Single ICI may have limited efficacy in the treatment of HCC, but combinations of ICIs, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4, have shown excellent efficacy in tumors such as non-small-cell lung cancer (40), melanoma (41), and colorectal cancer (42). Yau et al. (21) tested the safety of combination therapy with nivolumab (NIVO) and ipilimumab (IPI) in patients with advanced HCC and found that the ORR in the combination group was twice as high as that in the NIVO monotherapy group, with a DCR of 49% and 24-mo OS rate of 40%. This encouraging result led to accelerated FDA approval of this combination for the treatment of HCC patients after sorafenib. Single-agent pembrolizumab and nivolumab plus ipilimumab are now approved as second-line treatments for patients with disease progression from first-line TKIs (43). Liu et al. (37) found that the up-regulation of TIM-3 and/or PD-1 expression on tumor infiltrating lymphocytes (TILs) weakened their functions and was closely related to the disease progression of HBV-related HCC. The anti-tumor effect of TILs was restored after blocking TIM-3 and PD-1, which provided new ideas for the combination of immune checkpoint inhibitors. Currently, dual blockade of LAG-3 with anti-PD-1 treatment is being tested in a Phase I trial (NCT01968109) (44).

Hepatic arterial infusion chemotherapy (HAIC) is an emerging therapy that has attracted much attention due to its high response rate and favorable survival for advanced liver cancer. In the Asian region, HAIC has been used as a sorafenib replacement therapy for patients with advanced HCC (45, 46). As a locoregional interventional therapy, HAIC not only maximizes tumor cell killing but also reduces systemic toxicity of chemotherapy agents through first-pass effects in the liver (47). Despite the promising efficacy of HAIC against unresectable tumors, its ability to inhibit extrahepatic metastasis remains unsatisfactory.

Mei et al. (22) investigated the effect of HAIC combined with anti-PD-1 immunotherapy (HAICAP) in advanced HCC and showed that patients in the HAICAP group had significantly better survival outcomes than those in the HAIC group. Muscular steatosis refers to the abnormal distribution of adipose tissue between and within muscle cells, which leads to excessive fat deposition in muscle, resulting in the decline of muscle mass, limb function and physical fitness. Yi et al. (23) evaluated the effects of anti-PD-1 immunotherapy and HAIC in HCC patients with varying degrees of muscular steatosis. The median PFS was 7.1 months and OS was 15.6 months. It was also found that patients with muscular steatosis had accelerated disease progression, increased levels of complications, prolonged hospital stay and poor prognosis after receiving treatment. A phase II study (24) focused on the clinical benefit of combining the anti-PD-L1 antibody camrelizumab with oxaliplatin-based chemotherapy (FOLFOX or GEMOX) in HCC patients and found an ORR of 26.5%, a DCR of 79.4%, a median TTR of 2.0 months, and a median PFS of 5.5 months. Camrelizumab plus FOLFOX or GEMOX chemotherapy is well tolerated and may provide a new option for patients with advanced HCC.

HCC is a highly vascularized tumor (48), and clinical trials have successfully shown the critical role of targeting VEGF-driven angiogenesis in HCC (49). Anti-angiogenic agents inhibits tumor growth by normalizing tumor vasculature and disrupting the hypoxic tumor microenvironment (50). In 2020, atezolizumab-bevacizumab combination became the standard first-line systemic treatment for advanced HCC for its significant efficacy (43) and other combinations are being explored. Shigeta et al. (25) used orthotopic transplantation or induced mouse models of HCC to examine the effect of anti-PD-1/VEGFR-2 combination therapy on survival. VEGFR-2/PD-1 dual blockade can promote the normalization of blood vessels, reprogram the immune microenvironment, and promote the anti-tumor immunity of HCC. A nonrandomized, open-label, phase II trial by Xu et al. (28) evaluated the efficacy of camrelizumab plus apatinib in patients with advanced HCC who were naive or refractory/intolerant to first-line targeted therapy. The results showed that the combination of camrelizumab and apatinib achieved effective efficacy in terms of ORR and OS and met the primary endpoint in both the first- and second-line cohorts.

Deng et al. (26) explored the optimal combination of anti-PD-1 antibody and TKIs in patients with unresectable HCC and the possible mechanism of combined therapy. They found that lenvatinib can reduce the expression of PD-1, CTLA-4 and Tim-3 on T cells promoted by VEGFA and basic fibroblast growth factor, and improve the cytotoxicity of T cells. The combination of anti-PD-1 antibody and lenvatinib has a more effective anti-tumor effect than sorafenib or BGJ398. An Ib multicenter open-label study of 100 patients was designed to assess the tolerability, safety, and efficacy of lenvatinib plus pembrolizumab in the treatment of unresectable HCC (27). In this study, lenvatinib plus pembrolizumab produced a definitive response rate, with a PFS of 9.3 months and a median OS of 22.0 months. This suggests that lenvatinib plus PD-1 inhibition and pembrolizumab have promising antitumor effects.

Anti-angiogenic agents combined with PD-1 or PD-L1 inhibitors have shown promising survival results in the treatment of unresectable HCC tumors. HAIC has also attracted attention for its high response rate and favorable survival in patients with advanced HCC. The combination of these three factors provides a new option for personalized treatment of different HCC patients.

A retrospective study was designed to compare survival among patients with advanced HCC who received HAIC plus lenvatinib plus a PD-1 inhibitor (HPL) with survival among patients who received lenvatinib plus a PD-1 inhibitor (PL) (29). The results showed that the survival outcome of patients in the HPL group was significantly better than that in the PL group, with a median OS of 15.9 months and a median PFS of 8.8 months, which were almost twice of PL group. All adverse events were assessed as mild and manageable, and no toxicity-related deaths occurred during follow-up. Liu et al. (30) investigated the efficacy and safety of HAIC combined with anti-PD-1 immunotherapy versus TKIs. Finally, the median PFS was 10.6 months, ORR was 63.0%, and DCR was 92.6% in the patients who received triple therapy, which was satisfying. Song et al. (31) evaluated the efficacy and safety of pembrolizumab, lenvatinib plus HAIC (PLH) versus pembrolizumab and lenvatinib (PL) in selected treat-naive uHCC patients. At the final follow-up, the median OS was 17.7 months in PLH group and 12.6 months in PL group. And the median PFS in the PLH group (10.9 months) was also significantly higher than that in the PL group (6.8 months), confirming that the combination of PD-L1 inhibitor, HAIC and lenvatinib can also improve survival rate.

Recent studies have found that the use of local radiotherapy may stimulate antitumor immune responses by increasing apoptosis and necrosis of tumor cells and subsequently increasing the expression of antigen presentation and immunomodulatory genes (51). This may improve the response to immunotherapy, increase efficiency and reduce adverse effects. The strategy of combining immunotherapy with radiotherapy has shown promising results in clinical and basic studies. PD-L1 circulating tumor cells (CTCs) can be used as a predictive biomarker in patients with HCC who receive PD-1 inhibitors in combination with intensive radiation therapy (IMRT) and antiangiogenic therapy (32). Zhong et al. (33) explored the safety and clinical efficacy of PD-1/PD-L1 inhibitors combined with palliative radiotherapy and antiangiogenic agents in the treatment of BCLC stage C HCC. The final ORR was 40.0%, the median PFS was 140 days, and the median OS was 637 days, which proved that PD-1/PD-L1 inhibitors combined with palliative radiotherapy and anti-angiogenesis therapy were reliable without unexpected adverse events. Additional studies exploring clinical benefit are needed.

TILs are lymphocytes that leave the blood and enter the tumor, and have a wide range of antigen recognition roles in tumor cells. Among them, T cell populations such as CD8 and CD4 play a key role in tumor control through mechanisms such as production of proinflammatory cytokines and promotion of plasma cell production (55, 56).

Ding et al. (57) conducted a meta-analysis of 7905 patients from 46 studies to evaluate the prognostic impact of TILs in HCC patients. The results showed that for TILs subsets, the density of CD8+, FOXP3+, CD3+ and granzyme B+ lymphocytes was significantly associated with improved survival, and the density of FOXP3+ TILs in the intratumoral (IT) was the most important prognostic marker. Higher CD8+ TIL and granzyme B+ T lymphocyte infiltration rate in the IT of patients are associated with better OS, and high CD3+ density predicts worse OS. This indicates that some TIL subsets can be used as prognostic biomarkers for HCC. Huang et al. (58) evaluated the expression of FoxP3 regulatory T cells (Tregs), CD4, CD8, and CD34 in tumor and surrounding tissues of 54 HCC patients by immunohistochemistry. It was found that the density of Tregs within the tumor was significantly elevated, whereas the density of CD8+T cells was lower. Tumor-infiltrating Tregs may promote HCC progression by promoting angiogenesis and decreasing CD8 + T cells, and are considered to be poor prognostic indicators for HCC. Whether ILs can exert normal anti-tumor activity depends on the expression level of inhibitory receptors on their surface. Pfister et al. (59) reported the progressive accumulation of depleted CD8+ PD-1 T cells in NASH-affected livers, and elimination of enriched CD8+ PD-1 T cells reduced liver injury and HCC incidence. Overall, TIL has been shown to exhibit complex anti-tumor and pro-tumor properties in HCC. As a therapeutic strategy, TILs expand immune cells from the immunosuppressive tumor microenvironment and infuse them back into the patient. However, clinical studies on the therapeutic effect of TILs in HCC are still limited.

NK cells are unique cytotoxic lymphocytes that play a crucial role in fighting tumors and infections. The importance of NK cells and their activated receptor-ligand axis in the immune surveillance of HCC has been extensively studied. Major histocompatibility complex class I chain-related protein A (MICA) is the human ligand for the NKG2D receptor on NK cells, and binding of MICA triggers NK cells and enhances antigen-specific tumor immunity (60). Reduced MICA expression in HCC tissues is associated with lower PFS and OS in patients 53 (61).

A in vitro study conducted by Kim et al. (62) showed that HI CD56^bright NK cells could produce significant killing effects on the human HCC cell line SNU398, a result that supports the next step in the investigation of the immunotherapy potential of NK cells. The clinical efficacy of immunotherapy with irreversible electroporation (IRE) in combination with allogeneic NK cells for stage IV HCC was evaluated in 40 patients (63). The results showed a synergistic effect of IRE and NK cell therapy, which not only enhanced the immune function, but also reduced the expression of AFP, showing good clinical efficacy. Weng et al. (64) randomized 85 patients with HCC after TACE and radiofrequency ablation to immunotherapy or no adjuvant therapy. Autologous cytokine-induced killer (CIK) cells were infused through the hepatic artery. CD3+, CD4+, CD56+ cells and CD4+/CD8+ ratio were increased in the CIK treatment group, while the recurrence rates at 1 year and 18 months were significantly decreased, indicating the important role of CIK in the treatment of HCC.

The major advantage of CAR-T cells, which are genetically modified lymphocytes, is the ability to recognize extracellular antigens that are presented independently of HLA without the need for antigen presentation to the surface by MHC, making more cancer cells vulnerable to attack (65). The application of CAR-T in HCC has also received extensive attention in recent years.

Secretion of IL-7 and CCL19 (7×19) improves the infiltration and survival of mouse CAR-T cells in vivo (66). Pang et al. (67) co-transduced T cells with CAR vector and 7×19 lentivirus to further verify the anti-HCC efficacy of 7× 19-secreting CAR-T cells. It was found that the liver tumor lesions significantly shrank on day 10 after receiving CAR-T cell injection and completely disappeared after one month. However, the number of patients enrolled in this study is small, and larger studies are still needed to prove the safety and efficacy of CAR-T cells. While HCC tumor organoids and CD39 HBV-CAR-T cells were co-cultured, Zou et al. (68) discovered that the anti-tumor effects of the CD39 HBV-CAR-T cells were enhanced when the PD-1, TIM-3, and LAG-3 checkpoints were down-regulated. Glypican-3 (GPC3) is a cell membrane cancer fetoprotein that is highly expressed in various solid tumors (69). A large body of evidence has shown that CAR-T cells targeting GPC3 can inhibit the growth of HCC cells. In a preclinical study, CAR-T cells were generated on the basis of humanized YP7 (hYP7) and HN3 antibodies (70). They found that CAR (hYP7) T cells suppressed GPC3-positive HCC in a mouse model of HCC, possibly by inducing perforin and granzyme mediated apoptosis or reducing Wnt signaling in tumor cells. Expanded and modified peripheral blood V1 T cells were employed by KAkkouk et al. (71) to produce GPC3-specific CAR and sIL-15. The generated CAR/sIL-15 V1 T cells demonstrated improved antitumor activity and successfully slowed the development of the HCC tumor. Patients with HCC may benefit from treatment with CAR-T cells that target GPC3.

The α and β chains of the TCR are key components that determine T-cell antigen specificity, and the TCR recognizes antigens through enzymatically cleaved peptides that are presented on the cell surface by MHC molecules (72). In TCR-T therapy, T cells are edited to express the TCRα and β chains, giving them tumor-targeting specificity (73).

Zhu et al. (74) identified AFP-specific TCR genes in mice and transduced human T cells with mouse TCR genes to bind HLA-A2/AFP₁₅₈ tetamers. TCR-T cells specifically killed HLA-A2AFP HepG2 HCC tumor cells but had no obvious toxicity to normal primary hepatocytes in vitro. Meng et al. (75) studied the safety of HBV-TCR-T cell immunotherapy in eight patients with advanced HBV-HCC who did not qualify for liver transplantation. None of the patients showed acute adverse effects during or immediately after the injection, and they were subsequently well tolerated. The final median OS was 33.1 months, and the median TTP was 6.18 months. HBV-TCR T cells have the potential to treat HCC recurrence after liver transplantation, but their efficacy may be hindered by the immunosuppressive therapy required to prevent graft rejection. Hafezi et al. (76) engineered TCR-T cells molecularly to preserve their versatility in these patients while minimizing the risk associated with organ rejection. The results showed that patients with HBV-HCC after liver transplantation who received different immunosuppressive drugs showed different degrees of peripheral blood monocytes activation after HBV-TCR T cell infusion, and the disease progression was controlled.

The discovery of IFN provided the greatest impetus for research in all cytokine studies (100). IFN can be divided into three classes: I, II and III. IFN-I is mainly expressed by innate immune cells (101), IFN-II is produced mainly by T cells and NK cells (102), whereas IFN-III expression varies according to tissue (103).

IFN-I, represented by IFN-α and IFN-β, not only affects tumor cell development through cytotoxic, cytosuppressive and antiangiogenic effects (104), but also enhances TAA expression by up-regulating MHC class I proteins (105). A frequent hallmark of noninvasive cancers is loss of IFN-I signaling. Thus, the application of IFN-I is being widely explored. Wang et al. (91) investigated the combined effects of IFN-α and sorafenib on HCC and found that the combination therapy synergistically inhibited HCC cell viability, arrested cell cycle proliferation and induced apoptosis of HCC cells by regulating the expression levels of cyclin A and cyclin B as well as pro-survival Bcl-2 family proteins. Hagiwara et al. (92) investigated the anti-HCC effect of combination therapy with pegylated interferon (PEG-IFN)-α and 5-fluorouracil (5-FU), and found that the combination group had a reduction in tumor volume and a significant increase in tumor cell apoptosis compared with the monotherapy group. This is related to the increased expression of p53 protein and mRNA induced by PEG-IFN. Noguchi et al. (93) elucidated the combined effects of the clinically used angiotensin I converting enzyme (ACE) inhibitor perindopril (PE) and IFN-β on HCC development and angiogenesis in mice. It was found that both PE and IFN-β significantly inhibited HCC occurrence and tumor neovascularization, but the effect of low-dose IFN was weaker than that of PE. Additional preclinical and clinical research is required to examine more potent combo therapies.

IFN-γ, the only member of IFN-II, also plays an important role in tumor immune regulation. Patients with HCC have been found to have fewer mucosa-associated invariant T (MAIT) cells in the peripheral blood and liver than healthy controls, and these cells produce a corresponding reduction in IFN-γ (106). Evidence suggests that interferon signaling plays a key role in regulating the efficacy and sensitivity of ICIs against a variety of tumor types. Wu et al. (107) used Kaplan-Meier survival analysis based on HCC database and found that among the nine interferon regulatory factors (IRFs) that regulate interferon signaling, decreased expression of IRF8 was associated with poor prognosis in HCC patients. However, IFN-γ and PD-1 signaling pathways were significantly inhibited in HCC patients with low IRF8, which indirectly revealed the relationship between IFN-γ and HCC prognosis. Li et al. (94) found that the natural flavonoid pentamethylquercetin (PMQ) could partially inhibit HCC progression in obese mice by down-regulating adipocyte induced PD-L1 expression through IFN-γ signaling. However, some studies have shown that IFN-γ shows a pro-tumor effect in HCC mice model. The complex role of IFN-γ in TME needs to be investigated urgently to exert its antitumor effect.

ILs are key elements for orchestrating the TME and controlling tumor-immune-cell crosstalk, enabling both a conducive environment for cancer growth and critical for an effective tumor-directed immune response (90). Therefore, these properties of ILs can be exploited to improve immunotherapy to increase effectiveness and limit side effects.

IL-6 is a multifunctional inflammatory cytokine with very low expression in normal human cells and increased concentrations in the serum of patients with hepatitis and HCC (108). The mechanism of action of IL-6 in HCC has been extensively studied. The IL-6/STAT3 signaling pathway is involved in various physiological processes, including cell growth, differentiation, and immune regulation. Aberrant activation of the IL-6/STAT3 signaling pathway has been detected in HCC (109), with an impact on the proliferation, invasion, metastasis, immune escape, and drug resistance of HCC cells (110). IL-6/STAT3 signaling upregulates HNRNPC expression in HCC cells, and knockdown of HNRNPC significantly inhibits IL-6/STAT3-enhanced HCC metastasis (111). Yang et al. (95) explored the effect of IL-6 on the sensitivity of HCC cells treated with sorafenib and its mechanism, and found that siIL-6 further promoted sorafenib to impede proliferation and induce apoptosis, suggesting that blocking IL-6 could be used as a potential therapeutic approach for sorafenib sensitivity of HCC cells. Liu et al. (96) observed that IL-6 impaired anti-tumor immunity by suppressing TNF- and IFN-expression on tumor-infiltrating CD6 cells after the co-treatment of HCC with IL-6 inhibition and anti-PD-L1 checkpoint inhibitors. Anti-PD-L1 medication had a stronger effect on tumors when paired with anti-IL-6, and mice survived noticeably longer as a result.

The possible treatment of other types of ILs for HCC has also been discussed. Deng et al. (97) constructed a recombinant oncolytic virus vaccinia based on a vaccinia virus carrying the IL-24 gene (VG9-IL-24). Evaluation showed that VG9-IL-24 effectively infected HCC cell lines but not normal hepatocytes in vitro. In vivo, tumor growth was significantly inhibited and VG9-IL-24-treated mice lived longer. Interleukin-2 (IL-2) is an anticancer cytokine that triggers human innate and adaptive immunity by stimulating T cell proliferation and lymphocyte infiltration into tumor sites. Sun et al. (98) investigated the ability of recombinant adenovirus injection expressing IL-2 (rAd-IL-2) to inhibit tumor cell growth in HCC in HCC tumor model. The results showed that rAd-IL-2 significantly stimulated tumor-specific cytotoxic T lymphocyte responses by inducing the recruitment of CD4+ and CD8+ T cells to tumors, leading to tumor regression and long-term survival of mice during the 120-day treatment period. The study by El-Shemi et al. (99) examined the therapeutic efficacy of two armed oncolytic adenoviruses encoding the human TRAIL gene (Ad-ΔB/TRAIL) and the IL-12 gene (Ad-ΔB/IL-12) in a preclinical model of human HCC. It was found that the combination therapy exhibited profound anti-proliferative and cytopathic effects on human HCC cells and exerted potent tumor killing effect in vivo. More clinical trials are still in urgent need to apply ILs to broader usage.