HBV reprograms the tumor microenvironment in hepatocellular carcinoma: mechanisms and therapeutic implications.

Introduction​​
There are now around 296 million individuals living with hepatitis B virus (HBV) infection globally [1]. HBV infection is an important cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC), among many other complications. HBV is associated with more than 50% of global cases of HCC, particularly in high prevalence areas like Asia and Sub-Saharan Africa [2, 3]. The oncogenic potential of HBV involves many confounding factors, both direct and indirect, including integration and signaling via the HBx protein, along with host factors including chronic viral infection, inflammation, and immune dysregulation all leading to hepatocarcinogenesis [4]. The importance of the complex interplay of HBV with the tumor microenvironment (TME) is critical in understanding progression of HBV-related HCC [5]. Persistent HBV infection creates an immunosuppressive TME with the recruitment body immune cells including exhausted T cells, and regulatory T cells, stromal reorganization, and aberrant cytokine signaling that work together to facilitate tumor progression, growth, and immune evasion [5]. Alongside this interplay, tumorigenesis is enhanced through the suppression and modulation of key oncogenic signaling pathways through viral proteins like HBx (Wnt/β-catenin, NF-κB) and downstream epigenetic changes producing a tumor-permissive niche [4].
The purpose of this review is to provide a discussion and an overview of the complex relationships between HBV infection, HCC progression, and TME changes, with particular focus on the unique aspects of HBV persistence, including viral integration events, HBV-mediated oncogenic pathways, immune microenvironment remodeling, metabolic reprogramming, and epigenetic dysregulation. These mechanisms collectively contribute to the establishment of an immunosuppressive tumor microenvironment that facilitates immune evasion, chronic inflammation, and fibrosis—key drivers of hepatocarcinogenesis in HBV-infected individuals. Additionally, this review highlights emerging therapeutic strategies targeting these HBV-specific TME modifications to improve clinical outcomes in HCC.

HBV and hepatocarcinogenesis

Epidemiology of hepatitis B virus infection in liver cancer
Hepatitis B virus (HBV) infection, a major cause of hepatocellular carcinoma (HCC) worldwide, shows considerable differences in disease burden depending on geographic location (Fig. 1) [4]. In 2019, the Global Burden of Disease study estimated about 192,000 deaths from HBV-related HCC, threefold the estimated 156,000 deaths reported in 2010, up from 5995 HCC deaths reported in 1980 [1, 6]. Death rates vary, and the highest age-standardized death rates (ASDR) occur in sub-Saharan Africa and East Asia, with Mongolia reporting an ASDR of 28.23 deaths/100,000 population compared to 0.18 deaths/100,000 in Sweden [1]. The uneven incidence of HBV and age-standardized overall deaths is likely due to substantially greater regional or geographic HBV prevalence, as the WHO Western Pacific and WHO African Regions are home to 2/3 of all chronic HBV infections globally [7].
Changing demographics of hepatitis B infected populations will have various impacts on HCC epidemiology. In Hong Kong, for example, study investigators observed a proportionate increase of older HBV-infected patients; the proportion of chronic HBV patients aged ≥ 60 years increased from 11.6% for the years 2000–2004 to 35.4% for the years 2014–2017 [8]. This is likely due to advances in treatment promoting better long-term survival of patients with chronic HBV infection since most patients born before vaccination received normalizing HBsAg treatment, along with positive co-intervention databasing for programs promoting immunizing newborn children to limit the initiation of chronic HBV infection. Ultimately, because of the historical viral exposure time-framed long-term analyzed population; and presence of metabolic co-morbidities of this aging cohort (during time-cohorts) must be considered when evaluating their risk of developing HCC. Today, there remain global failures in the systematic management of HBV as only 10% of HBV-infected individuals are diagnosed, and of these only 5% of diagnosed individuals as of 2019 have received antiviral therapy [9]. In the USA, despite being a high-income country, as of 2018, HBV diagnosis among infected individuals was still only 19% in the USA and encouraged priority-setting system (e.g., HBV lifecycle tracking, hepatitis virus containment priority targets) interventions in 9/10 States in using limited health units to improve access to HBV treatment for acute infection and the majority of indeterminate HBV status patients [1]. Current projections estimate through direct proportionality to increase by 39% by 2030 in death/serious outcomes for patients with CID status [10]. As such, improving infant and youth vaccination coverage; increased birth-dose coverage; more downstream policy advanced simplification; or reduction of length for treatment protocols and algorithms may improve limited or diseased populations' access to care, expanding treatment caseload comprehensively in due time all, and are considered of a long-term approach toward timely insurance-mass intervention action. Further, introduction of HCC surveillance to HBV patients' pathways of care and the expansion of existing clinical metrics have overwhelming support as an adjunct to limit current metrics for “late-stage presentation” positioning the overwhelming majority of HCC as problematics in most resource deficient settings [10]. Multitudes, or multi-expertise multiplex-ranging evolving, patient sustainable adjustments, and follow-ups through social channels ultimately integrate virology, demographics, and health systems to counter the burden from HCC from chronic HBV infection [1].

Structure and replication cycle of hepatitis B virus
Hepatitis B virus (HBV) is an enveloped, hepatotropic DNA virus within the Hepadnaviridae family and is an enveloped, DNA virus with a complex structure that is entirely unique [11] (Fig. 2). The complete infectious particle is called the Dane particle, which has a diameter of approximately 42 nm, and it is composed of several components. The outer layer, the envelope, is formed from a lipid bilayer derived from the host, and the particle contains hepatitis B surface antigens (HBsAg), which appear in three forms (large, middle, and small or L, M, and S). The surface antigens are glycoproteins that play an important role in viral attachment and entry. Beneath the envelope lies a tetrahedral nucleocapsid made up of multiple copies of the hepatitis B core antigen (HBcAg), which contains the viral genetic material and the polymerase molecule itself [12]. The unique genomic structure of HBV is made up of partially double-stranded relaxed circular DNA (rcDNA) consisting of 3.0–3.2 kb of DNA, which has one complete negative strand and one incomplete positive strand. In addition to the infectious Dane particles, HBV produces a large number of non-infectious subviral particles that are 22 nm in diameter [11]. These spherical and filamentous structures are made up of excess HBsAg glycoproteins that do not contain nucleic acid. They are produced in much higher numbers than complete infectious virions during HBV infection. The HBV viral proteome is transcribed from four overlapping genes (P, preC/C, S, and X), and from each gene, multiple proteins are produced that play vital roles in HBV function due to complex transcriptional and translational regulation [4, 13]. The P gene encodes a multifunctional reverse transcriptase that has both reverse transcriptase, RNase H, and DNA polymerase functionalities that are required for viral replication. The C gene produces structured HBcAg that makes up the nucleocapsid and the secreted HBeAg that provides important immune modulation. The S gene produces three different types of HBsAg, which make up the envelope of the virus, while X encodes the regulatory HBx which is a multifunction factor involved in viral transcription, replication, and pathogenesis, including the potential role of HBx in promotion of hepatocarcinogenesis through oxidation and epigenetic modification. These structural complexities provide a framework for HBV to establish persistent infection and allow HBV to have unique infection characteristics compared to DNA viruses.
The hepatitis B virus (HBV) replication cycle is unique among DNA viruses in its use of a reverse transcriptase process, which shares some characteristics with retroviruses [14]. The infection cycle begins when HBV virions specifically engage with the sodium taurocholate co-transporting polypeptide (NTCP) receptor on the hepatocyte surface, followed by membrane fusion and subsequent nucleocapsid delivery into the host cytoplasm [15, 16]. After cellular entry, the virion nucleocapsid is transported to the nucleus and the relaxed circular DNA (rcDNA) genome is repaired by host enzymes to form covalently closed circular DNA (cccDNA) [17]. Once cccDNA is formed, it remains in the host nucleus as a stable minichromosome, with histones and viral proteins, serving as the persistent transcriptional template for all viral RNAs during the infection. cccDNA transcription by the host RNA polymerase generates multiple viral RNAs including pregenomic RNA (pgRNA) which serves as the dual template for reverse transcription and as mRNA for cellular translation of core protein and viral polymerase. Other subgenomic mRNAs encode the viral surface antigen (HBsAg), e antigen (HBeAg), and regulatory HBx proteins [11]. The pgRNA is packaged into newly forming nucleocapsids with the viral polymerase that copy the pgRNA through reverse transcription and produce new rcDNA genomes. While the majority of reverse transcription events result in rcDNA (90%), a smaller fraction results in double-stranded linear DNA (dslDNA, 10%) that could potentially integrate into the host genome and contribute to oncogenesis through mechanisms such as telomerase reverse transcriptase (TERT) activity [18]. Mature nucleocapsids containing rcDNA acquire their envelope, initiating new HBV virions, by budding into membranes of the endoplasmic reticulum and Golgi apparatus that are enriched with HBsAg. Once the enveloped virions are formed, they are secreted from the cell via the secretory pathway, but still some nucleocapsids may traffic back to the nucleus to maintain a pool of cccDNA. In addition, cells infected with HBV produce and secrete vast quantities of non-infectious subviral particles made of excess HBsAg [19]; the infectious virions produce at least several orders of magnitude less than subviral particles, which may not be protective against cell infection, and are used to evade the immune system by downregulating the interference response pathways [20, 21]. This multifaceted replication cycle, especially the creation and persistence of nuclear cccDNA, explains how HBV can persist in a course of chronic infection, and is a major obstacle for therapies that seek to eradicate HBV from the body.

HBV oncogenic mechanisms​​

Direct viral effects (integration, HBx activity)
Hepatitis B viruses (HBV) integrate their partially double-stranded DNA (dsDNA) into the genomes of their hosts in 75–90% of HBV-associated hepatocellular carcinoma (HCC) cases, which plays a contributory role in hepatocarcinogenesis in many ways [4]. Integration often occurs at common genomic loci such as the TERT promoter, where HBV binding brings transcription factor ELF4 to the promoter to induce telomerase activation and immortalize the cell [22]. Other common integration sites are MLL4, CCNE1, FN1, and CYP2C9 [23]. In general, HBV integration leads to insertional mutagenesis where tumor suppressor function is inactivated or a proto-oncogene is activated, and random integration induces chromosomal aberrations that produce chromosomal instability such as deletions, translocations, and other structural aberrations [24]. HBV-host chimeric transcripts including HBV-LINE1 fusions, would also promote oncogenesis by regulating Wnt/β-catenin signaling pathway production [25].
A common target of HBV integration is the viral X gene, leading to C-terminal truncated HBx proteins (ctHBx) such as HBx-120 and HBx-134 [26, 27]. These variants are commonly observed in HCC tissues, and they mirror and even augment the oncogenic capacity of full-length HBx. CtHBx promotes hepatocyte proliferation, motility, and invasiveness by enhancing mitogen-activated protein kinase signaling pathways such as ERK, JNK, and p38 [26, 27]. This then leads to increased phosphorylation of the cell cycle regulator cdc25C, and decreased phosphorylation levels of the tumor suppressor p53, leading to cell cycle progression and suppression of growth inhibition. CtHBx's actions would facilitate clonal expansion of HBV-infected hepatocytes and therefore expedite tumor formation.
In addition to the effects of its truncated forms, the full-length HBx protein itself functions as a potent 17 kDa chimeric oncoprotein and a key driver of HBV-related carcinogenesis [28]. HBx has many both oncogenic and epigenetic roles that it induces by itself or other known transcription factors to transcriptionally activate oncogenes (MYC, Cyclin D1) through NF-κB and AP-1, while inhibiting transcription of tumor suppressor genes, for instance, p53 and p16INK4a [29]. HBx also promotes mitochondrial dysfunction through binding with cytochrome c oxidase subunit III (COXIII), to stimulate production of reactive oxygen species (ROS) which leads to induction of DNA damage including 8-oxoguanine lesions [30]. HBx has been reported to also provide epigenetic regulation of genes by binding to and recruiting DNA methyltransferases (DNMTs), including DNMT3A, to aberrantly hypermethylate promoters of tumor suppressor genes (ASPP1, SFRP1) and silence their expression [31]. In addition to driver genes, HBx can modulate the regulatory roles of non-coding RNAs. Examples include regulating the over-expression of oncogenic long non-coding RNAs, or with a negative regulatory influence, to impact critical cellular pathways involved in proliferation, apoptosis, and immune suppression [32, 33].

Host genomic instability
The ability of HBV to induce hepatocellular carcinoma (HCC) is determined by viral genetic factors and host factors for susceptibility. Within HBV, genotypes such as C, D, and F1b have been shown to have a greater oncogenic potential based on viral characteristics, geographic distribution, and epidemiology than other genotypes [34]. The potential for an individual to develop HCC is also impacted by viral mutations, such as basal core promoter mutations (A1762T/G1764A) and a pre-core mutation (G1896A), all of which are associated through higher levels of viral replication and altered immune recognition [35, 36]. Variations of host factors associated with the induction of HCC include genetic polymorphisms, and ultimately, individuals will have varying susceptibility to HBV-induced hepatocarcinogenesis through these genetic factors. A very common and well-characterized mutation, TP53 R249S, when present in regions where aflatoxins are present, will act in channeling infection with HBV to malignant transformation [37]. Immune-related genes are much investigated, and variants such as HLA-DP would appear to discriminate by related disease progression perhaps, based on an ability to persist through viral clearance and immune surveillance pathways [38]. The dynamic interplay of viral and host factors particularly, genetic factors, make it difficult to appreciate the biological potential for liver carcinogenesis with HBV.

Epigenetic and epitranscriptomic dysregulation
HBV infection causes significant and widespread epigenetic reprogramming contributing to hepatocellular carcinoma (HCC) development. The mechanism for such epigenetic reprogramming is both a viral (HBx) and cellular response that is designed to stabilize the WDR5 subunit of a histone methyltransferase complex, which will increase marks of H3K4me3 activity at the promoters of pro-viral and oncogenic genes by recruiting epigenetic factors to cellular points of intersection [39]. Ultimately, this process will result in a more permissive chromatin state and increased expression of genes involved in proliferation and cell survival, and more importantly, HBV's ability to also epigenetically reprogram host RNA methylation pathways post-transcriptionally that enable oncogenesis, such as the m6A modification of PTEN mRNA by METTL3 which reduces mRNA stability and downregulates PTEN expression leading to persistent activation of the PI3K/AKT/mTOR signaling axis for oncogenesis [40, 41]. HBV-induced m6A, rather than removing the tumor suppressor brake in cellular growth and survival, is added to the promoted party [42]. However, in addition to PTEN, HBV infection is also known to contribute to widespread chromatin modifications and potentiating epigenetic paths through the recruitment of several epigenetic modifiers such as EZH2—the catalytically—active subunit of Polycomb repressive complex 2—that contribute deposited the repressive H3K27me3 histone mark onto tumor suppressor genes and implied a closed chromatin assumption that will steadily silence protective genes [43]. In fact, the aggregate of all the reprogrammed epigenomic changes that generate a permissive cellular landscape for malignant transformation will also be acted to stimulate oncogenic pathways while inhibiting cancer surveillance pathways [44]. In summary, the usually stable epigenetic program—both above (molecular markers, histone modification, RNA methylation) and below (more ordered chromatin structure, chromatin-remodelers)—is a highly self-perpetuating program that appears to facilitate the initiation of an HBV-dependent hepatocarcinogenic program.

Metabolic adaptation
Hepatitis B virus infection results in significant metabolic changes that are essential for tumor cell growth and immune evasion. A key viral message, HBx, drives the Warburg effect, a distinct feature of cancer metabolism where cells rewire to consume glucose through an enhanced glycolytic process even in the presence of oxygen [45]. HBx drives the Warburg effect through its HBx-dependent inhibition of pyruvate kinase M2 (PKM2) which it does by creating a glycolytic “bottleneck” leading to the accumulation of glycolytic intermediates that directly target biosynthetic pathways essential for cell proliferation [45]. HBV also drastically rewires hepatocyte lipid metabolism. HBV infection enhances both fatty acid oxidation pathways as well as cholesterol synthesis pathways providing substrates critical for generating membranes, building blocks for energy production, and supplies for supporting uncontrolled growth [46, 47]. These metabolic adaptations not only promote tumor cell metabolism but also could provide lipid-based resources for eventual virogenesis. (Lipid droplets may serve as sites for replication and assembly of HBV.) [48] HBV uses a clever strategy of immune metabolic suppression through the depletion of specific amino acids in the tumor microenvironment. The amino acid starvation of T cells mediated by the virus for arginine and tryptophan creates a nutrient deprived environment for T cell function and proliferation to occur [49]. This amino acid deprivation leads to a decrease in anti-tumor immunity while at the same time providing the anabolic needs for HBV-infected hepatocytes. The metabolic reprogramming of glycolysis, lipid metabolism, and amino acid utilization together provide a tumor-permissive microenvironment to promote both viral persistence and hepatocellular carcinogenesis.

Oncogenic pathway
The hepatitis B virus X protein (HBx) has a key role in inciting hepatocarcinogenesis, primarily through constitutive activation of many oncogenic pathways. Constitutive HBx-induced stabilization β-catenin is facilitated by inhibition of glycogen synthase kinase 3β (GSK3β) and promotes accumulation of β-catenin in the nucleus, where β-catenin activates downstream proliferation genes [50]. Additionally, HBx activates the PI3K/AKT/mTOR pathway in part through downregulation of the tumor suppressor PTEN (phosphatase and tensin homolog) and through promoting the action of the metabolic enzymes choline kinase α (CHKA) and glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1) to promote cell survival and metabolic reprogramming [51]. The MAPK/ERK pathway is another target of HBx [52]. Sustained ERK and p38 activation promotes unlimited proliferation in cells. HBx also induces oxidative stress by disrupting the homeostatic NRF2/KEAP1 anti-oxidant response system, forcing cells into a sustained pro-oxidative state that contributes to genomic instability and therefore drives malignant transformation [53]. Consequently, HBx and the activation of the pathways described above leads to coordinated activation providing a synergistic oncogenic network that promotes HBV-associated hepatocellular carcinoma. The combined pleiotropic effects of HBx on these multiple interconnected pathways further solidify its role as a driver of the transformed phenotype in HBV-infected hepatocytes.

Tumor microenvironment (TME) in HCC

Cellular components of the TME​​

Immune cell
T lymphocytes have important and distinct functions within the tumor microenvironment of hepatocellular carcinoma. Though CD8 cytotoxic T cells promote a vigorous anti-tumor effector immune response, a large percentage of them display an exhausted phenotype as indicated by persistent expression of inhibitory receptors (ex: PD-1, TIM-3, and LAG-3), inhibition by the engagement of inhibitory cytokines (ex: TGF-β and IL-10), and as a result, their effectiveness at tumor cell cytotoxic activity is greatly diminished [54–57]. The CD4 T lymphocyte subsets can also be relevant to the proliferation of these malignancies [58]; Th1 cells specifically lead to a successful anti-tumor immune effector response due to their production of interferon-γ [59], whereas regulatory T cells (FoxP3) are potent immunosuppressive cells, using multiple mechanisms (CTLA-4, IL-10, TGF-β, etc.) to exquisitely suppress immune responses and whose detection is strongly correlated with negative clinical outcome [60, 61]. The CD4 subset of T follicular helper (TFH) can also influence the immune suppression in the tumor microenvironment by influencing macrophages through the secretion of IL-21, which mediates the polarization of macrophages toward a pro-tumor M2 phenotype [62, 63]. The interactions and relevant contributions of these T lymphocyte subsets in the tumor microenvironment will play integral roles in both tumor proliferation and sensitivity to the effects of immunotherapy.
Natural killer (NK) cells play key roles as innate immune effectors in hepatocellular carcinoma (HCC) and exert immunologic attack through multiple mechanisms, primarily through direct cytotoxicity, which is mediated by engagement of activating receptors (NKG2D and NKp46) [64–66]. These cytotoxic lymphocytes employ granzyme/perforin-dependent mechanisms for elimination of malignant neoplastic cells and cytokine production, acting as a first line of defense against early tumor development [64]. Unfortunately, the immunosuppressive HCC tumor microenvironment causes NK cell activity to become severely suppressed from several inhibitory mechanisms. For instance, tumor-associated macrophages that are polarized to the M2 phenotype inhibit NK cell function and activity by expression of non-classical MHC molecules (HLA E/G) and the triggering receptor TREM-1 [67, 68]. Cancer-associated fibroblasts further contribute to NK cell dysfunction by also secreting prostaglandin E2 (PGE2) [69, 70]. Moreover, tumor hypoxia generates a metabolic barrier that hinders NK cell effector functions [71]. Collectively, the various multifactorial nets of NK cell activity suppression represent a significant and non-typical immune evasion strategy related to HCC progression and are likely a major contributor to the minimal responses generated by current immunotherapies.
B cells are highly diversified and context-dependent within the microenvironment of hepatocellular carcinoma (HCC). They are capable of both anti-tumor and pro-tumor functions. On the one hand, certain CD20-expressing (CD19 +) B cell subsets are predictive of improved clinical outcomes in HCC [72, 73]. This result is potentially driven by the ability of CD20 + B cells to activate antigen-presenting roles and support cytotoxic T cells [73, 74]. Supposedly, anti-tumor B cells may also possess the ability to create tertiary lymphoid tissue, which facilitate effective anticancer immune diseases [75]. On the other hand, regulatory B cells (Bregs) are strong immunosuppressive factors that facilitate immune tolerance during HCC progression [72]. Bregs are characterized by the release of inhibitory cytokines including interleukin (IL)-10 and TGF-β [76]. Bregs actively suppress effector T cell function and promote the increase of regulatory T cells in the tumor-permissive environment [77]. The B cell subset indicative of anti-tumor effects versus regulatory bimodal functions is important to disease progression and potentially drives patient immunotherapy responses.
Tumor-associated macrophages (TAMs) are crucial cellular constituents of the microenvironment of hepatocellular carcinoma (HCC), originating from both local embryonically derived progenitor cells, i.e., Kupffer cells (KCs), as well as from monocyte-derived macrophages (MoMs) recruited from the circulation via chemokine signaling pathways, including CCL2/CCR2 and CSF1/CSF1R [78–82]. These macrophage populations are heterogeneous and undergo further differentiation into functionally polarized subsets in response to environmental cues. These subsets have distinctly opposed effects on tumor progression. For example, the M1-like TAMs secrete cytokines such as IL-12 and TNF-α and chemokines including CXCL9/10 that promote the recruitment and activation of CD8 T cells [83], while the M2-like TAMs (characterized by the release of IL-10, TGF-β, and CCL17/22) adopt an immunosuppressive phenotype, contributing to pro-angiogenic and matrix remodeling activities associated with metastasis [78]. While terms like “M1” and “M2” may not capture the entirety of TAM heterogeneity and functional polarization, it is representative enough for the current state of the literature and introduces the key features of each function. In the early stages of hepatocarcinogenesis, TAMs help establish and maintain cancer stem cell (CSC) niches enabling tumor self-renewal and initiation [84]. As HCC progresses, the M2-polarized TAMs in the tumor microenvironment proliferate and broaden their immune evasion mechanisms, including the upregulation of immune checkpoint molecules such as PD-L1 and the production of systemic immunosuppressive metabolites such as adenosine [85, 86]. Additionally, TAMs promote metabolic reprogramming via fatty acid oxidation (FAO) and modulate resistance to targeted therapies by HGF-dependent induction of SCF/c-Met signaling that inhibits the effects of sorafenib for liver cancer cells [87–89]. The interplay of these macrophage subsets and their functional states in progression are critical in the establishment of an immunosuppressive HCC microenvironment, and this raises interest in them as practical targets for therapeutic clone strategies.
Myeloid-derived suppressor cells (MDSCs) are a collection of immature myeloid cells that are heterogeneous, found in hepatocellular carcinoma (HCC), and play an important role in tumor immune escape [90, 91]. MDSC has two major types: granulocytic MDSCs (G-MDSCs) that are phenotypically similar to neutrophils and monocytic MDSCs (M-MDSCs), which have phenotypic features of immature monocytes [91]. Both subsets have multiple mechanisms of anti-tumor immunity suppression, including arginine depletion through arginase, generating reactive oxygen species (ROS), and immune checkpoint molecules (i.e., PD-L1) [90, 91]. MDSC is recruited to the HCC microenvironment via chemotactic signals including CXCL2 from cancer-associated fibroblasts (CAFs) and IL-6 from tumor cells, creating a circuitous feed-forward loop that supports immunosuppression [92]. MDSCs contribute to the generation of an immune-tolerant tumor microenvironment, inhibiting effector T cell functions while enhancing regulatory T cell (Treg) expansion [93]; thus, they represent a significant barrier to anti-tumor immunity, and a potential target for HCC therapies.
Dendritic cells (DCs) are professional antigen-presenting cells, functioning to initiate and regulate anti-tumor immune responses. However, the roles of DCs in hepatocellular carcinoma (HCC) are seriously impaired and result in failure to prime effective T cell responses [94]. The primary reason for such dysfunction comes from the immunosuppressive tumor microenvironment [95]. In the HCC tumor microenvironment, tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) both act directly to inhibit DC function through cell–cell interactions and cytokine secretion [96]. Additionally, the HCC-derived vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) can block DC maturation and lead to enrichment of immature or tolerogenic DCs [97]. These defective DCs promote the induction of immune tolerance and never adequately present tumor antigens, which may contribute to tumor immune evasion in HCC [98]. The impaired DC function represents a significant mechanism underlying how HCC provides immunosuppression and supports the need for therapeutic options to restore DC immunocompetence.
Tumor-associated neutrophils (TANs) are remarkable because of their plasticity in the tumor microenvironment, demonstrating either an anti-tumor (N1) or pro-tumor (N2) phenotype based on contextual signals [99–101]. N1-polarized neutrophils have demonstrated anti-tumor potential mainly afforded through reactive oxygen species (ROS) production that can lead to direct cytotoxic outcomes upon malignant cells [102, 103]. Conversely, N2-polarized neutrophils can acquire a tumor-promoting role through various mechanisms. N2 TANs promote metastatic dissemination of tumor cells by releasing neutrophil extracellular traps (NETs) that can act as a scaffold for circulating tumor cells, while simultaneously producing matrix metalloproteinase-9 (MMP-9) to degrade components of the extracellular matrix, thus further promoting invasion of tumor cells, their subsequent proliferation, and their survival [102, 104–106]. N2 TANs also promote immune suppression through expression of programmed death-ligand 1 (PD-L1) that interacts with PD-1 expressed on T cells to inhibit their cytotoxic functions [107]. The important functional dichotomy of TANs illustrates the dynamic complexity of TANs in tumor progression, and the balance and signaling toward either N1 or N2 will have relevant consequences on final disease outcomes. The elevated numbers associated with N2 TANs in advanced malignancies allow for their consideration as possible targets for therapeutic interventions, as they could be used to ameliorate both tumor metastasis and immune evasion.

Non-immune cell
Cancer-associated fibroblasts, or cancer-associated fibroblasts (CAFs), are a diverse group of activated stroma cells, usually driven from hepatic stellate cells (HSCs), following persistent inflammatory exposure or through the epithelial–mesenchymal transition (EMT) of transformed tumor cells [108–110]. They help shape the tumor microenvironment largely by their secretion of numerous factors and by remodeling the physical characteristics of the extracellular environment. A basic property of CAFs includes remodeling the extracellular matrix (ECM) whereby they secrete large amounts of collagen, fibronectin, and matrix metalloproteinases (MMPs) represented in an ever-increasing tissue fibrotic weight and in elevated tissue stiffness which directly correlates to tumor progression and metastasis [111]. In addition to the structural influence of metastasis, CAFs contribute to immunosuppression by leukocyte chemokine signaling, e.g., CXCL12 recruits myeloid-derived suppressor cells (MDSCs) into the tumor space [112], and indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) inhibit natural killer (NK) cells essentially creating an immune-evasive variant of the microenvironment [113, 114]. Further, CAFs increase angiogenesis via CAF-derived vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF) via endothelial cell proliferation and neovascularization while ensuring tumor growth and metastasis are facilitated [115, 116]. In many ways, CAFs play a multitude of roles in tumor progression, and they are a real opportunity to initiate therapeutic approaches in clinical oncology.
Hepatic stellate cells (HSCs) are resident perisinusoidal cells located within the liver. HSCs undergo extensive phenotypic changes as they respond to chronic inflammation, when they are activated following transdifferentiation into myofibroblasts [117–119]. The activation process is a pivotal event in liver pathology as it provides the first cornerstone to establish a situation that alters the hepatic microenvironment. Following activation, HSCs also develop contractile properties and enhanced synthetic capabilities, largely through excessive production and deposition of ECM components such as collagen type I and III, which is responsible for progressive liver fibrosis [118, 120, 121]. The resultant fibrotic microenvironment produced by activated HSCs serves both to construct structural support for tumor establishment as well as create physical barriers for the infiltration of immune cells as well as drug delivery. In addition to its fibrogenic contribution, activated hepatic stellate cells are also critical in immune modulation as a component of the tumoral microenvironment. Hepatic stellate cells have shown to express immune checkpoint molecules such as PD-L1 that directly inhibit T cell function [122, 123]. They also produce and secrete the immunosuppressive cytokine, transforming growth factor-beta (TGF-β) which promotes regulatory T cell expansion and helps suppress effector immune functions [124, 125]. Overall, the role of hepatic stellate cells integrated in phase of development of fibrogenesis may restrain immune function and represent an acclimatizing environment for tumor growth; in addition, restricting the efficacy of immune therapies. The chronic activation of hepatic stellate cells as observed in chronic liver diseases emphasizes an uneasy self-reinforcing loop of inflammation, fibrosis, and immune evasion which ultimately poses a major therapeutic challenge for hepatocellular carcinoma.
Liver sinusoidal endothelial cells (LSECs) make up a unique vascular boundary in the hepatic microenvironment, and during tumor progression, these cells experience significant pathological remodeling [126, 127]. An important dysfunction of LSECs is capillarization, whereby structural alterations from specialized fenestrations are lost, and an organized basement membrane is formed [126, 127]. This damages the filtration function of the liver sinusoids and promotes localized hypoxia by further inducing fibrogenesis through hepatic (HSC) cell activation [116, 128]. These vascular pathological states deteriorate the exchange of nutrients and oxygen, form physical barriers for immune cells to traffic inside the tumor microenvironment, and limit the immune response [126]. Beyond their participation in the vascular remodeling of tumors, LSECs impact immune regulation in several different ways. LSECs are specialized endothelial cells that constitutively express programmed death-ligand 1 (PD-L1), through which LSECs provide T cell exhaustion and tolerance when PD-1 expressed on T cells bind PD-L1 [129]. LSECs release chemokines that preferentially recruit regulatory T cells to select for Tregs, creating a niche for immune suppression and preventing immune tumor response [130]. LSECs targeting both immune response and new vascular structures position LSECs as a driving force for tumor progression and immune therapy resistance in hepatocellular carcinoma (HCC). The unique phenotype of LSECs and adaption create functional and structural changes that perpetuate an immunosuppressive tumor microenvironment that include LSECs as a vital and active component of evolution and adaptation.

Non-cellular components​ of the TME​​
The network of cytokines and chemokines in the tumor microenvironment (TME) is essential to directing immune responses via signaling pathways. Immunosuppressive mediators hinder the expanding and activating Tregs and M2-polarized TAMs and MDSCs, such as transforming growth factor-β (TGF-β), interleukin-10 (IL-10), and interleukin-6 (IL-6) [97, 113, 131]. Collectively, these immunosuppressive mediators establish a milieu of immunosuppression that allows tumors to progress. Conversely, chemokines such as CCL2, CCL5, and CCL22 function as potent chemoattractants that recruit immunosuppressive cell populations into the tumor microenvironment (TME), further facilitating immune tolerance, or tumoral immune escape [132–134]. Pro-inflammatory cytokines exhibit anti-tumor activity by promoting cytotoxic immune responses. For example, interferon-γ (IFN-γ) and interleukin-12 (IL-12) elicit enhanced effector functions of T cells and natural killer (NK) cells with an outcome of tumor cell lysis and immune surveillance [135–137]. Chemokines such exemplified by CXCL9 and CXCL10 recruited activated T cells and NK cells into the tumor bed to directly inhibit immune suppression taking on an anti-tumor effector function [138, 139]. Collectively, the balance of cytokines and chemokines along the immunosuppressive and pro-inflammatory signal axis influence the functional state of the TME, impacting tumor progression and responsiveness to immunotherapeutics.
While most may view the extracellular matrix (ECM) as a structural component of the tumor microenvironment, it is also functional. The ECM consists primarily of collagen, fibronectin and hyaluronan, which provide mechanical stability to tissues, yet can regulate, and even promote tumor progression through physical and biochemical signals [140]. The most definitive physical property that represents ECM is the density of collagen and hyaluronan, when cross-linked has a density that physically excludes immune cell entry [141]. Thus, the dense ECM yields immune exclusion and only establishes “cold” tumors; tumors that elude onco-immune responses based on the absence of infiltrating, effector T cells [141]. ECM is more than a physical barrier; the stiffened ECM affects the behavior of the tumor cells, by activating mechanotransduction pathways [142]. Stiffness, as a measure of rigidness of the matrix, enhances the epithelial–mesenchymal transition (EMT) pathway, contributing tumor cells to be invasive and metastatic [143]. Stiffness of the matrix also contributes to tumor cell motility, along with providing directional, aligned collagen fibers, and thus, tracks for dissemination. The stroma is a dynamic microenvironment that can have the same composition one day, to an entirely different composition to the following day on account of ECM-remodeling enzymes that actively remove and insert components once released. This dynamic ability of the tumor microenvironment contributes to a tumor's capacity to adapt to growth and evade therapy.
Extracellular vehicles (EVs) are key messengers for intercellular communication in the tumor microenvironment and are continuously released throughout the tumor lifecycle by cells in the tumor microenvironment, such as cancer cells, tumor-associated macrophages (TAMs), and cancer-associated fibroblasts (CAFs) [144]. These bioactive membrane-bound nanoparticles then deliver a wide range of molecular cargo that is integral to tumor progression and dissemination through metastasis. One of the most actively studied EV components is microRNAs, specifically, miR-21 and miR-214, which recently have been implicated in the positive enhancement of the metastatic phenotype by modifying epithelial–mesenchymal transition and facilitating an invasion phenotype in the target cells [145, 146]. At present, very little is known about the protein cargo of EVs with some current research discussing the potential tumor-promoting effects of protein cargo through metabolic reprogramming. Pyruvate kinase M2 (PKM2), the most studied protein, is delivered via EVs derived from tumors and promotes glucose metabolism in adjacent and/or distal cells to create a tumor-enabling microenvironment, allowing for tumor growth [147, 148]. In summary, EVs that contain tumor-enabling cargo are used to facilitate the development of a pre-metastatic niche and systemic effects on the host, emphasizing their role in tumor biology and potential for therapeutic intervention. The diversity of the EV cargo, which represents the nature of the original cell and the tumor microenvironment state, provides differential and unique facets of both the development of the tumor and the mediators of pathological intercellular communication, which represent both a marker for the disease and pathological intercellular signaling.
Hypoxia is among the hallmark features of the tumor microenvironment and facilitates metabolic changes to promote immune evasion and tumor growth. In low oxygen conditions, hypoxia-inducible factors (HIFs), particularly HIF-1α, become stabilized and activate the expression of immunosuppressive molecules, such as PD-L1, and pro-angiogenesis molecules, such as VEGF [149–151]. The modulation of immunity and pro-angiogenesis not only causes an immune escape by suppressing the functions of cytotoxic T cells and NK cells but enhances vascularization and re-establishes hypoxia. Hypoxia can also cause metabolic changes that promote the accumulation of immunosuppressive metabolites such as lactate (substantially contributed by MDSCs), which affects the ability of T and NK cells to respond to tumor cells by altering their metabolic fitness [151–154]. Also, kynurenine another important metabolite produced by IDO, promotes T cell exhaustion, thereby creating an immunosuppressive environment in favor of tumor survival [155, 156]. There is an emerging interest in the gut microbiome as a key player in the development and progression of liver cancers by regulating inflammation and immunity [157, 158]. Better understanding of dysbiosis (alters in microbiome composition) in particular increases the number of harmful bacteria, such as Fusobacterium, which activate TLR4/NF-κB signaling, driving chronic inflammation [159–161]. This can develop an inflammatory cycle that supports tumorigenesis and promotes the activation of hepatic stellate cells (HSCs), which induce fibrosis and remodeling of the extracellular matrix. This remodeling can also recruit and induce MDSC expansion via LPS from the microbiome to affect anti-tumor immunity. The nuance interplay between gut microbiome, immunity of the host, and liver cancer progression highlights the potential of microbiome-targeted intervention in regulating the tumor milieu (Fig. 3).

HBV-Induced TME modifications

Immunosuppressive microenvironment​
Persistent replication of a virus and infringing immune responses relative to chronic hepatitis B virus (HBV) propagation leads to chronic hepatic inflammation, which develops basis immune responses to HBV-related liver cancers synonymous with hepatocellular carcinoma (HCC) [162]. Sensing of viral molecular patterns by pattern recognition receptors via toll-like receptor (TLR) and RIG-I signaling pathways relevant production of pro-inflammatory cytokine responses like TNF-α and IL-6, and downstream effects type I interferons (IFNs) [163–165]. HBV can indulge in breakdown in such systems and create their tolerant special context, however, via many mechanisms mainly HBx and HBeAg, that reduce the dictations of the IFN signaling pathway inhibiting antiviral host responses [166–168]. These chronic liver infections are problematic under chronic state as well, generally dysfunction of adaptive immune responses and exhaustion of virus-specific CD8 T lymphocytes and CD4 helper T cells, primarily limited frequency and functional defect of each. The chronic hepatitis HBV infection retains capacity to create unrestricted immune systems, such central, memory response as well as “acute infection” clearance ambient HBV limits [169]; memory immune responses and destabilization of useful frequency, comprehensive immune activations to competing invader [169]. Emerging context the few CD4 and CD8 present becomes exhausted CD8 and CD4 T cell tolerance capacity with upregulated inhibiting receptors, i.e., PD-1, CTLA-4, no adaptive mediating liver immune responses to limit the hepatocyte liver infection [170–172]. As chronic infection becoming stated by T cell exhaustion, as the trip relative fatigue, states the relevant PD-1 T cell exhausted states resides into the friendly bolstering attraction and state function faking synopsis tolerogenic tumor microenvironment (TME) capable hepatocarcinogenesis prime HBV-HCC [173]. It is not limited but emerged to the enrichment role for HBV to contribute and coerce proliferation and dominance of other immune suppressor cells type and populations like suppressive immune suppressor Tregs and myeloid-derived suppressor cells (MDSCs) to use the immune networks [4]. Additionally, chronic HBV infection perpetuates immune tolerance via TGF-β/IL-10-mediated suppression of anti-tumor responses, facilitating HCC development [174]. Cumulatively the chronic inflammation events and possibly unique entrepreneur immune compromised signals relative creator of chronic infection form the context for affectionate pro-cancerogenic behavioral outcomes and causes of hepatocarcinogenesis for individuals suffering chronic HBV infection (Fig. 4).

Immune evasion mechanisms​
HBV appears to have developed complex immune evasion mechanisms that are fundamental to viral persistence and hepatocellular carcinoma (HCC). One major immune evasion mechanism is the upregulation of immune checkpoint molecules (especially PD-L1 and CTLA-4) on HBV-infected hepatocytes and tumor cells that bind to ligands on cytotoxic T lymphocytes inhibiting their antiviral and anti-tumor activity, leading to an immunosuppressive tumor microenvironment [175]. This activation of immune checkpoints is an essential immune-evasive mechanism and represents a critical approach for both the virus and emerging tumor cells to evade immune recognition. The virus further compromises host immunity by interfering with antigen presentation pathways [176]. HBV infection leads to downregulation of MHC class I on hepatocytes which represents a severe impairment of the ability of CD8 T cells to recognize and eliminate infected or transformed cells [177]. Additionally, HBV utilizes the host ubiquitin–proteasome and autophagy systems to degrade or inactivate essential sensors and adaptors (MAVS, TRIF, cGAS, STING, NEMO) within the intracellular innate immune pathways, such as the RLR and cGAS/STING pathways [178]. HBx and HBV polymerase are primarily responsible for this targeted proteolysis, which enables immune evasion and promotes viral persistence. The innate defect in antigen presentation creates an immunological blind spot that facilitates viral persistence and tumor immune evasion. Early experimental evidence has demonstrated that HIV and HBV are capable of inducing tumors by promoting angiogenesis by infection [179, 180]. HIF-1α stabilization by HBx has been proposed to promote expression of angiogenic factors, namely VEGF and Ang-2, leading to a pro-angiogenic switch [181]. Therefore, it is likely that these immune-evasive mechanisms, namely checkpoint upregulation, impairment of antigen presentation, and angiogenesis, contribute to effective permissive environment for viral persistence and active primary tumor progression. This emphasizes the multifaceted nature of HBV in developing immune suppression in HCC.

Extracellular vesicle (EV)-mediated communication​
The hepatitis B virus (HBV) employs EV-mediated communication routes to alter the tumor microenvironment (TME) in hepatocellular carcinoma. HBV-infected hepatocytes release exosomes that are enriched with viral content such as HBx mRNA, viral proteins, and HBV-derived microRNAs (e.g., HBV-miR-3) to support the persistence of viruses and even induce oncogenic changes to the recipient cells [182, 183]. The exosomes containing viruses facilitate the intercellular transfer of viral genetic material and proteins, both increasing the viral pool and changing the behavior of host cells. HBV-positive exosomes have also been shown to communicate immune evasion by transferring interferon-induced transmembrane protein 2 (IFITM2) to dendritic cells [184]. This transfer diminishes the dendritic cell capacity to produce interferon-α (IFN-α), thereby inhibiting a functioning innate antiviral immune response [185]. The exchange of viral and host-derived components through exosome-mediated mechanisms generates an immunosuppressive microenvironment that nurtures HBV persistence, moderating immune responses for tumor immune evasion, and supporting hepatocellular carcinogenesis [186]. This demonstration of intercellular communication further illustrates the complex interaction between HBV infection and the tumor microenvironment as relevant to the development of HBV-associated liver cancer.

Chronic inflammation and cytokine dysregulation​
The chronic inflammation resulting from HBV infection is also a mechanism through which HBV infection drives hepatocellular carcinoma (HCC) development. The incredible dysregulation of cytokines has many different functions that contribute to HCC development, including responding to chronic inflammation. To mediate this damage HBV encodes HBx which has well-established functions in activating signaling pathways including NF-κB and JAK-STAT pathways sufficient to promote production of pro-inflammatory cytokines such as IL-6 and TNF-α [28, 167, 187]. These cytokines help maintain inflammation and promote proliferation to gain genomic instability for a prospective malignant transformation [188, 189]. Oxidative stress is also a mechanism through which HBV hysterically drives hepatocarcinogenesis. The virus-mediated mitochondrial dysfunction occurs through an interaction between HBx and COXIII followed by the buildup of misfolded HBV proteins (HBsAg and HBcAg) in the endoplasmic reticulum [30]. This alone would induce oxidative stress, and because of that stimulus, ROS increase to levels. Reactive oxygen species cause extensive damage to DNA beyond repair and create an oncogenic environment through accumulation of mutations [190]. The cytokines in HBV-associated HCC are out of balance toward increased presence of TNF-α, IL-6, and IL-10, which are fibrotic and oncogenic [174, 191]. TGF-β and IL-10 in the tumor microenvironment perpetuate an immunosuppressive mechanism that leads to inadequate DC stimulation and impaired T cell responses [192]. The imbalance of cytokines creates a process of immune evasion and a pro-fibrotic/pro-tumorigenic microenvironment where disease is vaccinated against chronic hepatitis, cirrhosis, and potentially HCC. The acute phase inflammation on the background of oxidative damage exacerbated by prolonged chronicity creates a perfect environment for hepatocarcinogenesis in a chronically HBV-infected person.

Fibrosis and stromal remodeling​
Hepatitis B virus-associated liver disease is characterized by significant fibrotic remodeling of the hepatic stroma through various interconnected mechanisms. Chronic HBV infection activates hepatic stellate cells (HSCs) which results in reactive oxygen species (ROS)-mediated NF-κB signaling resulting in upregulation of fibrogenic markers including α-smooth muscle actin (α-SMA) and excessive deposition of collagen in the extracellular matrix [193]. This activation of HSCs results in the transformation of quiescent HSCs into myofibroblast-like cells [120]. HSCs are primarily responsible for fibrotic response. The progression of fibrosis is exacerbated by HBV through intercellular communication mediated by exosome production. HBV-infected hepatocytes release exosomes containing microRNA-192-3p (miR-192-3p) that are taken up by HSCs and promote extracellular matrix (ECM) stiffening [194]. The movement of genetic material in the context of exosomes is a novel mechanism for HBV to activate the stroma away from the primary infection site and sustain fibrosis [182]. As fibrosis progresses into cirrhosis, the architectural distortion of the liver parenchyma creates a mechanically stiff and hypoxic microenvironment. The hypoxic environment promotes stabilization of hypoxia-inducible factors (HIFs), which in turn promotes angiogenesis as well as epithelial–mesenchymal transition [150]. Furthermore, the biomechanical changes of fibrotic stroma activate mechanosensitive signaling pathways in hepatocytes [195]. Collectively, these parallel fibrotic changes establish a tumor-promoting niche for the formation and development of hepatocellular carcinoma in chronic HBV infection. The interplay of viral factors and activated stromal cells as well as change to the extracellular matrix establishes a self-perpetuating cycle of fibrotic progression from chronic hepatitis to cirrhosis, and subsequently beyond into HCC.

Therapeutic strategies targeting TME in HBV-related HCC​​

Immune checkpoint blockade
Immune checkpoint blockade (ICB) is a critical therapeutic option for HBV-related HCC, due to its philosophy to counter cancer-induced immunosuppression through inhibition of the PD-1/PD-L1, CTLA-4, and TIGIT inhibitory pathways detrimental to T cell function [196]. Chronic HBV infection leads to T cell exhaustion which makes ICB an important consideration in this context PD-1/PD-L1 inhibition that blocks the binding between PD-1 on T cells and PD-L1 on tumor and immune cells to restore cytotoxic (CD8 +) T cell activity [197, 198]. PD-L1 expression is elevated in HBV-HCC, especially in tumor margins (such as LC11R cells), and correlates with immune evasion [198]. Several clinical agents such as nivolumab, pembrolizumab, and camrelizumab have been shown to be efficacious in HBV-HCC [199]. CTLA-4 inhibition depletes intratumoral regulatory T cells (Tregs) while enhancing effector T cells, thus offering an opportunity to complement PD-1 blockade through a non-redundant signaling pathways, as indicated by the ipilimumab and nivolumab combination therapy [200]. In addition, TIGIT is a novel immune checkpoint which is upregulated in HBV-HCC T cells and targeting by dual inhibition with PD-1 is a potential area of opportunity [201]. There is increasing clinical evidence for the use of ICB in HBV-HCC; however, ICB monotherapy retains important limitations. For instance, nivolumab (CheckMate 040) reported an overall objective response rate (ORR) of 20% in advanced HCC, including a subgroup of HBV-infected patients [202]; meanwhile, pembrolizumab (KEYNOTE-224) reported an 18.3% ORR as second-line therapy [203]. Nevertheless, mechanisms of primary resistance such as tumor microenvironment (TME) immunosuppression and metabolic dysregulation impact the rate of response to treatment contributing to overall low response rates (~ 20%) [204]. Combinations have fared more favorably, such as atezolizumab (anti-PD-L1) in addition to bevacizumab (anti-VEGF), both of which are FDA approved as a first-line standard of care, whose IMbrave150 trial reported median overall survival (OS) in HBV-HCC with a median OS of 24 months [205, 206]. Dual checkpoint inhibition (nivolumab and ipilimumab) achieved a 31% ORR in CheckMate 040 leading to FDA approval [207]. Immune checkpoint inhibitors (ICIs) in combination with reasonably effective systemic therapy, such as lenvatinib and pembrolizumab, have demonstrated improved T cell infiltration and vascular normalization resulting in therapeutic benefit [208]. It is clear from this research that combinatorial approaches will provide the most therapeutic benefit in HBV-related HCC to mitigate resistance mechanisms.

Adoptive cell therapy
Adoptive cell therapy (ACT) is a highly promising immunotherapy for HBV-associated hepatocellular carcinoma (HCC). ACT has, however, to employ multiple methods to overcome the immunosuppressive tumor microenvironment. Tumor-infiltrating lymphocytes (TILs) are a naturally occurring immune cell from the resected tumor that is enriched for tumor-specific T cell receptors and can demonstrate notable cytotoxicity targeting HBV-HCCs through IFN-γ, granzyme B, and caspase-3-mediated apoptosis [58, 209]. TILs are especially efficient because TILs are subjected to less immune suppression than tumor-associated or tumor-reactive T cells. Nevertheless, the use of TILs as a treatment for HCC presents several obstacles, including the technical difficulties involved in isolating TILs and expanding them, and the general heterogeneity of TIL dependent on the PD-L1-high margin-derived HCC cells being affected by immune suppression [210]. Peripheral blood T cells (PBTs) offer further aerodynamic move; nevertheless, because of HBV-HCC infection, autologous PBTs show features of exhaustion, including PD-1 upregulation from prolonged viral infection [211]. If HBV-HCC PBTs are stimulated with IL-2, they can partially regain cytotoxicity if they are harvested from immune suppression in the tumor rather than loose immunosuppression [212]. Allogeneic PBTs are sourced from HLA-matched healthy donors but can show strong activity toward the margin-derived HCC from the non-HBV-HCC [213]. This efficacy of engraftment is entirely contingent on HBV-HCC margin-derived or resident, non-HBV metastases having been sourcing from autologous T cells from recipient than those from the original donor with opposite side T cells from donors [214]. Engineered T cell therapies, such as chimeric antigen receptor T cells (CAR-T) and T cell receptor-modified T cells (TCR-T), further traverse the ability of ACT. CAR-T cells designed to target tumor-associated antigens like glypican-3 (GPC3) or alpha-fetoprotein (AFP) as well as HBV-specific proteins like HBsAg have shown evidence of primary tumor regression in early-phase clinical trials [215–217]. That said, CAR-Ts are still impeded by on-target/off-tumor toxicity and poor persistence in the immunosuppressive TME. The TCR-T is perhaps further employed for substantial possibility to elicit recognition HBV-derived antigens through MHC, giving that the prevalence of HBV-infected hepatocytes can be targeted as well as HCC [218]. Currently, clinical trial data tracked by multiple authors provide preliminary evidence of ACT in HBV-HCC. GPC3-targeted CAR-T therapy (NCT02395250) has reported individuals with advanced HCC regress with partial responses to GPC3 CAR-T [219], and HBsAg-specific CAR-T cells continue to be evaluated [220]. Researches identified patients with a reduced post-resection recurrence from cytokine-induced killer (CIK) cells as ACT intervention [221]. There remain hurdles in ACT manufacture complexity for efficacy based on heterogeneity in TME, as well as a range of predictive biomarkers to identify probable optimally responding patients. Potential future directions include combinatorial strategies of ACT used in combination with immune checkpoint blockade, oncolytic viruses, and next-generation engineering strategies, such as CRISPR-edited T cells to improve persistence and efficacy.

Targeting immunosuppressive cells
Myeloid-derived suppressor cells (MDSCs) provide a practical way for tumor cells to evade immune recognition through multiple mechanisms, including depletion of L-arginine through arginase-1 (ARG1) to prevent T cell proliferation, production of nitrous oxide (NO) and reactive oxygen species (ROS) inducing T cell apoptosis, and secretion of immunosuppressive cytokines (e.g., IL-10, TGF-β) promoting regulatory T cell (Treg) expansion in the immune-tolerant environment [222]. Evidence emerges in preclinical and clinical trials that therapies targeting MDSCs are effective. For example, CSF-1R inhibitors (e.g., pexidartinib) effectively inhibit the recruitment of MDSCs, while treatment with CCR2/CCR5 antagonists (e.g., maraviroc) blocks the chemokine sprout in the migratory process [223]. Other examples of therapeutic agents include STAT3 inhibitors (e.g., napabucasin), which prevent the survival of MDSCs, and all-trans retinoic acid (ATRA), which promotes the differentiation of MDSCs into mature myeloid cells that do not suppress T cell function [224]. Preclinical studies support evidence of CSF-1R blockade reducing growth of tumors in HCC models [225]. The CCR5 inhibitor, maraviroc, is currently being evaluated as a combined therapy with nivolumab, which highlights the translational aspect of inhibiting MDSCs [224]. Overall, there is a great need to eliminate the immunosuppressive effects of MDSCs to increase the efficacy of immunotherapies in HCC.
Tregs have the ability to suppress effector T cell function through various mechanisms; for example, Tregs can inhibit the activation of dendritic cells, and the priming of CD8 T cells in a CTLA-4- and ICOS-dependent manner, and produce and secrete suppressive cytokines, such as IL-10 and TGF-β, to promote immune tolerance, and express inhibitory receptors (PD-1 and LAG-3) to exhaust tumor-infiltrating lymphocytes (TILs) [212]. There are numerous treatment strategies that can overcome Tregs immune suppression in HCC. CTLA-4 blockade with agents such as ipilimumab has been demonstrated to result in depletion of Tregs in tumors [175]. Administering anti-ICOS antibodies, such as vopratelimab, can result in loss of viability of Tregs [226], and OX40 agonists, such as MEDI6469, can induce reprogramming of Tregs to a Th1-like phenotype [227]. The use of low-dose IL-2 treatment preferentially expands effector T cells with lower Tregs activation [212]. The clinical outcomes for these strategies are hopeful; dual PD-1/CTLA-4 inhibition (nivolumab, ipilimumab) improved objective response rates (31%), in comparison with nivolumab alone, in the CheckMate 040 clinical trial [228]. The additional clarified correlation between ICOS Tregs and poor prognosis led the investigator to further investigate ICOS-targeted therapies, as a limitation of Treg function, in current early-phase clinical trials [229]. Together, the clinical evidence provides justification for Tregs as an important therapeutic target for altering immune evasion in HCC and provides rationale for modifying Tregs immunosuppressive properties as a class of novel therapeutic agents. The success associated with the clinical use of checkpoint inhibitors in combination (i.e., nivolumab and ipilimumab) encourages the future consideration of therapies that target Treg function and activity as an innovative strategy to augment tumor immunity in HCC.
Tumor-associated macrophages (TAMs) have an arsenal of immunosuppressive capabilities, which include PD-L1 expression which directly inhibits the function of cytotoxic T cells, VEGF secretion to promote tumor angiogenesis, and remodeling of the extracellular matrix to promote metastasis [78]. The M2-polarized phenotype of TAMs almost exclusively works to establish an immune-permissive niche for tumor growth and immune evasion. Several approaches for targeting TAMs in HCC have evolved, including CSF-1R inhibitors like cabiralizumab to deplete M2-like TAMs, TLR agonists such as resiquimod to repolarize macrophages to a tumoricidal M1 phenotype, and CD40 agonists such as selicrelumab to promote cross priming between dendritic cells and T cells [230]. TGF-β inhibitors such as galunisertib can mitigate the fibrotic tumor microenvironment organized by TAMs [231]. The clinical experience is building with CSF-1R inhibitors combining with anti-PD-1 therapy in early-phase trials. A clinical trial is of interest as it looks at TGF-β inhibition with atezolizumab, which is clinically relevant with the context of TAM targeted modalities in HCC [205]. The studies previously mentioned begin to highlight the role that TAMs have in creating an immunosuppressive tumor microenvironment and confirm the treatment strategies that extend beyond immuno-oncology and instead target TAMs directly. The clinical evolution of TAM targeting immunotherapy that is already clinically established is a compelling option to reverse immune resistance in HCC. There is much potential and patients hope that studying macrophage biology and its interaction with other immune modulatory factors could offer increased therapeutic opportunities for patients with HCC.
Over the last few years, neutrophils, and neutrophil extracellular traps (NETs) have become important to the understanding of hepatocellular carcinoma (HCC) and the progression of tumors and tumor immunosuppression. So far, numerous therapeutic strategies have been reported in targeting neutrophil-mediated effects in HCC. The use of DNase I is a relatively direct way to degrade NETs that are already established [103]. Small molecule inhibitors of NDUFA4L2/IL33/PADI4 (e.g., GSK484) are another way of inhibiting the development of NETs at the site of formation [232]. S100A9 inhibitors like tasquinimod can also be useful in blocking the TLR4/ROS signaling pathway that sustains the immunosuppressive tumor microenvironment [233]. Most patients with HCC have shown significant association with disease progression. Indeed, in reports associating circulating NET markers (MPO-DNA complexes) with patient outcomes, elevated levels have been associated with significantly worse outcomes [103]. Preclinical studies working with these targets have established drugs that can provide significant therapeutic efficacy. For example, delivery of DNase I reduced HCC tumor growth in experiments and animal models [234]. The studies aimed at understanding neutrophil biology, in determining that neutrophils are important mediators of tumor progression and resistance to therapeutic interventions. The primary focus of any of the different therapeutic strategies has been on neutralization of neutrophil-derived factors such as NETs and other inflammatory mediators. In the future, it may be possible to find actionable targets in neutrophil activation pathways. For clinical application of the therapeutic strategies in targeting neutrophils in conjunction with immunotherapy, this approach may be easier because associations exist linking neutrophil activity and progression of disease in patients with HCC. Future studies investigating combination therapies while targeting neutrophils along with immunotherapies may enable improvements in treating this malignancy.

Stromal modulation
One of the most exciting strategies for stromal modulation in hepatocellular carcinoma (HCC) is the targeting of cancer-associated fibroblasts (CAFs). A central therapeutic strategy is the inhibition of TGF-β with either galunisertib (a TGF-βR1 inhibitor) or fresolimumab (an anti-TGF-β antibody), which together block the activation of CAFs and deposition of ECM and Treg suppression [97, 235]. Specifically, TGF-β inhibition will synergize with immune checkpoint inhibitors (ICIs), in active clinical trials (NCT02423343), as they will enhance T cell recruitment to tumors [236]. Another important therapeutic strategy is LOXL2 inhibition, with either simtuzumab (anti-LOXL2 antibody) or PXS-5505 (an oral LOXL2 inhibitor), which would impair collagen cross-linking and potentially lead to vascular decompression and better drug access into tumor tissue [237, 238]. Similarly, the exploration of FAP-targeted therapy with a preclinical focus on CAR-T cells or other radioligands for specifically depleting CAFs encourages research in HCC [239]. The concern of off-target toxicities because of FAP expression in non-malignant tissues is also a substantial consideration, so if designed appropriately, it will rely on appropriate design and patient selection. Each of these therapies is representative of the various ways researchers have targeted CAFs and modified the tumor stroma in HCC.
Reassigning the extracellular matrix (ECM) is a significant therapeutic target in hepatocellular carcinoma, particularly by targeting matrix metalloproteinases (MMPs). We considered MMP inhibition with both broad-spectrum MMP inhibitors like marimastat and MMP-9-specific inhibitors like andecaliximab [240]. MMP inhibitors derive their function, in part, from normalizing the tumor's ECM architecture, thereby reducing the potential for metastasis and facilitating the infiltration of immune effectors in the tumor microenvironment [241]. MMP inhibitors also revert the deposit of abnormal ECM material or stroma deposit seen in HCC, which constructs a physical barrier for both drug and immune effector access. Another, novel approach with some promise is the enzymatic degradation of key ECM components (e.g., hyaluronan, the glycosaminoglycan scaffold of the ECM) that results in stiffer tissue [242]. PEGylated hyaluronidase (PEGPH20) has been shown to effectively degrade hyaluronan and decrease the presumably PD-1-mediated mechanical resistance of the tumor stroma [243]. Collectively, reducing tissue stiffness allowing for better flux of drugs within the tumor and changing the biomechanical and electrical stimulation that drives the pathological malignant phenotype. In short, these myriad strategies for targeting the ECM include distinct biological modalities to bypass physical and chemical limits to improve the efficacy of both traditional therapies and newer immunotherapy-based strategies in the context of HCC.
Anti-angiogenic therapy has gained prominence in treatment of hepatocellular carcinoma with notable clinical contributions due to VEGF pathway blockade. Anti-VEGF strategies included agents like bevacizumab, a monoclonal antibody that targets VEGF, and lenvatinib, a multi-kinase inhibitor of VEGFR/FGFR where the effects, targeted through vascular abnormalization rather than destruction, when used in conjunction with immune-directed strategies, demonstrated enhanced perfusion to tumor vasculature and T cell infiltration into the tumor microenvironment that replicate the tumor, circumstances implicating favorable parameters for immune-mediated tumor destruction as a result of vascular normalization [244]. Translation of this strategy has been validated in the IMbrave150 regimen consisting of atezolizumab (anti-PD-L1) plus bevacizumab which ultimately became FDA approved due to establishment of a new standard of care by adding vascular deviation to immune checkpoint inhibition [245]. More recently, dual targeting has gained momentum in angiogenesis/stromal directed therapy with nintedanib, an inhibitor of VEGFR, PDGFR, and FGFR, which can inhibit tumor vasculature and cancer-associated fibroblasts (CAFs) targeting anti-angiogenic and immune checkpoints and significantly alter treatment response by targeting multiple dimensions of the tumor microenvironment [246]. When targeting multiple agents together such as anti-VEGF agents inhibiting angiogenic signaling and CAF activation simultaneously, the tumor microenvironment created is more supportive of immune cell functions while targeting communication supporting the underlying tumor. The exploration of these combination approaches signifies increasing recognition of dynamic interaction associated with targeting vascular biology, stromal morphology, and immune regulation in hepatocellular carcinoma which can broaden and enhance efficacy in treatment.

Cytokine therapy
Interferons (IFNs), and specifically IFN-α multifunctional medicine, are involved in the management of HBV-associated HCC by acting as both an antiviral and immunomodulatory agent and having direct anti-tumor effects. PEGylated IFN-α has strong antiviral effects and decreases HBV DNA and HBsAg, which correlates with a reduced risk of HCC recurrence after surgical resection [247, 248]. In addition to its antiviral properties, IFN-α also has essential immunomodulatory effects, enhancing antigen presentation through dendritic cell activation, enhancing cytotoxic CD8 T cell responses, and inhibiting MDSCs that mediate immune evasion [249]. IFN-α also has direct effects on tumor cells, inducing apoptosis and arresting the cell cycle, which gives IFN-α a unique anti-tumor mechanism of action. The therapeutic role of IFN-α has been confirmed clinically in HBV-HCC, with adjuvant treatment post-surgery demonstrating a significant improvement in median overall survival (63.8 vs. 38.8 months) as reported by Sun et al. [250]. More recently, studies have shown that IFN-α was able to work synergistically with PD-1 inhibitors by modifying glucose metabolism in the tumor milieu, which gave a 40% objective response rate in unresectable HCC [251]. The use of IFN-α in the clinic can be limited because of the toxicity, including fatigue or cytopenia, and heterogeneity in patient responses [252]. Given these challenges, a biomarker-driven strategy should be used to pinpoint optimal patients likely to benefit from IFN-α treatments while limiting adverse effects. A more detailed understanding of predictive biomarkers should aim to provide optimal treatments to improve patient outcomes.
Interferon-gamma (IFN-γ) is a major immunomodulatory cytokine within hepatocellular carcinoma (HCC) that has both immune-mediated activation and direct tumoricidal functions. The cytokine varies in many immunostimulatory roles including the activation of macrophages and upregulation of MHC class I and II, thereby helping antigen presentation and T cell recognition of tumor cells [253]. While these are immune-related effects, IFN-γ also has direct anti-tumor effects by inducing ferroptosis in HCC cells through oxidative stress pathways during tumor-infiltrating lymphocyte (TIL) co-culture systems [254]. These multifaceted roles have led researchers to examine IFN-γ as a preferential predictive biomarker of clinical responses to immune checkpoint inhibitors (ICIs), and it has been reported that high levels of IFN-γ are associated with improved clinical outcomes to PD-1/PD-L1 blockade therapies [255]. Since IFN-γ has diverse effects on both immune effector function and tumor cell viability, this cytokine is an important component in the HCC tumor microenvironment and is a possible therapeutic target.
Interleukin-2 (IL-2) is an important immunological agent that can influence immune interactions in the context of hepatocellular carcinoma by stimulating proliferation and activation of effector T cells and natural killer (NK) cells. Its therapeutic potential is especially relevant when administered at low dose, which seems to preferentially expand the cytotoxic CD8 T cell population while limiting expansion of the immunosuppressive regulatory T cells (Tregs) [64, 256]. This approach to selective immunomodulation has also rendered IL-2 of interest in combination therapies testing to augment anti-tumor immunity. Clinically, IL-2 has had meaningful use in adoptive cell therapy (ACT) where it is used to support and sustain activity of expanded tumor-infiltrating lymphocytes (TILs) or peripheral blood T cells (PBTs) in ACT protocols [257]. However, IL-2 has limitations as a therapeutic agent, especially when given at higher doses. Limitations to therapeutic IL-2 use can include paradoxical activation of Tregs (which would ultimately lead to inconsistent immune activation) to bothersome severe toxicities (including capillary leak syndrome), and all this has complicated the use of dosing schedules and heterologous dosing combinations to improve the therapeutic index of IL-2 in HCC.
Transforming growth factor-beta (TGF-β) is an important contributor to an immunosuppressive environment within the tumor through fibrosis, immune suppression, and regulatory T cell (Tregs) recruitment [258]. Collectively, these effects create hurdles to effective anti-tumor immunity and thwart e­fficacy of current therapies. Galunisertib, a selective TGF-β receptor inhibitor, has effects that limit the effects of TGF-β on modulating fibrosis in the tumor microenvironment and impede immunosuppressive pathways [259]. Preclinical and clinical studies have also suggested that inhibition of TGF-β can work in synergy with immune checkpoint inhibitors (ICIs) to overcome mechanisms of resistance and maximize the full benefit of single-agent immunotherapy [260]. Thus, the combination of TGF-β inhibition and ICIs will use TGF-β blockade for tumor stroma remodeling in conjunction to promote T cell-mediated anti-tumor immunity. Using this approach has the potential to improve clinical outcomes in patients with HCC.

Metabolic reprogramming of the TME

Glycolysis and lactate-driven immunosuppression​
HBV-related hepatocellular carcinoma (HCC) cells show extreme reliance on the Warburg effect, which is increased aerobic glycolysis producing excess lactate via lactate dehydrogenase (LDH) activity [261]. This metabolic pathway is associated with TME acidosis, which conveys severe inhibit in cytotoxic T cell and NK cell function in addition to the promoting of immunosuppressive M2 macrophage polarization. There are several treatment options, including LDH inhibitors such as FX11 decreasing lactate production and thereby limiting TME acidosis and immunosuppression, and hexokinase-2 (HK2), a rate-limiting glycolytic enzyme that is overly expressed in HCC cells [262]. Since HK2 is expressed in HCC, there are plenty of inhibitors that are suitable candidates for inhibition of glycolytic flux for anti-tumor treatment, particularly 2-deoxyglucose [263]. Interestingly, also using immune checkpoint inhibitors (ICIs) that target PD-1/PD-L1 offers a co-inhibitory mechanism in addition to glycolysis inhibitors like metformin and represents dual action treatment [251]. More evidence suggests that ICIs targeting PD-1/PD-L1 used in conjunction with glycolysis inhibitors were able to lessen lactate induced PD-L1 upregulation in the TME and this reduced expression enhanced T cell function [264]. Overall, these approaches are seeking to restore the TME metabolomic landscape by reprogramming it appropriately against the tumor and reinstating anti-tumor immunity.

Lipid metabolism and immune evasion
Chronic HBV infection leads to profound changes in lipid metabolism that facilitate immune evasion and contribute to tumor development in hepatocellular carcinoma (HCC). The HBV virus stimulates hepatic steatosis and promotes lipid loading in hepatocytes, creating a pro-tumorigenic context within the liver that allows for increased β-oxidation, which indirectly promotes HCC cell growth in the liver microenvironment while inhibiting the function of CD8 T cells [48, 265]. This reprogramming of hepatic lipid metabolism is compounded by the effect of the virus, which alters the liver and likely produces immunosuppressive lipid mediators like prostaglandin E2 (PGE2), and cholesterol esters to recruit more immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) into the immune contexture [266]. There are a variety of targeted therapies designed to offset the immunosuppressive properties of these lipids. FXR agonists like obeticholic acid have shown promise in treating HCC because it appears to reduce lipotoxicity and restore normal bile acid metabolism and may relieve HBV-induced metabolic dysfunction [267]. ACC/ACLY inhibitors like ND-630 inhibit hepatocyte lipogenesis and represents a strategy that blocks a tumor's lipid biosynthesis blocking key pathways that allow tumor cells to survive [268]. PPAR-α/δ antagonists inhibit the appearance of lipid droplets in cancer-associated fibroblasts (CAFs), which allows for a better understanding of how CAFs aid in immune dysfunction in HCC [269]. These interventions represent a multi-pronged approach to understanding this multifactorial disease that consists of virus-induced metabolic processes and impaired immune processes resulting in HBV liver cancer.

Amino acid deprivation and immune dysfunction
The tumor microenvironment in HBV-related HCC has extreme dysregulation of amino acid metabolism, which exacerbates immune dysfunction. Arginase-1 (ARG1) and indoleamine 2,3-dioxygenase 1 (IDO1) are often overexpressed in myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs), thereby locally depleting arginine and tryptophan and strongly suppressing T cell, natural killer (NK) cell, and other immune cell functions [270]. To mitigate these immune-suppressive effects, IDO1 inhibitors like epacadostat have been developed to restore tryptophan levels and improve T cell function, while PEGylated arginase inhibitors like CB-1158 selectively inhibits ARG1 activity and reverses T cell exhaustion [271, 272]. Besides arginine and tryptophan metabolism, glutamine represents an important energy source to HCC cells through catabolism by glutaminase (GLS), which supports energy production, nucleotide synthesis, and relies on glutamine via anaplerotic processes [273]. The dependency of tumor cells on glutamine metabolism presents a cancer therapeutic vulnerability that can be exploited through GLS inhibitors (such as telaglenastat) [274]. GLS inhibitors target tumor cell metabolism and have been shown to have some augmentative effects when administered with anti-PD-1 therapy [275]. This suggests that GLS inhibition may reverse resistance to immune checkpoint blockade therapies. The therapeutic targeting of these amino acid metabolic pathways will provide multiple opportunities for restoring immune surveillance while also limiting tumor cell viability in HCC.

Mitochondrial reprogramming and OXPHOS inhibition
Hepatocytes infected with HBV undergo significant mitochondrial reprogramming with increased OXPHOS activity generating excess ROS and leading to liver fibrosis and an immunosuppressive tumor microenvironment. The metabolic shift enhances HCC progression and leads to excess generation of ROS through oxidative stress-mediated pathways that inhibit anti-tumor immune responses [276]. Treatment options to target mitochondrial reprogramming events have been shown to be promising in preclinical and clinical settings [277]. Metformin, an established activator of the AMP-activated protein kinase (AMPK), exerts its anti-tumor activity signaling by the inhibition of mitochondrial complex I activity, preventing the proliferation and growth of HCC cells [278]. There are even ROS scavengers (e.g., N-acetylcysteine) that relieve oxidative stress and mitigate negative effects, promoting T cell infiltration and function in the tumor microenvironment [279]. These tactics take advantage of the metabolic sensitivities of HCC cells and immunologically restore effective tumor immune surveillance, by diminishing the immunosuppressive properties of aberrant metabolism [280]. Mitochondrial therapies added to current treatment strategies in HCC may also have additive effects and may be of particular benefit in the case for HBV-mediated HCC, whose etiologic agent is directly responsible for the reprogrammed metabolism. By targeting aberrant mitochondrial metabolism with anti-tumor and immune-suppressive implications, these strategies may present a unique opportunity to enhance treatment in HCC patients.

Hypoxia and HIF-1α signaling
The hypoxic tumor microenvironment (TME) in HBV-related HCC has activated hypoxia-inducible factor 1-alpha (HIF-1α) which causes elevated levels of vascular endothelial growth factor (VEGF) and programmed death-ligand 1 (PD-L1) that impair T cell activity [150]. The hypoxic signaling pathway generates an immunosuppressive niche for tumor immune evasion. Therapeutic strategies targeting hypoxia contain inhibitors of HIF-1α such as PT2977 which normalize TME oxygen levels and improve the response to immune checkpoint inhibitors (ICIs) [281]. Furthermore, utilizing anti-angiogenic agents such as bevacizumab (anti-VEGF) with ICIs, as shown in the IMbrave150 regimen, will allow for vascular normalization and increase T cell infiltration at the tumor sites [282]. Emerging metabolic targets in HBV-HCC include several solute carrier (SLC) transporters, primarily SLC35F1 (a nucleoside transporter) and SLC7A11 (a cystine/glutamate exchanger) both of which are frequently upregulated in HCC and contribute to tumor progression [283, 284]. SLC7A11 inhibition with agents such as sulfasalazine causes ferroptosis in HCC cells via redox dysregulation and de-initiation of glutathione (GSH) synthesis. Recently, it has been suggested by preclinical studies that ketogenic diets can mitigate the Warburg effect and enhance ICIs response by changing tumor metabolism and glucose availability [285]. These attacks on metabolic vulnerabilities to overcome resistant therapy and improve outcomes in HCC patients are novel strategies.

Combination immunotherapy
Chronic HBV infection worsens immune suppression through continued viral antigen presentation, and epigenetic silencing of immune genes. Monotherapy immunotherapies, in particular PD-1 inhibitors, show little clinical utility with objective response rates of only 15–20% due to intrinsic resistance mechanisms [286]. Combining immune strategies overcome limitations of monotherapies, through three interdependent mechanisms of action [287]. First, immune reactivation strategies combine immune checkpoint blockades with cytokine modulation to restore T cell function. Second, TME reprogramming approaches are to target both angiogenesis and stroma so the tumor ecosystem could be normalized. Third, HBV-specific strategies consist of antiviral agents and therapeutic vaccines which seeks to target the viral cause of HCC. These multidimensional strategies are considered in parallel by addressing the complex immunosuppressive networks limiting efficacy of monotherapy in the context of HBV-related HCC.
The synergistic use of immune checkpoint inhibitors (ICIs) with anti-angiogenesis therapies looks to be a promising synergistic approach to HCC therapy. For example, VEGF inhibitors (such as bevacizumab and lenvatinib) normalize the tumor vasculature and increase T cell infiltration into the tumor microenvironment (TME). In addition, PD-1/PD-L1 blockade paired with anti-angiogenesis therapy creates beneficial anti-tumor immunity through T cell exhaustion reversal. There are many trials, including atezolizumab with anti-VEGF therapy (bevacizumab), that show the combined adverse efficacy of anti-angiogenic and immunotherapy with HCC [288]. The IMbrave150 trial established the first-line standard treatment atezolizumab plus bevacizumab in patients with advanced HCC, with a median overall survival (OS) of 19.2 months vs 13.4 months on sorafenib treatment [289]. The subgroup of patients with HBV-HCC had a median OS of 24 months with atezolizumab plus bevacizumab compared with 13.4 months on sorafenib. The LEAP-002 trial showed a noted increase ORR (36% ORR) in patients using lenvatinib plus pembrolizumab vs (18% ORR) with lenvatinib alone [208]. Also, the increased efficacy with the dual blockade of PD-1 and CTLA-4 pathways will show better treatment strategies through a synergistic approach. CTLA-4 blockade will contribute to the anticancer response by decreasing immunosuppressive Tregs recruited into the tumor while continuously priming the T cell response, while PD-1 blockade only boosts the activity of T cells. In the CheckMate040 trial for second-line treatment of HCC patients on nivolumab plus ipilimumab, there was a reported ORR of 31% and the FDA approval followed228, while the oncologist has similarly huge data on the HIMALAYA study with tremelimumab plus durvalumab and improves OS (tremelimumab plus durvalumab [16.4 months] vs sorafenib [13.8 months]) [290] that can be shown to reinforce an important strategy in the clinic. The addition of ICIs plus locoregional treatments (or ablation) may be able to exploit the above-mentioned T cell and immune effector synergistic mechanisms, as locoregional treatment causes immunogenic death of the tumor cell inducing the release of tumor antigens that can run away with potentially systemic immune responses. Also, through the T cell activation mechanism inflammation is stimulated when ablation occurs to activate the dendritic cells required for T cell priming. In the CHANCE001 study, the combination of TACE and camrelizumab leads to a significantly improved PFS (10.5 months) vs TACE alone (5.1 months) [291, 292]. There are ongoing studies testing RFA and nivolumab and TACE and camrelizumab combination treatments for early-stage HCC testing recurrence. The combination of ICIs plus HBV-specific treatments will allow for targeting the distinct immune-suppressive effects of chronic HBV infection in the context of HCC. For example, nucleos(t)ide analogs (NAs) such as entecavir and tenofovir can decrease HBV replication, which decreases viral-driven immune dysfunction [293]. HBV-specific treatments with each known one using siRNA (JNJ-3989) showed patients had very low levels of HBsAg which allows T cells to regain restored function [294]. Clinically shown with HBV-HCC patients that CD8 T cell responses can be improved with pembrolizumab (anti-PD-1) and entecavir. To the extreme early-phase trials using the therapeutic HBV vaccine GS-4774 (therapeutic HBV vaccine) shows major immune activation with nivolumab showing persistent immune activation [295]. Lastly, combinations of ICIs with metabolic or stromal modulators target the TME to reprogram TME. For example, IDO inhibitors such as epacadostat may change the level of tryptophan depletion to reduce T cell immune suppression, while TGF-β inhibitors (galunisertib) alter recruitment of stromal CD68 + MDSC and reduce fibrosis [231]. Phase II studies with durvalumab plus galunisertib showed significant TME remodeling, and world even working on CCR5 inhibitors such as maraviroc (in combination with anti-PD-1 therapy) showed limited MDSC decrease recruitment in preclinical studies [231]. All the translational approaches suggest investigating dual mechanisms of action, for example, even in metabolic and stromal pathways, strengthens the opportunity for an integrated approach to support continued efficacy for immunotherapy in HCC (Table 1).

Challenges and future directions​​
Even with significant strides in knowledge regarding HBV-induced tumor microenvironment (TME) modifications and targeted therapies, several challenges persist. The heterogeneity of HBV-HCC tumors and immune microenvironments limits their potential treatment efficacy, thereby requiring patient-tailored approaches. Similarly, therapeutic resistance because of compensatory resistance pathways and persistence of HBV cccDNA emphasizes that combined antiviral and immunomodulatory approaches are critical. The dynamic evolution of inflammatory signatures and fibrotic signaling patterns in HBV-HCC further complicates treatment, with distinct molecular profiles emerging across disease stages [296]. Early fibrosis (F0-F1) contains dominant pro-inflammatory transcriptional modules that drive immune activation and on the diagonal fibrotic remodeling through expression of TGF-β1 and TIMP1 and is the first phase of initiating ECM deposition. In intermediate fibrosis (F2-F3), the inflammatory signature shifts from pro-inflammatory transcriptional modules to chromatin-remodeling factors that sustain chronic inflammation, which triggers accelerated fibrogenesis, while peak SMAD binding occurs to TGF-β1 and TIMP1, along with peak ECM accumulation that drives these processes. For advanced cirrhosis (F4), once dominant early pro-inflammatory modules are downregulated, PRC1/2 complex activity persists to create a fibro-inflammatory niche; despite reduced TGF-β1/TIMP1 signaling and continuing the signaling of ECM remodeling, permanent ECM deposition occurs that supports irreversible scarring.
The complex, heterogeneous nature of the TME, encompassing fibrosis, hypoxia, and immunosuppressive networks, presents additional challenges for drug delivery and immune activation, compounded by the current lack of reliable predictive biomarkers. Safety concerns further complicate treatment, especially for cirrhotic HBV-HCC patients receiving combination therapies. Future directions involve new combination agents using TGF-β inhibitors + ICIs; engineered immune cells that target HBV antigens, a component of the immune TME; metabolic approaches using immune-modulating agents to overcome immunosuppression; and potential microbiome modification and early prophylactic immunotherapy (for example, in immunotyping patients at high risk for HBV infections). Solving these issues will require multidisciplinary collaboration between basic, clinical scientists, and AI, as we aim to improve outcomes for patients with HBV-related HCC.

Conclusion
Persistent HBV infection induces hepatocellular carcinoma (HCC) mediated by the viral oncoproteins, immune escape, and metabolic reprogramming of the tumor microenvironment (TME). Currently, there are several options for anti-HCC therapies available that demonstrate clinical activity; however, they are hindered by the ability of HBV cccDNA to persist in liver cells and the heterogeneity of the TME. Future studies should consider combining anti-HCC immunotherapy with other models of TME metabolism, epigenetic therapy, and personalized biomarkers or approaches to improve potency and efficacy. Mechanisms involved in HBV-specific TME will be important for advancing therapy for HCC.