Chemokine (C-C motif) ligand (CCL) 5 and Chemokine (X-C motif) ligand (XCL)1/2 synthesized and secreted by NK cells are required for early intratumoral cDC1s accumulation and antitumor immunity, however, tumor-derived prostaglandin E2 (PGE2) can disrupt the NK-DC axis (26). In the mouse tumor model, it was found that PGE2 not only inhibited NK cells from secreting chemokines but also induced downregulated expression of chemokine receptors on cDC1, which limited the accumulation of cDC1 in tumor tissues and failed to activate sufficient anti-tumor immune responses, ultimately leading to tumor immune escape (26). However, whether the same mechanism exists in human HCC remains to be further verified, and the specific molecular mechanisms by which tumor-derived PGE2 interacts with NK cells or cDC1s also need to be further explored. In addition, hypoxia induces the production of hypoxia-inducible factor (HIF)-1, a protein that contributes to the heterogeneity of the TME and is linked to the evolution of malignancy in HCC (27). And the expression of the innate immune checkpoint CD47 molecule is regulated by HIF-1α (28). Commonly overexpressed in cancer cells, CD47 is known as a protein that transmits “do not eat me” signals, preventing phagocytosis by DCs and macrophages through the interaction with the signal regulatory protein (SIRP) (29). In addition, Shuai Wang et al. found that CD47 upregulation coincided with reduced CD103⁺ DC and NK cell counts and was linked to a poor prognosis (30). Consistently, the blockage of CD47 increased NK cell activation and recruitment in an orthotopic liver tumor model because of the secretion of chemokine (C-X-C motif) ligand (CXCL) 9 and IL-12 by CD103⁺ DCs and this effect was reversed by CD103⁺ DC depletion (Batf3-/-mice) and IL-12 blocking in vivo (30). CD47 may partly explain HCC immune evasion and is a promising therapeutic target.

Moreover, tumor cells-derived IL-10 and IL-6, transforming growth factor (TGF)-β as well as vascular endothelial growth factor (VEGF) prevent DCs maturation, showing a tolerant phenotype with downregulated expression of costimulatory molecules (31). VEGF, TGF-β, and alpha-fetoprotein (AFP) were discovered in the culture supernatant of Hepa1-6-1 expressing higher adhesion molecules, and the culture supernatant significantly suppressed the expression of CD86, CD80, and CD40 on DCs, especially CD86 (32). The cross-presenting capacities and immunomodulatory functions of these tolerogenic DCs with downregulated costimulatory molecules are impaired, failing to effectively activate effector T cells and thus resulting in tumor immune escape. Although the specific molecular mechanisms and signaling pathways that tumor-derived cytokines and growth factors induce downregulated expression of co-stimulatory molecules on DCs are currently unknown, it is undeniable that reversing tolerance DCs to functional DCs is indeed a potential immunotherapy approach.

Tregs can inhibit immune responses and are always as targets for the treatment of infectious diseases, autoimmune diseases, and cancers (33). Human leukocyte antigen-DR isotype (HLA-DR) expressed on the surface of cDC2 is a key antigen-presenting molecule for activating antitumor effector T cells. However, the level of HLA-DR on cDC2 significantly decreases in hypoxic HCC-TME, which impairs the antigen-presenting capacity of cDC2. Tumor tissue-derived cytokines such as CXCL5 and CCL2 make Tregs and cDC2s enrichment in hypoxic tumor tissue, and it is reported that direct interaction between Treg and cDC2 mediates the loss of HLA-DR on cDC2 (34). Notably, HLA-DR⁺ Tregs increased significantly along with the downregulation of HLA-DR on cDC2 surface and the levels of HLA-DR gene expression in both Treg and cDC2 were unchanged, which suggests that Tregs physically extract and ingest HLA-DR from cDC2s under hypoxia. Moreover, it has been demonstrated that these HLA-DR⁺ Tregs exhibit stronger immunosuppressive activity than HLA-DR^- Tregs in cervical carcinoma (35). Tregs-mediated downregulation of HLA-DR on cDC2 is a potential immunotherapeutic target for hypoxic HCC, and the antitumor effects of combination with other immunotherapies such as ICIs are expected.

According to studies, Tregs can directly downregulate costimulatory molecules CD80 and CD86 expression or prevent the upregulation of CD80 and CD86 on DC during DC maturation (36, 37), thus weakening the antigen-presenting ability of the DCs. The co-suppressive molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a transmembrane receptor on T cells, negatively regulates immune responses (38). The effect of CTLA-4 is achieved in part by competing for CD80 and CD86 mainly expressed on APCs with costimulatory molecules CD28 expressed on effector T cells to suppress antitumor immunity. It was previously found that Tregs could downregulate CD80 and CD86 by trans-endocytosis to downregulate their expression on DCs (39). Recent studies have shown that Tregs lacking CTLA-4’s extracellular fraction can also inhibit DCs expressing CD80 and CD86 and that extracellular CTLA-4 function is not crucial for downregulating CD86 and CD80 expression but essential for upregulating the expression of co-inhibitory receptor programmed cell death-ligand 2 (PD-L2) on DCs (40). This novel mechanism of Tregs-mediated DCs inhibition facilitates the discovery of new therapeutic methods to enhance antitumor immunity in HCC.

As intratumoral cDCs are also essential for T cell-based therapies, the low frequency and function of intratumoral cDCs may be partly responsible for the low response rate to ICIs in cancer patients. Hence, increasing the frequency and function of intratumoral DCs is the first step to trigger effective anti-tumor immunity and is probably feasible to combine with other ICIs for HCC treatment. Moreover, it is reported that DCs infiltration might predict the response to camrelizumab and apatinib and tumor recurrence in patients with resectable HCC (41).

Tumor cells occurring specific genetic mutations can reduce infiltration of CD8⁺T cells in TME through certain signaling pathways. For example, phosphatase and tensin homolog on chromosome ten (PTEN) inhibits the activation of PI3K signaling, and downregulation or deletion of PTEN leads to increasing PI3K-AKT pathway activity in multiple cancers, including HCC, thus accelerating tumor malignant progression (44, 45). Studies have found that PTEN loss in cancer cells suppressed the antitumor effect of CD8⁺T cells and reduced T cell transport to tumors in preclinical models of melanoma and was associated with reduced T cell infiltration at tumor tissue in patients (46). Reportedly, PTEN downregulation in the HCC mouse model reduced CD8⁺T cells infiltration in tumor tissue, along with increasing immunosuppressive Foxp3⁺CD4⁺CD25⁺Tregs and upregulating PD-L1 expression on tumor cells. Hence, recovering PTEN expression level in cancer cells can increase the infiltration degree and anti-tumor immune responses of CD8⁺T cells and reverse immunosuppressive TME.

Tumor-derived exosomes(TDEs) with PD-L1 on them inhibit CD8⁺T cells from proliferating and activating (47, 48). Lymphocyte function-related antigen-1 (LFA-1) is one crucial integrin on T cells, whose major ligand is intercellular adhesion molecule-1 (ICAM-1) (49). LFA-1 plays a crucial function in effector T cells destroying tumor cells by binding to related ligands expressed on tumor cells (50). ICAM-1 is present on tumor cell-derived exosomes as well, which can bind to leukocytes, thus preventing them from adhering to activated endothelial cells (51). Interferon (IFN)-γ upregulates ICAM-1 expression on tumor cells and ICAM-1 on TDEs mediates T cell inhibition principally by interacting with activated LFA-1 on CD8⁺ T cells (52). A previous study suggested that PD-L1 is of importance for TDEs-mediated CD8⁺T cells suppression (53). Nevertheless, there is a significantly reduced interaction between T cells and TDEs via PD-L1/PD-1 with the absence of ICAM-1, which indicates that the adhesion between tumor-derived extracellular vesicles (TEVs) and T cells mediated by ICAM-1/LFA-1 is a precondition for PD-1/PD-L1-mediated immunosuppression (52). Therefore, targeting TEV-derived ICAM-1 can improve the immune system of cancer patients and has the potential to greatly improve the efficacy of antitumor treatment. This mechanism exists in both melanoma and colon cancer models, while it requires further validation whether a similar mechanism exists in human HCC. Moreover, further analysis of TDEs is essential for understanding their protumor mechanisms and may contribute to developing TDEs-based therapeutic strategies.

In addition, tumor-repopulating cells (TRCs) are reported to promote programmed cell death-1 (PD-1) expression on CD8⁺T cells via transcellular kynurenine (Kyn)-aryl hydrocarbon receptor (AhR) signaling (54). Mechanically, TRCs are stimulated by interferon (INF)-γ to produce and secret more Kyn, the latter gets into neighboring CD8⁺T cells and activates AhR, consequently resulting in the upregulation of PD-1. Blockading Kyn-AhR pathway improves the antitumor effectiveness of adoptive T cell therapies.

MDSCs, a group of immature cells with high heterogeneity, originate from bone marrow and synthesize and secrete large amounts of immunosuppressive factors, playing crucial roles in suppressing antitumor immunity (55). Tumor-derived granulocyte-colony stimulating factor (G-CSF), IL-6, VEGF, and CCL2 cause MDSCs migration to HCC-TME (56). Two enzymes inducible nitric oxide synthase (iNOS) and arginase 1 (ARG1) are highly expressed in MDSCs and they cause the depletion of L-arginine, a conditionally essential amino acid related to T cells proliferation and differentiation (57, 58). Mechanically, L-arginine deficiency decreases the levels of CD3 ζ-chain indispensable for the assemble and stabilization of the TCR-CD3 complex on T cells, which weakens the antigen-recognition capability of T cells, as well as TAA-specific immune responses (59). In addition, arginine starvation impairs the formation of immune synapses between T cells and APCs through hindering the dephosphorylation of actin-binding protein cofilin (60). In general, arginine deprivation is one of the main mechanisms for MDSCs promoting HCC malignant progression.

A disintegrin and metalloprotease 17 (ADAM17), a membrane molecule expressed on MDSCs, prevents T cells from homing and being activated via interacting with L-selectin (CD62L) on T cells (61). Moreover, MDSCs express galectin(GAL)-9 which can interact with T cell immunoglobulin and mucin domain 3 (TIM-3) and consequently induce T cells apoptosis (62). MDSCs hamper the antitumor immunity of effector T cells through various methods, and controlling the expression of these MDSC-derived factors may greatly improve the antitumor immune responses and effects in HCC patients.

Innate lymphoid cells (ILCs) are a newly discovered family of immune cells that have similar cytokine-secreting profiles as T helper cell subsets and that are critical for host defense against infections and tissue homeostasis. It has been demonstrated that ILC3 lacking the natural cytotoxicity-triggering receptor (NCR^-ILC3) promoted the development of HCC in response to IL-23 highly expressed in HCC patients and associated with poor clinical outcomes. Furthermore, NCR^-ILC3 directly induced CD8⁺ T cell apoptosis and limited their proliferation by secreting IL-17 upon IL-23 stimulation (63).

A recent study has revealed that CD11b⁺F4/80⁺ macrophages in the liver metastatic TME are key drivers for inducing CD8⁺T cells apoptosis through Fas-FasL pathway (64). Whether similar mechanisms exist in primary liver cancer such as human HCC needs to be further explored. Reportedly, depleting FasL⁺CD11b⁺F4/80⁺ macrophages by using anti-CSF-1R is prospective but its clinical efficacy for immunomodulatory systemic therapies has not been demonstrated (65). The M2-polarization of macrophages induced by the CCL2 can suppress the proliferation of antitumor CD8⁺ T cells by secreting various cytokines, such as G-CSF, IL-6 and macrophage inflammatory proteins-2 (MIP-2) (66). Prospective studies are desperately needed to identify more reasonable strategies for combinatorial treatment to bypass hepatic resistance and improve the efficacy of systemic immunotherapy.

The research about the cell transplantation model established in immunocompetent mice suggests that HSCs prevent T cell infiltration in tumors (67). Furthermore, activated HSCs reduce responsiveness and cytotoxicity of T cells and increase apoptosis of them in vivo (68). Tregs have immunosuppressive activity and play key roles not only in maintaining body immune homeostasis but also in exhaustion of T cells and immune escape of HCC cells. As a kind of anti-inflammatory cells, Tregs can inhibit the response of T cells by producing IL-6, IL-17 and are connected with the poor prognosis of patients with HCC (69). Moreover, increased regulatory DCs induced by CAFs impaired T cell proliferation and promoted Treg expansion via indoleamine 2,3-dioxygenase (IDO) (70).

Previous study shows that circulating antigens are captured and cross-presented by LSECs, which contributes to CD8⁺ T cell tolerance rather than immunity (71). Subsequently, it is found that circulating carcinoembryonic antigen (CEA) was preferentially taken up in a mannose receptor-dependent manner and cross-presented by LSECs, but not DCs, to CD8⁺ T cells, which promoted the tolerization of CEA-specific CD8⁺ T cells in the endogenous T cell repertoire through the coinhibitory molecule B7-H1 (72). Moreover, a recent research demonstrates that overexpression of PD-L1 on LSECs inhibits the activation of CD8⁺ T cells and leads to immune evasion of HCC and poor prognosis (73).

In TME, complex cellular crosstalk leads to depletion and exhaustion of effector T cells, which weakens the effect of T cell-based therapies, such as ICI, chimeric antigen receptor T-cell immunotherapy. Hence, blocking various factors leading to T cell depletion and exhaustion is a prerequisite for effective treatment of HCC with other therapeutic methods.

HSCs are the main source of CAFs, which are crucial for HCC tumor development, metastasis, and treatment resistance (74, 75). It has been demonstrated that HSCs could be induced to transform to CAFs by HCC cells-derived exosomal miRNA-21 activating PDK1/AKT signaling that directly targets PTEN in HSCs (76). Furthermore, activated CAFs in turn promoted HCC malignant progression by secreting VEGF, matrix metalloproteinases (MMP) 2, MMP9, TGF-β and basic fibroblast growth factor (bFGF) (76). Similarly, exosomal miR-1247-3p derived from high-metastatic HCC cells (HMHs) in the lung metastatic niche reportedly triggered and stimulated β1-integrin/NF-κB signaling pathway in fibroblasts by directly targeting beta 1,4-galactosyltransferase, polypeptide 3 (B4GALT3) and activated CAFs in turn accelerated the development of HCC via producing IL-6 and IL-8 (77). A recent study reported that the palmitoylation of hexokinase 1 (HK1) is induced in HSCs after stimulated by TGF-β, thus more HK1 is secreted by forming large extracellular vesicles, which can be absorbed by HCC cells, causing enhanced glycolysis and HCC development (78).

It is found that the upregulation of connective tissue growth factor (CTGF), a matricellular protein secreted by hepatoma cells could activate nearby LX-2 cells (HSC line) and that the activated LX-2 cells promoted HCC cells proliferation by secreting IL-6 that activates STAT3 signaling in HCC cells (79). Similarly, it has been demonstrated that hepatoma cells induced LX-2 cells secreting more growth differentiation factor 15 (GDF15) in an autophagy-dependent manner to enhance hepatoma cells proliferation (80). Blocking the pro-tumor crosstalk between cancer cells and HSCs presents an opportunity for therapeutic intervention against HCC. It is well-known that forkhead box (FOX) proteins play critical roles in amplifying HCC malignancy. CAFs are found to induce FOXQ1 expression and FOXQ1/N-myc downstream-regulated gene 1 (NDRG1) axis is activated in tumor cells, which contributes to HCC initiation (81). Furthermore, the activation of FOXQ1/NDRG1 axis can recruit more HSCs to the TME as a supplement for CAFs via inducing pSTAT6/CCL26 signaling (81). The formation of positive feedback loop between CAFs and HCC cells unquestionably accelerates HCC initiation and development.

Nicotinamide N-methyltransferase (NNMT) modulates the metabolism of hepatoma cells and can be induced by activated HSCs. Reportedly, activated HSCs facilitate HCC invasion and migration through upregulating the expression of NNMT that alters the histone H3 methylation on 27 methylation pattern and transcriptionally activating CD44 in tumor cells (82). Although the molecular mechanism of HSCs inducing tumor cells to upregulate NNMT is still unclear, NNMT is a promising prognostic biomarker and therapeutic target for HCC. In addition, tissue inhibitors of metalloproteinases-1 (TIMP-1) secreted by HSCs is upregulated after stimulated by TGF-β, which triggers focal adhesion kinase (FAK) signaling by interacting with CD63 and contributes to proliferation and migration of HCC cells (83).

CD147, a transmembrane protein expressed highly in HCC is a key driver in the metastasis and development of tumor (84). A previous study revealed that CD147 highly expressed on HCC cells mediated the crosstalk between HCC cells and HSCs via activating HSCs characterized by high expression of α-smooth muscle actin (α-SMA), collagen I and TIMP-1 as well as increased secretion of MMP2, which in turn accelerated HCC malignant progression (85). STMN1 known as an oncogene is upregulated in breast cancer, non‐small cell lung cancer, and gastric cancer, which can induce cell differentiation, proliferation as well as invasion and migration in solid tumors (86, 87). Consistently, Rui Zhang et al. also found that STMN1 overexpression in HCC cells could promote cell proliferation, migration, drug resistance, and cell stemness in vitro as well as tumor growth in vivo (88). They also revealed that STMN1 is a bridge mediating complex crosstalk between HCC cells and HSCs by enhancing hepatocyte growth factor (HGF)/MET signal pathway and that STMN1‐induced PDGF secreted by HCC cells may be responsible for activating HSC to acquire CAF features and secrete more HGF (88). Thus, the positive feedback loop for mutual crosstalk between HCC cells and HSCs accelerates HCC malignant progression.

As a pro-inflammatory factor, Follistatin-like 1 (FSTL1) has been reported to promote various cancers malignant progression (89–91). Recent research found that FSTL1 mainly derived from CAFs in human HCC could promote tumor growth, metastasis, and therapy resistance by activating AKT/mTOR/4EBP1/c-myc pathway via binding to toll-like receptors 4 (TLR4) on HCC cells (92). It has been reported that c-myc plays a crucial role in hepatocarcinogenesis (93–95) and mTORC1 is vital for the progression of c-myc-driven HCC (96). FSTL1 expression is regulated by TGF-β1 in mouse pulmonary fibroblasts at both transcriptional and translational levels via Smad3/c-Jun pathway during fibrogenesis (97). The crosstalk between CAFs and HCC cells via TGF-β1 and FSTL1 signaling enhances HCC cells malignancy.

CXCL11 highly expressed by CAFs promoted HCC cells migration, whereas CXCL11 silencing decreased it (98). Concretely, CXCL11 stimulation upregulated circUBAP2 expression in tumor cells, and the later counteracted miR-4756-mediated inhibition on interferon-induced protein with tetratricopeptide repeats (IFIT)1/3 by sponging miR-4756, resulting in upregulation of IFIT1/3 expression that contributed to IL-17 and IL-1β expression, and elevated the migration capability of HCC cells. In addition, CAF-derived cardiotrophin-like cytokine factor 1 (CLCF1) improved HCC cells self-renewal ability through interacting with ciliary neurotrophic factor receptor (CNTFR) enhancing SOX2 signaling and increased CXCL6 and TGF-β expression in HCC cells via increasingly activating AKT-ERK1/2-STAT3 pathway (99). Moreover, CXCL6 and TGF-β induced CAFs to produce more CLCF1 via activating ERK1/2 signaling, thus forming a positive feedback loop to accelerate HCC malignant evolution (99).

Zhikui Liu et al. demonstrated that stiffness induced HSCs activation via CD36-AKT-E2F3 signaling pathway, driving activated HSCs to produce FGF2. Moreover, HSCs-derived FGF2 promoted HCC cells proliferation and metastasis through binding to FGFR1 on HCC cells to stimulate PI3K/AKT and MEK/ERK signaling pathways (100). Reportedly, Sox9/INHBB axis is upregulated in HCC and contributes to HCC development by driving the secretion of activin B to activate the peri-tumoral HSCs through activin B/Smad signaling (101). In addition, Keratin (KRT) 19 is positively associated with the aggressive phenotype of HCC and is upregulated by HSCs-derived HGF through activating c-MET and the MEK-ERK1/2 pathway in HCC cells (102).

The cross-talk between HCC cells and activated HSCs is considered to be important for modulating the biological behavior of tumor cells. We summarized the mediums or means and the corresponding results of the crosstalk between HCC cells and HSCs in Table 1 . It has been demonstrated that coculturing HCC cells with HSCs under hypoxic conditions enhanced their proliferation, migration, and resistance to bile acid-induced apoptosis compared to coculturing under normoxic conditions (103). How to block the crosstalk between HSCs and tumor and Inhibit cells and inhibit HSC activation deserve more attention in the future.

HSCs are important for MDSC-induced immunosuppression. A recent study found that activated HSCs could induce monocyte-intrinsic p38 MAPK signaling to enhance reprogramming for the development and immunosuppression of monocytic MDSCs (M-MDSCs) (104). MDSCs overexpressed MMP14 via CXCL10/TLR4 signaling. Several studies have demonstrated that overexpressed MMP14 contributed to tumor cells invasion and metastasis (105, 106). It is worth noting that Hui Liu et al. found a novel mechanism of M-MDSC motility that MMP14 regulated by CXCL10/TLR4 mediates M-MDSCs enrichment in liver graft, which promotes HCC recurrence after transplantation (107). Whereas it was found that blocking HSCs-induced intrinsic p38 MAPK signaling in monocytes inhibited the formation of MDSCs and their enrichment in fibrotic liver, which effectively inhibited HCC growth (104). Also, blocking CXCL10/TLR4/MMP14 signaling to inhibit MDSCs mobilization and tumor cells invasion and metastasis will present a great potential for developing novel treatment strategies against HCC malignant progression and recurrence. HSCs also play critical roles in regulating MDSCs migration in HCC. Another research about MDSC migration suggested that HSCs promoted MDSCs migration to HCC TME through SDF-1/CXCR4 axis (108). Hence, targeting activated HSCs in HCC is a potentially beneficial approach for modulating patients’ immune systems.

Endosialin, a transmembrane glycoprotein is demonstrated to mainly express in CAFs in HCC and allows CAFs to recruit macrophages though interacting with CD68 and induce M2 polarization of macrophages via regulating expression of GAS6 in CAFs (109). In addition, CAFs could induce macrophages polarize to M2-phenotype TAM (TAM2) and upregulate the expression of plasminogen activator inhibitor-1 (PAI-1) in them by secreting CXCL12, which augmented the malignant characteristic of HCC cells (110).

A lot of previous researches centered on linking various cells in TME with immunotherapy effectiveness. The latest study indicated that TME subtypes of HCC are associated to the immunotherapy efficacy by combining spatial transcriptomics with single-cell RNA sequencing (scRNA-seq) and multiplexed immunofluorescence of anti-PD-1-treated HCC patients (111). It suggested that the tumor immune barrier (TIB) structure consisting of SPP1⁺ macrophages and CAFs near the tumor boundary affected the therapeutic efficacy of ICIs. In addition, it further revealed that the crosstalk between SPP1⁺ macrophages and CAFs contributed to ECM remodeling and TIB formation, which led to reducing immune infiltration in the tumor tissue. Moreover, in vivo experiments have verified that SPP1 inhibition in mice with liver cancer resulted in better immunotherapy efficacy with anti-PD-1 (111). Although the molecular mechanism of SPP1-mediated crosstalk between SPP1⁺ macrophages and CAFs is not completely clear and the role of the crosstalk between SPP1⁺ macrophages and CAFs has not been verified in clinical trials, SPP1-blockading is promising for improving the efficacy of HCC treatment with ICIs.

As stroma cells in TME, activated HSCs and CAFs are the core factors that promote the formation of immunosuppressive microenvironment. They directly or indirectly promote the malignant progression of HCC and improve the resistance of tumor cells to immunotherapy. As the roles of HSCs and CAFs in HCC are extensive and complex, continue researches targeting them should not be slack in the future.