Immunosuppression In Hepatocellular Carcinoma Research Articles

Trojan-Horse Strategy Targeting the Gut-Liver Axis Modulates Gut Microbiome and Reshapes Microenvironment for Orthotopic Hepatocellular Carcinoma Therapy.

Reversing the hepatic inflammatory and immunosuppressive microenvironment caused by gut microbiota-derived lipopolysaccharides (LPS), accumulating to the liver through the gut-liver axis, is crucial for suppressing hepatocellular carcinoma (HCC) and metastasis. However, synergistically manipulating LPS-induced inflammation and gut microbiota remains a daunting task. Herein, a Trojan-horse strategy is proposed using an oral dextran-carbenoxolone (DEX-CBX) conjugate, which combines prebiotic and glycyrrhetinic acid (GA) homologs, to targeted delivery GA to HCC through the gut-liver axis for simultaneous modulation of hepatic inflammation and gut microbiota. In the orthotopic HCC model, a 95-45% reduction in the relative abundances of LPS-associated microbiota is observed, especially Helicobacter, caused by DEX-CBX treatment over phosphate-buffered saline (PBS) treatment. Notably, a dramatic increase (37-fold over PBS) in the abundance of Akkermansia, which is known to strengthen systemic immune response, is detected. Furthermore, DEX-CBX significantly increased natural killer T cells (5.7-fold) and CD8+ T cells (3.9-fold) as well as decreased M2 macrophages (59% reduction) over PBS treatment, resulting in a tumor suppression rate of 85.4%. DEX-CBX is anticipated to offer a novel strategy to precisely modulate hepatic inflammation and the gut microbiota to address both the symptoms and root causes of LPS-induced immunosuppression in HCC.

Treatment Options for Hepatocellular Carcinoma Using Immunotherapy: Present and Future.

Hepatocellular carcinoma (HCC) is a common cancer, and the body's immune responses greatly affect its progression and the prognosis of patients. Immunological suppression and the maintenance of self-tolerance in the tumor microenvironment are essential responses, and these form part of the theoretical foundations of immunotherapy. In this review, we first discuss the tumor microenvironment of HCC, describe immunosuppression in HCC, and review the major biomarkers used to track HCC progression and response to treatment. We then examine antibody-based therapies, with a focus on immune checkpoint inhibitors (ICIs), monoclonal antibodies that target key proteins in the immune response (programmed cell death protein 1, anti-cytotoxic T-lymphocyte associated protein 4, and programmed death-ligand 1) which have transformed the treatment of HCC and other cancers. ICIs may be used alone or in conjunction with various targeted therapies for patients with advanced HCC who are receiving first-line treatments or subsequent treatments. We also discuss the use of different cellular immunotherapies, including T cell receptor (TCR) T cell therapy and chimeric antigen receptor (CAR) T cell therapy. We then review the use of HCC vaccines, adjuvant immunotherapy, and oncolytic virotherapy, and describe the goals of future research in the development of treatments for HCC.

Interferon-α induces differentiation of cancer stem cells and immunosuppression in hepatocellular carcinoma by upregulating CXCL8 secretion

Interferon-alpha (IFN-α) is widely used in the clinical treatment of patients with chronic hepatitis B and hepatocellular carcinoma (HCC). However, high levels of CXCL8 are associated with resistance to IFN-α therapy and poorer prognosis in advanced cancers. In this study, we investigated whether IFN-α could directly induce the production of CXCL8 in HCC cells and whether CXCL8 could antagonize the antitumor activity of IFN-α. We found that IFN-α not only upregulated the expression of the inducible genes CXCL9, CXCL10, CXCL11 and PD-L1, but also significantly stimulated CXCL8 secretion in HCC cells. Mechanically, IFN-α induces CXCL8 expression by activating the AKT and JNK pathways. In addition, our results demonstrate that IFN-α exposure significantly increases the differentiation of HCC stem cells, but this effect is reversed by the addition of the CXCL8 receptor CXCR1/2 inhibitor Reparixin and STAT3 inhibitor Stattic. Besides, our study reveals that the cytokine CXCL8 secreted by IFN-α-induced HCC cells inhibits T-cell function. Conversely, inhibition of CXCL8 promotes TNF-α and IFN-γ secretion by T cells. Finally, liver cancer patients who received anti-PD-1/PD-L1 immunotherapy with high CXCL8 expression had a lower immunotherapy efficacy. Overall, our findings clarify that IFN-α triggers immunosuppression and cancer stem cell differentiation in hepatocellular carcinoma by upregulating CXCL8 secretion. This discovery provides a novel approach to enhance the effectiveness of HCC treatment in the future.

LINC00942 inhibits ferroptosis and induces the immunosuppression of regulatory T cells by recruiting IGF2BP3/SLC7A11 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common malignant tumor with a high recurrence rate and a poor prognosis. Long intergenic nonprotein coding RNA 942 (LINC00942) is reported to be related to ferroptosis and the immune response in HCC and serves as an oncogene in various cancers. This research aimed to explore the contribution of LINC00942 in HCC progression. Functional assays were used to evaluate the functional role of LINC00942 in vitro and in vivo. Mechanistic assays were conducted to assess the association of LINC00942 with insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) and solute carrier family 7 member 11 (SLC7A11) and the regulatory pattern of LINC00942 in HCC cells. LINC00942 was found to exhibit upregulation in HCC tissue and cells. LINC00942 facilitated HCC cell proliferation, suppressed ferroptosis, and converted naive CD4+ T cells to inducible Treg (iTreg) cells by regulating SLC7A11. Furthermore, SLC7A11 expression was positively modulated by LINC00942 in HCC cells. IGF2BP3 was a shared RNA-binding protein (RBP) for LINC00942 and SLC7A11. The binding between the SLC7A11 3′ untranslated region and IGF2BP3 was verified, and LINC00942 was found to recruit IGF2BP3 to promote SLC7A11 mRNA stability in an m6A-dependent manner. Moreover, mouse tumor growth and proliferation were inhibited, and the number of FOXP3+CD25+ T cells was increased, while ferroptosis was enhanced after LINC00942 knockdown in vivo. LINC00942 suppresses ferroptosis and induces Treg immunosuppression in HCC by recruiting IGF2BP3 to enhance SLC7A11 mRNA stability, which may provide novel therapeutic targets for HCC.

Crosstalk between myeloid and B cells shapes the distinct microenvironments of primary and secondary liver cancer.

The tumor microenvironment is distinctive in primary and secondary liver cancer. B cells represent an important component of immune infiltrates. Here, we demonstrated that B cells are an important regulator in hepatocellular carcinoma (HCC) and colorectal cancer liver metastasis (CRLM) microenvironments. B cells displayed distinct developmental trajectories in HCC and CRLM. Single-cell analysis revealed that IgG+ plasma cells preferentially accumulated in HCC while IgA+ plasma cells were preferentially enriched in CRLM. Mechanistically, IgG+ plasma cells in HCC were recruited by tumor-associated macrophages via the CXCR3-CXCL10 axis, whereas IgA+ plasma cells in CRLM were recruited by metastatic tumor cells via CCR10-CCL28 signaling. Functionally, IgG+ plasma cells preferentially promoted pro-tumorigenic macrophages formation in HCC, and IgA+ plasma cells preferentially induced granulocytic myeloid-derived suppressor cells activation in CRLM. Clinically, increased infiltration of IgG+ plasma cells and macrophages in HCC was correlated to worse survival, while increased intratumoral IgA+ plasma cells and neutrophils in CRLM indicated poor prognosis. Taken together, this study demonstrated plasma and myeloid cell-mediated immunosuppression in HCC and CRLM, suggesting that selectively modulating primary or secondary tumor-related immunosuppressive regulatory networks might reprogram the microenvironment and provide an immunotherapeutic strategy for treating liver cancer.

Lenalidomide Promotes Melarsoprol-Activated cGAS-STING-Mediated Immunotherapy for Hepatocellular Carcinoma via Attenuating TNF-α Activity

Current immunotherapy has limited efficacy in hepatocellular carcinoma (HCC) due to the immunosuppressive tumor microenvironment (TME). The activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of the interferon genes (STING) pathway demonstrates great potential to unleash the immunosuppressive TME. The capacity of melarsoprol (MEL; an arsenic-containing drug) on activating the cGAS-STING pathway in HCC cells for antitumor immunotherapy was confirmed for the first time in this study. When MEL (particularly at high doses) activated the cGAS-STING pathway, however, HCC growth was not fully inhibited, mainly due to the hyperactivation of tumor necrosis factor α (TNF-α; a cytokine associated with the cGAS-STING pathway). Accordingly, lenalidomide (LEN; a clinically approved TNF-α inhibitor) was used to alleviate pro-tumorigenic effects of the MEL-activated cGAS-STING pathway while maintaining immunotherapeutic effects. To modulate the cGAS-STING pathway in vivo, a poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticle (NP) was used for co-delivery of MEL and LEN, and the co-loaded NP was coated with aminoethyl anisamide (AEAA, a ligand for Sigma-1 receptor)-modified erythrocyte membrane, forming a co-formulation. In this study, co-formulation was able to modulate the cGAS-STING-mediated efficacy and reverse the immunosuppressive TME in allograft and carcinogen-induced orthotopic HCC mouse models, respectively. Our study reveals an intrinsic hurdle to DNA damaging drug-mediated cGAS-STING monotherapy and provides a promising combination strategy to tackle immunosuppression in HCC.

Myeloid cell-cycle-related-kinase drives tumor immunosuppression in hepatocellular carcinoma

Abstract Myeloid-derived suppressor cell (MDSC) comprises a heterogeneous population of immature myeloid cells featured by potent immunosuppression. We have uncovered a significant role of hepatoma-intrinsic cyclin-dependent-kinase 20 (CDK20), or cell-cycle-related-kinase (CCRK) in promoting tumor progression and immunotherapy resistance in hepatocellular carcinoma (HCC). As emerging evidence has highlighted the importance of immune cell intrinsic CDKs, we further explored the potentials of CCRK expressions and functions in different immune cells from HCC-bearing mice and patients. Of note, we uncovered specific CCRK overexpression in MDSCs but not lymphocytes, which functioned in regulating MDSC proliferation, immunosuppression, and differentiation. Intratumoral injection of bone-marrow-derived MDSCs with Ccrkknockdown resulted in impaired tumorigenicity, accompanied with increased CD8 +T cell responses. Mechanistically, myeloid CCRK upregulation was functional associated with IL-6-mediated MDSC expansion and immunosuppression. Inhibition of myeloid CCRK in turn suppressed STAT3 to revert E4-binding protein 4-dependent transcriptional alterations. Using a transgenic mouse bearing Ccrk-indel mutation (Ccrkindel/indel), we found that impaired Ccrksignal reprogramed the pro-tumoral MDSCs into an antitumoral phenotype, resulting in suppression of tumor growth in our established HCC orthotopic mouse models. As we also showed CCRK overexpression in HCC patient-derived MDSCs, the findings of our study not only unravel mechanistic insights in MDSC maintenance and immunosuppression, but also offer a novel therapeutic kinase-target in HCC therapies. The project is supported by RGC/GRF14108219. Supported by grants from general research fund (GRF)/research grants council (RGC) of Hong Kong, 14108219.

LRPPRC facilitates tumor progression and immune evasion through upregulation of m6A modification of PD-L1 mRNA in hepatocellular carcinoma.

LRPPRC is a newly discovered N6-methyladenosine (m6A) modification reader, which potentially affects hepatocellular carcinoma (HCC) progression. PD-L1 in tumor cells is essential for tumor immune evasion. This work investigated the LRPPRC-mediated m6A-modification effect on PD-L1 mRNA and immune escape in HCC. Expression and clinical implication of LRPPRC and PD-L1 were measured in human HCC cohorts. The influence of LRPPRC on malignant behaviors of HCC cells was investigated through in vitro assays and xenograft tumor murine models. The posttranscriptional mechanism of LRPPRC on PD-L1 and anti-tumor immunity was elucidated in HCC cells via RIP, MeRIP-qPCR, RNA stability, immunohistochemical staining, and so forth. LRPPRC exhibited the notable upregulated in human HCC tissues, which was in relation to advanced stage and worse overall survival and disease-free survival. Impaired proliferative capacity and G2/M phage arrest were found in LRPPRC-knockout cells, with increased apoptotic level, and attenuated migratory and invasive abilities. In HCC patients and murine models, LRPPRC presented a positive interaction with PD-L1, with negative associations with CD8+, and CD4+ T-cell infiltrations and chemokines CXCL9, and CXCL10. LRPPRC loss downregulated the expression of PD-L1 and its m6A level in HCC cells. Moreover, LRPPRC suppression mitigated tumor growth in murine models and improved anti-tumor immunity and immune infiltration in tumors. This work unveiled that LRPPRC may posttranscriptionally upregulate PD-L1 partially with an m6A-dependent manner for heightening mRNA stabilization of PD-L1 and provided a new mechanism for m6A regulator-mediated immunosuppression in HCC.

Identification of Prognosis-Related Oxidative Stress Model with Immunosuppression in HCC

For hepatocellular carcinoma (HCC) patients, we attempted to establish a new oxidative stress (OS)-related prognostic model for predicting prognosis, exploring immune microenvironment, and predicting the immunotherapy response. Significantly differently expressed oxidative stress-related genes (DEOSGs) between normal and HCC samples from the Cancer Genome Atlas (TCGA) were screened, and then based on weighted gene coexpression network analysis (WGCNA), HCC-related hub genes were discovered. Based on the least absolute shrinkage and selection operator (LASSO) and cox regression analysis, a prognostic model was developed. We validated the prognostic model's predictive power using an external validation cohort: the International Cancer Genome Consortium (ICGC).Then a nomogram was determined. Furthermore, we also examined the relationship of the risk model and clinical characteristics as well as immune microenvironment. 434 DEOSGs, comprising 62 downregulated and 372 upregulated genes (p < 0.05 and |log2FC| ≥ 1), and 257 HCC-related hub genes were recognized in HCC. Afterward, we built a five-DEOSG (LOX, CYP2C9, EIF2B4, EZH2, and SRXN1) prognostic risk model. Using the nomogram, the risk model was shown to have good prognostic value. Compared to the low risk group, HCC patients with high risk had poorer outcomes, worse pathological grades, and advanced tumor stages (p < 0.05). There were significant increases in LOX, EIF2B4, EZH2, and SRXN1 expression in HCC samples, while CYP2C9 expression was decreased. Finally, Real-time PCR (RT-qPCR) confirmed the mRNA expressions of five genes (CYP2C9, EIF2B4, EZH2, SRXN1, LOX) in HCC cell lines. Our study constructed a prognostic OS-related model with strong predictive power and potential as an immunosuppressive biomarker for HCC leading to improving prediction and providing new insights for HCC immunotherapy.

Association between HMGA1 and immunosuppression in hepatocellular carcinoma: A comprehensive bioinformatics analysis.

The high mobility group A1 (HMGA1) gene is overexpressed in malignant tumors, and its expression level correlates with the progression and metastasis of tumors. However, the specific role of HMGA1 in hepatocellular carcinoma (HCC) and relevant influencing approaches in tumor immunity remain unclear. In this study, the expression and clinical significance of HMGA1 in HCC immunity were analyzed. The expression levels of HMGA1 mRNA and protein in HCC tissue and normal liver tissue were analyzed based on the cancer genome atlas, the gene expression omnibus and the Human Protein Atlas databases. The correlation between HMGA1 and clinicopathological factors was analyzed, and survival was estimated based on the expression of HMGA1. Gene set cancer analysis and the TISIDB database were used to identify tumor-infiltrating immune cells and immune inhibitors. Gene set enrichment analysis was performed to determine the involved signaling pathway. The HMGA1 genetic alterations were identified with the cBioPortal for Cancer Genomics. The expression of HMGA1 mRNA and protein was significantly higher in HCC tissue and negatively correlated with survival. Neutrophils, Th17 cells, several immune inhibitors, and signaling pathways were positively correlated with the expression of HMGA1. Amplification was the main type of genetic alteration in HMGA1. These findings demonstrate that HMGA1 can be a therapeutic target and a potential biomarker to predict the prognosis of patients with HCC. HMGA1 may affect the progression of HCC by suppressing the immune function of these patients.

Overexpression of SMS in the tumor microenvironment is associated with immunosuppression in hepatocellular carcinoma.

Disorders of polyamine metabolism may contribute to the development of hepatocellular carcinoma (HCC), but the precise mechanism remains unknown. This study reports that spermine synthase (SMS), an enzyme involved in polyamine biosynthesis, is overexpressed in HCC and not associated with hepatitis virus infection in HCC patients. The results of analyzing the clinical data of HCC patients showed that SMS level as a categorical dependent variable was related to clinicopathological features of poor prognosis. Furthermore, the Kaplan-Meier survival analysis and ROC curve indicated that increased SMS level is associated with poor survival rate in HCC and may be a potential biomarker to discriminate HCC tissues. However, SMS overexpression limited the therapeutic effect of immune checkpoint blockade (ICB), which seemed to be related to the immunosuppressive effect of the HCC immune microenvironment formed by higher mRNA transcript levels of immune checkpoints and higher infiltration levels of immunosuppressive cells. In samples with high and low SMS expression, functional enrichment analysis of the differentially expressed genes (DEGs) showed that SMS may be linked to the occurrence and development of HCC by affecting a variety of immune-related pathways, such as Intestinal immune network for IgA production, Fc gamma R-mediated phagocytosis, Antigen processing and presentation, Th1 and Th2 cell differentiation. Subsequently, analysis of the co-expression network of SMS in the liver hepatocellular carcinoma (LIHC) cohort revealed that SMS has a broad impact on multiple important immune- and metabolic-related processes in HCC. In summary, SMS is a promising biomarker to differentiate the prognosis, immune characteristics, and holds promise as a potential target for ICB therapy to improve HCC.

Astragalus polysaccharide (APS) attenuated PD-L1-mediated immunosuppression via the miR-133a-3p/MSN axis in HCC

Context Astragalus polysaccharide (APS) is a new tumour therapeutic drug, that has an inhibitory effect on a variety of solid tumours. Tumour cell immunosuppression is related to the up-regulation of programmed death ligand 1 (PD-L1). However, whether APS exerts its antitumor effect by regulating PD-L1 remains unclear. Objective To explore whether APS exerts its antineoplastic effect via regulating PD-L1-mediated immunosuppression in hepatocellular carcinoma (HCC). Materials and methods SMMC-7721 cells were subcutaneous injected into BALB/C mice for HCC model establishment. Mice were intraperitoneally injected with 100, 200 and 400 mg/kg APS for 12 days. Immunohistochemistry (IHC) was performed to assess CD8+ T cells' rate and PD-L1 level in HCC tissues. HCC cells were pre-treated with 0.1, 0.5 and 1 mg/mL APS for 4 h, then were treated with 10 ng/mL IFN-γ 24 h. PD-L1 level and cell apoptosis was detected by flow cytometry. PD-L1 and Moesin (MSN) proteins were measured by western blot. MiR-133a-3p and MSN mRNA levels were assessed by qRT-PCR. The targets of miR-133a-3p were predicted by starBase, and which was verified by dual-luciferase reporter assay. Results Our findings illustrated that APS dose-dependently inhibited HCC growth tested with IC50 values of 4.2 mg/mL, and IFN-γ-induced PD-L1 expression and attenuated PD-L1-mediated immunosuppression in HCC cells. APS attenuated PD-L1-mediated immunosuppression via miR-133a-3p in HCC cells. Besides, miR-133a-3p targeted to MSN, and MSN inhibited the antitumor effect of APS by maintaining the stability of PD-L1. Moreover, APS attenuated PD-L1-mediated immunosuppression via the miR-133a-3p/MSN axis. Conclusions APS attenuated PD-L1-mediated immunosuppression via miR-133a-3p/MSN axis to develop an antitumor effect. APS may be an effective drug for HCC treatment.

PD-L1-Mediated Immunosuppression in Hepatocellular Carcinoma: Relationship with Macrophages Infiltration and Inflammatory Response Activity.

Immune dysfunction and pro-oncogenic inflammation play critical roles in malignant progression and non-response to immunotherapy for hepatocellular carcinoma (HCC). In particular, PD-1/PD-L1 blockade therapy could induce durable tumor remissions and improve the prognosis of patients to a certain extent. However, PD-L1, as a promising biomarker, has limited knowledge about its relevance to tumor microenvironment (TME) characterization and endogenous inflammatory immune responses. In this study, we systematically investigated and characterized the important intercommunication of PD-L1 with immunosuppressive TME and inflammatory response activity in HCC and predicted promising therapeutic drugs to improve the current therapeutic strategy for specific patients. We identified aberrant expression patterns of PD-L1 in HCC and completely different clinical and molecular characteristics among the PD-L1 subgroups. PD-L1 positively associated with immunosuppressive macrophages and macrophage-derived cytokines, which may contribute to the polarization of macrophages. Moreover, inflammatory response activity exhibited significant differences between high and low PD-L1 expression groups and had robust positive correlativity of the infiltration level of tumor-associated macrophages. Notably, given the immunosuppressive and inflammatory microenvironment in HCC, we screened four candidate drugs, including dasatinib, vemurafenib, topotecan and AZD6482, and corroborated in two pharmacogenomics databases, which might have potential therapeutic implications in specific HCC patients. Our results enhanced the understanding of linkage in PD-L1 expression patterns with macrophages and inflammation, which may provide new insight into the pathogenic mechanisms and potential therapeutic strategy for HCC.

Prognostic value of PD-L1 expression combined with hypoxia-associated immunosuppression in hepatocellular carcinoma.

Background: Hypoxia and immunosuppression are two properties of cancer. This study intends to establish the potential relationship between these two hallmarks in hepatocellular carcinoma (HCC). Materials & methods: A bioinformatics analysis of data obtained from the Cancer Genome Atlas and a retrospective single-center analysis based on a tissue microarray were utilized in this study. Results: We identified a hypoxia-high subtype of patients with immunosuppressive HCC which represented a poor prognosis in the Cancer Genome Atlas cohort. Immunohistochemical analysis of the tissue microarray showed that tumor PD-L1 expression was positively linked to HIF-1α expression, pro-tumor immunocyte infiltration and poor survival in HCC patients. Conclusion: This study provides evidence supporting the correlation between hypoxic signals and immunosuppression in HCC; the combined use of them might improve survival prediction and act as a potential predictor for PD-1/PD-L1 therapy.

Combining p53 mRNA nanotherapy with immune checkpoint blockade reprograms the immune microenvironment for effective cancer therapy

Immunotherapy with immune checkpoint blockade (ICB) has shown limited benefits in hepatocellular carcinoma (HCC) and other cancers, mediated in part by the immunosuppressive tumor microenvironment (TME). As p53 loss of function may play a role in immunosuppression, we herein examine the effects of restoring p53 expression on the immune TME and ICB efficacy. We develop and optimize a CXCR4-targeted mRNA nanoparticle platform to effectively induce p53 expression in HCC models. Using p53-null orthotopic and ectopic models of murine HCC, we find that combining CXCR4-targeted p53 mRNA nanoparticles with anti-PD-1 therapy effectively induces global reprogramming of cellular and molecular components of the immune TME. This effect results in improved anti-tumor effects compared to anti-PD-1 therapy or therapeutic p53 expression alone. Thus, our findings demonstrate the reversal of immunosuppression in HCC by a p53 mRNA nanomedicine when combined with ICB and support the implementation of this strategy for cancer treatment.

Plasmacytoid dendritic cells recruited by HIF-1α/eADO/ADORA1 signaling induce immunosuppression in hepatocellular carcinoma.

Plasmacytoid dendritic cells (pDCs) play immunosuppressive roles in the tumor microenvironment (TME). However, the molecular mechanisms underlying the recruitment and dysfunction of pDCs in the TME remain largely elusive, especially in hepatocellular carcinoma (HCC). In this study, we observed the accumulation of pDCs in the blood, tumor tissue, and ascitic fluid of HCC patients. A high density of tumor-infiltrating pDCs was correlated with poor prognosis in patients with HCC. Hypoxia-induced extracellular adenosine (eADO) significantly enhanced pDC recruitment into tumors via the adenosine A1 receptor (ADORA1). Mechanistically, hypoxia-inducible factor 1-alpha (HIF-1α) transcriptionally upregulated the expression of the ectonucleotidases CD39 and CD73 in HCC cells, both of which are essential for the generation of eADO. Moreover, eADO-stimulated pDCs promoted the induction of regulatory T cells and suppressed proliferation and cytotoxicity of CD8+ T cells. Depletion of pDCs using a monoclonal antibody or an ADORA1 antagonist significantly improved antitumor immunity and suppressed HCC growth in the immunocompetent HCC mouse model. Thus, targeting pDC recruitment may serve as a potential adjuvant strategy for immunotherapies in HCC.

MicroRNA-15a/16-1 Prevents Hepatocellular Carcinoma by Disrupting the Communication Between Kupffer Cells and Regulatory T Cells

Hepatocellular carcinoma (HCC) is characterized by intratumoral accumulation of regulatory T cells (Tregs), which suppresses antitumor immunity. This study was designed to investigate how microRNAs regulate immunosuppression in HCC. FVB/NJ mice were hydrodynamically injected with AKT/Ras or c-Myc and Sleeping Beauty transposon to induce HCC. The Sleeping Beauty system was used to deliver microRNA-15a/16-1 into livers of mice. Flow cytometry and immunostaining were used to determine changes in the immune system. Hydrodynamic injection of AKT/Ras or c-Myc into mice resulted in hepatic enrichment of Tregs and reduced cytotoxic T cells (CTLs) and HCC development. HCC impaired microRNA-15a/16-1 biogenesis in Kupffer cells (KCs) of AKT/Ras and c-Myc mice. Hydrodynamic injection of microRNA-15a/16-1 fully prevented HCC in AKT/Ras and c-Myc mice, while 100% of control mice died of HCC. Therapeutically, microRNA-15a/16-1 promoted a regression of HCC in both mouse models, impaired hepatic enrichment of Tregs, and increased hepatic CTLs. Mechanistically, a significant increase was observed in serum C-C motif chemokine 22 (CCL22) and transcription of Ccl22 in KCs of AKT/Ras and c-Myc mice. MicroRNA-15a/16-1 prevented KCs from overproducing CCL22 by inhibiting nuclear factor-κB that activates transcription of Ccl22. By reducing CCL22 binding to C-C chemokine receptor type 4 on Tregs, microRNA-15a/16-1 impaired Treg chemotaxis. Disrupting the interaction between microRNA-15a/16-1 and nuclear factor-κB impaired the ability of microRNA-15a/16-1 to prevent hepatic Treg accumulation and HCC. Depletion of cluster of differentiation 8+ T cells and additional treatment of CCL22 recovered growth of HCC that was fully prevented by microRNA-15a/16. MicroRNA-15a/16-1 attenuates immunosuppression by disrupting CCL22-mediated communication between KCs and Tregs. MicroRNA-15a/16-1 represents a potential immunotherapy against HCC.

KDM1A Promotes Immunosuppression in Hepatocellular Carcinoma by Regulating PD-L1 through Demethylating MEF2D.

Background Immune checkpoint inhibitor therapy targeting antiprogrammed cell death-1 (anti-PD-1) or its ligand (anti-PD-L1) is effective in the treatment of some hepatocellular carcinomas (HCC). Hence, further identification of biological targets related to PD-L1 regulation in HCC is beneficial to improve the clinical efficacy of immunotherapy. Some HCC cells express lysine-specific demethylase 1A (KDM1A), which is implicated in the reduced survival time of patients. Here, we studied whether the level of PD-L1 and the immunosuppression are regulated by KDM1A and its miRNA in HCC cells. Methods In the present study, we studied clinical data from The Cancer Genome Atlas (TCGA) database. We performed qPCR and western blotting assays to measure the expression level of genes of interest. PD-L1 expression was also analyzed by FACS. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate gene knockout cells to investigate the relationships of genes of interest. We also developed a reporter gene assay (RGA) to explore the changes in T cell-induced antitumor immunity relative to PD-L1 expression in HCC cells. The binding between proteins and promoters or miRNAs and their target genes was explored by luciferase reporter assays. Results The results showed that PD-L1 and KDM1A were increased in HCC patients and cells, and KDM1A promoted the expression of PD-L1 in HCC cells. Our findings showed that the enhancement of PD-L1 expression was not attributed to mitochondrial dysfunction caused by increases in KDM1A in HCC cells. Furthermore, we observed a lower level of MEF2D methylation in HCC cells than in normal human liver cells. Demethylated MEF2D could bind to the promoter of PD-L1 and activate its expression, while KDM1A interacted with MEF2D and acted as a demethylase to reduce its methylation. Moreover, a new miRNA, miR-329-3p, targeting KDM1A was found to regulate the PD-L1 expression profile in HCC cells. In the xenograft model, the tumors treated with miR-329-3p showed growth inhibition. Conclusions Mechanistically, miR-329-3p inhibits tumor cellular immunosuppression and reinforces the response of tumor cells to T cell-induced cytotoxic effect by targeting KDM1A mRNA and downregulating its expression, which contributed to MEF2D demethylation and activation of PD-L1 expression.

Toward a new landscape for the mechanism of immunosuppression in hepatocellular carcinoma

Toward a new landscape for the mechanism of immunosuppression in hepatocellular carcinoma

Hypoxic Characteristic in the Immunosuppressive Microenvironment of Hepatocellular Carcinoma.

Background: Generally, hepatocellular carcinoma (HCC) exists in an immunosuppressive microenvironment that promotes tumor evasion. Hypoxia can impact intercellular crosstalk in the tumor microenvironment. This study aimed to explore and elucidate the underlying relationship between hypoxia and immunotherapy in patients with HCC.Methods: HCC genomic and clinicopathological datasets were obtained from The Cancer Genome Atlas (TCGA-LIHC), Gene Expression Omnibus databases (GSE14520) and International Cancer Genome Consortium (ICGC-LIRI). The TCGA-LIHC cases were divided into clusters based on single sample gene set enrichment analysis and hierarchical clustering. After identifying patients with immunosuppressive microenvironment with different hypoxic conditions, correlations between immunological characteristics and hypoxia clusters were investigated. Subsequently, a hypoxia-associated score was established by differential expression, univariable Cox regression, and lasso regression analyses. The score was verified by survival and receiver operating characteristic curve analyses. The GSE14520 cohort was used to validate the findings of immune cell infiltration and immune checkpoints expression, while the ICGC-LIRI cohort was employed to verify the hypoxia-associated score.Results: We identified hypoxic patients with immunosuppressive HCC. This cluster exhibited higher immune cell infiltration and immune checkpoint expression in the TCGA cohort, while similar significant differences were observed in the GEO cohort. The hypoxia-associated score was composed of five genes (ephrin A3, dihydropyrimidinase like 4, solute carrier family 2 member 5, stanniocalcin 2, and lysyl oxidase). In both two cohorts, survival analysis revealed significant differences between the high-risk and low-risk groups. In addition, compared to other clinical parameters, the established score had the highest predictive performance at both 3 and 5 years in two cohorts.Conclusion: This study provides further evidence of the link between hypoxic signals in patients and immunosuppression in HCC. Defining hypoxia-associated HCC subtypes may help reveal potential regulatory mechanisms between hypoxia and the immunosuppressive microenvironment, and our hypoxia-associated score could exhibit potential implications for future predictive models.