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Fibrinogen-like protein 1 (FGL1) contributes to the proliferation and metabolism of hepatocytes; however, as a major ligand of the immune checkpoint, its role in the liver regional immune microenvironment is poorly understood. Hepatocytes specifically and highly expressed FGL1 under normal physiological conditions. Increases in hepatic CD8+ T and NK cell numbers and functions were found in Fgl1-deficient (Fgl1-/-) mice, but not in the spleen or lymph node, similar to findings in anti-FGL1 mAb-treated wild-type mice. Furthermore, Fgl1 deficiency or anti-FGL1 mAb blockade restrained liver metastasis and slowed the growth of orthotopic tumors, with significantly prolonged survival of tumor-bearing mice. Tumor-infiltrating hepatic CD8+ T and NK cells upregulated the expression of lymphocyte activation gene-3 (LAG-3) and exhibited stronger antitumor activities after anti-FGL1 treatment. The antitumor efficacy of FGL1 blockade depended on cytotoxic T lymphocytes and NK cells, demonstrated by using a cell-deficient mouse model and cell transfer in vivo. In vitro, FGL1 directly inhibited hepatic T and NK cells related to the receptor LAG-3. In conclusion, hepatocyte-derived FGL1 played critical immunoregulatory roles in the liver and contributed to liver metastasis and tumor growth by inhibiting CD8+ T and NK cell functions via the receptor LAG-3, providing a new strategy for liver cancer immunotherapy.

Background and Aims: The outcomes of HBV infections are related to complex immune imbalances; however, the precise mechanisms by which HBV induces immune dysfunction are not well understood., Methods: HBV transgenic (HBs-Tg) mice were used to investigate intrahepatic NK cells in two distinct subsets: conventional NK (cNK) and liver-resident NK (LrNK) cells during a chronic HBV infection., Results: The cNK cells, but not the LrNK cells, were primarily responsible for the increase in the number of bulk NK cells in the livers of ageing HBs-Tg mice. The hepatic cNK cells showed a stronger ability to produce IL-10, coupled with a higher expression of CD69, TIGIT and PD-L1, and lower NKG2D expression in ageing HBs-Tg mice. A lower mitochondrial mass and membrane potential, and less polarized localization were observed in the hepatic cNK cells compared with the splenic cNK cells in the HBs-Tg mice. The enhanced galectin-3 (Gal-3) secreted from HBsAg + hepatocytes accounted for the IL-10 production of hepatic cNK cells via ITGB1 signaling. For humans, LGALS3 and ITGB1 expression is positively correlated with IL-10 expression, and negatively correlated with the poor clinical progression of HCC., Conclusions: Gal-3-ITGB1 signaling shapes hepatic cNK cells but not LrNK cells during a chronic HBV infection, which may correlate with HCC progression.

Background and Aims: The innate-like mucosa-associated invariant T (MAIT) cells are enriched in human liver and have been linked to human HCC. However, their contributions to the progression of HCC are controversial due to the heterogeneity of MAIT cells, and new MAIT cell subsets remain to be explored., Approach and Results: Combining single cell RNA sequencing (scRNA-seq) and flow cytometry analysis, we performed phenotypic and functional studies and found that FOXP3 + CXCR3 + MAIT cells in HCC patients were regulatory MAIT cells (MAITregs) with high immunosuppressive potential. These MAITregs were induced under Treg-inducing condition and predominantly from FOXP3 - CXCR3 + MAIT cells, which displayed mild Treg-related features and represented a pre-MAITreg reservoir. In addition, the induction and function of MAITregs were promoted by β1 adrenergic receptor signaling in pre-MAITregs and MAITregs, respectively. In HCC patients, high proportion of the intratumoral MAITregs inhibited antitumor immune responses and was associated with poor clinical outcomes., Conclusions: Together, we reveal an immunosuppressive subset of MAIT cells in HCC patients that contributes to HCC progression, and propose a control through neuroimmune crosstalk., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies, whose initiation and development are driven by alterations in driver genes. In this study, we identified four driver genes (TP53, PTEN, CTNNB1, and KRAS) that show a high frequency of somatic mutations or copy number variations (CNVs) in patients with HCC. Four different spontaneous HCC mouse models were constructed to screen for changes in various kinase signaling pathways. The sgTrp53 + sgPten tumor upregulated mTOR and noncanonical nuclear factor-κB signaling, which was shown to be strongly inhibited by rapamycin (an mTOR inhibitor) in vitro and in vivo. The JAK-signal transducer and activator of transcription (STAT) signaling was activated in Ctnnb1 mut  + sgPten tumor, the proliferation of which was strongly inhibited by napabucasin (a STAT3 inhibitor). Additionally, mTOR, cytoskeleton, and AMPK signaling were upregulated while rapamycin and ezrin inhibitors exerted potent antiproliferative effects in sgPten + Kras G12D tumor. We found that JAK-STAT, MAPK, and cytoskeleton signaling were activated in sgTrp53 + Kras G12D tumor and the combination of sorafenib and napabucasin led to the complete inhibition of tumor growth in vivo. In patients with HCC who had the same molecular classification as our mouse models, the downstream signaling pathway landscapes associated with genomic alterations were identical. Our research provides novel targeted therapeutic options for the clinical treatment of HCC, based on the presence of specific genetic alterations within the tumor., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The highly variable response rates to immunotherapies underscore our limited knowledge about how tumors can manipulate immune cells. Here the membrane topology of natural killer (NK) cells from patients with liver cancer showed that intratumoral NK cells have fewer membrane protrusions compared with liver NK cells outside tumors and with peripheral NK cells. Dysregulation of these protrusions prevented intratumoral NK cells from recognizing tumor cells, from forming lytic immunological synapses and from killing tumor cells. The membranes of intratumoral NK cells have altered sphingomyelin (SM) content and dysregulated serine metabolism in tumors contributed to the decrease in SM levels of intratumoral NK cells. Inhibition of SM biosynthesis in peripheral NK cells phenocopied the disrupted membrane topology and cytotoxicity of the intratumoral NK cells. Targeting sphingomyelinase confers powerful antitumor efficacy, both as a monotherapy and as a combination therapy with checkpoint blockade., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background and Aims: Chronic HBV infection is the leading cause of HCC and a serious health problem in China, East Asia, and North African countries. Effective treatment of HBV-related HCC is currently unavailable. This study evaluated the therapeutic potential of T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade in HBV-related HCC., Approach and Results: A mouse model of spontaneous HBV-related HCC was generated by replacing wild-type hepatocytes with HBsAg + hepatocytes (namely HBs-HepR mice). The tumors in HBs-HepR mice were inflammation-associated HCC, similar to HBV-related HCC in patients, which was distinguished from other HCC mouse models, such as diethylnitrosamine-induced HCC, TGF-β-activated kinase 1 knockout-induced HCC, HCC in a stelic animal model, or NASH-induced HCC. HCC in HBs-HepR mice was characterized by an increased number of CD8 + T cells, whereas the production of IL-2, TNF-α, and interferon-gamma (IFN-γ) by intrahepatic CD8 + T cells was decreased. Increased expression of TIGIT on CD8 + T cells was responsible for functional exhaustion. The therapeutic effect of TIGIT blockade was investigated at the early and middle stages of HCC progression in HBs-HepR mice. TIGIT blockade reinvigorated intrahepatic CD8 + T cells with increased TNF-α and IFN-γ production and an increased number of CD8 + T cells in tumors, thereby slowing the development of HCC in HBs-HepR mice. Blocking PD-L1 did not show direct therapeutic effects or synergize with TIGIT blockade., Conclusions: Blockade of TIGIT alone enhanced the antitumor activity of CD8 + T cells during the progression of HBV-related HCC in a spontaneous HCC mouse model., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Objective: NK cells play crucial roles in the immune defense mechanisms against viral infections and transformed cells. However, the developmental progression, transcriptomic landscape, and functional subtypes of liver NK cells are not well defined. Hepatocellular carcinoma (HCC) accounts for approximately 80% of primary liver cancer worldwide, yet the biological characteristics of NK cells in the HCC environment are unclear. Therefore, we aimed to determine these cells' roles in tumorigenesis and prognosis., Methods: We compared the single-cell RNA sequencing profiles of NK cells purified from blood ( n = 1), healthy liver tissues ( n = 3), HCC tumor tissues ( n = 4), and peritumor liver tissues ( n = 1) to identify NK cell subsets. Furthermore, we performed bioinformatics analysis by using The Cancer Genome Atlas (TCGA) data to identify prognostic biomarkers simultaneously overexpressed in the blood and tumor tissues of patients with HCC., Results: Transcriptomic analysis revealed 5 NK cell subsets (L1-NK-CD56 bright , L2-NK-CD56 dim , L3-NK-HLA, L4-LrNK-FCGR3A, and L5-LrNK-XCL1) in the healthy liver tissues. However, the transitional L3 subset and the CXCR6 + CD16 + L4 subset with strong anti-tumor activity were absent in the HCC and peritumor liver tissues. Furthermore, 4 common prognosis-associated genes ( RHOB , TALDO1 , HLA-DPA1 , and TKT ) were significantly overexpressed in the paired tumor tissue and blood., Conclusions: Our study revealed 5 specific subsets of NK cells in healthy human liver tissues. However, only 3 of the 5 NK cell subsets were present in HCC and peritumor tissues. The cytotoxic NK cell subsets were absent in HCC tissues. Furthermore, we identified 4 potential non-invasive prognostic biomarkers in patients with HCC., Competing Interests: No potential conflicts of interest are disclosed., (Copyright © 2022 Cancer Biology & Medicine.)

Backgroud and Aims: Liver type 1 innate lymphoid cells (ILC1s), also known as liver-resident natural killer (LrNK) cells, comprise a high proportion of total hepatic ILCs. However, factors regulating their maintenance and function remain unclear., Approach and Results: In this study, we found high expression of retinoid-related orphan nuclear receptor alpha (RORα) in LrNK cells/ILC1s. Mice with conditional ablation of retinoid-related orphan nuclear receptor alpha (Rorα) in LrNK cells/ILC1s and conventional natural killer (cNK) cells had decreased LrNK cells/ILC1s but normal numbers of cNK cells. RORα-deficient LrNK cells/ILC1s displayed increased apoptosis and significantly altered transcriptional profile. Using a murine model of colorectal cancer liver metastasis, we found that RORα conditional deficiency resulted in more aggressive liver tumor progression and impaired effector molecule expression in LrNK cells/ILC1s. Consequently, treatment with the RORα agonist efficiently limited liver metastases and promoted effector molecule expression of LrNK cells/ILC1s., Conclusions: This study reveals a role of RORα in LrNK cell/ILC1 maintenance and function, providing insights into the harnessing of LrNK cell/ILC1 activity in the treatment of liver cancer., (© 2021 American Association for the Study of Liver Diseases.)

Hepatocellular carcinoma (HCC) is a world-wide health problem. Poor and delayed diagnoses as well as high recurrence rate resulting in high mortality rate. In this study, we established a patient-derived xenograft (PDX) model from HCC patient, and continuously maintained with subcutaneous passage more than 20 times. This HCC PDX tumor exhibited the same histological characteristics with the HCC patient and could be used to verify therapeutic effect of liver cancer. We further evaluated this PDX model by experimental chemotherapy, demonstrating that this HCC PDX model was sensitive to sorafenib treatment. Further, the potential of natural killer cell-based immunotherapy for HCC was tested using this model. We found that NK92 cells effectively suppressed the tumor growth in vivo and prolonged the survival time of HCC-bearing PDX mice. This study indicates that HCC PDX model is a good platform to testify the efficacy of preclinical chemotherapy and immunotherapy., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Natural killer (NK) cells have crucial roles in tumor surveillance. We found that tumor-infiltrating NK cells in human liver cancers had small, fragmented mitochondria in their cytoplasm, whereas liver NK cells outside tumors, as well as peripheral NK cells, had normal large, tubular mitochondria. This fragmentation was correlated with reduced cytotoxicity and NK cell loss, resulting in tumor evasion of NK cell-mediated surveillance, which predicted poor survival in patients with liver cancer. The hypoxic tumor microenvironment drove the sustained activation of mechanistic target of rapamycin-GTPase dynamin-related protein 1 (mTOR-Drp1) in NK cells, resulting in excessive mitochondrial fission into fragments. Inhibition of mitochondrial fragmentation improved mitochondrial metabolism, survival and the antitumor capacity of NK cells. These data reveal a mechanism of immune escape that might be targetable and could invigorate NK cell-based cancer treatments.

The discovery of CD49a + liver-resident natural killer (NK) cells in mice alters our view of NK cells and provides another opportunity to study NK cells. Although evidence has suggested roles for NK cells in liver diseases, whether and how CD49a + NK cells contribute to liver diseases remain unclear. In this study, we observed that accumulation of CD49a + tissue-resident NK cells in human hepatocellular carcinoma (HCC) was higher than in peritumoral tissues. We studied the exhausted and regulatory phenotypes of CD49a + tissue-resident NK cells by analysis of protein and mRNA. The proportion of CD49a + NK cells was positively correlated to the proportion of NK cells expressing inhibitory receptors. In addition, CD49a + NK cells expressed more of checkpoint molecules PD-1, CD96, and TIGIT. Transcriptomic analysis implicated CD49a + tissue-resident NK cells in the negative regulation of immune responses. Comparison of murine and human CD49a + NK cells revealed their distinct characteristics and functions. Finally, accumulation of tissue-resident CD49a + NK cells in liver tumor was correlated to deteriorating disease condition and poor prognosis. Our findings show that CD49a + NK cells accumulate in liver tumor and suggest a role for CD49a + NK cells in the negative regulation of immune responses and the development of HCC., (©2019 American Association for Cancer Research.)

Chronic hepatitis B virus (HBV) infection is one of the high-risk factors for human HCC. Despite the integration of virus DNA and the oncoprotein HBx, chronic necroinflammation and hepatocellular regeneration account for hepatocarcinogenesis. As a non-cytopathic virus, HBV is extensively recognized to mediate chronic liver damage through abnormal immune attack. However, the mechanisms driving HBV infection to HCC are poorly understood. During chronic HBV infection in humans, the adaptive immunity changes from immune tolerance to progressive immune activation, inactivation, reactivation and exhaustion, all of which may be the immune pathogenic factors for the development of HCC. Recently, the immunopathogenic mechanisms were described in mouse HBV-induced HCC models, which is absolutely dependent on the presence of HBV-specific T cell response and NK cell-derived IFN-γ, findings which are consistent with the observations from CHB and HCC patients. In this review, we summarize recent research progression on the HBV-specific CD8 + T cells, and also CD4 + T cells, B cells and non-specific immune cells and molecules underlying chronic HBV infection and eventual HCC development to demonstrate the pathogenesis of HBV-induced immune imbalance. Based on the progression, we discussed the potential of immune-based therapies and their challenges in the treatment of HBV-related HCC, including the checkpoint inhibition, genetically modified T cell transfer, therapeutic vaccines and metabolic modulation.

Background: Patients undergoing surgical resection of hepatocellular carcinoma (HCC) are at risk of recurrence; however, the underlying mechanism remains poorly understood., Methods: Through the analysis of gene expression profiles in tumour and matched normal tissues from patients with hepatocellular carcinoma (HCC), we identified differences in interleukin-11 (IL-11) expression. Further, we used genetic mouse, orthotopic tumour, chemically induced, and orthotopic allograft models to study the correlation between IL-11 and postsurgical recurrence. Additionally, we conducted a series of experiments, including histology and immunohistochemistry analysis, three-dimensional culture, immunofluorescence, western blotting, enzyme-linked immunosorbent assay (ELISA) and flow cytometry to investigate the role of IL-11-signal transducer and activator of transcription 3 (STAT3) signaling in HCC recurrence., Findings: We demonstrate that IL-11 levels increase after surgery, triggering HCC outgrowth. Accordingly, pharmacological blocking of IL-11-STAT3 signaling in model systems significantly alleviates tumour cell proliferation and suppresses postsurgical recurrence of HCC tumours., Interpretation: These data demonstrate that IL-11 has a central role in postsurgical HCC recurrence, and that inhibition of IL-11-STAT3 signaling is a potential therapeutic strategy to prevent recurrence. FUND: Natural Science Foundation of China., (Copyright © 2019. Published by Elsevier B.V.)

Immune checkpoint blockade has become a promising therapeutic approach to reverse immune cell exhaustion. Coinhibitory CD96 and T-cell immunoglobulin and ITIM domain (TIGIT), together with costimulatory CD226, bind to common ligand CD155. The balancing between three receptors fine-tunes immune responses against tumors. In this study, we investigated the expression of CD96, TIGIT, and CD226 in 55 fresh human hepatocellular carcinoma (HCC) samples, 236 paraffin-embedded HCC samples, and 20 normal human livers. The cumulative percentage, absolute count, and mean fluorescence intensity (MFI) of CD96 + NK cells are significantly increased in the intratumoral tissues of HCC and break the balance between three receptors. Human CD96 + NK cells are functionally exhausted with impaired interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production, high gene expression of interleukin (IL)-10 and transforming growth factor-beta 1 (TGF-β1), and low gene expression of T-bet, IL-15, perforin, and granzyme B. In addition, blocking CD96-CD155 interaction specifically increases lysis of HepG2 cells by NK cells. HCC patients with a high level of CD96 or CD155 expression within tumor are strongly associated with deteriorating disease condition and shorter disease-free survival (DFS) and overall survival times. Patients with a higher cumulative percentage of CD96 + NK cells within tumor also exhibit shorter DFS. High plasma level of TGF-β1 in HCC patients up-regulates CD96 expression and dynamically shifts the balance between CD96, TIGIT, and CD226 in NK cells. Blocking TGF-β1 specifically restores normal CD96 expression and reverses the dysfunction of NK cells. Conclusion: These findings indicate that human intratumoral CD96 + NK cells are functionally exhausted and patients with higher intratumoral CD96 expression exhibit poorer clinical outcomes. Blocking CD96-CD155 interaction or TGF-β1 restores NK cell immunity against tumors by reversing NK cell exhaustion, suggesting a possible therapeutic role of CD96 in fighting liver cancer., (© 2018 by the American Association for the Study of Liver Diseases.)

Chronic alcohol consumption increases the risk of hepatocellular carcinoma (HCC). However, little is known about the potential immunological mechanisms by which ethanol affects tumor progression. Here, adult male mice were administered multiple doses of diethylnitrosamine (DEN). Four and a half months later, the DEN-treated mice were placed on a liquid Lieber-DeCarli control diet or diet containing 5% ethanol for 2.5 months. At the end of the study, liver tissue samples were obtained to analyze pathology, gene expression, and hepatic mononuclear cells (MNCs). Results showed that ethanol feeding exacerbates the progression of hepatic tumors (characterized by the ratio of liver weight to body weight, and the tumor volume and diameter) in DEN-treated mice. Mechanistically, chronic alcohol consumption decreased the number of antitumor CD8 + T cells but increased the number of tumor-associated macrophages (TAMs) in the liver in DEN-initiated tumorigenesis. Besides, TAMs were prone to be M2 phenotype after alcohol consumption. Moreover, chronic alcohol consumption aggravated inflammation, fibrosis, and epithelial-mesenchymal transition (EMT) in the pathological process of HCC. These data demonstrate that chronic alcohol consumption exacerbates DEN-induced hepatocarcinogenesis by enhancing protumor immunity, impairing antitumor immunity and aggravating hepatic pathological injury. Targeting the immune system is a potential therapeutic regimen for alcohol-promoted HCC.

Hepatitis B virus (HBV) infection is a significant cause of liver disease pathogenesis, which results in the development of hepatic dysfunction, cirrhosis and hepatocellular carcinoma (HCC). Our previous studies showed that oncogene STAT3 might be an ideal target for HCC therapy. Here, we investigated whether targeting blockage of STAT3 signaling is efficient for HBV-related HCC. Based on the refractory of HCC and the persistence of HBV, in this study, we designed shRNAs targeting STAT3. The results showed that blocking STAT3 signaling by shRNAs could promote HBV positive HCC cell apoptosis and induce cell cycle arrest, resulting in HCC cell growth inhibition in vitro. Importantly, STAT3-shRNAs efficiently suppressed HBV replication, which would reduce HBV-derived stimulation to STAT3 signaling and augment STAT3-shRNAs-mediated anti-HCC effect. Finally, STAT3-shRNAs-mediated anti-HBV positive HCC effect was confirmed in xenograft nude mice. This study suggested that targeting STAT3 therapies such as STAT3-shRNAs may be an efficacious strategy for HBV-related HCC.

The increasing evidences suggest that Immunoscore(IS), a combinatorial density analysis of CD8+ and CD3+ cells in the centre and invasive margin of tumour (CT and IM), has an advantage over the currently used tumour staging methods in a variety of tumours; however, IS in hepatocellular carcinoma remains unreported. In this study, IS was performed on serial sections from two HCC cohorts (total 449) and compared with current tumour staging systems. Kaplan-Meier curves illustrate a positive association between a higher IS (IS≥2) and longer survival of HCC patients. Although the IS was highly related to the outcome of patients, however, IS seems not to be the optimal prognostic factor when compared with the CD8CT. As noted, among CD8CT, CD8IM, CD3CT, CD3IM and IS, CD8CT, as an independent indicator, demonstrated the highest prognostic impact on both DFS and OS in our Cox multivariate regression analysis (P< 0.0001). In our study, the minimum cut-off value was 93 CD8CT cells per mm2, to be used to divide the patients into CD8CTHi group and CD8CTLo group in clinical settings. Our results suggest that CD8CT densities analysis notably improved the accuracy of survival prediction with convenience of clinical manipulation in HCC.

Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer mortality and a common poor-prognosis malignancy due to postoperative recurrence and metastasis. There is a significant correlation between chronic hepatitis B virus (HBV) infection and hepatocarcinogenesis. As the first line of host defense against viral infections and tumors, natural killer (NK) cells express a large number of immune recognition receptors (NK receptors (NKRs)) to recognize ligands on hepatocytes, liver sinusoidal endothelial cells, stellate cells and Kupffer cells, which maintain the balance between immune response and immune tolerance of NK cells. Unfortunately, the percentage and absolute number of liver NK cells decrease significantly during the development and progression of HCC. The abnormal expression of NK cell receptors and dysfunction of liver NK cells contribute to the progression of chronic HBV infection and HCC and are significantly associated with poor prognosis for liver cancer. In this review, we focus on the role of NK cell receptors in anti-tumor immune responses in HCC, particularly HBV-related HCC. We discuss specifically how tumor cells evade attack from NK cells and how emerging understanding of NKRs may aid the development of novel treatments for HCC. Novel mono- and combination therapeutic strategies that target the NK cell receptor-ligand system may potentially lead to successful and effective immunotherapy in HCC.Cellular & Molecular Immunology advance online publication, 6 October 2014; doi:10.1038/cmi.2014.91.

Toll-like receptors (TLRs) play a crucial role in the innate immune response and subsequent induction of adaptive immune responses. Recently, it has been noted that TLRs on tumor cells are involved in tumor development, and several TLR agonists, such as the TLR3 agonist poly(I:C) and the TLR9 agonist CpG ODN, are being developed as vaccine adjuvants and cancer immunotherapeutics. In this study, we investigated whether combining poly(I:C) with a TLR9 agonist CpG ODN would result in a stronger anti-tumor effect on hepatocellular carcinoma cells (HCCs). Surprisingly, we found that simultaneous transfection of poly(I:C) and ODN M362 exhibited a lower pro-apoptotic effect on HCCs than transfection with poly(I:C) alone. Simultaneous co-transfection was accompanied by down-regulation of poly(I:C)-related innate receptors, pro-inflammatory cytokines and apoptotic genes induced by poly(I:C), indicating that ODN M362 blocked the activation of poly(I:C)-triggered intrinsic immune responses and cellular apoptosis. Further studies indicated that these effects were partly due to the phosphorothioate-modification of CpG ODN, which blocked the entry of poly(I:C) into tumor cells. This entry blockade was avoided by administering poly(I:C) after CpG ODN. Moreover, poly(I:C)-mediated pro-apoptotic effects were enhanced in vitro and in vivo by pre-treating HCC cells with CpG ODN. Our findings thus suggest that when combining poly(I:C) and CpG ODN for cancer therapy, these agents should be used in an alternating rather than simultaneous manner to avoid the blocking effect of phosphorothioate-modified TLR9 ligands., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

STAT3 is highly activated in a wide variety of cancers and functions to promote tumor survival. We previously reported that blocking STAT3 activation inhibited human hepatocellular carcinoma (HCC) growth in vitro, but whether this treatment also triggered antitumor immune responses in vivo remained unknown. In this study, we found that blocking the STAT3 pathway in HCC cells dramatically inhibited murine HCC growth in vivo and prolonged survival of tumor-bearing mice. Importantly, the presence of STAT3-blocked HCC augmented NK cell cytotoxicity against HCC and increased expression of molecules associated with NK cell activation and cytotoxicity. In T cell-deficient nude mice, a unique NK cell-mediated antitumor function against STAT3-blocked HCC was suggested. NK cells were shown to be necessary and sufficient in NK or T cell depletion experiments, or by adoptively transferring NK cells. Furthermore, regulatory T cells and immunosuppressive IL-10 and TGF-β cytokines were reduced in mice bearing STAT3-blocked HCC cells, suggesting that these factors may be involved in HCC-induced NK cell suppression. These findings indicate that blocking STAT3 in HCC cells can initiate innate immunity in vivo., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Genetic modification of NK cells may provide new possibilities for developing effective cancer immunotherapy by improving NK cell function and specificity. We previously established human interleukin-15 (hIL-15) gene-modified NKL cells (NKL-IL15) and demonstrated their therapeutic efficiency against human hepatocellular carcinoma (HCC) in vitro. To further assess the applicability of NKL-IL15 cells in adoptive cellular immunotherapy, we further investigated their natural cytotoxicity against HCC in vivo in the present study. NKL-IL15 cells exhibited strong inhibition on the growth of transplanted human HCC tumors in xenograft nude mouse models. Further investigation showed that NKL-IL15 cells expressed much higher levels of cytolysis-related molecules, including NKp80, TRAIL, granzyme B, IFN-γ, and TNF-α, than parental NKL cells in response to HCC stimulation. Moreover, soluble mediators secreted by NKL-IL15 cells decreased HCC cell proliferation; in particular, NKL-IL15-derived TNF-α and IFN-γ induced higher NKG2D ligand expression on target cells and resulted in the increased susceptibility of HCCs to NKL-mediated cytolysis. These results show that hIL-15 gene-modified human NK cells can augment the anti-tumor effect of NK cells on human HCC in vivo and suggest their promising applicability as a new candidate for adoptive immunotherapy against HCCs in the future., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Tumorigenesis is an immortalization process in which the growth of normal cells is uncontrolled and programmed cell death is suppressed. Molecular biologic and immunologic studies have revealed that the aberrant expression of some proto-oncogenes boosts proliferation and inhibits apoptosis, which is vital for tumor development. The hypofunction of the host immune system also drives the development and metastasis of malignant tumors. Pim-3, a member of the Pim family, is aberrantly expressed in several cancers. Data suggest that Pim-3 inhibits apoptosis by phosphorylating the proapoptotic BH3-only protein Bad. Here, we constructed a dual-function small hairpin RNA (shRNA) vector containing an shRNA targeting Pim-3 and a TLR7-stimulating ssRNA. Stimulation with this bi-functional vector in vitro promoted significant apoptosis of Hepa1-6 cells by regulating the expression of apoptosis-related proteins and induced secretion of type I IFNs. Most importantly, this bi-functional vector more effectively inhibited subcutaneous Hepa1-6 cell growth than did single shRNA and ssRNA treatment in vivo. Natural killer (NK), CD4(+) T, and CD8(+) T cells and macrophages were required for effective tumor suppression, and CD4(+) T cells were shown to play a helper role in the activation of NK cells, possibly by regulating the secretion of Th1 or Th2 cytokines. This ssRNA-shRNA bi-functional vector may represent a promising approach for tumor therapy., (©2014 American Association for Cancer Research.)

Toll-like receptors (TLRs) expressed on cancer cells are closely associated with tumor development. In this study, we investigated the biological functions of the TLR9 ligand, CpG oligodeoxynucleotide (CpG ODN), on TLR9 expressed in the cytoplasm of hepatocellular carcinoma (HCC) cells. In vitro, human HCC cell lines were transfected with phosphorothioate-modified oligodeoxynucleotides TLR9 agonist OND M362 and its negative control ODN M362 ctrl, which inhibited the proliferation of HCC cells by inducing apoptosis without altering the cell cycle. Interestingly, ODN M362 and ODN M362 Ctrl displayed a similar proapoptotic effect on HCC, possibly related to phosphorothioate modification of the structure of CpG ODN. Although both of them resulted in the upregulation of the TLR9 receptor, their effect on HCC apoptosis was independent of TLR9. They also upregulated inflammatory cytokines, but did not activate the NF-κB signaling pathway. Finally, the activities of ODN M362 and ODN M362 Ctrl were demonstrated in nude mice inoculated with HCC cells. These findings suggest that the phosphorothioate-modified TLR9 agonist ODN M362, and its control, elicit antitumor activity in HCC cells and may serve as a novel therapeutic target for HCC therapy.

STAT3 is an important transcriptional factor for cell growth, differentiation, and apoptosis. Although evidence suggests a positive role for STAT3 in cancer, the inhibitory effects of tumor STAT3 on natural killer (NK) cell functions in human hepatocellular carcinoma are unclear. In this study, we found that blocking STAT3 in hepatocellular carcinoma cells enhanced NK-cell antitumor function. In the case of STAT3-blocked hepatocellular carcinoma cells, NKG2D ligands were upregulated, which promoted recognition by NK cells. Importantly, the cytokine profile of hepatocellular carcinoma cells was altered; in particular, TGF-β and interleukin 10 (IL-10) expression was reduced, and type I interferon (IFN) was induced, thus facilitating NK-cell activation. Indeed, the cytotoxicity of NK cells treated with supernatant from STAT3-blocked hepatocellular carcinoma cells was augmented, with a concomitant elevation of molecules associated with NK cytolysis. Further experiments confirmed that the recovery of NK cells depended on the downregulation of TGF-β and upregulation of type I IFN derived from STAT3-blocked hepatocellular carcinoma cells. These findings demonstrated a pivotal role for STAT3 in hepatocellular carcinoma-mediated NK-cell dysfunction, and highlighted the importance of STAT3 blockade for hepatocellular carcinoma immunotherapy, which could restore NK-cell cytotoxicity in addition to its direct influence on tumor cells., (©2013 AACR.)

HMBOX1 was a novel transcription factor possibly involving in function of pancreas and cytotoxicity of NK cells. For function determination, recombinant human HMBOX1 protein was obtained and purified, and the monoclonal antibodies against HMBOX1 were prepared. The full-length cDNA fragment encoding HMBOX1 was amplified from NK-92 cells and inserted into prokaryotic expression vector pET22b. The pET22b-HMBOX1-6his vector was then transformed into E. coli Rosetta (DE3) and induced by 1 mM IPTG for 4 h at 37 degrees Celsius. The fusion HMBOX1 protein was mainly expressed in inclusion bodies, which was purified and refolded using Ni2+-affinity chromatography. With the purified fusion HMBOX1 protein as antigen, monoclonal antibodies against HMBOX1 were generated, providing a potentially useful tool for further study in HMBOX1 functions. Using these anti-HMBOX1 mAbs, we identified that HMBOX1 is located in both cytoplasm and nucleus and could be detected in 10 human normal tissues, including cerebrum, pancreas, kidney and liver tissues. Moreover, the expression in hepatic carcinoma was significantly lower than that in adjacent tissues.

Toll-like receptor 3 and RIG-I like receptors (RLRs; MDA5, RIG-I) are involved in cell growth inhibition and apoptosis. However, the toll-like receptor 3-related apoptotic pathway is insensitive to direct polyinosinic-polycytidylic acid (dsRNA analog) stimulation in hepatoma cells. To determine whether the strategy of transferring polyinosinic-polycytidylic acid into cells (polyinosinic-polycytidylic acid-liposome) could induce apoptosis in hepatoma cells through cytoplasm receptors, we examined the responses of innate immune receptors RLRs and toll-like receptor 3 in response to different stimulation. We found that the apoptosis could exclusively be detected under polyinosinic-polycytidylic acid-liposome stimulation, which involved the activation of the caspase pathway. Besides, the expression of RIG-I, MDA5, IFNbeta and interferon-stimulated gene 15 was increased significantly at an early stage. Moreover, the growth inhibition of polyinosinic-polycytidylic acid-liposome was confirmed in a mouse model. Taken together, these results suggest polyinosinic-polycytidylic acid-liposome could be used as a potential apoptotic agent in hepatocellular carcinoma cells and imply a potential therapeutic strategy.

More and more studies show that signal transducer and activator of transcription 3 (STAT3) is frequently constitutively activated in a wide number of malignancies and named as an attractive molecular target for tumor treatment. Here, we employed STAT3-decoy ODN, which specifically block over-activated STAT3, to treat human hepatocellular carcinoma (HCC) cells, and evaluated the cellular proliferation ability and investigated the molecular mechanisms in vitro. The results demonstrated that the proliferation of HCC cells was suppressed significantly by STAT3-decoy ODN, being associated with the increased apoptosis and cell arrest at G0/G1 to S phase transition. Further investigates showed the expression of STAT3-regulated genes including bcl-x1, cyclin D1 and c-myc, which involved in cell apoptosis and cell cycle progression, were down-regulated significantly both at transcription and translation levels. These data suggested that STAT3 may be potentially used as a molecular target in HCC therapy.

Antineoplaston A10 (3-phenylacetylamino-2,6-piperidinedion) is a naturally occurring substance and was the first antineoplaston in the human body to be chemically identified. The effect of antineoplaston A10 on human hepatocellular carcinoma cell lines HepG2 and HLE has been examined. Antineoplaston A10 displayed anti-proliferative action inhibiting cell growth in a dose- and time-dependent manner in vitro as measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. Incubation with antineoplaston A10 for 48 h induced apoptotic events such as a typical apoptotic morphology, formation of a characteristic ladder pattern of DNA migration and accumulation of sub-G1 phase cells. Next, hepatoma xenografts in nude mice were employed to study the antitumor effects of antineoplaston A10 in vivo. Oral administration of antineoplaston A10 delayed the growth of HepG2 and HLE cells in the mice without a reduction in body weight. A higher proportion of apoptotic cells in xenografts was observed by means of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. In addition, the level of expression of apoptotic marker p53 increased while that of anti-apoptotic protein bcl-2 decreased, as evaluated with immunohistochemical staining in the xenografts. These results suggested that antineoplaston A10 may inhibit the growth of human hepatoma cells through the induction of apoptosis.

Chronic HBV infection is associated with a 100-fold high risk of developing hepatocellular carcinoma. Tumor recognition is of the most importance during the immune surveillance process that prevents cancer development in humans. In the present study, the expressions of MHC class I molecules on hepatoplastoma cell line HepG2.2.15 were investigated to indicate the possible effects of HBV on the immune recognition during HBV-associated hepatocellular carcinoma. It was found that the expressions of MHC class I molecules HLA-ABC, HLA-E and MICA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class I molecules. Additionally, it was observed that in murine chronic HBsAg carriers the expression of classical MHC-I molecule on hepatocytes was down-regulated. These results demonstrated that HBV might affect the immune recognition during HBV-associated hepatocellular carcinoma such as the recognition of CD8+ T, NK-CTL and NK cells and prevent the immune surveillance against tumors. However, the effects of HBV down-regulation of MHC class I molecules on the target cells in vivo should be further studied.