Myeloid-derived suppressor cells
Myeloid-derived suppressor cells are a group of heterogeneous cells that can expand during cancer, inflammation and infection, and can suppress the functions of T-cells. 4 Based on morphological features, MDSCs are mainly divided into polymorphonuclear cells (PMN-MDSC) and monocytes (M-MDSC).Under pathological conditions, activated MDSCs are involved in regulating immune responses and are strongly associated with poor prognosis of HCC.MDSCs mediate immunosuppression through regulating arginase (ARG1), iNOS, TGFβ, COX2, IL10 and L-selectin. 5 MDSCs in the TME can boost tumorigenesis and progression through several mechanisms, such as induction of the generation immunosuppressive cells, blocking lymphocyte homing, production of ROS, exhausting metabolites essential for T cells, releasing hydrolases and negative immune checkpoint molecules. 4Its promoting effect on HCC can be reversed by depleting MDSC, blocking the transport and migration to the TME, and inhibiting the immunosuppressive function of MDSC. 6In a murine model, tadalafil, a PDE5 inhibitor, not only inhibited the accumulation of MDSCs, but also enhanced the efficacy of CIK cell-based therapy. 7nrichment and activation of MDSCs is a common characteristic of aggressive tumours and therefore targeting these cells allows for tumour therapy.Thus, it is crucial to incorporate the functional neutralization of different MDSCs with current therapeutic strategies to improve therapeutic outcomes.Although the findings of preclinical studies are promising, further clinical trials are needed to prove the synergistic effect of MDSC inhibition with existing immunotherapies.

| Tumour-associated macrophages
Tumour-associated macrophages (TAMs) are a cohort of macrophages in TME that promote tumour growth, angiogenesis, inhibit cancer cell apoptosis and induce tumour metastasis through releasing cytokines, chemokines, growth factors and matrix metalloproteinases. 8Based on surface receptors and functional characteristics, TAMs were divided into classically activated M1 macrophages (M1-type) and alternatively activated M2 macrophages (M2-type).M1-type cells have an antigen-presenting function and play an anti-tumour role.M1-type cells highly express HLA-DR and release inflammatory factors, such as TNF-α, IL1/6/12 and IFNγ-I.0][11]   can suppress anti-tumour immune responses involving T-cells and facilitate angiogenesis and cancer cell migration. 12M1-and M2-type cells are very plastic and able to interconvert depending on the TME or therapeutic interventions. 8eleased IL-6, IL-1 and TNF-α can activate signal transduction molecules (Smad, Wnt, PI3K/PKB, NF-κB, ERK and STAT3) and promote epithelial-mesenchymal transition (EMT) in tumours, resulting in a decrease of intercellular adhesion and easier metastasis. 13A recent study showed that CSF1 significantly induced AIF1 expression in M2-type macrophages and promoted the progression and microvascular invasion of HCC. 14 Apart from directly inducing TAMs to migrate to hypoxic regions, hypoxia also can enhance tumour metastasis by inducing tumour cells to secrete high mobility group protein B1 and exacerbate macrophage polarization to M2 type. 15VEGF, type IV collagenase, MMP2 and MMP9 secreted by M2-Ttype cells facilitate tumour growth, angiogenesis and cancer cell metastasis. 16M2-type cells recruited to the tumour site can secrete EGF and activate the corresponding signalling pathway, which consequently promotes tumour progression. 17As one of the most enriched immune cells in TME, TAMs are found in all phases of HCC and complex crosstalk between tumour cells and TAMs can occur.In addition, macrophages in the TME of HCC interact with other cell types by directing intercellular contacts and secreting effector molecules.Similarly, the interactions between tumour cells with immune cells can facilitate the recruitment and polarization of macrophages.Therefore, targeting TAMs has become one of the most popular immunotherapeutic strategies due to their critical role in the TME of HCC.

| Tumour-associated neutrophils
Tumour-associated neutrophils (TANs) are mainly originated from bone marrow haematopoietic stem cells and play important roles in tumour progression, tumour cell migration and immunosuppression.TANs are highly plastic and can be polarized to N1 or N2 phenotypes by IFN-I or TGF-β, respectively.In N1-TAN cells.high activity of NADPH oxidase promotes the generation of ROS with toxicity to tumour cells.In contrast, N2-TANs stimulate immunosuppression by causing DNA instability, and release cytokines and chemokines. 18The N1 and N2 phenotypes of TANs are interconvertible in different TMEs.
Neutrophils have a dual role in the progression of HCC.A previous study showed that the expression tumour-derived factors, such as GM-CSF and TNF, were significantly increased in the tumour areas of HCC.
Modulation of neutrophils to an immunosuppressed state can increase the expression of PDL-1, suppressing T cell functions. 19High-density neutrophils exert anti-tumour effects through either direct cytotoxicity or activating immune cells. 20argeted therapies against TANs are becoming an exciting field today.Considering their roles in the tumour progression, two treatment recommendations were proposed: (1) blocking TANs by targeting the CXCL-8/CXCR-1/2 axis; (2) targeting tumour-promoting molecules derived from polymorphonuclear cells.Since neutrophils are twosided in cancer immunotherapy, the more anti-tumour neutrophils accumulated in the tumour tissue, the better the therapeutic effect.

| Tumour infiltrating lymphocytes
TILs mainly include cytotoxic T lymphocytes (CTL), regulatory T cells (Tregs), mucosal-associated invariant T cells (MAIT), regulatory B cells (Bregs) and natural killer cells (NK).In the TME of HCC, CTLs are mainly composed of CD8 + T cells, which produce cytotoxic effects by recognizing specific antigens.It was found that IL-21 secreted by CD8 + CXCR5 + T could induce B cells to differentiate into IgG-producing cells. 21In addition, the infiltration of CD8 + T cells is not only associated with the prognosis of HCC patients but also impacts the immunotherapy effect.With the promising clinical results of PD-1/PD-L1 in various tumours, blockade therapy has been explored for HCC therapy. 22,23regs are a subset of T cells expressing CD4 and exhibit high immunosuppressive activity.CD4 + T cell has a distinct targeting and specificity to tumour cells and can generate long-term immune responses by recognizing and aggregating to a tumour antigen expression site to directly suppress or kill tumour cells.In a murine model, T cells promote the clearance of senescent hepatocytes to prevent hepatocarcinogenesis.However, its absence facilitates the development of HCC. 24Although CD4 + T cells have an essential role in immune responses, they secrete less granzyme A and granzyme B compared with CD8 + T cells, leading to a poorer clearance of cancer cells. 25In addition, Tregs play negative roles in HCC progression by immunosuppression by impairing CD8 + T cell functions.Tregs can decrease the expression of CD80/86 in DCs with a CTLA-4-dependent manner, suggesting that they act negatively on immune cells. 26Analysis of clinical specimens showed that CD4 + CD25 + T cells contribute to the migration of HCC cells by TGF-β1induced EMT. 27Migration of FOXP3 + Tregs to tumours may cause the recruitment to CCL20, indicating that inhibition of Tregs function by blocking the CCL20-CCR6 axis is a promising treatment for HCC. 28Activated TILs affect hepatocarcinogenesis by affecting the interactions between immune system and cytokines, and promoting neoangiogenesis.Therefore, quantification of TILs in solid tumours may help to understand their roles in hepatocarcinogenesis and can be used to monitor the therapeutic outcomes of immune checkpoint inhibitors.
Infiltrating B cells exert antitumor effects in a variety of tumours.Infiltration of high-density B cells is closely related to the overall survival of HCC patients. 29B cells can act as APCs and present tumour antigens directly to CD4 + T or CD8 + T cells.Using activated CD40 ligand B cells (CD40 B) can activate the function of CD8 + T cells and thus exert anti-HCC effects. 30B cells can facilitate the uptake of tumour antigens of TMAs and DCs by promoting antibodies and secreting related cytokines, thereby activating anti-tumour immunity or direct killing of tumour cells.In HCC, IgG + memory B cells accumulated at the margins of tumour infiltration can produce granzyme B, TRAIL, and IFN-γ, leading to a good prognosis. 31egulatory B cells (Bregs) are a subgroup of B cells with immunosuppressive properties.In a murine HCC model, TGF-β induces the transition of IgM-expressing B cells to IgA-expressing regulatory cells.IL-10-secreting IgA + PD-L1 + Breg cells directly inhibit CTL activity in tumours. 32his cell type is also correlated with a poor prognosis in HCC patients. 33In TME, various types of immune cells often require cooperation with each other to exert antitumour effects.In an HCC murine model, the decrease of B cells inhibited the secretion of CXCL10 of macrophages, increased anti-tumour responses of T cells, and suppressed HCC cell growth. 34Tight contacts of T cells and B cells in the infiltrated tumour suggest that they are functionally interacting, which contributes to enhancing the local immune effect and improving the prognosis of HCC patients.In addition, not all infiltrating B cells have positive roles in anti-HCC.The infiltration of Bregs (TIM-1 + type) was higher in the tumour tissue than that of paraneoplastic tissues.IL-10 released from TIM-1 + Bregs had a strong inhibitory effect on CD8 + T cells.The abundance of TIM-1 + Bregs was strongly associated with the late stage of HCC, and therefore be used to predict the survival rate of patients. 35he lack of NK cells was thought to be vital for the immune evasion of tumour cells.There was a remarkable positive correlation between the abundance and activity of NK cells and the prognosis of HCC patients.Compared with normal liver tissue, the infiltration of NK cells was significantly reduced in advanced HCC.NK cell dysfunction or depletion is associated with the development of HCC. 36NK cells co-cultured with sorafenib-treated macrophages could cause the death of cancer cells. 37Studies have shown that NK cells also have a negative regulatory role in HCC.When NK cells declined in HCC patients, the amount of antigen-specific T cells increased.These NK cells may cause a decrease in T cells, thus impairing the tumour's immune responses. 38ucosa-associated constant T cells (MAITs) are a subgroup of evolutionarily conserved, non-traditional T cells that are an important component of the immune system.In a murine model of HCC, co-administration of 5-OP-RU and CpG could activate MAIT cells, subsequently producing strong and extensive anti-tumour immune responses. 39uan et al 40 found a remarkable decrease in the number of MAITs in HCC patients, while the expression of PD-1, CTLA-4 and TIM-3 was significantly increased, suggesting that the decrease in MAITs was caused by immune depletion rather than apoptosis.In addition, MAIT cells in HCC patients were significantly infiltrated by tumour cells and closely associated with a poor prognosis.MAIT cells can secret more IL-8, but less IFN-γ, IL-17, granzyme and perforin, suggesting the function of MAITs was impaired and re-edited from anti-tumour to pro-tumour.Yan et al 41 found that MR1-expressing tumour cells could activate MAIT cells.Using IL-17A to inhibit the functions of T cells and NK cells may facilitate the growth and metastasis of tumour cells.
During the progression of HCC, TILs are connected by various signalling pathways and form a complex immune network.TILs not only act as prognostic cues but may also have a role in immunotherapy.Since mechanisms of each TILs subset in immunotherapeutic processes of HCC are unclear, future in-depth studies of TILs are necessary to reveal the composition of undiscovered TILs.In conclusion, studying the role and mechanism of TILs may help to better understand the occurrence and progression of HCC and provide a basis for the immunotherapy of HCC.
aHSCs are responsible for the synthesis and deposition of collagen, fibronectin and laminin, and involved in liver fibrosis and cirrhosis.ECM remodelling in HCC is manifested by increased the expression of ECM degrading enzymes.A previous study showed that MMP-2, MMP-9 and TIMP-1 were over-expressed in HCC tissues and were involved in tumour invasion. 42Interactions between HCC cells and aHSCs enhanced expression of COL1A1 and MMP9, promoting the migration of HCC cells. 43aHSCs also can induce a mesenchymal phenotype in HCC cells, which was characterized by up-regulated expression of Vimentin and decreased expression of Ecadherin, facilitating cancer cell migration. 44aHSCs can promote fibrosis and collagen deposition in HCC, leading to reduced drug penetration and increased therapeutic resistance. 45ngiogenesis is very critical during HCC tumour growth and metastasis.aHSCs promote the expression of VEGF and ANG-2 by activating the PI3K/Akt signalling. 46nteractions between aHSCs and HCC cells formed a proangiogenic environment by over-expression of VEGFα and MMP9. 43In addition, up-regulation expression of VEGF was tightly correlated with tumour grade and vascular invasion. 47FGF9 secreted by aHSC significantly enhances the proliferation and clonality of HCC cells via ERK-JNK signalling. 48Ang-1 derived from aHSC promotes vascular remodelling and maturation by activating tyrosine kinases in hepatic vascular endothelial cells, thereby maintaining vascular stability. 49aHSCs secrete hepatocyte growth factor to promot the expression of STMN1, which in turn promotes the activation of HSCs with a CAF phenotype.Therefore, blocking STMN1-mediated signalling between HCC cells and HSCs may be another target for future HCC therapy. 50ancer-associated fibroblasts (CAFs) are mainly derived from HSCs, bone marrow-derived mesenchymal stem cells and resting fibroblasts.CAFs can be activated by sonic hedgehog, TGF-beta, TNF-alpha and IL-1/-6/-10.Activated CAFs promote tumorigenesis, progression, invasion and metastasis by altering ECM, 51 reducing immune monitoring, 52 promoting EMT, 53 and affecting the microenvironment by releasing cytokines and angiogenic factors. 54VEGF released from CAFs plays a vital role in promoting angiogenesis.CAFs also promote the deposition of ECM by secreting various cytokines, recruiting inflammatory cells, and promoting the synthesis of collagen, laminin and fibronectin. 51GF from CAFs contributes to the proliferation of malignant cells in HCC. 55IL-6 secreted by CAFs could facilitate the proliferation of MDSCs through the activation of STAT3/Notch signalling. 56Up-regulated expression of lysine demethylase 1 (LDS-1) in CAFs drives Notch3-mediated self-renewal of HCC stem cells. 57In addition, CAFs could also promote to form of a suppressive immune microenvironment by up-regulating PD-L1 expression in DCs, macrophages, neutrophils through IL-6/STAT3 signalling, which further induces the differentiation of PDL1 + neutrophils and MDSCs, impairing the immune capacity of T cells. 58,59CAFs contribute to the progression of HCC by regulating the expression of FOXQ1 in HCC cells, which subsequently activates Nmyc downstream-regulated gene 1 (NDRG1).Enhanced STAT6/CCL26 signalling recruits more aHSCs, and thus eventually a positive feedback regulatory loop was established between HCC cells and CAFs. 60CCL2, CCL5/6/7 released by CAFs facilitate the metastasis of HCC cells by coordinating the activation of TGF-β and Hh signalling. 61CAFs also can alter the immune responses of HCC cells by modulating the activity of cytokines and chemokines.In addition, HSCs promote the recruitment of Tregs and MDSCs into the TME, causing immune tolerance and tumour progression. 62

| Endothelial cells
Liver sinusoidal endothelial cells (LSECs) are critical for the pathogenesis of liver fibrosis and the subsequent development of HCC.LSECs are highly efficient APCs with the capability to cross-activate T cells.Interactions of LSECs and Kupffer cells, lymphoid cells or DCs in the TME regulate the expressions of CD54, CD80, CD86, MHC I/II and CD40, thus modulating the response of T cells in the liver.LSECs can effectively present exogenous antigens to CD8 + T cells, resulting in the tolerance of antigen-specific T cells. 63LSECs constitutively express integrin, ICAM-1 and VCAM-1 that impair the function of activated CD8 + T cells. 64In pathological states, LSECs undergo morphological changes that stimulate the release of IL-6 and TNF-α, and simultaneously reduce the production of immunosuppressive cytokines IL-10 and TGF-β, thus interfering with the immune tolerance of the liver. 65,66In addition, αmelittin-NPs trigger the activation of LSECs and cause significant changes in the cytokine/chemokine environment, which opens a window for controlling liver metastasis through immune regulation of LSECs. 67n human and mouse HCC, LSECs sequentially lose marker stabilin-1/-2, LYVE-1 and CD32b during the development of HCC. 68In addition, LSECs from HCC patients showed a high level of integrins and a lower level of ICAM-1 compared with healthy individuals.CAFs in the TME can secrete MMP9, uPA, and tPA, degrading collagen, laminin, and fibronectin and destroying the basement membrane, thus promoting local infiltration of HCC cells.Possible mechanisms by which DCs promote the growth of HCC were summarized as follows: (1) reduced antigen presentation capacity caused by hypoxia and abnormal lipid accumulation.HIF-1α induces the expression of genes CD39 and CD73 and the generation of eADO.eADO-activated pDCs further induce Tregs and inhibit the proliferation and cytotoxicity of CD8 + T cells. 70Studies have shown that tolerant DCs are more likely to adapt to the environment of OXPHOS and FAO rather than glycolysis.This disorder of fatty acid metabolism can impair DC-mediated immune resistance 71,72 ; (2) Inducing tolerance to tumour antigens.It has been shown that a sharp decrease in the amount of regulatory DCs in HCC inhibits T cell proliferation and therefore increases immune tolerance. 73,746][77] However, it is not clear whether non-tumour cells in the TME are also able to affect the differentiation of regulatory DCs;

| OTHER ADVANCES ASSOCIATED WITH THE TME OF HCC
HCC development is a combined and cumulative dysfunction of a range of genetic and non-genetic factors.Due to the genetic instability of tumour genomes, most tumorigenic events occur randomly.These random events can establish conditions for the development of a heterogeneous TME.Additionally, competition for nutrients, treatment stress, or the evolution of oncogenes continually remodel the TME.The above factors create opportunities for tumour cells to escape immune monitoring, eventually resulting in cancer progression and cell metastasis.In this paper, we reviewed important studies and recent advances in transcriptome sequencing, epigenetics, non-coding RNA, and gut microbe in the immune microenvironment of live cancers (Figure 2).

| Bulk RNA sequencing
Bulk RNA transcriptome analysis provides an understanding of the overall gene expression of HCC cells.Integrated analysis of RNA sequencing data showed that the enrichment of TREM2 + LAM-like cells was not associated with poor clinical outcomes of HCC patients. 79Unsupervised clustering of gene expression data suggested a possible link between the increased fibrosis regulated through TGF-β, PDGFR, SHH signalling, and the immune phenotype of T cell rejection. 80ranscriptome analysis revealed that the activation of T cells was suppressed in the TME of HCC, while MDSCs were increased, with a significant expression alteration of immune-related genes.A three-gene panel (UPB1-SOCS2-RTN3) was identified as a candidate biomarker for HCC prognosis by the integration analysis of bulk transcriptome data derived from the TCGA database. 81However, the molecular mechanism of how they affect patient survival is not clear.Transcriptome analysis of pathological tissues revealed an overall down-regulation of immune-related gene expression in HCC cells when compared with the adjacent liver tissue.Further analysis of gene function and signalling pathways revealed impaired functions of T-and B-cells.This revealed that immune responses were suppressed in tumours.The TME with enriched T cells and CTL, lacking macrophages and Th2 cells was closely related to a good prognosis of HCC patients.Transcriptome analysis of the TME in HCC provided valuable insights into the molecular mechanisms of tumour progression and the development of new therapeutic strategies.However, two challenges must be addressed: (1) the inability to distinguish the heterogeneity of the TME in tumour tissues; (2) the need for clinical validation of new immunerelated signatures and therapeutic targets.

| Single-cell RNA sequencing
Some pioneering single-cell RNA sequencing (scRNA-seq) studies have investigated the immune cells or malignant cells of primary HCCs and early-relapse HCCs (Table 1).Compared with traditional bulk RNA-seq, the application of scRNA-seq has provided new insights into the composition of immune cells in the TME and has helped to reveal the TME heterogeneity of HCC.scRNA-seq was used to cluster T cell subsets in the TME including exhausted CD8 + T cells and Tregs.These subsets have been shown to play different roles in immune responses.Exhausted CD8 + T cells and Tregs could promote tumour growth, while CD4 + T cells boosted the anti-tumour immune responses. 82Zheng et al 83 identified 11 T cell clusters and found that exhausted CD8 + T cells and Tregs were enriched.By tracing the developmental trajectory of T cells as well as their activation states, it was found that most Tregs were possibly recruited from the peripheral blood, but exhausted CD8 + T cells seemed to be of hepatic origin.ScRNA-seq showed that macrophages in HCC differed in polarization state and gene transcription profiles.M1-type macrophages expressed inflammatory genes, such as TNF and IL-1β, whereas M2-type macrophages expressed antiinflammatory genes, such as IL-10 and TGFβ. 84In addition, an unbiased scRNA-seq analysis found a subset of circulating TREM2 + /CD9 + macrophages were clustered in the fibrotic niche of cirrhotic livers.MDSCs in HCC have distinct transcriptional profiles and functional properties.G-MDSCs expressed high levels of genes involved in oxygen consumption and glycolysis and promoted tumour angiogenesis and metastasis.While, M-MDSCs expressed high levels of genes involved in immunosuppression and tolerance, and inhibited T cell proliferation and activation. 85In addition, ScRNA-seq revealed that immune checkpoint inhibitors could increase the amount of activated T cells and decrease the number of depleted T cells and Tregs, leading to an improved anti-tumour immune response.

| Intestinal microbes
Perturbation of gut microbes has been shown to increase levels of pathogen-associated molecular patterns and results in uncontrolled inflammation. 86In a live cancer animal model, the activated LPS-TLR4 pathway promoted a massive release of inflammatory factors and caused stress and inflammatory responses, eventually leading to DNA damage and gene mutations. 87Stellate cells activated by this pathway secreted angiogenic factors.Under the joint influence of LPS, the migration of mesenchymal endothelial cells and the gradual generation of new blood vessels are induced. 88Currently, there are few studies on the pathogenesis of intestinal microecology and HCC.Therefore, there is a need to further elucidate the relationship between them to provide new ideas for HCC therapy.

| Epigenetics
DNA methylation analyses revealed the clinical significance of epigenetic regulation in HCC progression.In the early phase of HCC, the DNA methylation profile was significantly altered. 89Studies have shown that the key tumour-related gene UHRF1 can regulate DNA methylation. 90In addition, DNA demethylation of LINE-1, Filamin C, Hexokinase 2, hepatitis B virus X and CD147 has been identified in primary HCC. 91DNA methylation analysis of peripheral blood mononuclear cells and T cells showed that DNA methylation was enhanced with HCC development. 92Integrated epigenomic profiling showed that the recurrence of hypermethylated enhancers of CCAAT/ enhancer binding protein-β (C/EBPβ) as well as the overexpression of C/EBPβ was closely related to the poor prognosis of HCC patients. 86Highly elevated DNA methyltransferase 1 (DNMT1) was positively correlated with the over-expression of PD-L1 in sorafenib-resistant HCC cells.PD-L1 could subsequently induce DNMT1-dependent DNA hypomethylation and restore the expression of the suppressor gene Cadherin 1 in HCC. 93In addition, growing evidence showed that histone deacetylation could regulate the expression of immune checkpoint genes and play important roles in cancer progression.Inhibitors of histone deacetylases were shown to enhance NK cell functions. 94

| Non-coding RNA
Previous studies revealed that miRNAs play key roles in regulating gene expression and immune responses in HCC.Understanding the functions of miRNAs in the TME of HCC may help to develop new diagnostic and therapeutic strategies for HCC.MicroRNA-155-5p could promote HCC progression by inhibiting PTEN via the PI3K/Akt pathway. 95ver-expression of miR-182 may increase the cytotoxicity NK-cells by regulating the expression of gene NKG2D and NKG2A. 96miR-146a-5p suppressed the secretion of inflammatory cytokine and activation of HSC through inhibition of TLR4/NF-κB pathway. 97In addition, compared with the adjacent non-cancerous hepatic the expression of MicroRNA-146a was suppressed in HCC tissues, which was associated with TNM stage and metastasis. 98cir-cRNA_0005075 was shown to promote HCC development by modulating gene expressions associated with cell proliferation and apoptosis. 99Compared with paracancerous tissues, circRNA_100 338 was significantly up-regulated and closely correlated with the cumulative survival of HCC patients. 100lnc-EGFR promoted immune escape of HCC cells by stimulating Tregs differentiation. 101The up-regulated expression of lnc-Tim3 was negatively correlated with IFN-γ level in CD8 + T cells.The binding of Lnc-Tim3 and Tim-3 facilitated the release of Bat3 and inhibited NFAT1 and AP-1 in the downstream signalling pathway.In addition, the released Bat3 promotes the recruitment of p53 and RelA to p300, inhibiting CD8 + T cell death. 102lncRNA HOTAIR triggered epithelial-mesenchymal transition by inhibiting RNA binding motif protein 38.In addition, its interaction with microRNAs that serve as tumour suppressors could promote the oncogenic activity of HCC cells. 103LncRNA MALAT1 can affect the angiogenesis and immunosuppressive properties of HCC cells. 104

LYMPHOID STRUCTURES WITH HCC
In-depth studies of TME revealed that the regulations of antitumor defences occur not only in secondary lymphoid organs (SLOs) but also in tumour sites with aggregations of organized cells similar to SLOs -tertiary lymphoid structures (TLS).

| Composition and heterogeneity of TLS
TLSs are histologically identified structures mainly located in the peritumoral mesenchyme, invasive margins and/or core, where the maturation of TLS ranges from dense lymphocyte aggregates to primary and secondary follicularlike TLS. 105TLSs are composed of many immune cells, including B cells, follicular dendritic cells (FDCs), plasma cells, T cells, DCs, neutrophils, macrophages and fibroblast reticular cells (FRCs). 106

| Identification approaches of TLS
Different tumour types have varying TME due to multiple cell types and complex structures in the tumour microenvironment.Current approaches to identify TLS are HE staining, 107 immunohistochemistry, 108 quantitative pathology, 109 immunofluorescence analysis, laser capture fibre cutting TLS selection gene expression profiles. 110The above methods suffered from the following drawbacks: time-consuming, subjective, poor repetition and standardization.Therefore, they are difficult to be widely used in clinical practices.In the future, there is a need to identify specific markers from peripheral tissues to reduce damage and improve detection efficiency.

| Relationship between TLS and prognosis of HCC
TLS was found to be a prognostic factor independent of tumour TNM stage in almost all solid tumours and associated with recurrence-free survival (RFS) and overall survival (OS). 111However, the prognostic value of TLS in HCC remains a controversial issue.Finkin et al 112 found that higher TLS scores in HCC patients were associated with an increased risk of late recurrence and decreased postoperative overall survival.Pathological analysis of 273 HCC patients revealed that TLS in the non-tumour core was not associated with early and late recurrence of HCC, while maturation of TLS within the tumour was associated with a reduced risk of early recurrence. 113Studies have shown that TLS was present in ~48% of patients with HCC.The prognostic value of TLSs was limited to earlystage HCC with surgical resection (BCLC 0-A), but no correlation between intratumoral TLSs and OS or relapse-free survival (RFS) was observed in advanced stages (B-C). 114i Hui analyzed the TLS infiltration in 360 HCC patients and found a high density of TLS within the tumour.Joint analysis of intra-and extra-tumour TLS showed that a high density of TLS was associated with a good prognosis. 115Immunohistochemical analysis revealed that high CD20 + , CD208 + and CD8 + cell densities in HCC patients were favourable prognostic factors for overall survival.In contrast, high CD3 + , CD20 + , CD208 + and CD8 + cell densities were significantly associated with a reduction in early recurrence. 116The differences in these results may be related to the study population, sample size, statistical methods, criteria for TLS identification, TLS heterogeneity and location.

| Targeting chronic inflammation
As mentioned above, TAM usually presents an M2type phenotype.Therefore, targeting M2-type cells or reprogramming M2-type to type cells may inhibit liver cancer progression.Currently, apart from sorafenib, no other drugs related to the inhibition of M2-TAMs have been put into clinical trials.As a multikinase inhibitor, olafenib has been widely used to treat patients with advanced HCC, and their average survival is 3 months.Sorafenib is not as effective, which may be related to the infiltration of TAMs. 117Sorafenib-induced hypoxia may increase the expression of CSF1, HIF and CCR4, which further affect TAM invasion and result in tumour progression.In addition, macrophage infiltration promotes the overexpression of PD-L1 in HCC cells, which may also contribute to sorafenib resistance.In a mouse model of HCC, a natural product from Abies georgei, structurally similar to kaempferol, can inhibit the recruitment of TAMs by inhibiting CCR2 and enhances the therapeutic effect of sorafenib, indicating that the combination of an immunomodulator with a chemotherapeutic drug could be a new approach for treating HCC. 118

| Targeting cancer-related fibroblasts
CAFs secrete a variety of growth factors and cytokines, and degrade extracellular matrix, thereby affecting tumour cell proliferation, metastasis and chemotherapy resistance.Therefore, blocking the downstream effects of CAF or inhibiting CAF-secreted factors that promote tumorigenesis and progression has become a potential strategy for HCC intervention.Targeting CAF may be achieved by killing CAF, inhibiting CAF activation, or interfering with CAF function.Nanocarrier-targeted cytotoxic drugs (acridine yellow, paclitaxel) and tumour vaccines can selectively kill CAF. 119The activation of CAF can be inhibited by blocking TGFβ/Smad/ROS activation signalling and reversing the phenotype. 13Antifibrotic drugs and cytokine blockers can interfere with the secretion of CAF cytokines.A previous study showed that liposome-mediated DNA and siRNA can block the information exchange between CAF and Wnt signalling and CXCL1 of HCC cells and inhibit the production of IL-6 and granulocyte colony-stimulating factor, thus inhibiting HCC progression. 120

| Targeted tumour angiogenesis
Signalling imbalances caused by angiogenic and antiangiogenic molecules lead to vascular abnormalities that allow tumours to evade the immune system, in which VGEF and angiopoietin-2 are notably increased.Therefore, blocking VEGF/VEGFR and reducing angiogenesis in tissues are considered to be new ideas for targeted therapy in HCC.Currently, anti-VEGF-VEGFR drugs include: (1) antibodies directly target VEGF and VEGFR proteins, such as ramucirumab. 121(2) Inhibitors of signalling pathways mediated by the tyrosine kinase, such as sorafenib. 122However, the clinical benefits of angiogenesis inhibitors are not obvious and have some limitations.
The possible explanations are that the network regulating angiogenesis involves multiple proteins or signalling pathways.Compensatory angiogenesis occurs when a target is inhibited.Additionally, the newly generated blood vessels are structurally incomplete with poor permeability, which prevents some drugs from effectively reaching their targets.

| Targeting extracellular components
Acidosis is a fundamental feature of the tumour microenvironment, which directly regulates tumour cell invasion by affecting immune cell function and drug resistance.Therefore, reducing local acidity may interfere with tumour growth and metastasis.The application of selective antibodies and chimeric antigen receptor T (CAR-T) lymphocytes based on an acidic environment is a novel and promising therapeutic strategy. 123Targets for CAR-T therapy in HCC include fetoprotein, phosphatidylinositol proteoglycan-3, melanoma antigen gene-1/3, telomerase reverse transcriptase, VEGF, epithelial cell adhesion molecules, and D-ligand of NK-2.A recent study showed that HCC cell-derived exosomes can trigger the HGF/c-Met/ Akt signalling pathway, which plays a crucial role in inhibiting sorafenib-induced apoptosis and promoting drug resistance in HCC. 124The good biocompatibility, low immunogenicity and low toxicity of exosomes make them promising for the treatment of HCC.

| Tumour vaccine
Currently, tumour vaccines used for HCC treatment and research mainly include nucleic acid vaccines, peptide vaccines, dendritic cell (DC) vaccines and oncolytic virus (OV) vaccines.Nucleic acid vaccine refers to the recombination of a gene (DNA or RNA) encoding a certain tumour antigen with a vector, and then injecting it into the patient.In a mice model of HCC, a DNA vaccine targeting fibroblast-activated proteins altered the tumour mesenchyme and presented an anti-tumour effect. 125Alpha-fetoprotein can mediate HCC immune escape by altering the proportion of CD4 + T/CD8 + T cells and inhibiting DCs and NK cells.These features with the high expression in HCC cells make it a promising target for vaccine-based therapy.Exosomes derived from AFP-expressing DCs induced more antigen-specific immune responses in mouse models, leading to a prolonged survival time and an ameliorative tumour microenvironment. 126One clinical research of AFP-derived peptides vaccine (AFP 357 and AFP 403 ) had a good anti-tumour effect, prolonging the overall survival time of patients. 127OVs can not only directly kill tumour cells but also stimulate immune responses and enhance the anti-tumour effect.Most Ovs are modified from herpes simplex virus (HSV), reovirus (RV), vaccinia virus (VV), vesicular stomatitis virus (VSV), influenza virus (IV), oncolytic adenovirus (OAd) and Newcastle disease virus (NDV).A recent study showed that NDV HK84 has a better tumour-inhibiting effect than cisplatin by activating IFN-related signalling pathway. 128Measles virus combined with CD8NKG2D cells could enhance the activation and infiltration of CD8NKG2D cells. 129lthough promising, the strains of OVs, the selection of combined strategies and the timing of the combination still need further research.

| Immune checkpoint inhibitors
As a typical inflammation-associated tumour, immune evasion is one of the important features in the development and progression of HCC.A variety of immunosuppressive ligands expressed on the surface of tumour cells bind to inhibitory receptors on activated T cells, reducing the strength of antitumor immune responses and thus evading immune monitoring.Drugs that block these tumour immune evasions are called immune checkpoint inhibitors (ICI), and their targets include programmed cell death proteins and their ligands (PD-1, PD-L1), cytotoxic T-lymphocyte protein 4 (CTLA-4), lymphocyte activation gene 3 protein (LAG-3) and mucin-containing structural domain molecule 3 (Tim-3).PD-1 is a regulatory immunoglobulin that regulates the expression of CD4 + /CD8 + T cells, B cells and NK cells and plays a significant role in maintaining the immune tolerance of T lymphocyte and suppressing the activities of immune cells.TME can induce high expression of PDL1 in tumour cells, which is a member of the CD28 family.The binding of PDL1 to PD1 on the surface of T cells can negatively regulate T cell function and suppress the proliferation and activity of T cells, thus promoting immune evasion of tumours. 130,131PD-1 inhibitors in current clinical studies, such as nabumab, pembrolumab, and dulvestrant, have been shown to be effective in HCC.
CTLA-4, a homologue of CD28, is a suppressive coreceptor expressed by activated T cells that inhibits the development of immune responses primarily by regulating the development and proliferation of T cells.HCC-derived Tregs have been shown to suppress the expression of CD80/86 in DCs in a CTLA-4-dependent manner, and thereby inhibition of CTLA-4 may prevent Treg-mediated suppression of antitumor immune responses.In addition, down-regulation of CD80/CD86 could inhibit DC maturation and suppress antigen presentation from DCs to T cells. 132,133Currently, CTLA-4 antibodies, epirimizumab and trametinib, have been tested in clinical phase I/II trials for progressive HCC.
Tim-3 is a transmembrane protein expressed on the surface of various immune cells, such as CD4+/CD8+ T cells, Tregs, tumour-infiltrating resident NK (LrNK) cells, and classical NK (cNK) cells.It can act as a receptor of galectin-9 (Gal-9), phosphatidylserine (PtdSer), high mobility group box-1protein (HMGB1), carcinoembryonic antigenassociated cell adhesion molecule-1 (CEACAM-1).Tim-3-mediated PI3K/mTOR1 signalling pathway enhances HCC growth by inhibiting NK cell function.Tim-1 was significantly up-regulated in tumour-derived exosomeactivated B cells, thereby inhibiting the function of CD8+ T cells. 134Exogenous high mobility group proteins activate B cells and promote the expansion of Tim-1Breg cells via Toll-like receptor 2/4 and mitochondria-activated protein kinase signalling pathways.HCC patients have a high infiltration of Tim-1Breg cells in tumour tissue, showing a CD5 + + -CD24 − -CD27 − -CD38 + phenotype.Expressed high levels of the immunosuppressive cytokine IL-10 and exhibited strong inhibitory activity against CD8T cells. 35 recent study found that IL-27R signalling inhibits the ability of hepatic NK cells as well as ILC1 to infiltrate into the tumour and cytotoxic function, promoting HCC development in an animal model.Knockdown or inhibition of IL-27 or IL-27R with drugs could enhance anti-tumour immune responses and arrest HCC development. 135espite the remarkable success of ICI immunotherapy, some patients have experienced immune-related adverse events (irAEs).The incidence of skin toxicity in patients receiving CTLA-4 inhibitors was over 40%.Among patients treated with PD-1/PD-L1 inhibitors, skin toxicity was found in about 40% of patients.In addition, PD-1 inhibitors can cause mucositis.The incidence of gastrointestinal toxicity caused by PD-1 or PD-L1 inhibitors alone was 10-20%, while the incidence was 30-40% when CTLA-4 inhibitors were used alone or combined with PD-1 inhibitors.The incidence of liver toxicity was 15% in patients treated with CTLA-4 inhibitors and 5-10% in patients treated with PD-1/PD-L1 inhibitors.The current strategy for irAEs is to discontinue the drug and provide symptomatic treatment.Although the forms and degree of toxicity of irAEs are diverse, the majority can be reversed and controlled.There is still a need to focus on the occurrence of irAEs that may lead to serious consequences.

AND FUTURE PERSPECTIVES
The TME of HCC is a complex cell network that plays crucial roles in tumour progression, metastasis, recurrence and treatment resistance.A better understanding of the crosstalk between components of the HCC TME and potential targets can help predict immunotherapy responses, determine the efficacy of immunotherapy, identify prognostic markers and guide personalized immunotherapy protocols.Key issues that need to be addressed are as follows: (1) Finding targets in the TME is the first step, how can they be applied in the clinic?(2) How to select suitable therapeutic targets without affecting their normal immune functions?(3) How to use joint immunotherapy for multiple targets to improve the efficacy?(4) How to overcome the low drug penetration and heterogeneous expression of therapeutic targets caused by the complex dynamic changes of TME? Nanotechnology-based platforms for drug delivery may be potential promising approaches.

2. 7 |
Dendritic cellsDendritic cells are found in parenchymal and nonparenchymal cells and are involved in regulating immune responses to various stimuli.Based on morphology and function, DCs are divided into conventional dendritic cells (cDCs) [subtype cDC1: CD141 + /CD14 − ; subtype cDC2: CD1c + /CD14 − ] and plasmacytoid dendritic cells (pDCs) (CD303 + /CD304 + , type I IFN-secreting) and monocytederived DCs (moDCs) (SIGN + ).DCs are powerful specialized APCs and prime activators of CD4 + T and CD8 + T cells.DCs can capture, process, and present antigenexpressing co-stimulatory molecules such as CD40 and CD70, and migrate to lymphoid organs to activate effector T cells and induce memory T cells.Therefore, the DC-mediated malfunction of antigen cross-presentation and T-cell suppression is important for immune evasion.

( 3 )
Expression of inhibitory ligands suppresses T cell function.It was shown that DCs positively induce tolerance responses in human HCC by expressing inhibitory ligands PD-L1, Gal9, MHC-II, and CD86/CD80, down-regulating T-cell incompetence or Tregs, and subsequently inhibiting CTL responses through IL-10.In addition, the interaction of Treg cells with tumour-associated CD11c + DCs reduces the expression of DCs co-stimulatory ligands, thereby inhibiting T cell proliferation. 78As a result, clearance of Treg cells can recover the immunogenicity of tumourassociated DCs and the expression of co-stimulatory molecules, suppressing tumour cell growth.

F I G U R E 2
Multiple methodologies for exploring molecular mechanisms of the immune microenvironment and pathogenesis of liver cancer.
Sha Liu and Rongyang Dai designed this study.Sha Liu drafted the manuscript.Rongyang Dai and Man Jia revised the manuscript.All authors have read and agreed to the published version of the manuscript.
Current studies involving single-cell RNA sequencing in hepatocellular carcinoma.
T A B L E 1