Hepatocellular carcinoma (HCC) is a prevalent malignant neoplasm worldwide, with the third highest fatality rate and increasing incidence and mortality rates (1–3). Despite substantial advancements in contemporary medical knowledge regarding the diagnosis and treatment of HCC, its incidence and mortality rates remain high. Given the high heterogeneity of HCC, early diagnosis and treatment play a crucial role in improving patients’ survival (4, 5). Alpha-fetoprotein (AFP) is an important tumor marker for diagnosing and treating liver cancer. It is also an important indicator for clinical diagnosis of liver cancer metastasis (6, 7). AFP, a glycoprotein primarily synthesized by the yolk sac and fetal liver during embryogenesis, is critically involved in sustaining embryonic development and placental functionality (8). However, the expression of AFP is extremely low in normal adults (9, 10). Nevertheless, when hepatocytes undergo malignant transformation, AFP expression exhibits a substantial increase (11, 12). Consequently, elevated AFP levels serve as a reliable indicator for the development and progression of HCC. Generally, high levels of AFP indicate the presence of HCC, and dynamic changes in AFP level can be used to predict the prognosis and response to treatment in HCC (7). However, there are also some patients with liver cancer who have normal AFP levels or only mildly elevated AFP levels, which may be related to differences in the type, location, and size of the tumor and the regulatory mechanism of AFP secretion (11).

The tumor microenvironment (TME) refers to the local tissue surrounding the tumor, including tumor cells, blood vessels, immune cells, stromal cells, and matrix molecules (13). The interaction between the TME and tumor cells plays an important role in tumorigenesis (14–16). HCCs caused by different etiologies (hepatitis B virus (HBV), hepatitis C virus (HCV), and chronic alcohol drinking) exhibit notable differences in their TME. Alcohol intake has inhibitory effects on immune cells in the TME, decreasing immune surveillance and enabling tumor cells to evade the immune response (17, 18). The expression level of AFP significantly increases during the progression of alcoholic liver disease to HCC (19). However, the molecular mechanisms underlying this process are still poorly understood. Chronic infections with HBV and HCV can lead to immune tolerance, allowing the viral infection to persist and cause hepatocyte damage, finally leading to carcinogenesis (20, 21). Studies have found that in the TME, the X protein of HBV can stimulate the expression of reprogramming-related proteins by increasing AFP expression, thus inducing the proliferation of liver cancer stem cells (LCSCs) (22). HCV infection, on the other hand, upregulates AFP expression, which decreases the activity of NK cells and reduces CD4⁺ cells in the TME (23, 24). The immune dysregulation in the TME of HCV-related HCC patients is closely related to the elevated levels of AFP. Although there are significant differences in the TME, the function of AFP in the TME remains consistent. AFP promotes the formation of an immune-tolerant microenvironment and facilitates immune evasion of tumor cells.

As a marker of HCC, AFP plays an important regulatory role in the TME. However, the current role of AFP in the TME of HCC is not fully understood. Therefore, a deeper insight into the role of AFP in the TME is of great significance for the early diagnosis and treatment of HCC. This article summarizes the role of AFP in the TME of HCC, especially discussing its effect on liver cancer stem cells, tumor-associated macrophages, cancer-associated fibroblasts (CAFs), endothelial cells, mesenchymal stem cells, tumor immune escape, etc., providing a reference for the clinical diagnosis and treatment of HCC.

AFP is a fetal-specific alpha-globulin synthesized during fetal development and present in fetal blood and tissues. It can also be detected in adult liver and some malignant tumors (25). Human AFP is similar to albumin, composed of a single polypeptide chain with a molecular weight of nearly 69 kDa (25, 26). The single-chain polypeptide of AFP consists of 609 amino acid residues. These amino acid residues are arranged in a specific order to form three structural domains, namely the N-terminal domain (1-210 amino acid residues; domain I), the central domain (211-402 amino acid residues; domain II), and the C-terminal domain (403-609; domain III) (27) ( Figure 1A ). These three domains of AFP are linked by disulfide bonds to form a V-shaped structure ( Figure 1B ). AFP possesses three distinct structural domains, each exhibiting diverse biological activities. The N-terminal domain can interact with other molecules, thereby affecting the function of AFP. For example, previous studies have found that AFP domain I can bind to the phosphatase domain of PTEN, thereby affecting PTEN activity (25). The central domain (domain II) consists of approximately 192 amino acid residues, possesses a high degree of flexibility, and is easily digested by proteases. The C-terminal domain (domain III) is the last domain of AFP, which is the most conserved domain, consisting of about 207 amino acid residues. This domain possesses a continuous sequence of several hydrophobic amino acids forming a leucine zipper-like structure. Domain III is responsible for binding to signaling proteins and receptors, thereby regulating their biological activity (26–28). Studies also found that AFP domain III can bind to the C2 domain of PTEN, forming the AFP-PTEN complex, inhibiting PTEN, activating the PI3K/AKT signaling pathway, and promoting HCC progression (25). Domain III can also bind to mucin, scavenger receptors, chemokines, etc. After binding to its receptor, AFP is endocytosed and packaged with the receptor and transported to the cell organelles via the Golgi complex, where it is degraded or activates/blocks cell signaling pathways (26–28).

AFP secretion and expression are not consistent with HCC development. One reason is that AFP has two basic forms: native AFP (nAFP) and tumor-derived AFP (tAFP). nAFP is mainly produced by the fetal yolk sac and fetal liver and secreted into fetal circulation. It is a normal plasma protein and does not promote HCC development. Sometimes, adult individuals express nAFP, which is mainly observed during physiological cell regeneration and hematopoiesis. However, tAFP expressed in TME mainly originates from HCC and can support tumor growth and metastasis. Interestingly, tAFP is not a mutated form of nAFP and differs only slightly in terms of glycosylation and lipid-binding characteristics (29, 30).

AFP described in this article specifically refers to tAFP. AFP exhibits numerous variability, thereby presenting considerable challenges in comprehending its biological activity with HCC (31). AFP variability relies on the types of species, tissues, isoforms, binding ligands, binding partners, and post-translational modifications such as N-glycosylation ( Figure 1B ). Different variants of AFP possess different biological functions (29); for example, it was found that the serum levels of fucosylated AFP variant increased in patients with HCC but were undetectable in the serum of normal patients (32). Polyunsaturated fatty acids binding AFP variants play an important role in the TME and affect dendritic cells (DCs) and natural killer cells (NK cells) activation (33). Despite extensive studies on the structure of AFP, its biological functions remain unclear. Further studies are imperative to unveil its precise association with various physiological and pathological processes.

The interplay among cells within the TME is a crucial factor in tumor progression, metastasis, and treatment (34, 35). AFP, as a tumor biomarker, directly modulates tumor cell behavior and the TME by interacting with tumor-associated cells and molecules.

AFP is as a serological biomarker for HCC screening, diagnosis, treatment response monitoring, and prognosis. The sensitivity and specificity of AFP for diagnosing HCC are 41–65% and 80–94%, respectively. However, nearly 50% of patients with HCC are AFP-negative and AFP may be elevated in benign liver diseases, such as hepatitis and cirrhosis (72, 73).

AFP-negative and AFP-positive patients display differential proteomic profiles and metabolomic profiles. The symptoms of AFP-negative patients are generally mild and they benefit more from treatment compared with AFP-positive patients. AFP level has strong relationships with malignant features of HCC (72, 73). The analysis of tumor-infiltrating lymphocytes in AFP-positive TME reveals an enrichment of Tregs and exhausted CD8⁺ T cells. Accordingly, the number of cytotoxic T-lymphocyte associated protein 4 (CTLA4) and programmed death 1 (PD-1) T cells is higher in AFP-positive patients than in AFP-negative patients. Also, high abundances of regulatory T cells were found in AFP-positive patients, which drive cytotoxic T cell dysfunction in the TME (74).

TAMs, DCs, and monocytes were more abundant in the adjacent normal tissues of AFP-positive patients. AFP upregulates IL-6, transforming growth factor beta 1 (TGF-β1), CXCR4 and NF-κB, to recruit TAMs into TME. TAMs promote the expression of angiogenesis-associated genes and downregulate phagocytosis and lymphocyte response, which may recruit fibroblasts and induce extracellular matrix remodeling to promote tumorigenesis (75).

Elevated serum levels of AFP typically signify the existence or recurrence of HCC, thus rendering AFP a valuable tool for diagnosis and treatment (76).Some patients with HCC are AFP-negative preoperatively; however, AFP may increase after surgery. AFP can acutely elevate for about 5 days after partial hepatectomy (77). Rising concentrations of AFP are closely associated with hepatocyte regeneration. AFP can be synthesized and released by hepatocytes undergoing proliferation and mitosis after operation (78). AFP expression was also seen in the regenerative phase after liver injury, which was associated with hepatocyte proliferation. During proliferation, AFP is produced by hepatocytes and released into the microenvironment and circulation. It was also found that AFP is not only related to cell proliferation but also related to cellular death and apoptosis (52). After surgery, AFP level will decline with healing. However, in postoperative recurrence, AFP is reproduced by HCC cells and cancer stem cells. AFP production during postoperative recurrence is related to fast-growing, poorly differentiated, and malignant HCC cells (44). HCC is an extraordinarily heterogeneous malignant liver cancer. Preoperative and postoperative recurrence of HCC are different in molecular characteristics, signal transduction, and genomic instability. They are characterized by great heterogeneity. AFP can arise from cancer stem cells, which are silent in patients before surgery. These cells are more active in the TME during hepatocyte necrosis, myeloid cell migration, immune evasion, and matrix remodeling after postoperative recurrence (44, 79, 80). Thus, AFP not only functions as a marker but also aids in the interpretation of imaging in liver cancer. The value of AFP can be associated with the dimensions, progression, and prognosis of HCC. Elevated levels of AFP may serve as an indicator of a larger or more aggressive tumor and an unfavorable prognosis (81). Moreover, post-treatment monitoring of AFP levels in patients with liver cancer can be employed to assess treatment efficacy and disease recurrence. A substantial decrease in AFP level following treatment generally signifies successful intervention, whereas an increase in AFP level may suggest tumor relapse or progression (60). Thus, AFP plays a crucial role in the diagnosis and management of HCC. As a marker, it can be used for early screening, diagnosis, and follow-up of patients with liver cancer. In addition, AFP levels can also be used to evaluate the therapeutic effect and predict the prognosis.

Recently, AFP has increasingly gained interest in HCC immunotherapy. AFP can be applied in immunization. Particularly, it can be a marker for immunotherapy and predict the efficacy of immunotherapy. AFP is highly expressed in nearly 65% of patients with HCC, and serum AFP is as a biomarker for diagnosing and treating HCC because it is inversely correlated with prognosis (2, 82). HCC patients cannot tolerate to high serum levels of AFP which induce significant tumor burdens. AFP is a tumor-associated antigen, and cytotoxic T lymphocyte (CTL) epitopes for AFP were identified during tumorigenesis (83, 84). Patients with high circulating levels of AFP have strong T cell responses. Both murine and human T cells can recognize the self-antigen AFP, indicating that it can serve as a tumor rejection antigen. AFP-based CAR-T cells and AFP-based vaccines have shown an antitumor immune response in vivo and in clinical trials (85, 86).

AFP is produced intracellularly and secreted by HCC, making it “untargetable” for chimeric antigen receptor T (CAR-T) cell immunotherapy. However, peptides derived from AFP are processed and presented by class I MHC on the surface of HCC cells. Therefore, antibodies against AFP-derived peptides/MHC complexes can be designed and engineered into CAR-T cells. CAR-T cell therapy was successful in liver cancer xenograft models in vivo (85). Also, CD8+ T cells were found in transgenic mice that recognized AFP-derived peptide epitope on human HCC cells. Adoptive transfer of AFP-derived peptide-specific CD8⁺ T cells eradicated HepG2 tumor xenografts in mice (87).

Preclinical studies found that AFP-derived peptide-specific TCR-engineered T cells are unlikely to cause severe off-target toxicity (88). AFP peptide-based CAR-T therapy in advanced HCC is being tested in a phase I clinical trial (NCT03132792) to find the safety of adoptive transfer of engineered T cells, which target AFP-positive liver cancer (89).

Dysfunction of DCs is also the main mechanism of tumor immune escape. Vaccination can promote the activity of DCs and stimulate antitumor responses in HCC. The beneficial immune responses of CD8⁺ and CD4⁺ T cells can be generated if AFP is presented by more AFP-engineered DCs. It was found that AFP-pulsed DCs can shift specific cytotoxic T lymphocytes toward AFP-producing HCC cells (90, 91).

Using AFP-coding adenoviruses to transduce DCs can induce the antitumor immune response, delay tumor growth, and improve long-term survival in vivo subcutaneous HCCs. The combination of the AFP-DCs vaccine with CD40 ligand-DCs vaccine has shown synergistic results. It changes the TME, enhances the tumor infiltration of cytotoxic T lymphocytes and DCs, and upregulates Th1-derived cytokines, leading to tumor apoptosis and regression (92, 93). A phase I/II trial measuring immunization in HCC patients found that AFP-DCs vaccine can produce AFP-specific T cell responses with increasing IFN-γ in the environment of high oncofetal antigen (86). Nevertheless, more comprehensive studies are imperative to ascertain the efficacy and safety of these approaches and vaccines before their implementation in clinical settings.

TME is a complex ecosystem comprising tumor cells, blood vessels, immune cells, stromal cells, and matrix molecules. AFP and its downstream pathways play a key role in this system, making them targets for novel tumor treatment. The expression level of AFP is associated with tumorigenesis and tumor prognosis (94, 95). Hence, therapeutic strategies that target AFP or its downstream pathways may be beneficial for treating these tumors. One potential therapeutic approach is to inhibit the expression or activity of AFP. For instance, using small interfering RNA (siRNA) or antisense oligonucleotides (ASO) technology to reduce AFP expression. These technologies can specifically inhibit the synthesis of AFP or prevent its interaction with target cells, thereby suppressing tumor growth and diffusion (96, 97). Secondly, the use of recombinant AFP, combined with anticancer drugs, has been widely explored. Besides, recombinant AFP fragment derived from AFP domain-3 was found to suppress tumor cell growth. Various anticancer drugs exhibited therapeutic prospects when combined with AFP or AFP fragments, such as carminomycin, doxorubicin, paclitaxel, sorafenib, and maytansinoid (98–103). Moreover, AFP can activate downstream signaling pathways, such as PI3K/Akt in tumor cells, promoting proliferation, survival, and migration (22, 104). Targeting these pathways with small-molecule inhibitors or antibodies can inhibit tumor growth. The complexity of TME makes a single therapy ineffective. Combining multiple strategies, like targeting AFP and its downstream pathways with immunotherapy and radiotherapy, may more effectively control tumor growth and invasion.

In the TME, AFP-targeted therapies still face challenges and limitations. Firstly, the expression level of AFP has limited sensitivity and specificity for HCC patients, it is not a marker for all HCC, and about one-third of the patients with advanced HCC have no AFP expression (105, 106). Moreover, patients with acute and chronic liver without evidence of HCC may have high AFP elevation. Therefore, therapeutic strategies targeting AFP may not be applicable to all HCC patients. Secondly, the heterogeneity of the tumor microenvironment is another challenge. AFP expression varies among different types and stages of HCC (7, 105), and the accuracy value with advanced HCC varies according to patient characteristics (7). Due to the significant differences in cell types, gene expression, and signal pathway activation among different regions of a tumor (106). Drug resistance is another concern. Tumor cells may adapt to treatment pressure by activating other signaling pathways or mutations, leading to treatment failure (107). Overall, there are challenges in AFP-targeted therapies, and future studies are needed to understand the mechanism by which AFP is involved in tumorigenesis and assess the efficacy and safety of AFP-based treatment strategies.

AFP utility is limited and challenges, however, targeting the AFP-TME interaction in HCC treatment has significant benefits to AFP positive patients (75). Blocking the function of AFP or inhibiting its interaction with TME can disrupt the connection between liver cancer cells and TME, thereby inhibiting tumor growth and diffusion. This strategy may provide a new approach for treating HCC. Secondly, AFP is also involved in the interaction between liver cancer cells and the immune microenvironment (29). AFP overexpression can promote the immune evasion of HCC cells, protecting them from the immune response. Targeting AFP may help attenuate this immune evasion, enhancing the immune recognition and response, thereby improving the efficacy of immunotherapy. In summary, targeting the AFP-TME interaction in HCC can disrupt the link between liver cancer cells and the TME, inhibiting tumor growth, invasion, and immune evasion.

In conclusion, AFP holds significant value as an HCC biomarker, and its high expression is intricately linked to the development and progression of HCC. AFP can stimulate the expansion of LCSCs in the TME by activating the PI3K/Akt signaling pathway. Additionally, AFP can facilitate HCC progression by upregulating the expression of genes associated with LCSCs. Furthermore, AFP can undermine macrophage-mediated phagocytosis of tumor cells by interacting with macrophage receptors and inducing tumor immune escape. Simultaneously, AFP may also suppress the function of NK cells, macrophages, DCs, CAFs, endothelial cells, and mesenchymal stem cells, and impair the ability of cytotoxic T lymphocytes to eliminate tumor cells, thus facilitating tumor immune evasion ( Figure 4 ). Recent findings have demonstrated that AFP inhibition can effectively inhibit the malignant behaviors of HCC cells, suppressing their proliferation, invasion, and metastasis and inducing cancer cell apoptosis. Consequently, AFP plays a crucial role in facilitating the malignant progression of HCC. Nevertheless, AFP is a double-edged sword in the development, diagnosis, and treatment of HCC ( Figure 5 ). For clinical practice, measuring AFP levels holds immense significance for the early detection, prognosis, and treatment of HCC. By monitoring the fluctuations in AFP levels, medical professionals can monitor disease progression and treatment response, guiding clinical decision-making. Furthermore, clinicians can use AFP vaccines to generate AFP-specific CD8⁺ T cells and kill cancer cells. In addition, AFP combined with immunotherapy, can improve the therapeutic efficacy.

In summary, AFP plays a significant role in the TME of HCC. It modulates the function of tumor-associated macrophages, increases the population of tumor stem cells, and facilitates the immune evasion of liver cancer cells. Further studies are needed to gain a comprehensive understanding of the underlying mechanisms through which AFP is involved in the TME of HCC and provide new approaches for treatment and prognosis prediction in HCC.

YL: Data curation, Resources, Supervision, Writing – original draft, Writing – review & editing. BL: Supervision, Writing – original draft, Writing – review & editing. ML: Funding acquisition, Investigation, Supervision, Writing – review & editing.