Pre-metastatic niche-targeted nanoparticle-loaded myeloid cell as well as preparation method and application thereof

Abstract

The invention discloses a pre-transfer ecological niche targeted nanoparticle-loaded myeloid cell as well as a preparation method and application thereof, and particularly relates to synthesis of a PMN microenvironment MMP2 enzyme responsive connecting bond, and the structure of the connecting bond does not affect expression of LNP loaded ATRA drugs and genes in the cell. And stable loading of the AIG-LNP on the surface of the MCs cell is realized through a connecting bond in a normal-temperature mild environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a pre-metastatic niche-targeted nanoparticle-loaded myeloid cell, a preparation method thereof, and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance some understanding of the overall background of the invention and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death worldwide. The latest cancer statistics in the United States show that the survival rate for liver cancer patients is only 22%. Improving the cure rate and survival rate of HCC patients has become a pressing task in current treatment research. Surgery is the best treatment for HCC, but the metastasis and recurrence rate within five years after surgery remains as high as 70%, which is the main cause of death. The pre-metastatic niche (PMN) promotes the formation of lung metastases in hepatocellular carcinoma (HCC) by maintaining an immunosuppressive microenvironment rich in myeloid-derived suppressor cells (MDSCs).

[0004] PMNs are characterized by an immunosuppressive microenvironment and extensive remodeling of the extracellular matrix, which are key biological foundations of tumor metastasis. MDSCs maintain an immunosuppressive microenvironment in PMNs, leading to a reduction in the number of dendritic cells (DCs) and impaired maturation. Furthermore, MDSCs suppress the proliferation of CD4+ T cells and attenuate the effector function of cytotoxic T lymphocytes (CTLs) by producing reactive oxygen species (ROS) and arginase-1 (Arg-1), thereby enabling CTCs to evade immune surveillance, colonize PMNs, and ultimately metastasize. Remodeling the immunosuppressive microenvironment in PMNs can prevent the colonization of circulating tumor cells (CTCs), providing a promising strategy for inhibiting tumor metastasis.

[0005] Current approaches include depleting factors that induce PMN formation (such as exosomes and cytokines) to prevent PMN formation and eliminating CTCs by polarizing immune cells (such as macrophages and neutrophils). However, these therapies are limited by the lack of effective delivery strategies to target PMNs and reshape the persistent immunosuppressive microenvironment within them, hindering effective inhibition of tumor metastasis. Summary of the Invention

[0006] In response to the shortcomings of existing technologies and the promoting role of PMNs in tumor metastasis formation, a combination of drug-loaded nanoparticles and surface-engineered myeloid cells (MCs) was designed and prepared for the targeted delivery of therapeutic drugs to PMNs to inhibit HCC lung metastasis.

[0007] The technical solution adopted in the present invention is as follows: In a first aspect of the present invention, a method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells is provided, the method comprising the following steps: (1) Preparation of distearoylphosphatidylethanolamine-polyethylene glycol 5000-matrix metalloproteinase-2 sensitive peptide-N-hydroxysuccinimide ester (DSPE-PEG5000-MMP2 sensitive peptide (pep)-NHS): a. Thiol-matrix metalloproteinase-2 sensitive peptide-carboxyl (SH-MMP2 sensitive peptide-COOH) and succinimidyl ester-polyethylene glycol 5000-maleimide (DSPE-PEG 5000 After the reaction, the mixture was dialyzed and freeze-dried to obtain distearoylphosphatidylethanolamine-polyethylene glycol 5000-matrix metalloproteinase-2 sensitive peptide-carboxyl (DSPE-PEG 5000 -MMP2-sensitive peptide-COOH) products; b. the DSPE-PEG 5000 -MMP2 sensitive peptide-COOH, NHS and EDC were dissolved in dimethylformamide (DMF) for reaction. After the reaction, DSPE-PEG5000-MMP2 sensitive peptide-NHS was obtained by dialysis and freeze-drying. (2) Preparation of AIG-LNP nanoparticles co-loaded with all-trans retinoic acid (ATRA), interleukin-12 (IL-12) mRNA and glypican 3 (GPC3) mRNA: ((4-Hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) ALC-0315, dioleoylphosphatidylethanolamine (DOPE), β-sitosterol, DSPE-PEG5000-MMP2 sensitive peptide-NHS, cholesterol, and ATRA were dissolved in ethanol to obtain an oil phase solution. IL-12 mRNA and GPC3 mRNA were added to the aqueous phase solution, and AIG-LNPs were prepared using microfluidic technology. (3) Preparation of AIG-LNP@MC: AIG-LNP and myeloid cells (MCs) were incubated to obtain AIG-LNP@MC.

[0008] In one or more embodiments of the present invention, in step (1) a, the SH-MMP2 sensitive peptide-COOH and DSPE-PEG 5000 The molar ratio of -Mal is (1~2):1; the reaction conditions are: time 10~15h, temperature 20~25℃, reaction solvent is PBS buffer, and nitrogen atmosphere protection.

[0009] In one or more embodiments of the present invention, in step (1) b, DSPE-PEG 5000 The molar ratio of -MMP2 sensitive peptide -COOH, NHS and EDC is 1:1~2:1~2; the reaction conditions are: time is 20~28h, temperature is 20~25℃, and the reaction solvent is DMF.

[0010] In one or some embodiments of the present invention, in step (2), the mass ratio of ALC-0315, DOPE, β-sitosterol, DPMN, cholesterol, and ATRA is (0.2-0.3): (0.5-0.6): (0.02-0.04): (0.5-0.8): (0.05-0.1): (0.05-0.1).

[0011] In one or some embodiments of the present invention, in step (2), the aqueous phase solution is an enzyme-free citric acid buffer.

[0012] In one or some embodiments of the present invention, in step (2), GPC-3 mRNA and IL-12 mRNA are produced by conventional techniques, and those skilled in the art can conventionally know and obtain GPC-3 mRNA and IL-12 mRNA.

[0013] In one or some embodiments of the present invention, in step (2), the microfluidic conditions are: the flow rate ratio of the oil phase solution to the aqueous phase solution is 1:(1.5~3), and the total flow rate is 12~24 mL / min.

[0014] In one or some embodiments of the present invention, in step (3), the incubation temperature is 2-6° C., and the incubation time is 0.5-1.5 h.

[0015] In one or some embodiments of the present invention, in step (3), MCs are present at a concentration of 1×10 6 Cells were incubated with AIG-LNPs at a final ATRA concentration of 10-80 μg / mL.

[0016] In a second aspect of the present invention, provided are pre-metastatic niche-targeted nanoparticle-loaded myeloid cells prepared by the above method.

[0017] In a third aspect of the present invention, there is provided a use of the pre-metastatic niche-targeted nanoparticle-loaded myeloid cells in the preparation of a drug for inhibiting hepatocellular carcinoma metastasis.

[0018] Compared with the related art known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) The present invention co-loads the drug and gene mRNA into LNPs and modifies the LNP surface with an MMP2 enzyme-sensitive linker. The modified linker can anchor the LNP on the surface of MCs cells, effectively delivering the drug to the PMNs of the tumor, significantly increasing the accumulation of the drug in PMNs and improving its anti-metastatic therapeutic effect.

[0019] (2) The present invention significantly improves the efficacy of anti-lung metastasis of liver cancer. AIG-LNP delivered to PMN induces MDSC to differentiate into mature antigen-presenting cell DC cells, thereby reducing the number of MDSC, increasing the proportion of DC in PMN, and reversing the immunosuppressive microenvironment maintained by MDSC. GPC-3 and IL-12, as hepatocellular carcinoma (HCC)-related antigens and adjuvants, respectively, promote DC maturation and activate cytotoxic T lymphocytes (CTLs) to eliminate CTC. The anti-tumor metastasis effect shows that AIG-LNP@MC can effectively target PMN and promote 86.10% of MDSCs to differentiate into dendritic cells (DCs), increasing the maturity of DCs by 1.96 times, thereby reconstructing PMN. In the reconstructed PMN, AIG-LNP@MC increased the activation of CTLs by 2.05 times and achieved an 88.23% lung metastasis inhibition rate in an in situ liver cancer mouse model.

[0020] (3) The drug ratio and phospholipid concentration in the present invention are optimized for MDSCs, resulting in a good therapeutic effect. The raw materials used in the present invention are simple, safe, and reliable. The carrier phospholipids are endogenous substances in the human body, safe, non-toxic, biodegradable, and have good biocompatibility.

[0021] (4) The lipid nanoparticles prepared by the present invention have an appropriate particle size (100-200 nm), the process is stable, simple and feasible, and easy to industrialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 : DSPE-PEG5000-MMP2 sensitive peptide-COOH structure and synthesis process 4 1 H NMR spectrum.

[0024] Figure 2 : Schematic diagram of the preparation of LNPs by nanoprecipitation method.

[0025] Figure 3:AIG-LNPs particle size distribution (a); ζ potential (b); transmission electron microscopy (TEM) (c).

[0026] Figure 4 : Preparation flow chart of AIG-LNP@MC.

[0027] Figure 5 :Screening of drug loading conditions for AIG-LNP@MC.

[0028] Figure 6 : Preparation and characterization of AIG-LNP@MC.

[0029] Figure 7 : PMN targeting ability of AIG-LNP@MC.

[0030] Figure 8 : Anti-tumor metastasis ability of AIG-LNP@MC. DETAILED DESCRIPTION

[0031] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0032] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.

[0033] In this study, targeting the role of PMNs in promoting tumor metastasis, a combination of drug-loaded nanoparticles and surface-engineered myeloid cells (MCs) was designed and prepared for targeted delivery of therapeutic drugs to PMNs to inhibit HCC lung metastasis. As precursors of MDSCs, MCs can naturally be recruited and aggregated within PMNs. This study demonstrated that intravenously injected allogeneic MCs exhibit a tropism for PMNs. Based on this, the present invention utilized the free amines on the surface of MCs to conjugate lipid nanoparticles (AIG-LNPs) co-loaded with all-trans retinoic acid (ATRA), glycoprotein glycan-3 (GPC-3) mRNA, and interleukin-12 (IL-12) mRNA to MCs, forming a drug-loaded nanoparticle and cell combination (AIG-LNP@MCs). The AIG-LNP@MCs of the present invention are first specifically recruited and accumulated in the pre-metastatic niche, where they then specifically release AIG-LNPs and deliver them to MDSCs. The drug regulates the differentiation of MDSCs into mature antigen-presenting DCs, and the expression of GPC-3 and IL-12 further activates microenvironmental immune cells, thereby overcoming the PMN drug delivery barrier and reversing the immunosuppressive microenvironment within the PMN. Compared to existing anti-metastatic nanoformulations, the present invention adds β-sitosterol to enhance the DC transfection ability of LNPs without affecting their in vivo safety. Furthermore, the present invention synthesizes an MMP2 enzyme-sensitive linker that allows LNPs to be loaded onto the surface of MCs at room temperature and under mild conditions. The PMN homing ability of MCs is then utilized to promote drug accumulation in PMNs, enhancing the anti-metastatic therapeutic effect.

[0034] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0035] Sources of reagents and materials: In the examples, the ionizable cationic lipid ALC-0315 was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.; DSPE-PEG 5000-MAL was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; all-trans retinoic acid was purchased from Solebao, Shanghai Biochempartner Company; dioleoylphosphatidylethanolamine DOPE was purchased from Avituo Pharmaceutical Technology Co., Ltd.; cholesterol was purchased from Sigma-Aldrich, SH-MMP2 sensitive peptide-COOH refers to SH-Cys-Pro-Val-Gly-Leu-Ile-Gly-COOH (SH-CPVGLIG-COOH), purchased from Dangang Bio; IL-12 mRNA: IL-12 in this example consists of two fragments, one p40 and one p35 fusion expression, with NCBI numbers NM_001303244.1 and NM_008351.3, respectively; the NCBI number of GPC3 mRNA is NM_016697.3.

[0036] Example 1 Synthesis of DSPE-PEG5000-MMP2 Sensitive Peptide-NHS Preparation flow chart Figure 1 , SH-MMP2 sensitive peptide-COOH and DSPE-PEG 5000 -Mal was dissolved in PBS at a molar ratio of 1.5:1, and then nitrogen was added for protection and stirred at 20°C for 12 hours. In order to remove the free MMP2 sensitive peptide, DSPE-PEG was dialyzed with deionized water (molecular weight cutoff value of 3000 Da) and freeze-dried to obtain DSPE-PEG. 5000 -MMP2-sensitive -COOH products.

[0037] Accurately weigh DSPE-PEG 5000 100 mg of MMP2-sensitive peptide-COOH, 3.3 mg of NHS, and 3.5 mg of EDC were dissolved in 5 mL, 0.2 mL, and 0.2 mL of anhydrous DMF, respectively, and sonicated until dissolved. The reaction was allowed to proceed at room temperature (24°C) for 24 hours. Free NHS was removed by dialysis (molecular weight cutoff 1000 Da), and DSPE-PEG was finally obtained by freeze-drying. 5000 -MMP2-sensitive peptide-NHS.

[0038] Example 2 Preparation of AIG-LNP Nanoparticles by Nanoprecipitation Prepare lipid-loaded nanoparticles AIG-LNP, the process is as follows Figure 2, all-trans retinoic acid, and immune-activating mRNA-loaded nanoparticles. ALC-0315 (20 mg), DOPE (10 mg), β-sitosterol (12.5 mg), DPMN (11.1 mg), cholesterol (12.5 mg), and ATRA (5 mg) were precisely weighed and dissolved in 1 mL of ethanol to prepare concentrated stock solutions. The solutions were sonicated until dissolved. According to the recipe, 0.138 mL, 0.054 mL, 0.027 mL, 0.065 mL, 0.060 mL, and 0.156 mL of the concentrated stock solutions were pipetted and mixed to obtain a 0.5 mL mixture. The mixed oil phase was then added to 1 mL of enzyme-free citrate buffer (pH 4.5) containing IL-12 and GPC3 mRNA (40 μg / mL, respectively) in a microfluidic device at an oil / water flow rate ratio of 1:2 (total flow rate of 12 mL / min). The organic solvent ethanol was removed by ultrafiltration centrifugation to obtain nanoparticles AIG-LNP, which were stored in the dark. The hydrodynamic diameter, PDI, and zeta potential of AIG-LNP were measured using a Malvern Zeta Sizer Nano-ZS instrument. Transmission electron microscopy (TEM) showed the morphology of AIG-LNP. Figure 3 .

[0039] Table 1 Particle size, zeta potential, drug loading and encapsulation efficiency of AIG-LNP

[0040] Example 3 Preparation of AIG-LNP@MC The AIG-LNP@myeloid cell drug delivery system (AIG-LNP@MC) was prepared by incubation method. The process is as follows: Figure 4 Myeloid cells were gently blown off, resuspended, counted, and seeded into 6-well plates at 1.0×10 cells per well. 6 Cells were incubated with AIG-LNPs at various ATRA concentrations for a specific time in PBS at 4°C. After incubation, the AIG-LNP@MCs were washed three times with PBS at 1200 rpm to remove unattached AIG-LNPs. This is the AIG-LNP@MC obtained.

[0041] Single-factor analysis was used to investigate the effects of different AIG-LNP concentrations (using ATRA concentration as the standard: 4, 10, 25, 50, and 75 μg / ml, obtained by demulsifying the AIG-LNP prepared in Example 2 and measuring the ATRA concentration, followed by dilution) and different incubation times (0.5 h, 1 h, and 1.5 h) on the drug loading capacity of myeloid cells to determine the optimal drug loading conditions. The formulation with the appropriate drug loading capacity for myeloid cells was selected as the optimal formulation.6 The cells were added with LNPs with a final concentration of ATRA of 50 μg / mL and incubated at 4°C for 1 h to obtain AIG-LNP@MC. Figure 6 The AIG-LNP@MC was washed three times in PBS at 1200 rpm to remove the unattached AIG-LNP. Subsequently, the obtained AIG-LNP@MC was demulsified with ethanol, and the ATRA drug loading was detected by high performance liquid chromatography at 340 nm. Figure 5 .

[0042] Example 4 Following the methods described in Examples 2 and 3, the nanoparticles AIG-LNP were diluted (DIR (cell membrane deep red fluorescent probe) was substituted for ATRA at a concentration of 50 μg / ml) to prepare DIR-LNP (without ATRA, IL-12 mRNA, and GPC3 mRNA). These were then co-incubated with MCs to prepare DIR-LNP@MCs. Six female mice were intraperitoneally injected with hepatocellular carcinoma cell culture supernatant (300 μl per mouse) for 10 consecutive days. On day 7, the mice were injected with H22 (1×10 per mouse) via the tail vein. 5 On days 11 and 12, PMN model mice were injected with 5×10 6 DIR-LNP@MCs and the control preparation DIR-LNP were used to slice lung tissues on day 13 for DAPI staining to observe the accumulation ability of the preparations in PMNs. Figure 7 As shown, the modified linker can anchor LNP on the surface of MCs cells, effectively deliver the drug to the PMN of the tumor, significantly increase the accumulation of the drug in PMN, and improve its anti-metastatic therapeutic effect.

[0043] Example 5 To investigate the effect of AIG-LNP@MC on liver cancer metastasis, each mouse was treated with 300 μL of HCC cell culture supernatant for 10 days. On the third day, the mice were divided into 10 groups: (1) control group, (2) MC group, (3) IL-12 mRNA-LNP@MC group, (4) GPC3 mRNA-LNP@MC group, (5) IL-12 mRNA+GPC3 mRNA-LNP@MC group, (6) ATRA-LNP@MC group, (7) ATRA mRNA+IL-12 mRNA-LNP group, (8) ATRA+IL-12 mRNA-LNP@MC group, (9) ATRA+IL-12 mRNA+GPC3 mRNA-LNP group, and (10) ATRA+IL-12 mRNA+GPC3 mRNA-LNP@MC group. Each mouse in each group received a dose of 100 μg of ATRA. The dose of IL-12 mRNA and GPC3 mRNA was 10 μg per mouse, and the drug was administered four times over 20 days. On day 7, 5×10 6 H22 cells. On the 20th day, the lung tissues of the mice were collected, and the nodules were photographed and counted. Figure 8 As shown, the anti-tumor metastasis effect showed that AIG-LNP@MC could effectively target PMNs and promote the differentiation of 86.10% of MDSCs into dendritic cells (DCs), increasing the maturation of DCs by 1.96 times, thereby reconstructing PMNs. In the reconstructed PMNs, AIG-LNP@MC increased the activation of CTLs by 2.05 times and achieved an 88.23% lung metastasis inhibition rate in an orthotopic liver cancer mouse model.

[0044] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells, characterized by: The method comprises the following steps: (1) Preparation of distearoylphosphatidylethanolamine-polyethylene glycol 5000-matrix metalloproteinase-2 sensitive peptide-N-hydroxysuccinimide ester (DSPE-PEG5000-MMP2 sensitive peptide-NHS): a. Thiol-matrix metalloproteinase-2 sensitive peptide-carboxyl (SH-MMP2 sensitive peptide-COOH) and succinimidyl ester-polyethylene glycol 5000-maleimide (DSPE-PEG 5000 After the reaction, the mixture was dialyzed and freeze-dried to obtain distearoylphosphatidylethanolamine-polyethylene glycol 5000-matrix metalloproteinase-2 sensitive peptide-carboxyl (DSPE-PEG 5000 -MMP2-sensitive peptide-COOH) products; b. the DSPE-PEG 5000 -MMP2 sensitive peptide-COOH, NHS and EDC were dissolved in dimethylformamide (DMF) for reaction. After the reaction, DSPE-PEG5000-MMP2 sensitive peptide-NHS was obtained by dialysis and freeze-drying. (2) Preparation of AIG-LNP nanoparticles co-loaded with all-trans retinoic acid (ATRA), interleukin-12 (IL-12) mRNA and glypican 3 (GPC3) mRNA: ((4-Hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) ALC-0315, dioleoylphosphatidylethanolamine (DOPE), β-sitosterol, DSPE-PEG5000-MMP2 sensitive peptide-NHS, cholesterol, and ATRA were dissolved in ethanol to obtain an oil phase solution. IL-12 mRNA and GPC3 were added to the aqueous phase solution, and AIG-LNPs were prepared using microfluidic technology. (3) Preparation of AIG-LNP@MC: AIG-LNP and myeloid cells (MCs) were incubated to obtain AIG-LNP@MC.

2. The method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 1, wherein: In step (1) a, the SH-MMP2 sensitive peptide-COOH and DSPE-PEG 5000 The molar ratio of -Mal is (1~2):1; the reaction conditions are: time 10~15h, temperature 20~25℃, reaction solvent is PBS buffer, and nitrogen atmosphere protection.

3. The method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 1, wherein: In step (1)b, DSPE-PEG 5000 The molar ratio of -MMP2 sensitive peptide -COOH, NHS and EDC is 1:1~2:1~2; the reaction conditions are: time is 20~28h, temperature is 20~25℃, and the reaction solvent is DMF.

4. The method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 1, wherein: In step (2), the mass ratio of ALC-0315, DOPE, β-sitosterol, DPMN, cholesterol, and ATRA is (0.2~0.3):(0.5~0.6):(0.02~0.04):(0.5~0.8):(0.05~0.1):(0.05~0.1).

5. The method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 1, wherein: In step (2), the aqueous phase solution is an enzyme-free citric acid buffer.

6. The method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 1, wherein: In step (2), the microfluidic conditions are: the flow rate ratio of the oil phase solution to the aqueous phase solution is 1:(1.5~3), and the total flow rate is 12~24mL / min.

7. The method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 1, wherein: In step (3), the incubation temperature is 2-6°C and the incubation time is 0.5-1.5h.

8. The method for preparing pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 1, wherein: In step (3), MCs were 1×10 6 Cells were incubated with AIG-LNPs at a final ATRA concentration of 10-80 μg / mL.

9. Pre-metastatic niche-targeted nanoparticle-loaded myeloid cells prepared by the method of any one of claims 1 to 8.

10. Use of the pre-metastatic niche-targeted nanoparticle-loaded myeloid cells according to claim 9 in the preparation of a drug for inhibiting hepatocellular carcinoma metastasis.

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