bims-drudre Biomed News
on Targeted drug delivery and programmed release mechanisms
Issue of 2021‒03‒07
twenty-two papers selected by
Ceren Kimna
Technical University of Munich

  1. Adv Sci (Weinh). 2021 Feb;8(4): 2002746
      The low immunogenicity, insufficient infiltration of T lymphocytes, and dismal response to immune checkpoint blockade therapy pose major difficulties in immunotherapy of pancreatic cancer. Photoimmunotherapy by photodynamic therapy (PDT) can induce an antitumor immune response by triggering immunogenic cell death in the tumor cells. Notwithstanding, PDT-driven oxygen consumption and microvascular damage can further aggravate hypoxia to exaggerates glycolysis, leading to lactate accumulation and immunosuppressive tumor microenvironment. Herein, a supramolecular prodrug nanoplatform codelivering a photosensitizer and a prodrug of bromodomain-containing protein 4 inhibitor (BRD4i) JQ1 for combinatory photoimmunotherapy of pancreatic cancer are demonstrated. The nanoparticles are fabricated by host-guest complexation between cyclodextrin-grafted hyaluronic acid (HA-CD) and adamantine-conjugated heterodimers of pyropheophorbide a (PPa) and JQ1, respectively. HA can achieve active tumor targeting by recognizing highly expressed CD44 on the surface of pancreatic tumors. PPa-mediated PDT can enhance the immunogenicity of the tumor cells and promote intratumoral infiltration of the cytotoxic T lymphocytes. Meanwhile, JQ1 combats PDT-mediated immune evasion through inhibiting expression of c-Myc and PD-L1, which are key regulators of tumor glycolysis and immune evasion. Collectively, this study presents a novel strategy to enhance photoimmunotherapy of the pancreatic cancer by provoking T cells activation and overcoming adaptive immune resistance.
    Keywords:  glucose metabolism; immunogenic cell death; pancreatic cancer; photoimmunotherapy; prodrug nanoparticles
  2. ACS Nano. 2021 Mar 02.
      Simultaneously targeting tumor cells and nonmalignant cells represent a more efficient strategy for replacing the traditional method of targeting only tumor cells, and co-delivery nanocarriers have inherent advantages to achieve this goal. However, differential delivery of multiple agents to various types of cell with different spatial distribution patterns remains a large challenge. Herein, we developed a nanocarrier of platinum(IV) prodrug and BLZ-945, BLZ@S-NP/Pt, to differentially target tumor cells and tumor-associated macrophages (TAMs). The BLZ@S-NP/Pt undergoes shrinkage to small platinum(IV) prodrug-conjugating nanoparticles under 660 nm light, resulting in deep tumor penetration to kill more cancer cells. Meanwhile, such shrinkage also enables the rapid release of BLZ-945 in the perivascular regions of tumor to preferentially deplete TAMs (enriched in perivascular regions). Therefore, BLZ@S-NP/Pt differentially and precisely delivers agents to TAMs and tumor cells located in different spatial distribution, respectively, eventually having synergistic anticancer effects in multiple tumor models.
    Keywords:  cancer chemoimmunotherapy; deep tumor penetration; photoinduced shrinkable nanocarrier; phototriggered drug release; targeting “the soil and the seed” strategy
  3. ACS Nano. 2021 Mar 01.
      For systemic delivery of small interfering RNA (siRNA) to solid tumors, the carrier must circulate avoiding premature degradation, extravasate and penetrate tumors, enter target cells, traffic to the intracellular destination, and release siRNA for gene silencing. However, existing siRNA carriers, which typically exhibit positive charges, fall short of these requirements by a large margin; thus, systemic delivery of siRNA to tumors remains a significant challenge. To overcome the limitations of existing approaches, we have developed a carrier of siRNA, called "Nanosac", a noncationic soft polyphenol nanocapsule. A siRNA-loaded Nanosac is produced by sequential coating of mesoporous silica nanoparticles (MSNs) with siRNA and polydopamine, followed by removal of the sacrificial MSN core. The Nanosac recruits serum albumin, co-opts caveolae-mediated endocytosis to enter tumor cells, and efficiently silences target genes. The softness of Nanosac improves extravasation and penetration into tumors compared to its hard counterpart. As a carrier of siRNA targeting PD-L1, Nanosac induces a significant attenuation of CT26 tumor growth by immune checkpoint blockade. These results support the utility of Nanosac in the systemic delivery of siRNA for solid tumor therapy.
    Keywords:  immune checkpoint blockade; noncationic; polyphenol nanocapsules; siRNA; soft; solid tumors; systemic delivery
  4. Adv Mater. 2021 Mar 06. e2007426
      Subcellular organelle-targeted nanoformulations for cancer theranostics are receiving increasing attention owing to their benefits of precise drug delivery, maximized therapeutic index, and reduced off-target side effects. Herein, a multichannel calcium ion (Ca2+ ) nanomodulator (CaNMCUR+CDDP ), i.e., a cisplatin (CDDP) and curcumin (CUR) co-incorporating calcium carbonate (CaCO3 ) nanoparticle, is prepared by a facile one-pot strategy in a sealed container with in situ synthesized polydopamine (PDA) as a template to enhance Ca2+ -overload-induced mitochondrial dysfunction in cancer therapy. After systemic administration, the PEGylated CaNMCUR+CDDP (PEG CaNMCUR+CDDP ) selectively accumulates in tumor tissues, enters tumor cells, and induces multilevel destruction of mitochondria by the combined effects of burst Ca2+ release, Ca2+ efflux inhibition by CUR, and chemotherapeutic CDDP, thereby observably boosting mitochondria-targeted tumor inhibition. Fluorescence imaging of CUR combined with photoacoustic imaging of PDA facilitates the visualization of the nanomodulator. The facile and practical design of this multichannel Ca2+ nanomodulator will contribute to the development of multimodal bioimaging-guided organelle-targeted cancer therapy in the future.
    Keywords:  calcium-ion overload; cancer theranostics; mitochondrial dysfunction; multichannel calcium-ion nanomodulators; multimodal bioimaging
  5. Nano Lett. 2021 Mar 02.
      Compared with traditional chemotherapeutics, vascular disruption agents (VDAs) have the advantages of rapidly blocking the supply of nutrients and starving tumors to death. Although the VDAs are effective under certain scenarios, this treatment triggers angiogenesis in the later stage of therapy that frequently leads to tumor recurrence and treatment failure. Additionally, the nonspecific tumor targeting and considerable side effects also impede the clinical applications of VDAs. Here we develop a customized strategy that combines a VDA with an anti-angiogenic drug (AAD) using mesoporous silica nanoparticles (MSNs) coated with platelet membrane for the self-assembled tumor targeting accumulation. The tailor-made nanoparticles accumulate in tumor tissues through the targeted adhesion of platelet membrane surface to damaged vessel sites, resulting in significant vascular disruption and efficient anti-angiogenesis in animal models. This study demonstrates the promising potential of combining VDA and AAD in a single nanoplatform for tumor eradication.
    Keywords:  Platelet membrane; combination therapy; nanoparticle; self-assembled tumor accumulation; tumor vessels
  6. Sci Adv. 2021 Mar;pii: eabe7853. [Epub ahead of print]7(10):
      Immunotherapies controlling the adaptive immune system are firmly established, but regulating the innate immune system remains much less explored. The intrinsic interactions between nanoparticles and phagocytic myeloid cells make these materials especially suited for engaging the innate immune system. However, developing nanotherapeutics is an elaborate process. Here, we demonstrate a modular approach that facilitates efficiently incorporating a broad variety of drugs in a nanobiologic platform. Using a microfluidic formulation strategy, we produced apolipoprotein A1-based nanobiologics with favorable innate immune system-engaging properties as evaluated by in vivo screening. Subsequently, rapamycin and three small-molecule inhibitors were derivatized with lipophilic promoieties, ensuring their seamless incorporation and efficient retention in nanobiologics. A short regimen of intravenously administered rapamycin-loaded nanobiologics (mTORi-NBs) significantly prolonged allograft survival in a heart transplantation mouse model. Last, we studied mTORi-NB biodistribution in nonhuman primates by PET/MR imaging and evaluated its safety, paving the way for clinical translation.
  7. Adv Sci (Weinh). 2021 Feb;8(4): 2002834
      Mitochondria are highly involved in the metastasis of cancer cells. However, low permeability of mitochondria impedes the entry of anti-cancer drugs. Here, a self-assembled nanoparticle platform is designed that not only targets the DNA-intercalating agent doxorubicin to mitochondria but also enhances the specific penetration by opening the mitochondrial permeability transition pores (MPTPs). With drastic improvement in mitochondrial uptake, the drug delivery system results in substantial mitochondrial impairment leading to amplified induction of apoptosis, depletion of energy supply, and inhibition of numerous metastasis-associated proteins. As a consequence, the drug delivery system significantly inhibits the orthotopic tumor growth, and suppressed the metastasis of cancer cells detached from primary tumors. Additionally, the nanoparticle exhibits a potent effect on eradicating the metastasis of disseminated tumor cell from blood to lung. The results show that strategies of targeting mitochondria and unlocking MPTP are feasible and beneficial to mitigate both tumorigenesis and metastasis.
    Keywords:  MPTP opening; cancer therapy; mitochondria targeting; self‐assembly nanoparticle; tumor metastasis suppression
  8. ACS Nano. 2021 Mar 01.
      Autoimmune diseases like multiple sclerosis (MS), type 1 diabetes, and lupus occur when the immune system attacks host tissue. Immunotherapies that promote selective tolerance without suppressing normal immune function are of tremendous interest. Here, nanotechnology was used for rational assembly of peptides and modulatory immune cues into immune complexes. Complexes containing self-peptides and regulatory nucleic acids reverse established paralysis in a preclinical MS model. Importantly, mice responding to immunotherapy maintain healthy, antigen-specific B and T cell responses during a foreign antigen challenge. A therapeutic library isolating specific components reveals that regulatory nucleic acids suppress inflammatory genes in innate immune cells, while disease-matched peptide sequences control specificity of tolerance. Distinct gene expression profiles in cells and animals are associated with the immune signals administered in particulate and soluble forms, highlighting the impact of biophysical presentation of signals. This work provides insight into the rational manipulation of immune signaling to drive tolerance.
    Keywords:  immune tolerance and regulation; microparticles and nanotechnology; multiple sclerosis and autoimmunity; polyelectrolyte multilayer capsule; toll-like receptor ligands
  9. Nat Commun. 2021 03 02. 12(1): 1394
      N6-methyladenosine (m6A) is a reversible mRNA modification that has been shown to play important roles in various biological processes. However, the roles of m6A modification in macrophages are still unknown. Here, we discover that ablation of Mettl3 in myeloid cells promotes tumour growth and metastasis in vivo. In contrast to wild-type mice, Mettl3-deficient mice show increased M1/M2-like tumour-associated macrophage and regulatory T cell infiltration into tumours. m6A sequencing reveals that loss of METTL3 impairs the YTHDF1-mediated translation of SPRED2, which enhances the activation of NF-kB and STAT3 through the ERK pathway, leading to increased tumour growth and metastasis. Furthermore, the therapeutic efficacy of PD-1 checkpoint blockade is attenuated in Mettl3-deficient mice, identifying METTL3 as a potential therapeutic target for tumour immunotherapy.
  10. Sci Adv. 2021 Mar;pii: eabe8671. [Epub ahead of print]7(10):
      Basal-like breast cancer (BLBC) shows brain metastatic (BM) capability and overexpresses EGFR and death-receptors 4/5 (DR4/5); however, the anatomical location of BM prohibits efficient drug-delivery to these targetable markers. In this study, we developed BLBC-BM mouse models featuring different patterns of BMs and explored the versatility of estem cell (SC)-mediated bi-functional EGFR and DR4/5-targeted treatment in these models. Most BLBC lines demonstrated a high sensitivity to EGFR and DR4/5 bi-targeting therapeutic protein, EVDRL [anti-EGFR VHH (EV) fused to DR ligand (DRL)]. Functional analyses using inhibitors and CRISPR-Cas9 knockouts revealed that the EV domain facilitated in augmenting DR4/5-DRL binding and enhancing DRL-induced apoptosis. EVDRL secreting stem cells alleviated tumor-burden and significantly increased survival in mouse models of residual-tumor after macrometastasis resection, perivascular niche micrometastasis, and leptomeningeal metastasis. This study reports mechanism based simultaneous targeting of EGFR and DR4/5 in BLBC and defines a new treatment paradigm for treatment of BM.
  11. Nat Commun. 2021 Mar 05. 12(1): 1472
      Modules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a 'cargo' molecule reversibly to a permanent membrane 'anchor'; and (ii) creating a 'membrane-avidity switch' that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.
  12. Nat Commun. 2021 Mar 05. 12(1): 1482
      Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.
  13. Adv Healthc Mater. 2021 Feb 28. e2002200
      Gliomas remain difficult to treat because of their metastatic and recurrent nature and the existence of the blood-brain barrier (BBB), which impedes drug delivery. Microglia, the resident macrophages in the CNS, can be recruited by gliomas and can penetrate the tumor. In this study, microglia (BV2 cells) are used as transport vectors to deliver paclitaxel for the treatment of glioma. To avoid paclitaxel toxicity in microglia, liposomes are first employed to isolate the drug from BV2 cells. Dipalmitoyl phosphatidylserine (DPPS), as an "eat me" signal, is doped into liposomes to amplify their phagocytosis by microglia. This study demonstrates that engineered microglia can cross the BBB, independently migrate toward gliomas, and transfer cargo to glioma cells. Of note, extracellular vesicles and tunneling nanotubes are found to offer unique modes of cargo transportation between microglia and glioma cells. In vivo, the engineered drug-loaded microglia has a high ability to target the brain, penetrate glioma, and suppress tumor progression, supporting the notion that the use of engineered microglia is a potential strategy for the treatment of glioma. These findings present new opportunities for exploration into the use of microglia as transport vectors to deliver therapeutic agents through specific membrane nanotubes and vesicles.
    Keywords:  engineered microglia; extracellular vesicles; glioma; phagocytosis; tunneling nanotubes
  14. Adv Sci (Weinh). 2021 Feb;8(4): 2002667
      MnO2 nanoparticles have been widely employed in cancer immunotherapy, playing a subsidiary role in assisting immunostimulatory drugs by improving their pharmacokinetics and/or creating a favorable microenvironment. Here, the stereotype of the subsidiary role of MnO2 nanoparticles in cancer immunotherapy is challenged. This study unravels an intrinsic immunomodulatory property of MnO2 nanoparticles as a unique nutrient-responsive immunogenic cell death (ICD) inducer, capable of directly modulating immunosurveillance toward tumor cells. MnO2 nanoflowers (MNFs) constructed via a one pot self-assembly approach selectively induce ICD to nutrient-deprived but not nutrient-replete cancer cells, which is confirmed by the upregulated damage associated molecular patterns in vitro and a prophylactic vaccination in vivo. The underlying mechanism of the MNFs-mediated selective ICD induction is likely associated with the concurrently upregulated oxidative stress and autophagy. Built on their unique immunomodulatory properties, an innovative nanomaterials orchestrated cancer starvation-immunotherapy is successfully developed, which is realized by the in situ vaccination with MNFs and vascular disrupting agents that cut off intratumoral nutrient supply, eliciting potent efficacy for suppressing local and distant tumors. These findings open up a new avenue toward biomedical applications of MnO2 materials, enabling an innovative therapeutics paradigm with great clinical significance.
    Keywords:  MnO2 nanoparticles; autophagy; immunogenic cell death; immunotherapy
  15. Pharmaceuticals (Basel). 2021 Feb 17. pii: 161. [Epub ahead of print]14(2):
      Evolution of nanomedicine is the re-design of synthetic and biological carriers to implement novel theranostic platforms. In recent years, bacteriophage research favors this process, which has opened up new roads in drug and gene delivery studies. By displaying antibodies, peptides, or proteins on the surface of different bacteriophages through the phage display technique, it is now possible to unravel specific molecular determinants of both cancer cells and tumor-associated microenvironmental molecules. Downstream applications are manifold, with peptides being employed most of the times to functionalize drug carriers and improve their therapeutic index. Bacteriophages themselves were proven, in this scenario, to be good carriers for imaging molecules and therapeutics as well. Moreover, manipulation of their genetic material to stably vehiculate suicide genes within cancer cells substantially changed perspectives in gene therapy. In this review, we provide examples of how amenable phages can be used as anticancer agents, especially because their systemic administration is possible. We also provide some insights into how their immunogenic profile can be modulated and exploited in immuno-oncology for vaccine production.
    Keywords:  biopanning; drug delivery; gene therapy; phage display; targeting
  16. Mol Cancer. 2021 03 03. 20(1): 48
      mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature's core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient.For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless.As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application.
    Keywords:  Antibody; Cancer; Cytokine; Dendritic cell; Nanoparticle; T cell; Tumor microenvironment; mRNA
  17. Nat Commun. 2021 03 03. 12(1): 1426
      Metastatic prostate cancer (mPC) comprises a spectrum of diverse phenotypes. However, the extent of inter- and intra-tumor heterogeneity is not established. Here we use digital spatial profiling (DSP) technology to quantitate transcript and protein abundance in spatially-distinct regions of mPCs. By assessing multiple discrete areas across multiple metastases, we find a high level of intra-patient homogeneity with respect to tumor phenotype. However, there are notable exceptions including tumors comprised of regions with high and low androgen receptor (AR) and neuroendocrine activity. While the vast majority of metastases examined are devoid of significant inflammatory infiltrates and lack PD1, PD-L1 and CTLA4, the B7-H3/CD276 immune checkpoint protein is highly expressed, particularly in mPCs with high AR activity. Our results demonstrate the utility of DSP for accurately classifying tumor phenotype, assessing tumor heterogeneity, and identifying aspects of tumor biology involving the immunological composition of metastases.
  18. Nat Rev Drug Discov. 2021 Mar 01.
      Achieving sufficient delivery across the blood-brain barrier is a key challenge in the development of drugs to treat central nervous system (CNS) disorders. This is particularly the case for biopharmaceuticals such as monoclonal antibodies and enzyme replacement therapies, which are largely excluded from the brain following systemic administration. In recent years, increasing research efforts by pharmaceutical and biotechnology companies, academic institutions and public-private consortia have resulted in the evaluation of various technologies developed to deliver therapeutics to the CNS, some of which have entered clinical testing. Here we review recent developments and challenges related to selected blood-brain barrier-crossing strategies - with a focus on non-invasive approaches such as receptor-mediated transcytosis and the use of neurotropic viruses, nanoparticles and exosomes - and analyse their potential in the treatment of CNS disorders.
  19. Adv Sci (Weinh). 2021 Feb;8(4): 2003263
      Evaluating the therapeutic response and survival of lung cancer patients receiving first-line chemotherapy has always been difficult. Limited biomarkers for evaluation exist and as a result histology represents an empiric tool to guide therapeutic decision making. In this study, molecular signatures associated with response and long-term survival of lung cancer patients receiving first-line chemotherapy are discovered. Whole-exome sequencing is performed on pretherapeutic tissue samples of 186 patients [145 non-small cell lung cancer (NSCLC) and 41 small cell lung cancer (SCLC)]. On the basis of genomic alteration characteristics, NSCLC patients can be classified into four subtypes (C1-C4). The long-term survival is similar among different subtypes. SCLC patients are also divided into four subtypes and significant difference in their progression free survival is revealed (P < 0.001). NSCLC patients can be divided into three subtypes (S1-S3) based on TMB. A trend of worse survival associated with higher TMB in subtype S3 than in S1+S2 is found. In contrast, no significant correlations between molecular subtype and therapeutic response are observed. In conclusion, this study identifies several molecular signatures associated with response and survival to first-line chemotherapy in lung cancer.
    Keywords:  NSCLC; SCLC; TMB; chemotherapy; lung cancer; subtype
  20. Adv Sci (Weinh). 2021 Feb;8(4): 2003338
      Radiotherapy (RT) based on DNA damage and reactive oxygen species (ROS) generation has been clinically validated in various types of cancer. However, high dose-dependent induced toxicity to tissues, non-selectivity, and radioresistance greatly limit the application of RT. Herein, an oxygen-enriched X-ray nanoprocessor Hb@Hf-Ce6 nanoparticle is developed for improving the therapeutic effect of RT-radiodynamic therapy (RDT), enhancing modulation of hypoxia tumor microenvironment (TME) and promoting antitumor immune response in combination with programmed cell death protein 1 (PD-1) immune checkpoint blockade. All functional molecules are integrated into the nanoparticle based on metal-phenolic coordination, wherein one high-Z radiosensitizer (hafnium, Hf) coordinated with chlorin e6 (Ce6) modified polyphenols and a promising oxygen carrier (hemoglobin, Hb) is encapsulated for modulation of oxygen balance in the hypoxia TME. Specifically, under single X-ray irradiation, radioluminescence excited by Hf can activate photosensitizer Ce6 for ROS generation by RDT. Therefore, this combinatory strategy induces comprehensive antitumor immune response for cancer eradication and metastasis inhibition. This work presents a multifunctional metal-phenolic nanoplatform for efficient X-ray mediated RT-RDT in combination with immunotherapy and may provide a new therapeutic option for cancer treatment.
    Keywords:  hemoglobin; immunotherapy; metal‐phenolic coordination; nanomedicine; radiodynamic therapy; radiotherapy
  21. Adv Sci (Weinh). 2021 Feb;8(4): 2001961
      The incidence of bone metastases in hepatocellular carcinoma (HCC) has increased prominently over the past decade owing to the prolonged overall survival of HCC patients. However, the mechanisms underlying HCC bone-metastasis remain largely unknown. In the current study, HCC-secreted lectin galactoside-binding soluble 3 (LGALS3) is found to be significantly upregulated and correlates with shorter bone-metastasis-free survival of HCC patients. Overexpression of LGALS3 enhances the metastatic capability of HCC cells to bone and induces skeletal-related events by forming a bone pre-metastatic niche via promoting osteoclast fusion and podosome formation. Mechanically, ubiquitin ligaseRNF219-meidated α-catenin degradation prompts YAP1/β-catenin complex-dependent epigenetic modifications of LGALS3 promoter, resulting in LGALS3 upregulation and metastatic bone diseases. Importantly, treatment with verteporfin, a clinical drug for macular degeneration, decreases LGALS3 expression and effectively inhibits skeletal complications of HCC. These findings unveil a plausible role for HCC-secreted LGALS3 in pre-metastatic niche and can suggest a promising strategy for clinical intervention in HCC bone-metastasis.
    Keywords:  LGALS3; RNF219; bone metastasis; hepatocellular carcinoma; skeletal‐related events