Effects of the subtypes of apolipoprotein E on immune inhibition and prognosis in patients with Hepatocellular Carcinoma

Primary liver cancer (PLC) is a common cancer worldwide, with the sixth highest overall morbidity and the third highest mortality (Chidambaranathan-Reghupaty et al. 2021; Sung et al. 2021). HCC, which accounts for the largest proportion of PLC, presented with more than 900,000 new cases and 830,000 deaths in 2020 globally (Sung et al. 2021). The intrinsic biological characters of metabolic function, relatively tolerant immunological features and the prevalence of viral hepatitis have made the HCC an unique cancer for insight into the tumour biology (Hanahan 2022).

As a type of cancer occurs in liver, which is the metabolic centre of human body, the research of HCC could provide deep insight into the relationship between metabolism and cancer. Abnormalities in lipid metabolism have now been found not only in metabolic disorders but also chronic liver diseases such as hepatitis C virus (HCV) infection, alcoholic liver disease, etc. (Chidambaranathan-Reghupaty et al. 2021; Hu et al. 2020). The key molecule in the current investigation, apolipoprotein E (apoE), mainly synthesized by the liver, possesses a basic function as a composition of lipoproteins transporting lipid. Besides, the roles of apoE in immunoregulation (Tavazoie et al. 2018), lipid metabolic homeostasis and cell cycle(Du et al. 2020) and many other biological process have also been demonstrated. It participates in the biological processes of inflammatory pathology, oxidative stress (Li et al. 2022) and so on. Abnormal lipid metabolic pathways induce liver inflammation through processes such as lipid peroxidation, a molecular mechanism common to various chronic liver diseases contributing to similar cirrhotic outcome (Lawrence et al. 2019), which is a widely shared background for the oncogenesis of HCC, for the infection of HBV and HCV itself and the immune clearance could cause liver damage. Increasing frequency of genetic instability is shown in the process of proliferation and repairment of hepatocytes. In addition, immune cells including NK cells, Mucosal-associated-invariant-T lymphocytes (MAIT), NKT lymphocytes, cytotoxic T lymphocytes, T helper lymphocytes (Th), memory B lymphocytes and regulatory B lymphocytes (Bregs) in liver and relevant cytokines as well as chemokines contribute to the inflammation in various pathological conditions (Yuksel, Nazmi, et al., 2022). The persistence of chronic inflammations could cause microscopic histological abnormalities, along with the change of circulation state leading to severe destruction of liver parenchyma (Meng et al. 2021), and ultimately liver fibrosis(Goessling 2012; Yang et al. 2021).

Recently, although immunotherapies represented by anti-CTLA-4 antibodies and PD1/PD-L1 antibodies have made their debut in anti-cancer clinical practice(Xin Yu et al. 2020), the lack of universally accepted paradigm for the immune escape of cancer is one of the reasons limiting the effect of immunotherapies. To significantly improve the effect of immunotherapy for cancer, mechanism for immune escape of cancer is urgently needed. The immune tolerant nature of liver (Heymann and Tacke 2016) should be taken into account in the study of immune suppression and escape in HCC (Xin et al. 2022). The role of MDSCs in the current study, has received increasing attention in the immune escape mechanism of tumours in recent years(Zhao et al. 2023). MDSCs contain myeloid progenitors cells and immature myeloid cells released from the bone marrow, which then could be differentiated into immunosuppressive cell subpopulations in response to tumour-secreted chemokines and cytokines, and can be further classified into CD11b⁺CD14⁻CD15⁺CD66b⁺LOX1⁺ PMN-MDSCs and CD14⁺CD15⁻HLA-DR^−/low M-MDSCs (Gabrilovich and Nagaraj 2009). Existing studies have shown that MDSCs are highly involved in every stage of tumour and clinical outcomes (Cole et al. 2021).

A study showed that apoE could act on the LRP8 receptor on the surface of MDSCs, promoting their apoptosis and alleviating the suppressive effect of MDSCs on CD8⁺ T lymphocytes(Tavazoie et al. 2018). Another research in melanoma patients found that different apoE isoform allele carriers had significantly different tumour immunological biology and clinical outcomes, which was the first to give evidence that pre-existing genes in the genome can influence malignancy progression and patients’ prognosis (Ostendorf et al. 2020). However, melanoma happens in skin, which is an immunologically defensive and relatively aggressive organ, in contrary to the immune tolerant background of liver. Besides, lipid metabolism plays a much more important role in the development of HCC than in melanoma. Therefore, whether a similar phenomenon will be found in HCC remains unknown.

It is of interest how apoE, MDSCs and the relationship between them affect the progress and prognosis of HCC. In this study, we firstly performed flow cytometry on the peripheral blood samples from 31 patients treated with hepatectomy for HCC to seek the relationship between apoE and immune cells with suppressive phenotypes. We subsequently built a second cohort of 360 HCC patients to investigate how apoE subtypes, expression and infiltration of MDSCs in the HCC tissues affect patients’ survival, to examine its potential as a biomarker for HCC patients’ prognosis and as a therapeutic target. Other factors, especially lipid metabolism, were also analysed. We tried to construct a preliminary theoretical basis for the future application of apoE and MDSCs in clinic for diagnosis and treatment strategy selection, and in addition exploring the different biology of malignancies brought about by organ-specificity.

Recently, it was found that the relationship between lipid metabolism and immune state was capable of affecting the progress of the malignancy and response to immunotherapy in melanoma patients (Ostendorf et al. 2020). However, there is no data to indicate whether this conclusion can be applied to the liver, one of whose core functions is metabolism. In addition, liver is a tolerogenic organ, its immune status is opposite to that possessed by the skin. Phenomenologically, the vast majority of HCC occurs on the basis of liver fibrosis, which is a common pathological result of chronic liver diseases such as chronic HBV and HCV infection, metabolic dysfunction-associated steatotic liver disease (MASLD) and alcoholic liver disease (ALD), etc(Fattovich et al. 2004). Abnormal lipid metabolism is one of the shared mechanisms in the transition from many chronic liver diseases to liver fibrosis and liver cancer(Paul et al. 2022).

As one of the important molecules in the lipid metabolism pathways, apoE has been found to have key roles in various liver diseases leading to HCC. For instance, apoE is an essential composition of mature HCV particle engaging in the infection process and able to protect the virus from immune clearance(Fukuhara et al. 2014; Hueging et al. 2014; Lavie and Dubuisson 2017). In addition, low apoE expression is associated with malignant transformation of hepatocytes (Hirao-Suzuki et al. 2018), and decreased apoE levels in peripheral blood was found to be related to the metastasis of HCC (Fu et al. 2009; Yokoyama et al. 2006). These studies suggest that apoE and its associated biomolecules play multiple roles in the pathogenesis of various liver diseases and in the progression to cirrhosis and HCC in an extremely complex manner (Nascimento et al. 2020).

Liver is also well-known for its unique immune characteristics in the co-existence of clearance of pathogens and tolerance of macromolecular antigens from gut (Gao 2016; Heymann and Tacke 2016; Meijenfeldt and Jenne 2020; Stamataki and Swadling 2020). The immune suppression and tolerance make it the most easily accepted xenograft in transplantation (Inal 2014), and increases the success rate of other organs when transplanted together (Bogdanos et al. 2013). In addition, a sustained inflammatory response, including infectious inflammation and aseptic lipid inflammation leading to inflammatory-cancerous transformation is one of the most important mechanisms in the development of liver cancer (Heymann and Tacke 2016; Robinson et al. 2016; Yang et al. 2019).

Melanoma patients’ apoE subtypes are found strongly associated with disease progression and efficacy of immunotherapy, which is considered as the first-time demonstration that pre-existing genes in the genome can influence malignant progression and patients’ response to immunotherapy(Ostendorf et al. 2020). Given the metabolic function of the liver, it is a question worth exploring how apoE is involved in the immunosuppressive phenotype of HCC and affects patients’ prognosis.

To investigate the effect of apoE levels in HCC patients’ immune system, we firstly built a cohort containing 31 patients with HCC and performed flow cytometry to analyse their immune cell subsets in the peripheral blood. The results showed that the abundance of immunosuppressive cells in tumour immunity, such as CD11b⁺LOX⁻ neutrophils, CD14⁺HLA-DR⁻ M-MDSCs, CD11b⁺CD33⁺CD14⁻HLA-DR⁻ e-MDSCs and CD14⁺HLA-DR⁺ monocytes, was significantly higher in the group with low levels of apoE; also, the abundance of CD3⁺CD4⁺PD-1⁺ T lymphocytes was significantly higher in the abnormal level apoE patient groups than in the normal level apoE group, while the abundance of CD3⁺CD8⁺CTLA-4⁺ T lymphocytes was significantly higher in the low level apoE group than in the normal level apoE group. These results suggested that apoE is related to the immunosuppressive status in patients with HCC, increasing the abundance of peripheral blood MDSCs and lymphocytes expressing suppressive molecules such as PD-1 and CTLA-4. This on the one hand reflects the potential of apoE as a biomarker to assess the immune status of patients and predict their response to immunotherapies such as anti-PD-1/PD-L1 and anti-CTLA-4 antibodies, and on the other hand indicates the potential of therapeutic strategies that target apoE to regulate the immune status of patients. The application of the antibodies of apoE has been reported to enhance the effect of anti-PD-1 antibody immunotherapy in the animal models of colorectal cancer, gastric cancer and HCC(Hui et al. 2022). However, the anti-PD-1/PD-L1 or anti-CTLA-4 antibodies were not approved for HCC patients in 2012, therefore we were not able to analyse the relationship of the response to immunotherapy and apoE in HCC patients, which was a limitation of this research.

Although the results of flow cytometry preliminarily suggested the association between apoE and immunosuppressive cells in blood, it has been reported that there are more CD8⁺ T lymphocytes and CD56⁺ NK cells but fewer CD4 ⁺ T lymphocytes in liver(Norris et al. 1998; Xystrakis et al. 2015; Yuksel, Demirbas, et al., 2022), indicating the diverse phenotype profiles inside and outside livers. Therefore, to explore its application value in clinical practice, we built the second cohort to evaluate the subtypes and expression of apoE in HCC tissues. Among the results of this study, OS was better in the group with higher apoE2 expression in tumour tissue, while apoE4 was a risk factor for OS in patients with HCC. As a heterozygote can express both apoE2 and apoE4, we also analysed the impact of the intensity ratio of apoE2 to apoE4 expressed in tumour tissue on patients’ prognosis. The result that the cut-off for the value of apoE2/apoE4 was not 1, not only demonstrated the different impact of two subtypes of apoE in HCC, but also reflected the different strength of apoE2 and apoE4 on HCC patients’ prognosis. However, this finding is contrary to the findings in melanoma, where apoE4 carriers have slower tumour progression and metastasis, and a stronger response to immunotherapy(Ostendorf et al. 2020). Currently, there is still no universal, accepted systematic theory of immune escape of tumours (Bruttel and Wischhusen 2014; Hanahan 2022; Hanahan and Weinberg 2011; Villalba et al. 2013). This finding in HCC, in contrast to that in melanoma, is of great importance, the different roles of apoE2 and apoE4 in HCC and melanoma may suggest that the organ difference of tumorigenesis results in different immune mechanisms, which may partly account for the reasons why it is currently difficult to construct a widely accepted systematic theory in the immune escape of cancers. Besides, the property of apoE in regulating the inflammation could be another possible hypothesis for the contradiction(Li et al. 2015; Vitek et al. 2009). It has been reported that apoE plays a critical role in inflammatory process, in which the properties of anti-oxidation and anti-inflammation of apoE2, apoE3 and apoE4 successively decrease(Jofre-Monseny et al. 2008; Shen et al. 2023). The individuals with apoE4 usually present stronger inflammation responses than those with apoE3 (generally considered as the wild type of apoE), showing higher level of pro-inflammatory cytokines such as TNFα, IL-6 and IL-12p40(Vitek et al. 2009). And the key role that inflammation plays in the occurrence and development of HCC has been widely accepted for a long time(Galun 2016). The lower count of PMN-MDSCs and the higher proportion of M-MDSCs in the total cell count in tumour tissues showed a tendency related to longer OS. Similarly, the sum of H-scores for apoE pronounced trend in the K-M survival curves, with shorter OS in those with higher apoE expression in tumour tissue. These tendencies were consistent with past reports in the literatures, and thus correlations with statistically significant differences will be likely to be found if a larger sample size study is conducted in the future. Besides, the effects of the amount of apoE protein in tumour tissues on the OS of patients has been discussed in different cancer types before(Bancaro et al. 2023; Chen et al. 2013; Nan et al. 2021). Therefore, the result of this study suggests its potential as a possible tumour biomarker that can be used for accurate molecular diagnosis of liver cancer and prediction of patients’ OS, and may also provide a potential strategy to enhance the effect of immunotherapy for HCC. A drug design strategy called ‘small molecule structure correctors’ has been reported, through which could convert the function of apoE4 into apoE3-like. This strategy was originally developed in neurology in vitro(Chen et al. 2012), but it could be a replenishment in immunotherapy plan for HCC, after determining the genotype of apoE in patients, shedding light on personal and precise medicine for HCC. Furthermore, in the survival analysis of OS, higher γ-GT level and AFP level in peripheral blood were both significantly associated with shorter OS, consistent with the effect of both on the prognosis of patients with HCC as reported in the past(Tian et al. 2021).

In the survival analysis of RFS, higher PMN-MDSCs count and lower proportion of M-MDSCs were both significantly associated with shorter RFS. Although multivariate COX regression analysis failed to obtain results with statistical significance, differences could be seen in the survival curves for H-score of apoE3, H-score of apo4, H-score ratio of apoE2 and apoE4, total apoE H-score and H-score of iNOS in tumour tissues, suggesting that these indicators were associated with recurrence of HCC and also consistent with the trend of OS. Conversely, the H-scores of apoE2 and ARG showed no significant effect on RFS in this study. Among other clinical tests and pathological variables in this study, RFS was significantly shorter in those with a greater total maximum tumour diameter and those with moderate and severe cirrhosis compared to those without cirrhosis, while RFS in patients with mild cirrhosis was close to that of those without cirrhosis. Among the variables related to lipid metabolism, it is particularly noteworthy that RFS was significantly shorter in patients with higher N-HDL level. N-HDL comprises the sum of lipoproteins other than HDL, and was introduced to the academic community as an indicator of cardiovascular disease at first, but has recently gained attention for its value and clinical significance in oncology(Guaita-Esteruelas et al. 2017; Kong et al. 2022). The effect of elevated N-HDL on patients’ OS reflects the complexity of lipid metabolism in HCC, and the mechanisms through which different types of lipoproteins are involved in the development of HCC, the body’s anti-tumour immune response and the immune escape process of HCC are particularly worthy of further investigation.

The lower level of apoE in peripheral blood was associated with the immunosuppressive status in HCC patients. The high expression of apoE2 was a protective factor for patients’ OS, while patients with high expression of apoE4 subtype had shorter OS; the density of infiltrated PMN-MDSCs in tumour tissues was negatively correlated with patients’ OS and RFS, suggesting that it was related to patients’ tumour recurrence and poor prognosis.

In addition, the striking finding of this study is that the prognostic impact of different apoE subtypes on HCC is opposite to that in melanoma, emphasizing the variability and complexity in the mechanisms of tumour immune escape among different cancer species and organ backgrounds.