TROVE2 regulated invasion and migration of hepatocellular carcinoma cells via heparanase

Background:The role of TROVE domain family member 2(TROVE2)has been well-demonstrated in autoimmune diseases;however,its involvement in liver cancer remains unclear.Therefore,this study aimed to explore the biological function and clinical significance of TROVE2 in hepatocellular carcinoma(HCC).Methods:The expression level of TROVE2 was analyzed in HCC and paired adjacent tissue samples using real-time reverse transcription-quantitative polymerase chain reaction.The impact of TROVE2 on migration and invasion in HCC cells was analyzed through Transwell assays and Western blotting.High-throughput transcriptome sequencing and bioinformatics analyses were performed to identify downstream target genes.Back-complementation experiments were employed to verify the influence of downstream proteins on TROVE2-induced invasion and migration of HCC cells.Results:TROVE2 exhibited significant overexpression in liver cancer tissue,correlating with shorter overall survival.Overexpression of TROVE2 facilitated the invasion,metastasis,and epithelial-mesenchymal transition(EMT)process of HCC cells,whereas TROVE2 knockdown restrained migration,invasion,and EMT in these cells.Transcriptome sequencing and bioinformatics analysis identified heparanase(HPSE)as a downstreamtarget protein of TROVE2.Subsequent back-complementation experiments provided evidence that HPSE overexpression promoted TROVE2-mediated prometastasis effects.Moreover,the study revealed that TROVE2 was capable of regulating the EMT pathway through GSK-3βphosphorylation.Conclusions:TROVE2 facilitated the invasion,migration,and EMT process ofHCC cells through phosphorylation of the HPSE/GSK-3βaxis,indicating its significance as an important protein in tumor progression.

Profiling of hepatocellular carcinoma neoantigens reveals immune microenvironment and clonal evolution related patterns

Objective: Neoantigens derived from tumor-specific genomic alterations have demonstrated great potential for immunotherapeutic interventions in cancers. However, the comprehensive profile of hepatocellular carcinoma(HCC) neoantigens and their complex interplay with immune microenvironment and tumor evolution have not been fully addressed.Methods: Here we integrated whole exome sequencing data, transcriptome sequencing data and clinical information of 72 primary HCC patients to characterize the HCC neoantigen profile, and systematically explored its interactions with tumor clonal evolution, driver mutations and immune microenvironments.Results: We observed that higher somatic mutation/neoantigen load was associated with better clinical outcomes and HCC patients could be further divided into two subgroups with distinct prognosis based on their neoantigen expression patterns. HCC subgroup with neoantigen expression probability high(NEP-H) showed more aggressive pathologic features including increased incidence of tumor thrombus(P=0.038), higher recurrence rate(P=0.029),more inclined to lack tumor capsule(P=0.026) and with more microsatellite instability sites(P=0.006). In addition,NEP-H subgroup was also characterized by higher chance to be involved in tumor clonal evolution [odds ratio(OR)=46.7, P<0.001]. Gene set enrichment analysis revealed that upregulation of MYC and its targets could suppress immune responses, leading to elevated neoantigen expression proportion in tumor cells. Furthermore, we discovered an immune escape mechanism that tumors could become more inconspicuous by evolving subclones with less immunogenicity. We observed that smaller clonal mutation clusters with higher immunogenicity in tumor were more likely to involve in clonal evolution. Based on identified neoantigen profiles, we also discovered series of neoantigenic hotspot genes, which could serve as potential actionable targets in future.Conclusions: Our results revealed the landscape of HCC neoantigens and discovered two clinically relevant subgroups with distinct neoantigen expression patterns, suggesting the neoantigen expression should be fully considered in future immunotherapeutic interventions.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity

Objective:To investigate the effects of Pien Tze Huang(PZH) on the migration and invasion of HCC cells and underlying molecular mechanism.Methods:Cell counting kit-8(CCK-8) was applied to evaluate the cell viabilities of SMMC-7721,SK-Hep-1,C3A and HL-7702(6 × 10^(3)cells/well) co-incubated with different concentrations of PZH(0,0.2,0.4,0.6,0.8 mg/mL) for 24 h.Transwell,wound healing assay,CCK-8and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration,invasion,proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells(650 μg/mL for SK-Hep-1 cells and 330 μg/mL for SMMC-7721 cells),respectively.In vivo,lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH.SK-Hep-1 cells(10^(6)cells/200 μL per mice) were injected into B-NDG mice via tail vein.Totally 8 mice were randomly divided into PZH and control groups,4 mice in each group.After 2-d inoculation,mice in the PZH group were administered with PZH(250 mg/kg,daily) and mice in the control group received only vehicle(PBS) from the 2nd day after xenograft to day 17.Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH.Quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot were applied to verify RNA-seq results.Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein(YAP).Results:PZH treatment significantly inhibited the migration,invasion,proliferation and promoted the apoptosis of HCC cells in vitro and in vivo(P<0.01).Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH.Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta(PDGFRB),YAP,connective tissue growth factor(CCN2),N-cadherin,vimentin and matrix metallopeptidase 2(MMP2,P<0.01).Meanwhile,the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo.Furthermore,PZH played roles in inhibiting the transcriptional activity of YAP.Conclusion:PZH restrained migration,invasion and epithelialmesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

Tumor Microenvironment Cascade‑Responsive Nanodrug with Self‑Targeting Activation and ROS Regeneration for Synergistic Oxidation‑Chemotherapy

Carrier-free nanodrug with exceptionally high drug payload has attracted increasing attentions.Herein,we construct a pH/ROS cascade-responsive nanodrug which could achieve tumor acidity-triggered targeting activation followed by circularly amplified ROS-triggered drug release via positive-feedback loop.The di-selenide-bridged prodrug synthesized from vitamin E succinate and methotrexate(MTX)self-assembles into nanoparticles(VSeM);decorating acidity-cleavable PEG onto VSeM surface temporarily shields the targeting ability of MTX to evade immune clearance and consequently elongate circulation time.Upon reaching tumor sites,acidity-triggered detachment of PEG results in targeting recovery to enhance tumor cell uptake.Afterward,the VSeM could be dissociated in response to intracellular ROS to trigger VES/MTX release;then the released VES could produce extra ROS to accelerate the collapse of VSeM.Finally,the excessive ROS produced from VES could synergize with the released MTX to efficiently suppress tumor growth via orchestrated oxidation-chemotherapy.Our study provides a novel strategy to engineer cascade-responsive nanodrug for synergistic cancer treatment.

RBC Membrane Camouflaged Semiconducting Polymer Nanoparticles for Near-Infrared Photoacoustic Imaging and Photothermal Therapy

Semiconducting conjugated polymer nanoparticles(SPNs)represent an emerging class of phototheranostic materi-als with great promise for cancer treatment.In this report,low-bandgap electron donoracceptor(DA)-conjugated SPNs with sur-face cloaked by red blood cell membrane(RBCM)are developed for highly e ective photoacoustic imaging and photothermal therapy.The resulting RBCM-coated SPN(SPN@RBCM)displays remarkable near-infrared light absorption and good photosta-bility,as well as high photothermal conver-sion e ciency for photoacoustic imaging and photothermal therapy.Particularly,due to the small size(<5 nm),SPN@RBCM has the advantages of deep tumor penetration and rapid clearance from the body with no appreciable toxicity.The RBCM endows the SPNs with prolonged systematic circulation time,less reticuloendothelial system uptake and reduced immune-recognition,hence improving tumor accumulation after intravenous injection,which provides strong photoacoustic signals and exerts excellent photothermal therapeutic e ects.Thus,this work provides a valuable paradigm for safe and highly e cient tumor pho-toacoustic imaging and photothermal therapy for further clinical translation.

Red Blood Cell-Mimic Nanocatalyst Triggering Radical Storm to Augment Cancer Immunotherapy

Red blood cells(RBCs)have recently emerged as promosing candidates for cancer treatment in terms of relieving tumor hypoxia and inducing oxidative damage against cancer cells,but they are still far from satisfactory due to their limited oxygen transport and reactive oxygen species generation rate in tumor tissue.Herein,artificial RBCs(designated FTP@RBCM)with radical storm production ability were developed for oncotherapy through multidimensional reactivity pathways of Fe-protoporphyrin-based hybrid metal-organic frameworks(FTPs,as the core),including photodynamic/chemodynamic-like,catalase-like and glutathione peroxidase-like activities.Meanwhile,owing to the advantages of long circulation abilities of RBCs provided by their cell membranes(RBCMs),FTP with a surface coated with RBCMs(FTP@RBCM)could enormously accumulate at tumor site to achieve remarkably enhanced therapeutic efficiency.Intriguingly,this ROS-mediated dynamic therapy was demonstrated to induce acute local inflammation and high immunogenic cancer death,which evoked a systemic antitumor immune response when combined with the newly identified T cell immunoglobulin and mucin-containing molecule 3(Tim-3)checkpoint blockade,leading to not only effective elimination of primary tumors but also an abscopal effect of growth suppression of distant tumors.Therefore,such RBC-mimic nanocatalysts with multidimensional catalytic capacities might provide a promising new insight into synergistic cancer treatment.

A novel BODIPY-based nano-photosensitizer with aggregation-induced emission for cancer photodynamic therapy

The discovery of aggregation induced enmission(AIE)effect provides opportunities for the rapid development of fuorescence imaging-guided photodynamic therapy(PDT).In this work,a boron dipyrromethene(BODIPY)-based photosensitizer(ET-BDP-O)with AIE characteristics was developed,in which the two linear arms of BODIPY group were linked with triphenylamine to form an electron Donor-Acceptor-Donor(D-A-D)architecture while side chain was equipped with triethylene glycol group.ET-BDP-O was able to directly self-assemble into nanoparticles(NPs)without supplement of any other matrices or stabilizers due to its amphiphilic property.The as-prepared ET-BDP-O NPs had an excellent colloid stability with the size of 125 nm.Benefiting from the AIE property,ET-BDP-O NPs could generate strong fluorescence and reactive oxygen species under light-emitting diode light rradiation(60mW/cm^(2)).After inter-nalized in cancer cells,ET-BDP-O NPs were able to emit bright red fuorescence signal for bioimaging.In addition,the cell viability assay demonstrated that the ET-BDP-O NPs exhibited excellent photocytotoxicity against cancer cells,while negligible cytotoicity under dark envi-ronment.Thus,ET-BDP-O NPs might be regarded as a promising photosensitizer for fluores-cence imaging-guided PDT in future.

Genetic variants in promoter region of TFR2 is associated with the risk of non-alcoholic fatty liver disease in a Chinese Han population:a case–control study

Background Iron overload is frequently observed in non-alcoholic fatty liver disease(NAFLD).Transferrin receptor 2(TFR2)is an important key factor in iron regulation.We aimed to investigate whether TFR2 single nucleotide polymorphisms(SNPs)contribute to susceptibility to NAFLD in a Chinese Han population.Methods Five tag SNPs(rs10247962,rs4434553,rs2075672,rs1052897,and rs3757859)in the TFR2 gene were selected and genotyped in a case–control study on participants who visited two affiliated hospitals of Fujian Medical University between June 2011 and August 2017.Propensity score matching and inverse probability of treatment weighting analyses were used to verify the risk associated with TFR2 SNPs.Results Logistic regression analyses suggested that subjects with the rs4434553 GA or GG genotype had a lower risk of NAFLD than those carrying the AA genotype(odds ratio=0.630,95%confidence interval=0.504–0.788).Moreover,the rs4434553 GA or GG genotype was negatively correlated with body mass index,hepatic steatosis index,and serum ferritin(b=-0.363,P=0.008;b=-1.040,P=0.009;b=-35.258,P=0.015,respectively),and positively associated with serum hepcidin level(b=35.308,P<0.001).Moreover,rs10247962 and rs1052897 had multiplicative interactions with age in relation to the risk of NAFLD(P for interactions,0.041 and 0.034,respectively).The cumulative effects of the rs10247962,rs1052897,and rs4434553 SNPs were positively associated with the risk of NAFLD(adjusted P_(trend)=0.012).Conclusions In this Chinese Han population,the rs4434553 polymorphism in TFR2 may be an independent influencing factor associated with the susceptibility to NAFLD.The ageing effect on the development of NAFLD may be inhibited by SNPs rs10247962 and rs1052897.

HIF-1αand HDAC1 mediated regulation of FAM99AmiR92a signaling contributes to hypoxia induced HCC metastasis

Dear Editor,Hypoxic microenvironment is clinically associated with metastasis and poor prognosis of numerous cancers.Previous studies have shown that many protein-coding genes and microRNAs are regulated upon hypoxia and involved in the progression of cancer.However,the roles of lncRNAs in the hypoxia-responsive gene networks and how lncRNA-related signaling network regulates hypoxia induced tumor metastasis is still not clear.

Quantitative Secretome Analysis Reveals Clinical Values of Carbonic Anhydrase Ⅱ in Hepatocellular Carcinoma

Early detection and intervention are key strategies to reduce mortality,increase longterm survival,and improve the therapeutic effects of hepatocellular carcinoma(HCC)patients.Herein,the isobaric tag for relative and absolute quantitation(iTRAQ)-based quantitative proteomic strategy was used to study the secretomes in conditioned media from HCC cancerous tissues,surrounding noncancerous tissues,and distal noncancerous tissues to identify diagnostic and prognostic biomarkers for HCC.In total,22 and 49 dysregulated secretory proteins were identified in the cancerous and surrounding noncancerous tissues,respectively,compared with the distal noncancerous tissues.Among these proteins,carbonic anhydrase II(CA2)was identified to be significantly upregulated in the secretome of cancerous tissues;correspondingly,the serum concentrations of CA2 were remarkably increased in HCC patients compared with that in normal populations.Interestingly,a significant increase of serum CA2 in recurrent HCC patients after radical resection was also confirmed compared with HCC patients without recurrence,and the serum level of CA2 could act as an independent prognostic factor for time to recurrence and overall survival.Regarding the mechanism,the secreted CA2 enhances the migration and invasion of HCC cells by activating the epithelial mesenchymal transition pathway.Taken together,this study identified a novel biomarker for HCC diagnosis and prognosis,and provided a valuable resource of HCC secretome for investigating serological biomarkers.

Scattering Inversion Study for Suspended Label-Free Lymphocytes with Complex Fine Structures

Objective and Impact Statement.Distinguishing malignant lymphocytes from normal ones is vital in pathological examination.We proposed an inverse light scattering(ILS)method for label-free suspended lymphocytes with complex fine structures to identify their volumes for pathological state.Introduction.Light scattering as cell’s“fingerprint”provides valuable morphology information closely related to its biophysical states.However,the detail relationships between the morphology with complex fine structures and its scattering characters are not fully understood.Methods.To quantitatively inverse the volumes of membrane and nucleus as the main scatterers,clinical lymphocyte morphologies were modeled combining the Gaussian random sphere geometry algorithm by 750 reconstructed results after confocal scanning,which allowed the accurate simulation to solve ILS problem.For complex fine structures,the specificity for ILS study was firstly discussed(to our knowledge)considering the differences of not only surface roughness,posture,but also the ratio of nucleus to the cytoplasm and refractive index.Results.The volumes of membrane and nucleus were proved theoretically to have good linear relationship with the effective area and entropy of forward scattering images.Their specificity deviations were less than 3.5%.Then,our experimental results for microsphere and clinical leukocytes showed the Pearson product-moment correlation coefficients(PPMCC)of this linear relationship were up to 0.9830~0.9926.Conclusion.Our scattering inversion method could be effectively applied to identify suspended label-free lymphocytes without destructive sample pretreatments and complex experimental systems.

2D graphene oxide-L-arginine-soybean lecithin nanogenerator for synergistic photothermal and NO gas therapy

Nitric oxide(NO) gas therapy has been regarded as a promising strategy for cancer treatment. However,its therapeutic efficiency is still unsatisfying due to the limitations of monotherapy. Previous preclinical and clinical studies have shown that combination therapy could significantly enhance therapeutic efficiency. Herein, a graphene oxide(GO)-L-arginine(L-Arg, a natural NO donor) hybrid nanogenerator is developed followed by surface functionalization of soybean lecithin(SL) for synergistic enhancement of cancer treatment through photothermal and gas therapy. The resultant GO-Arg-SL nanogenerator not only exhibited good biocompatibility and excellent endocytosis ability, but also exhibited excellent photothermal conversion capability and high sensitivity to release NO within tumor microenvironment via inducible NO synthase(i NOS) catalyzation. Moreover, the produced hyperthermia and intracellular NO could synergistically kill cancer cells both in vitro and in vivo. More importantly, this nanogenerator can efficiently eliminate tumor while inhibiting the tumor recurrence because of the immunogenic cell death(ICD) elicited by NIR laser-triggered hyperthermia and the immune response activation by massive NO generation. We envision that the GO-Arg-SL nanogenerator could provide a potential strategy for synergistic photothermal and gas therapy.