Targeting Immune Cells in the Tumor Microenvironment of HCC: New Opportunities and Challenges

1Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for the sixth most common type of cancer and the second leading cause of death among all cancers. Due to changes in environmental factors, immunization and people’s lifestyle, the incidence of HCC and HCC-related mortality are increasing all over the world. The latest research indicates that HCC accounts for approximately 85% of patients diagnosed with liver cirrhosis. Its 5-year survival rate is only 18%, second only to pancreatic cancer. With the improvement of the treatment level for HCC, a variety of treatment options such as liver transplantation, surgical removal, systemic therapy and liver targeted therapy are constantly enhanced and created. At present, only surgical treatment is considered as a potential radical treatment for HCC. But only 15% of HCC patients have the opportunity to have surgery, most patients are found in the advanced stage. Sorafenib is the only systemic medication approved by the FDA for advanced HCC. However, because of the overexpression of dihydropyrimine dehydrogenase, the multi-drug resistance gene MDR-1and p-glycoprotein gene products, HCC is regarded a chemotherapy-resistant tumour, and how to execute effective chemotherapy is still a major difficulty.

2Seven essential characteristics of cancer have been identified as crosstalk between cells and immune cells, self-sufficiency of signals for growth, unrestricted replication potential, apoptosis avoidance, growth signals insensitivity and continuous angiogenesis and invasion/metastasis of tissue. Tumor cells, immune cells, stromal cells, endothelial cells, and cancer-related fibroblasts are all found in the tumour microenvironment (TME), according to current research. Malignant tumour cells can evade immune monitoring and kill, as well as impair the human body, via a range of intricate ways. Due to the limitations of traditional chemotherapy regimens in the treatment of HCC, a variety of immunotherapy methods for HCC have been developed. Immunotherapy mostly employs immune cells within or outside of the TME to specifically target and assault cancer cells, with the benefits of high specificity and low side effects. More crucially, thanks to advances in tools such as mass spectrometry and single-cell RNA sequencing, We can map immunological cells in TMEs at the single-cell level. We outline the significance of tumor-associated immune cells in the HCC tumour microenvironment and highlight their relevance in HCC cancer immunotherapy in this study.

3Tumor-associated immune cells are broadly classified into two types: tumor-promoting immune cells and tumor-antagonistic immune cells. At different stages of tumour formation, these two types of cells play different functions and impact each other (Figure 1). Because the significance of tumor-associated B cells in tumour growth is debatable, we shall introduce B cells additionally.

4Current studies suggest that CD8+ cytotoxic T cells (CTLs) are the main lymphocytes that kill cancer cells. When CD8+ T cells recognize antibodies on DC, CD80−CD86 and CD70 ligands on DC connect to CD27 and CD28 receptors on CD8+ T cells, and CD8+ T cells are modified to become cytotoxic effector CD8+ T cells. Furthermore, CD4+ T cells can activate CD8+ T cells through CD40−CD40L interaction, and CD4+ T cells can produce IL-2 to enhance CD8+ T cell proliferation. CD4+ T cells are also important in the maturation of CD8+ T cells into memory cells. CTL kills target cells through granular exocytosis and apoptotic induction mediated by FasL ligand (FasL) in working state. CTL can also produce interferon- (IFN-) and tumour necrosis factor (TNF-) to cause cancer cell cytotoxicity. Activation and regulation of CTL requires signals from T cell receptors (TCR) and immune checkpoints. For example, cancer cells inhibit CTL activity through the expression of a ligand that binds to an inhibitory checkpoint, such as PD-L1. A significant number of studies have established the function of CD8+ T cells and CD4+ T cells in the formation and progression of HCC, including diagnosis/treatment/prognosis, and so on.

5Chang et al. 's study confirmed that NanoMnSor improves the effectiveness of anti-PD-1 antibodies and whole-cell cancer vaccine immunotherapy by encouraging macrophage polarization to an immunostimulating M1 phenotype, decreasing hypoxica-induced tumor infiltration of tumor-associated macrophages, and raising the number of CD8 cytotoxic T cells in tumors, thereby reprogramming immunosuppressive TME. Xie et al. 's research proposed that PD-L1 overexpression is mostly triggered by pre-existing activated CD8 (+) cytotoxic T cells in the HCC environment, rather than being produced constitutively by tumor cells, and that it is a good prognostic factor for HCC. The frequency of circulating PD-1 (+) CD8 (+) T cells increases as the illness develops from LC to HCC. PD-1 expression was shown to be much higher in tumor-infiltrating CD8 (+) T cells. In vitro, CD8 (+) T cells promoted the production of PD-L1 on HCC cells in an IFN-dependent way, increasing CD8 (+) T cell death, whereas inhibiting PD-L1 reversed this effect.

6Both in vitro restimulation and in vivo depletion studies have indicated that CD4+ and CD8+ lymphocytes contribute to anticancer activity. ASPH activation resulted in considerable production of antigen-specific CD4+ T cells in PBMC from healthy volunteers and HCC patients. Lack of recovered CD19+, CD3+, CD4+, and especially CD8+ T cells is associated with poor survival in patients. Zhou et al. ‘s revealed that antibodies against CD274 (PD-L1), LAG3, or TIM3 boost CD4+ and CD8+ TIL proliferation and cytokine secretion in response to polyclonal antigens or TAA stimulation. More research results on the effect of Effector T cells in HCC are summarized in Table 1. It is clear that Effector T cells play a critical role in the immunological milieu of HCC. Many studies have shown that targeting these cells is effective in patients with HCC.

7NK cells are an essential anti-tumor immune cell that primarily mediates immune surveillance of malignancies. It performs a similar function as CD8+ T cells: NK cells regulate the killing response of tumor cells by releasing perforin and granulein, triggering apoptosis in target cells. In addition, to improve their anticancer activity, NK cells can produce proinflammatory cytokines and chemokines. Existing studies have confirmed the value of NK cells in the development, targeted therapy, prognosis of HCC. Sprinzl et al. 's research confirmed that Sorafenib can promote the pro-inflammatory response of tumor-associated macrophages in HCC, and then activate the anti-tumor NK cell response through the cytokine and NF-κB pathway. Senescence monitoring necessitates the recruitment and maturation of CCR2 myeloid cells, and CCR2 deficiency promotes HCC growth. Conversely, HCC cells suppress the maturation of recruited myeloid progenitors, which promotes mice HCC growth and worsens prognosis and survival in human HCC patients via NK cell inhibition. Kohga et al. 's revealed that natural killer (NK) cells had stronger cytolytic activity on ADAM9KD-HCC cells than on control cells, and that this cytotoxicity is enhanced by the MICA/B and NK group 2, D pathways. Sorafenib treatment resulted in a decrease in ADAM9 expression in HCC cells, an increase in membrane-bound MICA expression, and a decrease in the quantity of soluble MICA. Sorafenib increased HCC cell NK sensitivity by boosting the expression of membrane-bound MICA. Table 2 summarizes current research on NK cells in HCC, confirming the importance of NK cells in immune escape and anti-HCC therapy.

8DC cells, as specialized antigen-presenting cells in the human body, can present antigens to T cells and produce costimulatory signals for T cell activation. According to the current study, mature DC cells can penetrate tumor cells and limit tumor incidence and progression. Under many severe conditions, this inhibition effect will be avoided by tumors through certain means. Therefore, targeting at DC cells, some studies have reported its role in the occurrence, development, immunotherapy, diagnosis and prognosis of HCC. For example, In mice, combining DC vaccination and PD-L1 inhibitor treatment can result in longer overall life, reduced tumor volume, and increased tumor cell apoptosis. As a new therapy method for HCC, combined treatment with DC vaccination and PD-L1 inhibitor may offer promising results. Ali et al. 's research clarified that the combination of PEI or RFTA with active antigen-specific immunotherapy using DCS is a promising approach to induce a sustained anti-tumor immune response aimed at reducing tumor recurrence and metastasis in patients with HCC. Table 3 summarizes the current role of DC cells in HCC.

9Another important type of immune cell in TME is macrophages derived from circulating monocytes, which can generally be divided into M2 polarization and M1 polarization (Figure 2). M1-polarized macrophages, for example, can create pro-inflammatory cytokines and reactive oxygen species/nitrogen to prevent the formation and progression of malignancies. There are limited investigations on the function of M1-polarized macrophages in HCC at the moment. Studies have shown that M1-polarized macrophages in the S3 subclass in HCC increased, and the prognosis was good. Memory B cells, Total B cells, M1 macrophages and T follicle helper cells, were linked with strong total immune cell infiltration into HCC, whereas resting mast cells, neutrophils, and NK cells were associated with poor infiltration. Table 4 summarizes the current role of M1-polarized macrophages in HCC.

10In the immune microenvironment of HCC, some cells could promote the occurrence and development of HCC, and we will review them one by one.

11Tregs play a vital role in immunological homeostasis and immune self-tolerance, and they can express the CD4+ marker and the Foxp3 marker. Foxp3+ Treg acts as a switch for all levels of immune response, and its effects appear to be two-sided. First, Treg can inhibit harmful immune responses and thus inhibit the occurrence of autoimmune diseases. Second, Treg suppresses protective immune responses against invading pathogens or tumors, leading to further progression of the disease. How does Treg play a role in tumors, including how does Treg infiltrate and metastasize to tumor sites or how does Treg help tumors evade immune monitoring, has become a hot research topic in recent years. Many ideas have been put forward. Tregs were found in much higher numbers in HCC patients than in healthy controls. In addition, patients with high Treg(III) levels after TACE had a significantly lower progression-free survival than patients with low Treg(III) levels after TACE. Other studies have shown that tumor Treg upregulated the expression of the glucocorticoid-induced tumor necrosis factor receptor (GITR). Treatment with soluble GITR ligand (GITRL) reduced inhibition caused by activated tumor infiltrating Treg and restored CD4+ CD25-T cell proliferation and cytokine production.. In addition to that, the proportion of Tregs cells in HCC patients was significantly higher than that in healthy and cirrhosis controls, and was related to various clinical indicators of HCC patients. In HCC patients with BCLC stage C, the proportion of Treg cells was more pronounced than in BCLC stage B patients. One to 2 weeks after surgery, the fraction of Treg cells was much lower than before GSMS-TACE. Three to 5 weeks following surgery, the proportion of Treg cells continued to decline. Table 5 summarizes the most credible studies related to the role of Treg in HCC.

12MDSC is thought to be a cancer-promoting immune cell in the HCC tumor microenvironment and was discovered a decade ago. MDSC is divided into granulocyte or multinuclear MDSC(PMN MDSC) and mononuclear MDSC(M-MDSC). MDSCs enhance angiogenesis by producing vascular endothelial growth factor (VEGF), actuator 2 and MMP9. They can also cause cancer cells to migrate to endothelial cells and encourage metastasis. MDSC also suppresses T cell activity by secreting immunosuppressive cytokines, inducible nitric oxide synthase, and argininase. MDSCs can have a dual influence on immune cells via distinct methods. B cells, T cells, DCs and NK cells, are all inhibited by MDSCs. MDSCs, on the other hand, can stimulate Th17 cells, Tregs, and TAMs, as well as tumor angiogenesis and metastasis (Figure 3). There has been a great deal of research on the processes through which MDSC supports the advancement of HCC, and many therapeutic pathways have been developed for MDSC as a target of HCC. For example, myeloid-derived suppressor cells (MDSCs) are drawn to the tumor microenvironment by PIWIL1-overexpressed HCC cells. MDSCs consumption reduced the proliferation and growth of PIWIL1-overexpressed HCC tumors. Complement C3 stimulates HCC cell secretion via PIWIL1 and mediates the contact between HCC cells and MDSC via p38 MAPK activation in MD38, and then initiates the expression of immunosuppressive cytokine IL10. PIWIL1, which is expressed by tumor cells, could be a viable target for the development of new HCC treatments. Tumor-infiltrating LY6G MDSCs from orthotopic liver tumors treated with sorafenib dramatically increased CD4 T cells expressing IL-10 and TGF-and decreased CD8 T cell cytotoxicity. In vitro, IL-6 protects LY6G MDSC from sorafenib-induced cell death. Combining sorafenib and anti-IL-6 antibody or anti-LY6G antibody dramatically decreased the cell proportion of LY6G MDSCs in orthotopic liver tumors, synergistically boosted sorafenib’s therapeutic efficacy and increased T cell proliferation. Icaritin blocks MDSC production by blocking the attenuation of EMH, thereby inhibiting the immunosuppressive effect of the tumor, thereby triggering an anti-tumor immune response. Therefore, Icaritin can be used as a new adjuvant or even as an independent therapeutic agent for the effective treatment of HCC. In Table 6, we show the relevant role of MDSC in HCC immune microenvironment in recent years.

13CAF is not an immune cell, but it plays an important role in the tumor microenvironment, so we will introduce it in detail here. CAF exists as a prominent component of the tumor stroma between various inflammatory cells and components in the tumor microenvironment. As a result, CAF possesses functions that normal fibroblasts do not. A vast number of prior studies have demonstrated that CAF plays a critical function in changing the tumor microenvironment and driving the development of a variety of cancers. CAF is also significant in HCC. Many studies have revealed the great role of CAF in the pathogenesis, progression, prognosis, treatment and other aspects of HCC. CAF-derived cardioctonutrient-like cytokine 1 (CLCF1) has been found in studies to stimulate the release of TGF-β and CXCL6 in tumor cells, consequently increasing tumor stem cell development in HCC-TME. Other research have found that CCN2, EMA, and FAP expression may be involved in the activation of CAF in HCC, resulting in aggressive behavior. The substantial association between EMA-expressing tumor cells and FAP-expressing CAF, as well as their topographical proximity, suggests that there may be interplay between tumor epithelial and stromal cells in the HCC tumor microenvironment. Table 7 summarizes the current role of CAF in HCC.

14M2 polarized macrophages, as opposed to M1 polarized macrophages, have anti-inflammatory and pro-tumor actions (Figure 4). M2 macrophages are further differentiated into M2a, M2b, M2c, and M2d subsets. Th2 cytokines such as IL-13 and IL-4 can trigger macrophage transformation to the M2A phenotype, whereas TLR and immune complex activation induces M2B macrophages, the M2C subtype polarized by IL-10. Despite the fact that there have been few research on M2-polarized macrophages in HCC, the function of them in the occurrence and development of HCC has been confirmed. According to several studies, arsenite raises miR-15b levels and causes M2 polarization in THP-1 cells. Increased miR-15b in Evs transfer from arsenite-treated THP-1 (AS-THP-1) cells to HCC cells via miR-15b. By targeting LATS1, it can reduce Hippo pathway activation while still accelerating the invasion and metastasis of growing HCC cells. The potential therapeutic potential of M2 polarized macrophages has also been pointed out that Tumor cell-derived Wnt ligands induce M2-like polarization of TAM via traditional Wnt/-catenin signaling, resulting in tumor migration, development, immunosuppression and metastasis in HCC. We summarize the current relevant research progress in Table 8.

15One type of immune cells that cannot be ignored in the HCC tumor microenvironment is B cells. According to the current relevant studies, it is not clear whether B cells are “good” or “bad”. Some studies have reported that B cells promote HCC, while others have reported the opposite effect. B cells, on the one hand, release cytokines that comport with CTL activity and serve as potent antigen-presenting cells (APCs). On the other hand, they may be tumorigenic due to the production of cytokines that attract MDSC and promote angiogenesis. Studies have shown that CCL20 derived from tumor cells interacts with CD19CD5 B cells overexpressed by CCR6 to promote the development of HCC, possibly through enhanced angiogenesis. Liu et al. 's study confirmed that Selective recruitment of CXCR3 (+) B cells Bridges the pro-inflammatory interleukin-17 response and the polarization of tumorigenic macrophages in the tumor environment, and blocking the migration or function of CXCR3+ B cells may help to overcome HCC. Therefore, B cells are involved in both the development and inhibition of HCC. Table 9 highlights the most recent reliable research on the involvement of B cells in HCC.

16With the development of single-cell sequencing and other technologies, we have the opportunity to further explore TME. Immunotherapy, a new tumor treatment method, also has a better and broader application prospect. However, at present, it is still too early for immunotherapy to replace traditional chemotherapeutic therapy, and there is still a long way to go in the process of clinical application. However, immunotherapy can be regarded as a good alternative therapy for patients with chemotherapy resistance. As mentioned in this paper, immunotherapies for tumor suppressor related immune cells, such as effect DCs, as well as tumor promoting immune cells, such as MDSC/Treg, are being developed one after another.

17Compared with traditional chemotherapy, immunotherapy has many advantages. For example, immunotherapy has fewer overall side effects than chemotherapy and, once effective, may lead to long-term survival and even clinical cure. In addition, immunotherapy can be used as an important anti-tumor adjuvant therapy in addition to chemotherapy, radiotherapy and surgery. Appropriate immunotherapy can kill the tiny residual tumor cells after chemotherapy or some tumor cells resistant to chemotherapy. Immunotherapy should be considered for patients who are intolerant to chemotherapy or have extensive metastasis and cannot undergo surgery, radiotherapy or chemotherapy.

18At present, the mainstream immunotherapy mainly includes CAR T therapy/immune checkpoint inhibitor therapy (PD-1, PD-L1, etc.)/tumor vaccine. CAR-T is the T cells, biological engineering, when the cancer has an immune deficiency, immune surveillance, give play to the role of the case, through the biological engineering to determine the targets of leukemia, it specifically chimeric in T cells, to attack the leukemia cells, the effect is significant, but easy to appear “storm” cells, serious and even cause death. Immune checkpoint inhibitor therapy has the advantage of long-term survival and relatively small adverse reactions. This therapy activates tumor-specific immune cells in the body by removing or attenuating the negative regulatory factors of immunoreactive cells, but it is not suitable for all patients. The higher the mutation load, the better the treatment response. Therefore, biomarkers should be used to screen the dominant population. The most common predictive indicators of PD-1/PD-L1 immune checkpoint inhibitors are microsatellite instability, PD-L1 and tumor mutation load. The treatment of cancer vaccines is still incomplete.

19Immunotherapy is not the end of tumor therapy; on the contrary, tumor immunotherapy represented by immune checkpoints has just opened a new chapter in tumor therapy. Combined with the basic and characteristics of immunotherapy, with the deepening of human understanding of tumors, tumors as a chronic disease that can be cured are no longer so far out of reach.