Targeting myeloid-derived suppressor cells promotes antiparasitic T-cell immunity and enhances the efficacy of PD-1 blockade (15 words).

Immune exhaustion corresponds to a loss of effector function of T cells that associates with cancer or chronic infection. Here, our objective was to decipher the mechanisms involved in the immune suppression of myeloid-derived suppressor cells (MDSCs) and to explore the potential to target these cells for immunotherapy to enhance checkpoint blockade efficacy in a chronic parasite infection. We demonstrated that programmed cell-death-1 (PD-1) expression was significantly upregulated and associated with T-cell dysfunction in advanced alveolar echinococcosis (AE) patients and in Echinococcus multilocularis-infected mice. PD-1 blockade ex vivo failed to reverse AE patients' peripheral blood T-cell dysfunction. PD-1/PD-L1 blockade or PD-1 deficiency had no significant effects on metacestode in mouse model. This was due to the inhibitory capacities of immunosuppressive granulocytic MDSCs (G-MDSCs), especially in the liver surrounding the parasite pseudotumor. MDSCs suppressed T-cell function in vitro in an indoleamine 2, 3 dioxygenase 1 (IDO1)-dependent manner. Although depleting MDSCs alone restored T-cell effector functions and led to some limitation of disease progression in E. multilocularis-infected mice, combination with PD-1 blockade was better to induce antiparasitic efficacy. Our findings provide preclinical evidence in support of targeting MDSC or combining such an approach with checkpoint blockade in patients with advanced AE. (200 words).

Diagnosis and treatment experience of atypical hepatic cystic echinococcosis type 1 at a tertiary center in China.

BACKGROUND: Some hydatid cysts of cystic echinococcosis type 1 (CE1) lack well-defined cyst walls or distinctive endocysts, making them difficult to differentiate from simple hepatic cysts. AIM: To investigate the diagnostic methods for atypical hepatic CE1 and the clinical efficacy of laparoscopic surgeries. METHODS: The clinical data of 93 patients who had a history of visiting endemic areas of CE and were diagnosed with cystic liver lesions for the first time at the People's Hospital of Xinjiang Uygur Autonomous Region (China) from January 2018 to September 2023 were retrospectively analyzed. Clinical diagnoses were made based on findings from serum immunoglobulin tests for echinococcosis, routine abdominal ultrasound, high-frequency ultrasound, abdominal computed tomography (CT) scan, and laparoscopy. Subsequent to the treatments, these patients underwent reexaminations at the outpatient clinic until October 2023. The evaluations included the diagnostic precision of diverse examinations, the efficacy of surgical approaches, and the incidence of CE recurrence. RESULTS: All 93 patients were diagnosed with simple hepatic cysts by conventional abdominal ultrasound and abdominal CT scan. Among them, 16 patients were preoperatively diagnosed with atypical CE1, and 77 were diagnosed with simple hepatic cysts by high-frequency ultrasound. All the 16 patients preoperatively diagnosed with atypical CE1 underwent laparoscopy, of whom 14 patients were intraoperatively confirmed to have CE1, which was consistent with the postoperative pathological diagnosis, one patient was diagnosed with a mesothelial cyst of the liver, and the other was diagnosed with a hepatic cyst combined with local infection. Among the 77 patients who were preoperatively diagnosed with simple hepatic cysts, 4 received aspiration sclerotherapy of hepatic cysts, and 19 received laparoscopic fenestration. These patients were intraoperatively diagnosed with simple hepatic cysts. During the follow-up period, none of the 14 patients with CE1 experienced recurrence or implantation of hydatid scolices. One of the 77 patients was finally confirmed to have CE complicated with implantation to the right intercostal space. CONCLUSION: Abdominal high-frequency ultrasound can detect CE1 hydatid cysts. The laparoscopic technique serves as a more effective diagnostic and therapeutic tool for CE.

Grain Growth Mechanism of Lamellar-Structure High-Purity Nickel via Cold Rolling and Cryorolling during Annealing.

High-purity (99.999%) nickel with lamellar-structure grains (LG) was obtained by room-temperature rolling and cryorolling in this research, and then annealed at different temperatures (75 °C, 160 °C, and 245 °C). The microstructure was characterized by transmission electron microscopy. The grain growth mechanism during annealing of the LG materials obtained via different processes was studied. Results showed that the LG high-purity nickel obtained by room-temperature rolling had a static discontinuous recrystallization during annealing, whereas that obtained by cryorolling underwent static and continuous recrystallization during annealing, which was caused by the seriously inhibited dislocation recovery in the rolling process under cryogenic conditions, leading to more accumulated deformation energy storage in sheets.

Quantitative evaluation of range and metabolic activity of hepatic alveolar echinococcosis lesion microenvironment using PET/CT and multi-site sampling method.

BACKGROUND: Alveolar echinococcosis (AE) lesion microenvironment (LME) is crucial site where parasite-host interactions happen and of great significance during surgery and obtaining liver samples for basic research. However, little is known about quantification of LME range and its' metabolic activity regarding different lesion characteristics. METHODS: A prospective and retrospective analysis of LME from surgical AE patients was performed. Patients (n = 75) received abdominal computed tomography (CT) and position emission tomography/computed tomography using 18F-fluodeoxyglucose (18F-FDG-PET/CT) within 1 week prior to surgery. Semiquantitatively, calcification was clustered with 0%, < 50% and ≥ 50% degrees at lesion periphery; liquefaction was clustered with 0%, < 50%, 50 ~ 75%, ≥75% degrees at lesion center using volumetric ratio. Tumor to background ratio (TBR) of 18F-FDG standard uptake value (SUV, n = 75) was calculated, and range of 18F-FDG uptake area was measured; Multi-site sampling method (MSS, n = 35) was introduced to obtain histological slides to evaluate immune cell infiltrative ranges. RESULTS: Altogether six major lesion groups have been identified (A: 0% calcified, 0% liquefied; B: ≥50% calcified, 0% liquefied; C: < 50% calcified, < 50% liquefied; D: ≥50% calcified, < 50% liquefied; E: < 50% calcified, 50 ~ 75% liquefied; F: ≥50% calcified, ≥75% liquefied). Statistically, TBR values respectively were 5.1 ± 1.9, 2.7 ± 1.2, 4.2 ± 1.2, 2.7 ± 0.7, 4.6 ± 1.2, 2.9 ± 1.1 in groups A ~ F, and comparisons showed A > B, A > D, A > F, E > B, E > D, E > F, C > B, C > D, C > F (P < 0.05); LME ranges indicated by PET/CT respectively were 14.9 ± 3.9, 10.6 ± 1.5, 12.3 ± 1.1, 7.8 ± 1.6, 11.1 ± 2.3, 7.0 ± 0.4 mm in groups A ~ F, and comparisons showed A > B, A > D, A > F, A > E, C > B, C > D, C > F, E > D, E > F, B > D, B > F (P < 0.05); LME ranges indicated by MSS respectively were 17.9 ± 4.9, 13.0 ± 2.7, 11.9 ± 2.6, 6.0 ± 2.2, 11.0 ± 4.1, 6.0 ± 2.2 mm in groups A ~ F, and comparisons showed A > C, A > D, A > F, B > D, B > F, C > D, C > F (P < 0.05). Generally, less calcifications indicated higher TBR values and wider LME ranges; and, severer liquefactions indicated smaller LME ranges. Additionally, patients with previous medication history had lower TBR values. CONCLUSIONS: PET/CT and MSS method showed distinct TBRs and LME ranges for different calcifications and liquefactions. This study would be able to provide references for both surgical resections of lesions and more accurate sample acquisitions for basic research targeted to immunology.

The application of laparoscopy combined with indocyanine green fluorescence imaging technique for hepatic cystic echinococcosis.

BACKGROUND: With the mature application of laparoscopy in hepatobiliary surgery, laparoscopic treatment of hepatic cystic echinococcosis (CE) has made certain progress. But, due to the inherent limitations of laparoscopy and the growth characteristics of cystic echinococcosis, distinguishing the boundary between cystic lesion and normal hepatic parenchyma is pivotal importance for successful surgery. Indocyanine green (ICG) fluorescence imaging technology can view the boundary of lesion and normal tissue during the treatment of hepatic cystic echinococcosis. Applied laparoscopy combined with ICG fluorescence imaging technique for hepatic cystic echinococcosis may be an effective surgical strategy. METHODS: The clinical data contained nine patients with hepatic cystic echinococcosis who underwent laparoscopic surgery with indocyanine green fluorescence imaging technique in authors' institution from December 2018 to December 2019 were retrospectively analyzed. Indocyanine green was administered intravenously three days prior to surgery. The fluorescence acquisition system for real-time imaging was used during the surgery and the patients were followed up after surgery. RESULTS: Of reported nine patients, six are male and the remaining three are female. The average age is (36.4 ± 7.6) years. For all subjects, surgical procedures were performed under laparoscopy with indocyanine green fluorescence system. This technique showed the clear boundary of the hepatic cyst with normal liver parenchyma. Total cystectomy in six patients, subtotal cystectomy in two patients and partial hepatectomy in one patient were performed respectively. The average operation time was 3.8 ± 0.9 h, blood loss 206.0 ± 120.7 ml. Neither blood transfusion nor post-operative complication was experienced. The average abdominal drainage time was 3.4 ± 0.9 days with hospital stay 5.7 ± 2.1 days. During the 6-12 months follow-up period, neither recurrence nor intraperitoneal implantation was found. CONCLUSIONS: Applied laparoscopy combined with ICG fluorescence imaging technique for hepatic cystic echinococcosis is safe and feasible. Enhanced boundary image can assist surgeons to complete radical resection and reduce complications.

Immune Exhaustion of T Cells in Alveolar Echinococcosis Patients and Its Reversal by Blocking Checkpoint Receptor TIGIT in a Murine Model.

BACKGROUND AND AIMS: The cestode Echinococcus multilocularis infection, a serious health problem worldwide, causes alveolar echinococcosis (AE), a tumor-like disease predominantly located in the liver and able to spread to any organs. Until now, there have been few studies that explore how T-cell exhaustion contributes to the parasite's escape from immune attack and how it might be reversed. APPROACH AND RESULTS: In this study, we found that liver T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) expression was significantly enhanced and positively correlated with lesion activity in AE patients. High TIGIT expression in both liver-infiltrating and blood T cells was associated with their functional exhaustion, and its ligand CD155 was highly expressed by hepatocytes surrounding the infiltrating lymphocytes. In co-culture experiments using human blood T cells and hepatic cell line HL-7702, CD155 induced functional impairment of TIGIT+ T cells, and in vitro blockade with TIGIT antibody restored the function of AE patients' T cells. Similar TIGIT-related functional exhaustion of hepatic T cells and an abundant CD155 expression on hepatocytes were observed in E. multilocularis-infected mice. Importantly, in vivo blocking TIGIT prevented T-cell exhaustion and inhibited disease progression in E. multilocularis-infected mice. Mechanistically, CD4+ T cells were totally and CD8+ T cells partially required for anti-TIGIT-induced regression of parasite growth in mice. CONCLUSIONS: This study demonstrates that E. multilocularis can induce T-cell exhaustion through inhibitory receptor TIGIT, and that blocking this checkpoint may reverse the functional impairment of T cells and represent a possible approach to immunotherapy against AE.

Dual Role of Hepatic Macrophages in the Establishment of the Echinococcus multilocularis Metacestode in Mice.

Echinococcus multilocularis larvae, predominantly located in the liver, cause a tumor-like parasitic disease, alveolar echinococcosis (AE), that is characterized by increased infiltration of various immune cells, including macrophages, around the lesion that produces an "immunosuppressive" microenvironment, favoring its persistent infection. However, the role of hepatic macrophages in the host defense against E. multilocularis infection remains poorly defined. Using human liver tissues from patients with AE and a hepatic experimental mouse model of E. multilocularis, we investigated the phenotype and function of hepatic macrophages during the parasite infection. In the present study, we found that a large number of CD68+ macrophages accumulated around the metacestode lesion in the liver of human AE samples and that both S100A9+ proinflammatory (M1 phenotype) and CD163+ anti-inflammatory (M2 phenotype) macrophages were significantly higher in close liver tissue (CLT) than in distant liver tissue (DLT), whereas M2 macrophages represent the dominant macrophage population. Furthermore, E. multilocularis-infected mice exhibited a massive increase in macrophage (F4/80+) infiltration in the liver as early as day 5, and the infiltrated macrophages were mainly monocyte-derived macrophages (CD11bhi F4/80int MoMFs) that preferentially differentiated into the M1 phenotype (iNOS+) at the early stage of E. multilocularis infection and then polarized to anti-inflammatory macrophages of the M2 phenotype (CD206+) at the chronic stage of infection. We further showed that elimination of macrophages by treatment of mice with clodronate-liposomes before E. multilocularis infection impaired worm expulsion and was accompanied by a reduction in liver fibrosis, yielding a high parasite burden. These results suggest that hepatic macrophages may play a dual role in the establishment and development of E. multilocularis metacestodes in which early larvae clearance is promoted by M1 macrophages while persistent metacestode infection is favored by M2 macrophages.

The local immune response during Echinococcus granulosus growth in a quantitative hepatic experimental model.

The local immune mechanisms responsible for the establishment and development of Echinococcus granulosus sensu stricto infection in the liver, have been little explored. We developed a suitable experimental model that mimics naturally infected livers using portal injection of protoscoleces. Opposite to Echinococcus multilocularis infection which is dose-dependent, fully mature hydatid cysts can be established in the liver whatever the injection dose; although most of the infection sites were seen at the establishment phase as inflammatory granulomas associated with fibrosis, they never matured into cysts. At the establishment phase, a strong immune response was composed of T and B cells, with T1-type, T2-type cells and cytokines and IL-10-secreting CD8+ T cells in the liver. At the established phase, results suggested a local production of antibodies by B cells, and an involvement of NK and NKT cells. Infection outcome and local immune response in the liver, were different in the mouse models of Echinococcus granulosus sensu stricto and Echinococcus multilocularis respectively; however, only early specificities at the microenvironment level might explain the major differences found between the lesions induced by the two species. Our quantitative experimental model appears fully appropriate to further study this microenvironment and its relationship with each cestode species.

Thioredoxin peroxidase secreted by Echinococcus granulosus (sensu stricto) promotes the alternative activation of macrophages via PI3K/AKT/mTOR pathway.

BACKGROUND: Larvae of Echinococcus granulosus (sensu lato) dwell in host organs for a long time but elicit only a mild inflammatory response, which indicates that the resolution of host inflammation is necessary for parasite survival. The recruitment of alternatively activated macrophages (AAMs) has been observed in a variety of helminth infections, and emerging evidence indicates that AAMs are critical for the resolution of inflammation. However, whether AAMs can be induced by E. granulosus (s.l.) infection or thioredoxin peroxidase (TPx), one of the important molecules secreted by the parasite, remains unclear. METHODS: The activation status of peritoneal macrophages (PMs) derived from mice infected with E. granulosus (sensu stricto) was analyzed by evaluating the expression of phenotypic markers. PMs were then treated in vivo and in vitro with recombinant EgTPx (rEgTPx) and its variant (rvEgTPx) in combination with parasite excretory-secretory (ES) products, and the resulting activation of the PMs was evaluated by flow cytometry and real-time PCR. The phosphorylation levels of various molecules in the PI3K/AKT/mTOR pathway after parasite infection and antigen stimulation were also detected. RESULTS: The expression of AAM-related genes in PMs was preferentially induced after E. granulosus (s.s.) infection, and phenotypic differences in cell morphology were detected between PMs isolated from E. granulosus (s.s.)-infected mice and control mice. The administration of parasite ES products or rEgTPx induced the recruitment of AAMs to the peritoneum and a notable skewing of the ratio of PM subsets, and these effects are consistent with those obtained after E. granulosus (s.s.) infection. ES products or rEgTPx also induced PMs toward an AAM phenotype in vitro. Interestingly, this immunomodulatory property of rEgTPx was dependent on its antioxidant activity. In addition, the PI3K/AKT/mTOR pathway was activated after parasite infection and antigen stimulation, and the activation of this pathway was suppressed by pre-treatment with an AKT/mTOR inhibitor. CONCLUSIONS: This study demonstrates that E. granulosus (s.s.) infection and ES products, including EgTPx, can induce PM recruitment and alternative activation, at least in part, via the PI3K/AKT/mTOR pathway. These results suggest that EgTPx-induced AAMs might play a key role in the resolution of inflammation and thereby favour the establishment of hydatid cysts in the host.

Echinococcus multilocularis inoculation induces NK cell functional decrease through high expression of NKG2A in C57BL/6 mice.

BACKGROUND: Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis (E. multilocularis), and considered as public health issue. Parasite-host immune interaction is pivotal during infection. As a subset of innate lymphoid cells, NK cells are known to play an important role during virus, bacteria, intra/extracellular parasitic infections and tumor progression. However, the possible role of NK cells in E. multilocularis infection in both human and murine is little known. Herein, the functional alteration of hepatic NK cells and their related molecules in E. multilocularis infected mice were studied. METHODS: 2000 protoscoleces (PSCs) were injected to C57BL/6 mice via the portal vein to establish secondary E. multilocularis infection. NK cells population and their related molecules (CD69, Ly49D, Ly49G2, Ly49H, Ly49I, NKG2A, NKG2D, granzyme B, IFN-γ, TNF-α) were assessed by using fluorescence-activated cell sorter (FACS) techniques and qRT-PCR. NK cell depletion was performed for further understanding the possible function of NK cells during infection. RESULTS: The total frequencies of NK cells and NK-derived IFN-γ production were significantly reduced at designated time points (2, 4, 12 weeks). The liver resident (CD49a+DX5-) NK cells are decreased at 4 weeks after inoculation and which is significantly lower than in control mice. Moreover, in vivo antibody-mediated NK cell depletion increased parasitic load and decreased peri-parasitic fibrosis. Expression of the inhibitory receptor NKG2A was negatively related to NK- derived IFN-γ secretion. CONCLUSIONS: Our study showed down regulates of NK cells and upper regulates of NKG2A expression on NK cells during E. multilocularis infection. Reduction of NK cell frequencies and increased NKG2A might result in low cytotoxic activity through decreased IFN-γ secretion in E. multilocularis infection. This result might be helpful to restore NK cell related immunity against E. multilocularis infection to treat alveolar echinococcosis.

Author Correction: T-cell tolerance and exhaustion in the clearance of Echinococcus multilocularis: role of inoculum size in a quantitative hepatic experimental model.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Identification of Functional MKK3/6 and MEK1/2 Homologs from Echinococcus granulosus and Investigation of Protoscolecidal Activity of Mitogen-Activated Protein Kinase Signaling Pathway Inhibitors In Vitro and In Vivo.

Cystic echinococcosis is a zoonosis caused by the larval stage of Echinococcus granulosussensu lato There is an urgent need to develop new drugs for the treatment of this disease. In this study, we identified two new members of mitogen-activated protein kinase (MAPK) cascades, MKK3/6 and MEK1/2 homologs (termed EgMKK1 and EgMKK2, respectively), from E. granulosussensu stricto Both EgMKK1 and EgMKK2 were expressed at the larval stages. As shown by yeast two-hybrid and coimmunoprecipitation analyses, EgMKK1 interacted with the previously identified Egp38 protein but not with EgERK. EgMKK2, on the other hand, interacted with EgERK. In addition, EgMKK1 and EgMKK2 displayed kinase activity toward the substrate myelin basic protein. When sorafenib tosylate, PD184352, or U0126-ethanol (EtOH) was added to the medium for in vitro culture of E. granulosus protoscoleces (PSCs) or cysts, an inhibitory and cytolytic effect was observed via suppressed phosphorylation of EgMKKs and EgERK. Nonviability of PSCs treated with sorafenib tosylate or U0126-EtOH, and not with PD184352, was confirmed through bioassays, i.e., inoculation of treated and untreated protoscoleces into mice. In vivo treatment of E. granulosussensu stricto-infected mice with sorafenib tosylate or U0126-EtOH for 4 weeks demonstrated a reduction in parasite weight, but the results did not show a significant difference. In conclusion, the MAPK cascades were identified as new targets for drug development, and E. granulosus was efficiently inhibited by their inhibitors in vitro The translation of these findings into in vivo efficacy requires further adjustment of treatment regimens using sorafenib tosylate or, possibly, other kinase inhibitors.

A Comprehensive Classification System for Breast Cancer Diagnosis Based on Dynamic Optical Breast Imaging.

The Dynamic Optical Breast Imaging technology is a promising breast cancer diagnosis approach based on tumor angiogenesis or vascular change detection which generally causes an increased blood volume in tumor. By applying sustained pressure to breast tissue under red light, the tissue with abnormal vascularization exhibits different dynamic behaviors of optical properties compared with normal breast tissue. In this paper, we explore a comprehensive classification method to discriminate malignant from benign lesions based on the Dynamic Optical breast Imaging technology. Firstly, we propose an automatic Point of Reference (POR) and Point of Interest (POI) selection algorithm from input images for following comparison procedures. Secondly, an automatic Margin Shape Patterns (MSP) recognition algorithm for Region of Interest (ROI) is explored. Furthermore, we define a new significant temporal and contextual feature named Temporal Curves Similarity Index (TCSI), with the aim of better characterizing and quantifying the difference inside the same breast. Finally, Support Vector Machine (SVM) is utilized for comprehensive classification. Experimental results, sensitivity of 91% and specificity of 71%, on our clinical database verify the efficiency of the proposed method.

T-cell tolerance and exhaustion in the clearance of Echinococcus multilocularis: role of inoculum size in a quantitative hepatic experimental model.

The local immune mechanisms responsible for either self-healing or sustained chronic infection are not clear, in the development of E. multilocularis larvae. Here, we developed a suitable experimental model that mimics naturally infected livers, according to the parasite load. We demonstrated that local cellular immunity and fibrogenesis are actually protective and fully able to limit metacestode growth in the liver of low or medium dose-infected mice (LDG or MDG), or even to clear it, while impairment of cellular immunity is followed by a more rapid and severe course of the disease in high dose-infected mice (HDG). And recruitment and/ or proliferation of memory T cells (including CD4 Tem, CD8 Tcm and CD8 Tem) and imbalance of T1/T2/T17/Treg-type T cells in liver were not only associated with clearance of the parasite infection in LDG, but also with increased hepatic injury in HDG; in particular the dual role of CD8 T cells depending on the parasite load and the various stages of metacestode growth. Besides, we first demonstrate the association between LAG3- or 2B4-expressing T cells exhaustion and HD inocula in late stages. Our quantitative experimental model appears fully appropriate to study immunomodulation as a therapeutic strategy for patients with Alveolar Echinococcosis.

MiR-144 suppresses cell proliferation, migration, and invasion in hepatocellular carcinoma by targeting SMAD4.

BACKGROUND/AIM: Increasing evidence show microRNAs (miRNAs) are engaged in hepatocellular carcinoma (HCC). The aim of this study was to investigate the role of miR-144 in HCC, as well as to identify its underlying mechanism. METHODS: The expression levels of miR-144 were assessed in multiple HCC cell lines, as well as in liver tissues from patients with HCC. We further examined the effects of miR-144 on HCC. The molecular target of miR-144 was identified using a computer algorithm and confirmed experimentally. RESULTS: We found that the levels of miR-144 were frequently downregulated in human HCC tissues and cell lines, and overexpression of miR-144 dramatically inhibited HCC metastasis, invasion, cell cycle, epithelial-mesenchymal transition, and chemoresistance. We further verified the SMAD4 as a novel and direct target of miR-144 in HCCs. CONCLUSION: Taken together, overexpression of miR-144 or downregulation of SMAD4 may prove beneficial as therapeutic strategies for HCC treatment.

MiR-502-3P suppresses cell proliferation, migration, and invasion in hepatocellular carcinoma by targeting SET.

BACKGROUND/AIM: Increasing evidences show that microRNAs are engaged in hepatocellular carcinoma (HCC). The aim of this study was to investigate the role of miR-502-3P in HCC and to identify its underlying mechanism. METHODS: The expression levels of miR-502-3P were assessed in multiple HCC cell lines and in liver tissues of patients with HCC. We further examined the effects of miR-502-3P on malignant behavior of HCC. The molecular target of miR-502-3P was identified using a computer algorithm and confirmed experimentally. RESULTS: Downregulation of miR-502-3P was found in both HCC cell lines and human samples. Overexpression of miR-502-3P dramatically inhibits HCC proliferation, metastasis, invasion, and cell adhesion. We further verify the SET as a novel and direct target of miR-502-3P in HCCs. CONCLUSION: Taken together, overexpression of miR-502-3P or downregulation of SET may prove beneficial as a therapeutic strategy for HCC treatment.