Comparison of NK cell subsets, receptors and functions induced by radiofrequency ablation and microwave ablation in HBV-associated primary hepatocellular carcinoma.

Background: Topical therapy has been shown to induce an immune response in patients with hepatocellular carcinoma (HCC). In this study, a prospective parallel group control experiment was conducted to compare the differences between radiofrequency ablation and microwave ablation in inducing the immune regulation of NK cells. Methods: Sixty patients with clinically and pathologically confirmed hepatitis B-associated hepatocellular carcinoma (HCC) were selected for thermal ablation. Patients were randomly assigned into the MWA group (n = 30) and the RFA group (n = 30). Patient's peripheral blood was isolated on days D0, D7, and month M1. NK cell subsets, receptors, and killing function were detected by flow cytometry and LDH. Student t test and rank sum test were used to compare the statistical differences between the RFA (radio frequency) and MWA (microwave) groups. The Kaplan-Meier curve and log-rank test were used to calculate the difference between the two survival curves. Results: Comparison of the frequency of CD3-CD56+ and CD3-CD56+CD16+ in NK cells between the RFA and WMA groups showed that there was no difference in the D0, D7, M1, D7-D0, M1-D0, and M1-D7 groups. The changes of the inhibitory NK cell receptor CD159A were significantly different at D7 (P<0.05). CD107a were compared between the RFA and WMA groups, indicating that CD107a changes induced by NK cells were significantly different at D7-D0 (P<0.05). Comparison of NK cell lysis activity of target K562 cells between the RFA and WMA groups showed that there was no difference at D0, D7, D7-D0. There was no difference in recurrence-free survival (RFS) between the RFA and WMA groups (P=0.11). Conclusions: The difference between MWA and RFA-induced NK cell changes was mainly manifested in the inhibitory receptors CD159a and CD107a 1 week after surgery, with microwave-induced changes being more severe. Comparison of the NK cell lysis activity of the target K562 cells between the RFA and WMA groups showed that there was no difference in D0, D7, D7- D0. Survival analysis showed that these differences did not affect the recurrence-free survival (RFS) in the two groups.

Distinct tumour antigen-specific T-cell immune response profiles at different hepatocellular carcinoma stages.

BACKGROUND: Cancer-testis antigens (CTAs) and tumour-associated antigens (TAAs) are frequently expressed in hepatocellular carcinoma (HCC); however, the role of tumour-antigen-specific T cell immunity in HCC progression is poorly defined. We characterized CTA- and TAA-specific T cell responses in different HCC stages and investigated their alterations during HCC progression. METHODS: Fifty-eight HCC patients, 15 liver cirrhosis patients, 15 chronic hepatitis B patients and 10 heathy controls were enrolled in total. IFN-γ ELSPOT using CTAs, including MAGE-A1, MAGE-A3, NY-ESO-1, and SSX2, and two TAAs, SALL4 and AFP, was performed to characterize the T-cell immune response in the enrolled individuals. The functional phenotype of T cells and the responsive T cell populations were analyzed using short-term T-cell culture. RESULTS: T cell responses against CTAs and TAAs were specific to HCC. In early-stage HCC patients, the SALL4-specific response was the strongest, followed by MAGE-A3, NY-ESO-1, MAGE-A1 and SSX2. One-year recurrence-free survival after transcatheter arterial chemoembolization plus radiofrequency ablation treatment suggested the protective role of CTA-specific responses. The four CTA- and SALL4-specific T cell responses decreased with the progression of HCC, while the AFP-specific T cell response increased. A higher proportion of CD4+ T cells specific to CTA/SALL4 was observed than AFP-specific T cell responses. CONCLUSIONS: The IFN-γ ELISPOT assay characterized distinct profiles of tumour-antigen-specific T cell responses in HCC patients. CTA- and SALL4-specific T cell responses may be important for controlling HCC in the early stage, whereas AFP-specific T cell responses might be a signature of malignant tumour status in the advanced stage. The application of immunotherapy at an early stage of HCC development should be considered.

Transarterial chemoembolization combined with radiofrequency ablation for solitary large hepatocellular carcinoma ranging from 5 to 7 cm: an 8-year prospective study.

PURPOSE: This prospective study was performed to investigate long-term (8-year) survival in patients with solitary large hepatocellular carcinoma (HCC) ranging from 5 to 7 cm who underwent transarterial chemoembolization (TACE) combined with radiofrequency ablation (RFA) and identify factors that significantly affected outcomes. METHODS: Forty-eight patients with large HCC (36 men, 12 women; mean age, 57.0 ± 11.2 [range, 37-82] years) without fever or signs of infection were enrolled. All patients were treated with TACE + RFA. Overall survival (OS) and disease-free survival (DFS) were calculated using the Kaplan-Meier method. Prognostic factors were assessed using the Cox hazards regression method. RESULTS: The median OS duration was 47.0 months, and the 1-, 3-, 5-, and 8-year OS rates were 73%, 57%, 53%, and 27%, respectively. The median DFS duration was 9.05 (3.99-12.01) months, and the 1-, 3-, and 5-year DFS rates were 35%, 9%, and 0%, respectively. Cox hazards regression analysis revealed that the Child-Pugh class, platelet count, lymphocyte-to-monocyte ratio (LMR), and DFS were independent predictive factors of OS (p = 0.000, 0.003, 0.020, and 0.000, respectively). The LMR and platelet-to-lymphocyte ratio (PLR) were independent predictive factors of recurrence (p = 0.046 and 0.016, respectively). CONCLUSION: TACE + RFA may be a safe and effective treatment for selected solitary large HCC ranging from 5 to 7 cm. Measurement of the LMR (> 4) and PLR (≤ 100) in peripheral blood before the intervention might help to identify which patients with solitary large HCC are suitable for TACE + RFA. Registration number: ChiCTR-TRC-12002768 ( https://www.chictr.org.cn ).

Combination therapy of TACE and CT-guided partial hepatic segment ablation for liver cancer.

Aim: This study aimed to investigate the safety and efficacy of combination therapy of transcatheter arterial chemoembolization (TACE) and CT-guided percutaneous partial hepatic segment ablation. Material and methods: Liver cancer patients undergoing TACE plus partial segment ablation from 2012 to 2016 were retrospectively analyzed. Patient characteristics were collected. TACE was performed for all patients before ablation. After ablation, CT imagings were used to evaluate treatment and follow-up. Overall survival (OS) and local tumor progression free survival (LTPFS) were calculated. Results: Twenty-three patients with 68 liver tumors were included. The median tumor maximum diameter was 55 mm (range, 24 to 91 mm). The complete response rate of combination therapy was 87%. The one-, two-, three- and four-year overall survival rates were 78%, 65%, 54% and 54%, respectively. The three-year survival rate for BCLC stage C HCC patients was 53%. The one-, two- and three-year LTPFS rates were 54%, 35% and 35%, respectively. Tumor maximum diameter, vascular invasion and partial treatment response were independent risk factors for LTPFS. Only one patient suffered a major complication. Conclusion: TACE combined with partial hepatic segment thermal ablation is a safe and effective treatment for liver cancer patients, especially for those with more advanced disease.

High-Content Functional Screening of AEG-1 and AKR1C2 for the Promotion of Metastasis in Liver Cancer.

Liver cancer is one of the most lethal cancer types in humans, but our understanding of the molecular mechanisms underlying this process remains insufficient. Here, we conducted high-content screening of the potential genes involved in liver cancer metastasis, which we selected from the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, based on the SAMcell method and RNA interference technology. We identified two powerful genes in the liver cancer metastasis process, AEG-1 and AKR1C2, both of which proved to be positive regulators in promoting metastasis in liver cancer. Further clinical results verified their roles in liver cancer. In summary, these findings could provide new insight into the liver cancer mechanism and potentially therapeutic novel targets for liver cancer therapies in the future.

A Huaier polysaccharide reduced metastasis of human hepatocellular carcinoma SMMC-7721 cells via modulating AUF-1 signaling pathway.

TP-1 is a polysaccharide from one famous fungus Huaier. Treatment with TP-1 significantly inhibited the cell growth, adhesion, migration, and motility of SMMC-7721 cells in a dose-dependent manner. Real-time quantitative RT-PCR revealed a dose-dependent decrease in RNA-binding factor 1 (AUF-1) and astrocyte elevated gene-1 (AEG-1) messenger RNA (mRNA) levels in TP-1-treated SMMC-7721 cells, which is consistent with their protein expression detected by Western blotting. On the contrary, microRNA-122 (miR-122) expression increased in SMMC-7721 cells following TP-1 treatment. Moreover, TP-1 treatment at three doses apparently increased epithelial marker E-cadherin protein expression but decreased the mesenchymal marker N-cadherin protein level. In addition, the hematoxylin-eosin (H & E) staining showed that the TP-1 significantly inhibited the lung metastasis of liver cancer in mice orthotopic implanted with SMMC-7721 tumor tissue. Taken together, these findings proved the inhibitory effect of TP-1 on the growth and metastasis of SMMC-7721 cells, and TP-1 might be offered for future application as a powerful chemopreventive agent against hepatocellular carcinoma (HCC) metastasis.

A Huaier polysaccharide restrains hepatocellular carcinoma growth and metastasis by suppression angiogenesis.

Hepatocellular carcinoma (HCC) is a highly metastatic cancer. Huaier polysaccharide (TP-1) is a naturally occurring bioactive macromolecule, found in Huaier fungus and has been shown to exert in vitro antitumor and antimetastasis for HCC, but no study has addressed in vivo efficacy and mechanisms of action. Presently, we found that TP-1 at doses of 0.5, 1 and 2mg/kg significantly inhibited tumor growth and metastasis to the lung in mice bearing HCC SMMC-7721 tumors without toxicity. The analysis of tumors by immunohistochemistry demonstrated that TP-1 inhibited PCNA expression, increased the number of TUNEL-positive cells and reduced microvessel density (MVD) to achieve this effect. Furthermore, TP-1 administration reduced the protein expression of hypoxia-inducible factor (HIF)-1alpha, vascular endothelial growth factor (VEGF), AUF-1 and AEG-1, in tumor tissues. Taken together, our data suggested that the antitumor and anti-metastatic activities of TP-1 might be at least partially through down-regulation of HIF-1alpha/VEGF and AUF-1/AEG-1 signaling pathways.

A Huaier polysaccharide inhibits hepatocellular carcinoma growth and metastasis.

This study was carried out to evaluate the effects of a Huaier polysaccharide (TP-1) on the tumor growth and immune function in hepatocellular carcinoma (HCC) H22-based mouse in vivo. Results showed that TP-1 was capable of repressing transplanted H22 solid hepatic tumor cell growth in vivo, prolonging the live time of mice bearing ascetic H22 tumors, and repressing the pulmonary metastasis of H22-bearing mice. Moreover, the relative weight of immune organ (spleen and thymus) and lymphocyte proliferation were improved after TP-1 treatment. Furthermore, the treatment with TP-1 could promote immune-stimulating serum cytokines, such as IL-2 and IFN-γ, but inhibit immune-suppressing serum cytokines IL-10 secretion in H22-bearing mice. Besides, the percentage of CD4+ T cells and NK cells was increased, whereas the number of CD8+ T cells decreased in tumor-bearing mice following TP-1 administration. In addition, this compound displayed little toxic effects to major organ of tumor-bearing mice at the therapeutic dose, such as the liver and kidney. This experimental finding suggested that TP-1 exhibited prominent antitumor activities in vivo via enhancement of host immune system function in H22 tumor-bearing mice. This product could be developed individually as a safe and potent biological response modifier for HCC therapy.

Complete remission of multiple lung metastases after ablation of hepatocellular carcinoma by transarterial infusion with the p53 gene.

We describe the case of a 68-year-old man who presented with a massive lesion in the right liver. It was confirmed by preoperative aspiration biopsy to be a case of moderately differentiated hepatocellular carcinoma, and the patient was shown by immunohistochemistry to have a mutation in the p53 gene. The hepatic lesion showed complete necrosis after arterial embolization combined with microwave ablation. During a re-examination 3 months after ablation, the α-fetoprotein level was found to have increased markedly. Bilateral pulmonary metastases were shown by a lung computed tomography scan, with a focal diameter smaller than 1 cm. Hepatic and bronchial intra-arterial infusion with the recombinant adenovirus p53 gene (rAd-p53) was performed twice. The second time the infusion was administered, interleukin-2 was used in combination with rAd-p53. After 2 months of treatment, the bilateral pulmonary lesions had almost disappeared. After 7 months of treatment, the bilateral pulmonary metastases disappeared completely, and no further recurrence has been identified in the lungs and liver.

Huaier polysaccharides suppresses hepatocarcinoma MHCC97-H cell metastasis via inactivation of EMT and AEG-1 pathway.

We have recently reported that astrocyte elevated gene-1 (AEG-1) might be an epithelial-mesenchymal transition (EMT) associated biomarker in human hepatocellular carcinoma (HCC), and play an important role in the progression of hepatocellular carcinoma. To extend our study, we examined here the anti-invasive and metastatic effects of Huaier polysaccharide (HP) on human HCC cell line MHCC97-H and explored its possible mechanism of action. Treatment with HP dose-dependently inhibited the proliferation, adhesion, migration and invasion of MHCC97-H cells in vitro. This was achieved not only by reducing the expression of AEG-1 and N-cadherin, but also by enhancing E-cadherin expression. Therefore, these data suggested that HP can inhibit the growth and metastatic potential of MHCC97-H cells through modulation of the AEG-1/EMT pathway.

Astrocyte elevated gene-1 (AEG-1) shRNA sensitizes Huaier polysaccharide (HP)-induced anti-metastatic potency via inactivating downstream P13K/Akt pathway as well as augmenting cell-mediated immune response.

Astrocyte elevated gene-1 (AEG-1) is an important force in the development and progression of hepatocellular carcinoma (HCC). To extend our study, we examined here the role of AEG-1 in anti-metastatic effects of Huaier polysaccharide (HP) on the human HCC MHCC97-H cell line. AEG-1 shRNA contributed to the anti-proliferation effect of HP on MHCC97-H cells. Furthermore, results of Transwell insert chambers showed that low expression of AEG-1 could effectively facilitate HP to suppress MHCC97-H cell migration and invasion. We achieved this by reducing phosphoinositide 3-kinases (P13K) and phosphorylated Akt (pAkt) expression as well as enhancing natural killer (NK) cell activity. Taken together, our data strongly suggested that AEG-1 shRNA could block the carcinogenesis and progression of MHCC97-H cells and highlight the therapeutic potential of HP in HCC treatment, at least by part, by inhibiting the activation of the PI3K/Akt pathway and enhancing the NK cell-mediated immune response. These findings may provide a new strategy for HCC treatment.

Astrocyte elevated gene-1 is a novel biomarker of epithelial-mesenchymal transition and progression of hepatocellular carcinoma in two China regions.

Astrocyte elevated gene-1 (AEG-1) is involved in important biological processes including cell invasion, metastasis, and carcinogenesis. However, its clinical significance has remained largely unknown in hepatocellular carcinoma. Here, specimens from 144 patients with hepatocellular carcinomas in Beijing and Heilongjiang regions were investigated by immunohistochemical staining for AEG-1, vimentin, and E-cadherin expressions. A clinicopathological study revealed that AEG-1 expression level in tumor cells was significantly correlated with TNM stage (P = 0.001) and Edmonson grade (P < 0.0001). In addition, AEG-1, vimentin, and E-cadherin (epithelial-mesenchymal transition (EMT) biomarker) expressions were correlated with each other. These findings suggest that AEG-1 may be an epithelial-mesenchymal transition-associated biomarker in human hepatocellular carcinoma and play important roles in the progression of hepatocellular carcinoma. In addition, the AEG-1 gene is a potential target for elimination of hepatocellular carcinoma in the future.

AEG-1 Promotes Metastasis Through Downstream AKR1C2 and NF1 in Liver Cancer.

Liver cancer is one of the most lethal cancers, but our knowledge of the molecular mechanism underlying this process remains insufficient. Through deep sequencing and expression regulation analysis in liver cancer cells, we identified two novel factors, AKR1C2 (positive factor) and NF1 (negative factor), as the AEG-1 downstream players in the process of metastasis in liver cancer. They were experimentally validated to have the capacities of regulating cell migration, cell invasion, cell proliferation, and EMT. Further clinic expression and animal model evidence confirmed their functions. Together, our findings provide a new insight into the pharmaceutical and therapeutic use of AEG-1 and downstream AKR1C2 and NF1.