
==== Front
Hepatol Commun
Hepatol Commun
HC9
Hepatology Communications
2471-254X
Lippincott Williams & Wilkins Hagerstown, MD

39225682
HEP4-24-0719
10.1097/HC9.0000000000000516
00011
3
Review
The immune microenvironment of steatotic hepatocellular carcinoma: Current findings and future prospects
https://orcid.org/0009-0007-0931-5200
Cheu Jacinth Wing-Sum 12wscheu@connect.hku.hk

https://orcid.org/0000-0001-5866-4705
Wong Carmen Chak-Lui 12345cclwong@hku.hk

1 Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong
2 Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong
3 State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong
4 Department of Clinical Oncology, Shenzhen Key Laboratory for Cancer Metastasis and Personalized Therapy, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China
5 Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-sen University, Guangzhou, China
Correspondence Carmen Chak-Lui Wong, Department of Pathology, T8-010, Block T, Queen Mary Hospital, 102 Pokfulam Road, Pokfulam, Hong Kong. Email: carmencl@pathology.hku.hk, cclwong@hku.hk
9 2024
03 9 2024
8 9 e05169 7 2024
15 7 2024
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0/

Hepatocellular carcinoma (HCC), the major type of primary liver cancer, is notorious for its resistance to systemic treatments. The field has made a great leap in the past decade, with the number of FDA-approved therapies for advanced HCC increasing from 1 to 9. Although tyrosine kinase inhibitors remain the most common first-line option as monotherapy treatment, the clinical success of immune checkpoint inhibitors, especially when used in combination with anti-VEGF/VEGFR in HCC will likely transform the treatment landscape. While immune checkpoint inhibitors represent an exciting therapeutic revenue for HCC, recent studies have revealed that nonviral HCC, which is primarily caused by metabolic dysfunction–associated steatotic hepatitis (MASH), has a distinct and less favorable response to the immune checkpoint inhibitors. MASH is the most rapidly increasing etiology for HCC. The immune microenvironment of MASH-HCC is greatly affected by the intertwined pathological processes of steatosis-induced iterative cycles between steatohepatitis and liver injury. Here, we present a timely summary of the immune microenvironment of MASH-HCC. We will delve into the use of cutting-edge technologies, such as single-cell RNA sequencing, spatial transcriptomics, and mass cytometry imaging, to deconvolute the complexity of the immune ecosystem in MASH-HCC. We will also discuss the novel therapeutic innovations for MASH-HCC in preclinical models, such as the metabolic inhibitor, epigenetic inhibitor, and immunomodulator. These inhibitors all have the ability to subvert the immune microenvironment of MASH-HCC, improving the efficiency of anti-PD-1. While awaiting new drugs to be tested in clinical trials, the knowledge gained from these investigations is crucial for the development of personalized and effective treatment strategies for MASH-HCC.

OPEN-ACCESSTRUE
Read-alongYES
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pmcINTRODUCTION

Primary liver cancer ranks as the sixth most common cancer and the third deadliest cancer in the world. HCC arises from hepatocytes and accounts for 90% of all primary liver cancer. 1,2 The development of HCC typically occurs in a stepwise manner, from chronic inflammation progressing to cirrhosis and ultimately leading to HCC. The major causes of chronic liver inflammation are HBV infection, HCV infection, and metabolic dysfunction–associated steatotic hepatitis (MASH), previously known as NASH. Some other HCC risk factors include heavy alcohol consumption, coinfection of HBV and HDV, and aflatoxin intake. Since the launch of the vaccination programs and the introduction of direct-acting antivirals against HCV, MASH has risen to be the fastest-growing etiology for HCC. MASH contributes to 15%–20% of HCC in the Western countries. 2 MASH is developed from metabolic dysfunction–associated steatotic liver disease (MASLD), formerly known as NAFLD. The incidence of MASLD is surging worldwide. As of 2019, 1.24 billion individuals were affected by MASLD in the world, 3 affecting close to 20% of the entire population. 4 Around 170 thousand deaths worldwide are attributed to MASLD annually. 3 MASLD refers to the clinical situation when the liver has more than 5% hepatic steatosis, while MASH is a more serious condition when the liver further has a hepatocellular injury. 20% of patients with MASLD will develop MASH. It is worth noting that HCC can also occur in the absence of cirrhosis. In these patients without cirrhosis, MASH is the primary cause of HCC. 5,6 Starting from 2023, the new nomenclatures MASLD and MASH have been introduced to replace the previous terms NAFLD and NASH, respectively. 7,8 It is important to note that MASLD, in addition to the 5% steatosis, also requires the presence of at least 1 cardiometabolic risk factor. Recently, a study using the NAFLD database and the National Health and Nutrition Examination Survey (NHANES III) showed that 99.8% of patients with NAFLD fulfill the criteria of the MASLD definition, confirming the interchangeability of these terminologies. 9 Steatotic liver diseases and steatohepatitis are used as broader terminologies. 7,8 Consistent nomenclatures are recommended to be used in animal studies. 10–13 This review includes information from many former studies, which rely on animal models before the implementation of the new nomenclatures. Due to variations in the experimental designs in different research groups, we have employed nomenclatures as described by authors in their original papers, with NASH and NAFLD being converted to MASH and MASLD, respectively. Readers are recommended to adhere to the original papers to study the experimental conditions for accurate interpretation. There is a recent review on MASH-HCC nicely summarizing the mutational landscape and molecular mechanisms of MASH development 14 ; this review focuses on the immune microenvironment of MASH-HCC, particularly how the application of cutting-edge technologies and animal models enhances our understanding of the immune landscape of MASH-HCC and identifies novel therapeutic approaches for MASH-HCC.

CURRENT TREATMENT FOR MASH-HCC

MASH-HCC currently shares the same treatment guidelines with HCC of other etiologies. Early HCC is treated with surgeries, while advanced HCC can only be treated with systemic therapies. There are 9 FDA-approved systemic treatment regimens for patients with advanced HCC, involving the use of tyrosine kinase inhibitors and immune checkpoint inhibitors. First-line treatments include monotherapy of sorafenib, lenvatinib, and combination treatment of atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF) or tremelimumab (anti-CTLA-4) and durvalumab (anti-PD-L1). Second-line treatments are single treatment of regorafenib, cabozantinib, ramucirumab, or pembrolizumab (anti-PD-1) and combined treatment of nivolumab (anti-PD-1) and ipilimumab (anti-CTLA-4). 15

Growing evidence suggests differential treatment responses between viral and nonviral HCC. This highlights the importance of personalized treatments for HCC from different etiologies. A meta-analysis revealed that patients with MASH-HCC have better overall and disease-free survival upon surgical resection when compared to patients with HCC with other etiologies. 16 However, the absence of cirrhosis and reduced sensitivity of ultrasound in fatty liver often lead to delayed diagnosis, precluding patients from surgeries 17,18 . Meanwhile, a seminal study from Pfister et al 19 provided evidence that MASH-HCC contains T cells with distinctive features, leading to a lack of response to immune checkpoint inhibitors. In their meta-analysis, authors combined data from 3 clinical trials showing that anti-PD-1/PD-L1 treatment is not effective in patients with nonviral HCC (MASH/NASH and alcohol intake) as compared to patients with viral HCC (HBV and HCV). 19 Furthermore, patients with HCC with MASLD have shorter overall survival after anti-PD-1/PD-L1 treatment as compared to patients with HCC with other etiologies.

Although MASH-HCC has no specific treatment, a recent milestone has been achieved for MASH with the FDA approval of the first drug, resmetirom. Resmetirom is a thyroid hormone receptor beta agonist that activates thyroid hormone receptor beta to promote mitochondrial activity for the β-oxidation of lipid, leading to the resolution of fibrosis. The treatment of patients with MASH with resmetirom has resulted in a significant improvement in MASH resolution and fibrosis. 20 Although resmetirom is expected to prevent MASH-HCC, more experimental and clinical evidence is needed. While the field is actively exploring more MASH treatments, it remains an open question whether the newly developed MASH treatments can be given on a long-term basis due to the chronic nature of the disorder. In addition, the potential negative effects of immune checkpoint inhibitors on MASH-HCC further highlight the significant clinical need for effective therapies. Addressing these needs requires a thorough understanding of the immunological aspects that contribute to the pathology of MASH-HCC.

IMMUNE DYSREGULATION WITHIN THE MICROENVIRONMENT OF MASH PRIMING HCC

MASH is a severe form of MASLD in which steatosis is accompanied by inflammation and liver damage. The immune cells play a key role in disease progression. The immune cell populations undergo immense changes when lipid starts to accumulate in the liver. These changes play crucial roles in priming the liver for the development of HCC with unique immune characteristics, further affecting the response to immunotherapies. Here, we summarize the changes observed in various immune cell types, such as Treg cells, CD4+ T cells, CD8+ T cells, NK cells, macrophages, dendritic cells (DCs), and neutrophils during the development of MASH or MASH-HCC (Figure 1).

FIGURE 1 Features of immune cells in steatotic HCC. Abbreviation: MDSC, Myeloid-derived suppressor cell.

Lymphoid cells

An immunosuppressive CD4+ T-cell population, Treg cells, was increased in the livers of mice with MASH. 21,22 Treg depletion by anti-CD25 was able to reduce MASH-associated HCC in a murine model. Earlier studies showed that steatotic liver is characterized by the loss of hepatic CD4+ T cells. 23,24 Linoleic acid generated from lipid-loaded hepatocytes from steatotic liver inhibited the mitochondrial functions of CD4+ T cells, leading to apoptosis of CD4+ T cells in steatotic liver diseases, accelerating HCC development. 24 Interestingly, antioxidant, N-acetylcysteine, was able to prevent the loss of CD4+ T cells and restore the antitumor functions of CD4+ T cells. This suggested that lipid generated from hepatocytes induced reactive oxygen species in CD4+T cells, leading to their malfunctioning. Another interesting study demonstrated that CD8+ T cells experienced functional changes in MASH-HCC. scRNA seq and mass cytometry revealed that CD8+PD-1+CXCR6+ T cells were significantly increased in mouse and human MASLD/MASH. Interestingly, this population of CD8+ T cells failed to elicit antitumor functions but instead contributed to liver damage, which led to the development of HCC. 19 Mechanistically, IL-15 induced CXCR6 expression on hepatic CD8+ PD-1+ T cells, which, upon exposure to short-chain fatty acid acetate in the MASH liver, produced abundant TNF, further amplifying the inflammation. It was also demonstrated that ATP released from damaged hepatocytes upregulated FasL on CXCR6+ CD8+ T cells to exert auto-aggression. These auto-aggressive CXCR6+ CD8+ T cells killed hepatocytes in an MHC-class-I independent manner, a distinctive mechanism from antigen-specific CD8+ T-cell killing. 25 These studies offer novel mechanistic insight into why further activation of this CD8+PD1+CXCR6+ T cells by anti-PD-1/PD-L1 would lead to a worse prognosis in patients with MASLD/MASH. 19,25 In a recent study, it was discovered that gastrointestinal B cells played a crucial role in the activation of auto-aggressive T cells and the promotion of fibrosis in MASH. These B cells activated macrophages through immunoglobulins, leading to the progression of the disease. Interestingly, patients with MASH have a higher number of activated intestinal B cells, and there were positive correlations between their levels of IgG, activated hepatic myeloid cells, and the severity of liver fibrosis. However, it was found that depletion of gastrointestinal B cells effectively decelerated the progression of MASH and fibrosis. 26 Apart from CD8+ T cells, NKT also promoted liver damage in the murine MASH model. NKT cells produced high levels of LIGHT ligands, which activated the LTβR and canonical NF-kB signaling axis in hepatocytes to further promote steatosis and the progression to steatotic HCC. 27 Another recent study showed that the liver from patients with MASLD expressed increased NKG2D ligands and IL-17A expressions. 28 In mice, it was shown that a MASH diet caused hepatocytes to induce NKG2D ligands, which activated the γδT cells through the NKG2D receptor to induce IL-17A. 28 IL-17A further induced hepatocytes to produce proinflammatory cytokines to recruit proinflammatory macrophages, neutrophils, and eosinophils to induce fibrosis in MASH. 28 Apart from the seminal studies showing MASH-HCC is not responding to anti-PD-1 treatment, 19,25 another study interestingly demonstrated that liver tumors, even liver metastases, became resistant to immunotherapies in mice with steatotic hepatitis. 23 While the RNA vaccine (M30) and OX40 agonistic antibody were highly effective in reducing the growth of liver metastases originating from melanoma and colorectal cancer on a regular diet, these treatments had no effect on liver metastases in mice with steatotic hepatitis induced by different high-fat diets (HFDs). 23 However, the administration of the antioxidant N-acetylcysteine restored the infiltration of CD4+ T cells and effector memory cells, making the liver metastases responsive to immunotherapies. This suggests that reactive oxygen species may play a role in suppressing the immune system in livers with steatotic hepatitis. 23

Myeloid cells

Neutrophils were increased in the mouse and human MASH and MASLD. 22,29,30 A study demonstrated that the neutrophils in MASH-HCC expressed CXCR2. 29 Neutrophils release neutrophil extracellular trap (NET), which contains chromatin, granules, and other proteins that could “trap” and eliminate microbes. 31 The release of chromatin by neutrophils is mediated by chromatin decompensation due to the activation of myeloperoxidase and protein-arginine deiminase. Interestingly, myeloperoxidase-DNA complexes were elevated in the serum of patients with MASH, suggesting an increase in NET formation in patients with MASH. DNase, a drug that could disintegrate NET, and KO of protein-arginine deiminase in mice suppressed the progression of MASH to HCC in mice. 30 NET was able to mediate macrophage recruitment as well as to promote Treg differentiation in MASH. 22,30 Mechanistically, NET was shown to promote the oxidative phosphorylation of Treg cells through toll-like receptor 4 to favor their immunosuppressive activity against effector T cells. 22

An increase of hepatic conventional dendritic cells (cDCs) was observed in mice and humans with MASLD/MASH. 32 These cDCs express chemokines and maturation markers, which indicate a high antigen presentation signature. Interestingly, it was found that these cDCs might be originated from pre-DC progenitors in the bone marrow as a result of liver damage–induced hematopoiesis. Interestingly, by studying the DC-T-cell doublets, it was found that cDCs directly interacted with T cells in liver-draining lymph nodes to promote liver inflammation in MASH. 32 Cross-presentation which frequently occurs in cancer is an effective way by which cDC1s present antigens using MHCI to CD8+ T cells and this is characterized by the presence of XCR1, a chemokine receptor on DCs. XCL1 is the ligand for XCR1. 33 cDC1 depletion by XCL1 antibody or XCR1 genetic knockout was able to alleviate overall inflammatory responses caused by T and NK cells in MASH-associated liver damages. 32 ​ Macrophage populations in the liver are highly heterogeneous, and it is oversimplified to define a protective or promoting role of macrophages in MASH development. Liver macrophages consist of KCs, the resident hepatic macrophages, which are the most abundant macrophage population in the liver, and the macrophages recruited from bone marrow. On the one hand, macrophages mediated efferocytosis, the clearance of injured hepatocytes without inducing inflammation or tissue damage, 34 thus protecting the liver from MASH development. On the other hand, macrophages could be stimulated to secrete proinflammatory cytokines that further recruit other immune cell types, contributing to MASH progression.( 35 ) Recent study revealed that there was a reduction in KCs in the liver with MASLD and they were replaced by a recruited macrophage population termed hepatic lipid-associated macrophages. Lipid-associated macrophages expressed high levels of Gpnmb and Trem2. 36 A similar lipid-associated macrophage population was identified in humans and was increased in patients with MASLD. 37 Another scRNA seq analysis in normal and MASH murine livers showed that a similar macrophage population with high expressions of Gpnmb and Trem2 was significantly augmented in MASH livers and the hepatic expressions of GPNMB and TREM2 were positively correlated to MASH pathologies in human patients. 38 Interestingly, a recent study showed that Trem2 expression on liver macrophages was essential for efferocytosis of lipid-loaded hepatocytes. A loss of Trem2 on macrophages led to the accumulation of dying hepatocytes and promoted inflammation and MASH progression. 39 Whether the accumulation of Trem2+ liver macrophages plays a protective or promoting role in MASH development requires further investigation. Another interesting study showed that type 2 immunity, which is typically featured by high levels of IL-4, IL-5, and IL-13, promoted the progression of MASLD to MASH and fibrosis. 40 The lymphocytes of patients with MASH expressed higher levels of these type 2 cytokines, which have a significant positive correlation with the severity of fibrosis. Eosinophils, which are often associated with type 2 immunity, could also be detected in MASH liver.

The collective data suggested that MASH creates a unique immune microenvironment for HCC, characterized by a combination of proinflammatory and immunosuppressive elements. It is reasonable to speculate that in the early stages of MASH, proinflammatory immune cells initiate the priming and contribute further to liver damage. However, as the disease progresses, immunosuppressive cell populations such as Treg cells establish an immunosuppressive microenvironment, leading to evasion of immune surveillance and potentially immunotherapy resistance.

ADVANCED MULTIDIMENSIONAL ANALYSIS OF THE ESTABLISHED MASH-HCC IMMUNE LANDSCAPE

Previous studies on MASH-HCC were limited by a relatively narrow focus on only 1 or 2 cell types. However, the advent and rapid growth of single-cell and spatial omic technology platforms coupled with tools that enable high-dimensional analysis of immune cells have overcome these limitations and remarkably enhanced our understanding of the immune microenvironment in MASH-HCC. These recent studies have provided a more comprehensive and in-depth view of the dynamic microenvironment in MASH-HCC (Figure 1). Between 2023 and the third quarter of 2024, 5 studies independently used different state-of-the-art technologies, including scRNA seq, spatial transcriptomics, CyTOF, and mass cytometry imaging, to unravel the dynamic tumor microenvironment (TME) in human steatotic HCC 41–44 (Figure 2).

FIGURE 2 Advanced technologies accelerate the knowledge expansion in the microenvironment of steatotic HCC.

Murai et al 43 first performed RNA sequencing in 113 patients with nonviral HCC and found that these tumors could be categorized into classes I, II, and III with class I enriched in TP53 mutations and class III enriched in CTNNB1 mutations. Forty-three out of 113 patients, mostly belonging to Class II, are considered immune class, which exhibits a high expression of immune-related genes such as cytotoxic T lymphocytes. Patients with nonviral HCC that are grouped as an immune class are less likely to display CTNNB1 mutations, aligning with existing literature indicating that CTNNB1 mutations are correlated with a “cold” tumor phenotype. 45

By interpreting the gene signature, authors found that steatotic HCC, which constitutes 23% of all nonviral HCC in this study, showed increased immune cells but also higher T-cell exhaustion signature (T-cell inhibitory receptors and transcription factors associated with exhaustion) as well as immunosuppressive TGFβ signaling. Furthermore, steatotic HCC also displayed gene signatures enriched in M2 macrophage and CAFs. Murai et al 43 further employed spatial transcriptomics and revealed that areas with high exhausted cytotoxic T lymphocytes, as marked by CD8A and NR4A1 double-positive spots, have enriched M2 macrophage gene and CAF gene as indicated by CD163 and VIM, respectively. The authors suggested that M2 macrophage and CAF might cause cytotoxic T lymphocytes to be exhausted in steatotic HCC; however, this causal relationship needs to be further substantiated. Lipidomic analysis revealed that palmitic acid (PA) was enriched in steatotic HCC as compared to other nonviral HCC. The authors further demonstrated that PA could induce the expressions of PDL1 in an HCC cell line. Furthermore, the coculturing experiment demonstrated that PA-treated HCC cells could induce CD206 and IL10 in macrophages and TGFβ in HSCs. These genes are known to be associated with immunosuppressive features of macrophages and HSCs.

Giraud et al 41 employed scRNA seq to provide a comprehensive landscape of innate cells in tumors and nontumorous tissues from 10 patients with HCC with various etiologies, including 5 steatotic HCC. Innate cells were enriched for scRNA analysis by fluorescence-activated cell–sorted CD45+panTCRαβ−CD19−, which specifically eliminated T and B cells, respectively, in the CD45+ leukocytes. THBS1+ myeloid cells expressed a high level of TREM1. The THBS1+ myeloid cell gene signature was associated with poor survival in patients with HCC. Further subclustering of THBS1+ myeloid cells identified a specific population called THBS1+ Mreg cells, which were further increased in steatotic HCC. Spatial transcriptomics showed that THBS1+ Mreg cells were situated in fibrotic tissues and had colocalization and interactions with carcinoma-associated fibroblasts (CAFs). The therapeutic administration of a TREM1 antibody elevated the CD8+/FoxP3+ T-cell ratio and enhanced the activity of CD8+ T cells in murine steatotic HCC tumors, suggesting the immunosuppressive roles of TREM1 in steatotic HCC.

Li et al employed mass cytometry imaging with 35 markers to compare the immune landscape of 27 human patients with HCC, including 16 MASH-HCC, 6 HBV-HCC, 5 HCV-HCC, and 6 healthy donors. 42 These 35 markers cover general immune populations for myeloid and lymphoid cells, as well as a few markers that indicate cytotoxicity (granzyme B) and immune exhaustion (PD-1, PD-L1, and ICOS). The study showed that HCC generally has increased immune cells than normal liver tissues. MASH-HCC has more polymorphonuclear-myeloid-derived suppressor cells (MDSCs) and less CD8+ T cells and NKT cells than viral HCC. Interestingly, the authors further dissected the immune distribution in the peritumor region, the nontumorous region at the boundary, the tumorous region at the boundary, and the tumorous region in MASH-HCC. The authors found that various immune subsets displayed unique spatial patterns across the 4 regions, with CD8+ T cells gradually decreasing across the tissues into the tumor region in MASH-HCC. While MDSCs and TAMs showed a negative correlation with T cells in MASH-HCC, the authors unraveled a differential distribution of immunosuppressive cells in these regions. Nontumorous tissues were occupied with Treg cells, whereas tumorous tissues were occupied with MSDCs and TAMs in patients with MASH-HCC patients. This implicated the importance of Treg cells in immune tolerance in the liver, while MDSCs and TAMs are the major expanded populations during pathological conditions such as MASH-HCC development. This study further supported the findings of Pfister and colleagues about the elevated levels of PD-1+CD8+ T cells in MASH-HCC. Moreover, it emphasized the interaction of these PD-1+CD8+T cells with PD-L1+ICOS+ MDSCs/TAMs in MASH-HCC, a phenomenon not observed in viral HCC. In MASH-HCC, cancer cells exhibited low expression of PD-L1 and ICOS, and there was a low connection score between T and HCC cells. These findings suggested that T-cell exhaustion in MASH-HCC is primarily attributed to MDSCs rather than HCC cells.

Interestingly, a very recent study revealed a potential role of Treg in the establishment of immunosuppressive TME in steatotic HCC.( 44 ) Prawira and colleagues first performed scRNA seq in 11 tumors from patients with steatotic HCC (5 cases) and nonsteatotic HCC (6 cases). They observed a general depletion of CD8+ T cells in steatotic HCC but an enrichment of lipid-associated Treg and CAF. They then validated these observations using CyTOF in 17 steatotic HCC and 27 nonsteaotic HCC. They further employed spatial transcriptomic analysis and demonstrated a preferential interaction of Treg and CAF at the tumor margin in steatotic HCC. They identified TNFSF14-TNFRSF14 as a specific ligand-receptor interaction between Treg and CAF in steatotic HCC and showed that this interaction potentially contributed to the unresponsiveness to immunotherapy in steatotic HCC. How TNFSF14-TNFRSF14 contributed to immunotherapy resistance warrants further investigation and could be a potential therapeutic target for steatotic HCC.

Collectively, these immune profiling studies showed that MASH-HCC is characterized by the increased presence of immunosuppressive cells, including M2-like macrophages, THBS1+ myeloid cells, CAFs, and Treg that potentially suppress CD8+ T-cell functions.

EXPERIMENTAL MODELS FOR MASH-HCC

Studying MASLD, MASH, and MASH-HCC in vitro is relatively challenging because of the lack of microenvironmental factors. Although there were studies using HCC cell lines derived from patients with MASLD-HCC, 46,47 whether these cell lines maintain their phenotypes as they were in the lipid-rich TME is unclear. Some studies showed that culturing primary hepatocytes or HCC cell lines in the presence of a mixture of free fatty acids could recapitulate certain MASLD/MASH phenotypes. Gomez-Lechon et al 48 demonstrated that culturing primary human hepatocytes or HepG2, a human HCC cell line, in a mixture of oleic acid and palmitic acid (2:1 ratio), the 2 most abundant free fatty acids in the human liver, induced lipid accumulation in liver cells to a comparable level to the steatotic liver. However, it is hard to mimic the inflammatory microenvironment in MASLD/MASH that involves multiple cell types in vitro. Ouchi et al 49 developed liver organoids that recapitulated steatohepatitis development upon free fatty acid exposure. Multicellular liver organoids were established from induced pluripotent stem cells. These liver organoids are composed of hepatocyte-like, stellate-like, and Kupffer-like cells. Exposure to oleic acid induced steatosis, inflammation cytokine production, and fibrosis in liver organoids. It was further shown that these steatotic liver organoids could be used in drug response testing.

Given the difficulties of studying steatotic HCC in vitro, current research heavily relies on in vivo models. Multiple murine models have been established for studying MASLD, MASH, and MASH-HCC. Dietary intervention, genetic manipulation, and carcinogen administration are the 3 major approaches that have been used in combination with each other to induce steatotic HCC. 50,51 Each has its advantages and limitations (Table 1). Dietary-based models are the most common ones that mimic the natural development of MASH-HCC in humans. Different types of diets are used, mainly derived from methionine/choline-deficient diet (MCD), HFD, and western diet (WD). Still, not all types of diets can recapitulate MASLD/MASH. For example, MCD induces steatohepatitis and fibrosis in a relatively short period of time, but it causes weight loss and increased insulin sensitivity, failing to reflect the metabolic dysfunctions in human MASLD. Compared to MCD, HFD and WD better mimic human MASLD development; however, it is time-consuming, and the incidence of HCC is low. To increase the tumor incidence and shorten the latency, MASH-inducing diets are combined with liver-specific genetic manipulation. Liver-specific knockout of Pten has been shown to accelerate MASH-HCC development in combination with HFD feeding. Another example is the use of MUP-uPA transgenic mice plus an HFD. The overexpression of urokinase plasminogen activator in hepatocytes induces ER stress and liver damage, promoting the development of MASH-HCC. However, these genetic alterations are not related to the mutational landscape in human MASH-HCC. Diethylnitrosamine (DEN), a carcinogen, is also used to increase MASH-HCC incidence in mice. A single dose of DEN is usually injected when the mice are 2-week-old followed by long-term feeding of an HFD to induce MASH-HCC. Recently, some researchers also combined MASH-inducing diets with orthotopic tumor implantation to establish murine MASH-HCC models. Researchers are recommended to employ multiple models to demonstrate the consistencies of their findings.

TABLE 1 Summary of mouse models for studying MASLD/MASH/MASH-HCC

Approach	Description	Pros and cons	
Dietary intervention only	Methionine/choline-deficient diet (MCD)	Pros: better mimics the natural development of human MASH-HCC
Cons: low HCC incidence and long latency	
	High-fat diet (HFD)		
	Western diet (WD)		
Dietary intervention and genetic manipulation	Hepatocyte-specific Pten null mice fed with HFD (AlbCre × Pten flox/flox)	Pros: improved HCC incidence and latency
Cons: not related to human MASH-HCC mutational landscape	
	MUP-uPA transgenic mice fed with HFD		
Dietary intervention and carcinogen administration	HFD and diethylnitrosamine (DEN)	Pros: improved HCC incidence and latency
Cons: HCCs are not driven by MASH	
Dietary intervention and orthotopic implantation	MCD/HFD/WD and orthotopic implantation of murine HCC cell lines		
Abbreviations: MASH, metabolic dysfunction–associated steatotic hepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease.

NOVEL THERAPEUTIC INNOVATIONS FOR MASH-HCC

Animal models play a crucial role in MASH-HCC research, particularly in unraveling the underlying mechanistic details that can lead to the discovery of novel treatments. Here, we will highlight several animal studies that have identified treatments that target the immune microenvironment in MASH-HCC. Wabitsch and colleagues studied how MASH influenced tumor-infiltrating CD8+ T cells. MASH was induced in immunocompetent mice by 3 different diets, including an MCD, a choline-deficient L-amino–defined diet, and a WD, respectively, for 2 weeks to 6 months. Then, orthotopic implantation was performed on these mice to establish MASH-HCC tumors. The authors demonstrated that anti-PD-1 only suppressed orthotopic HCC tumors in mice fed with a normal diet but not those with MASH-inducing diets. 52 Consistently, they validated the increase of CD8+ PD-1+ CXCR6+ T cells in diet-induced MASH liver. Intravital imaging revealed that the mobility of tumor-infiltrating CD8+ T cells was impaired in the MASH liver. This was attributed to the reduced mitochondrial fitness of CD8+ T cells in MASH liver as reflected by reduced glucose uptake, mitochondrial polarization, and mitochondrial mass. Metformin is an FDA-approved drug to regulate blood glucose levels. Metformin has been demonstrated to also alter the mitochondrial activity and metabolism of T cells. Interestingly, the authors showed that metformin was able to rescue the metabolic dysfunction and mobility impairment in CD8+ T cells. Demonstrated in the MASH-HCC animal models, it was shown that metformin and anti-PD-1 worked synergistically in suppressing MASH-HCC. This study suggested that resistance to anti-PD-1 in MASH-HCC maybe due to metabolic impairment in CD8+ T cells which could be rescued by the use of a metabolic drug.

Recent immune landscape profiling of MASH-HCC revealed that myeloid cells played an important role in creating an immunosuppressive TME. Leslie and colleagues identified neutrophils as the potential population responsible for anti-PD-1 resistance in MASH-HCC. In addition to orthotopic tumors implanted on MASH liver induced by WD in mice, an autochthonous model using DEN and American lifestyle–induced obesity syndrome diet (ALIOS) was also established. It was shown that CXCR2+ neutrophils were enriched in MASH-HCC in both murine models and human patients with HCC. 29 In patients with human HCC, neutrophil expression signatures were highly enriched in MASH-HCC as compared to HCC with other etiologies. The number of neutrophils in tumors increased with MASH severity. This CXCR2+ neutrophil population was further increased upon anti-PD-1 treatment in both murine MASH-HCC models, suggesting that this population could be a potential therapeutic target. Indeed, the use of CXCR2 antagonist (AZD5069) improved anti-PD-1 efficacy in MASH-HCC. CXCR2 antagonist and anti-PD-1 reprogrammed neutrophils to an immature state with enhanced phagocytosis and antigen presentation pathways. Mass cytometry imaging revealed that combination treatment of anti-PD-1 and CXCR2 antagonists also increased interactions between neutrophils, DCs, and CD8+ T cells, accompanied by enhanced activation of XCR1+ cDC1 and CD8+ T cells, as reflected by their CD86 and granzyme B expression respectively. This study not only identified CXCR2+ neutrophils as the contributor to anti-PD-1 resistance in MASH-HCC but also indicated the influence of immune cell interactions in establishing the immune landscape in MASH-HCC. This study highlighted the therapeutic opportunities of drugs that can enhance the immune cell interaction networks in suppressing MASH-HCC.

Pan et al 53 performed a CRISPR-Cas9 screening to identify druggable candidates that were essential to MASLD-HCC cell survival. The authors identified METTL3, an m6A methyltransferase, as the top candidate which was also overexpressed in patients with MASLD-HCC. 53 The authors established MASH-HCC by DEN and choline-deficient HFD in wild-type or liver-specific Mettl3 knock-in (Mettl3LKI) or knockout mice. Interestingly, these mouse models demonstrated that Mettl3 directly dictated the immune composition in MASH-HCC. Therefore, the authors performed scRNA seq on CD45+ cells isolated from MASH-HCC tumors of Mettl3LKI or wild-type mice. Tumor-infiltrating T cells were significantly reduced in Mettl3LKI mice, in particular GZMB+ and IFNG+ CD8+ T cells, suggesting that METTL3 suppressed CD8+ T-cell cytotoxicity. Mechanistically, METTL3 stabilized SCAP mRNA through m6A modification and promoted cholesterol production by cancer cells. Cholesterol secreted by cancer cells reduced granzyme B and IFN-γ production by CD8+ T cells. Therapeutically, METTL3 inhibitor (STM2457) alone inhibited MASH-HCC growth and worked in synergy with anti-PD-1 in suppressing MASH-HCC progression through enhancing CD8+ T-cell cytotoxicity.

DISCUSSION

Patients with HCC are treated in a one-size-fits-all manner regardless of their etiologies; however, viral and nonviral HCC respond to treatments differently. With the advancement of multi-omic platforms, accumulating evidence reveals that MASH-HCC has a distinct immune landscape that renders it unresponsive to immune checkpoint blockade. MASH-HCC is not only characterized by an increase of exhausted CD8+ T cells but also the immunosuppressive cells interacting with CD8+ T cells. With the support of a broad spectrum of murine MASH-HCC models, the observations in human MASH-HCC can be validated and further explored mechanistically. Moreover, these murine MASH-HCC models offer a great experimental platform for testing treatment efficacy in MASH-HCC. This review underscores the growing comprehension of the immune microenvironment in MASH-HCC. Furthermore, this review ignites several compelling scientific and clinical points about MASH-HCC that merit deeper exploration. For example, while CD8+ T cells are well-known for their cancer-killing role, it is also the contributor to liver damage and MASH development. Current studies suggest that CD8+ PD-1+ CXCR6+ T cells are the auto-aggressive CD8+ T-cell population contributing to MASH development. While PD-1 and CXCR6 are also highly expressed in tumor-infiltrating CD8+ T cells, treatments that can selectively activate other antigen-specific T cells while avoiding activation of auto-aggressive T cells should be explored. Moreover, it is becoming more apparent that myeloid cells play a crucial role in suppressing CD8+ T in MASH-HCC. Myeloid cells emerge as tantalizing targets for MASH-HCC. Therefore, more translational efforts should be devoted to studying the treatments that can block the immunosuppressive myeloid cells in MASH-HCC. In addition, metabolic dysfunction is frequently observed in CD8+ T cells in MASH-HCC. It is crucial to investigate how MASH-HCC alters the metabolism of other immune cell types, such as MDSCs and TAMs, that can potentially hinder the efficiency of treatments for MASH-HCC. Finally, the response of MASH-HCC to other standard treatment regimens in HCC, such as combination treatment of atezolizumab (anti-PD-L1) and bevacizumab (anti-VEGF), should also be investigated as this is going to replace tyrosine kinase inhibitors to be the first-line treatment for advanced HCC.

We will continue to leverage advanced multi-omic technologies in the analysis of mouse and human MASH-HCC tissues to address the points. In addition to knowing the numbers and functional status, the field should employ scRNA seq analysis for the identification of new subsets, including rare populations that are preferentially present in MASH-HCC. The immune cells undergo different transitional status. scRNA seq technology is especially useful for identifying the fine details of cellular subsets and transitional states in MASH-HCC, which may not be easily identified by conventional methods like flow cytometry. In the past, scRNA seq analysis requires isolated cells from fresh specimens. However, with the rapid development of technologies, it is now feasible to perform scRNA seq analysis on fixed tissues. This advancement provides more flexibilities in the collection of more rare samples and expands the potential applications to a wider range of specimens. In addition to scRNA seq, the use of spatial transcriptomics will expand our knowledge about how regional factors, such as hypoxia, tumor boundaries, tertiary lymphoid structures, and distance to blood vessels, affect the distribution of immune cells and their interactions in MASH-HCC. Moving forward, single-cell omic technologies will continue to bloom including single-cell epitranscriptomics (ATAC sequencing for chromatin accessibility and CUT&Tag sequencing for various histone modifications), proteomics, and metabolomics. These technologies will further propel the field to understand the immune microenvironment of MASH-HCC from different dimensions.

Finally, the most direct way to understand the differences in the TME of viral and nonviral HCC is to compare the 2 types of HCC. Notably, there are limitations in doing such a comparison in preclinical mouse models due to the lack of HBV mouse models that could faithfully recapitulate the pathogenesis of viral HCC. Comparison of viral and nonviral HCC is feasible in humans; however, the geographic distribution of viral HCC and nonviral HCC is uneven. International cross-center collaborations and a systemic collection of published scRNA seq and spatial transcriptomics data containing HCC of various etiologies will facilitate us in building a comprehensive roadmap that outlines how the microenvironments are caused by hepatitis virus and steatosis idiosyncratically.

AUTHOR CONTRIBUTIONS

Jacinth Wing-Sum Cheu and Carmen Chak-Lui Wong drafted the manuscript.

FUNDING INFORMATION

This work is supported by the National Natural Science Foundation of China—the Excellent Young Scientist Fund (Hong Kong and Macau) (Project#82022077), the Research Grant Council Collaborative Research Fund (RGC CRF) (C7008-22G) (C5106-23G), the Research Grant Council Research Impact Fund (RGC RIF) (R5008-22), the Research Grant Council Theme Based Research Scheme (RGC TBRS) (T12-716/22-R), and the Shenzhen Science and Technology Program (ZDSYS20210623091811035). This work is supported by the Centre for Oncology and Immunology under the Health@InnoHK initiative funded by the Innovation and Technology Commission, the Government of Hong Kong SAR, China. Carmen Chak-Lui Wong is the recipient of the University of Hong Kong Outstanding Research Supervisor Award.

CONFLICTS OF INTEREST

The authors have no conflicts to report.

Abbreviations: ALIOS, American lifestyle–induced obesity syndrome diet; CAF, carcinoma-associated fibroblast; cDC, conventional dendritic cell; DC, dendritic cell; DEN, diethylnitrosamine; HFD, high-fat diet; MASH, metabolic dysfunction–associated steatotic hepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; MCD, methionine/choline-deficient diet; MDSC, myeloid-derived suppressor cell; NET, neutrophil extracellular trap; NHANES III, National Health and Nutrition Examination Survey; PA, palmitic acid; TME, tumor microenvironment; WD, western diet.
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