Consensus statement on extracellular vesicles in liquid biopsy for advancing laboratory medicine.

Extracellular vesicles (EVs) represent a diverse class of nanoscale membrane vesicles actively released by cells. These EVs can be further subdivided into categories like exosomes and microvesicles, based on their origins, sizes, and physical attributes. Significantly, disease-derived EVs have been detected in virtually all types of body fluids, providing a comprehensive molecular profile of their cellular origins. As a result, EVs are emerging as a valuable addition to liquid biopsy techniques. In this collective statement, the authors share their current perspectives on EV-related research and product development, with a shared commitment to translating this newfound knowledge into clinical applications for cancer and other diseases, particularly as disease biomarkers. The consensus within this document revolves around the overarching recognition of the merits, unresolved questions, and existing challenges surrounding EVs. This consensus manuscript is a collaborative effort led by the Committee of Exosomes, Society of Tumor Markers, Chinese anti-Cancer Association, aimed at expediting the cultivation of robust scientific and clinically applicable breakthroughs and propelling the field forward with greater swiftness and efficacy.

A pan-cancer analysis revealing the role of LFNG, MFNG and RFNG in tumor prognosis and microenvironment.

BACKGROUND: Fringe is a glycosyltransferase involved in tumor occurrence and metastasis. However, a comprehensive analysis of the Fringe family members lunatic fringe (LFNG), manic fringe (MFNG), radical fringe (RFNG) in human cancers is lacking. METHODS: In this study, we performed a pan-cancer analysis of Fringe family members in 33 cancer types with transcriptomic, genomic, methylation data from The Cancer Genome Atlas (TCGA) project. The correlation between Fringe family member expression and patient overall survival, copy number variation, methylation, Gene Ontology enrichment, and tumor-infiltrating lymphocytes (TILs) was investigated by using multiple databases, such as cBioPortal, Human Protein Atlas, GeneCards, STRING, MSigDB, TISIDB, and TIMER2. In vitro experiments and immunohistochemical assays were performed to validate our findings. RESULTS: High expression levels of LFNG, MFNG, RFNG were closely associated with poor overall survival in multiple cancers, particularly in pancreatic adenocarcinoma (PAAD), uveal melanoma (UVM), and brain lower-grade glioma (LGG). Copy number variation analysis revealed that diploid and gain mutations of LFNG was significantly increased in PAAD and stomach adenocarcinoma (STAD), and significantly associated with the methylation levels in promoter regions. Significant differential genes between high and low expression groups of these Fringe family members were found to be consistently enriched in immune response and T cell activation pathway, extracellular matrix adhesion pathway, RNA splicing and ion transport pathways. Correlation between the abundance of tumor-infiltrating lymphocytes (TILs) and LFNG, MFNG, and RFNG expression showed that high LFNG expression was associated with lower TIL levels, particularly in PAAD. In vitro experiment by using pancreatic cancer PANC1 cells showed that LFNG overexpression promoted cell proliferation and invasion. Immunohistochemical assay in 90 PAAD patients verified the expression level of LFNG and its relationship with the prognosis. CONCLUSIONS: Our study provides a relatively comprehensive understanding of the expression, mutation, copy number, promoter methylation level changes along with prognosis values of LFNG, MFNG, and RFNG in different tumors. High LFNG expression may serve as a poor prognosis molecular marker for PAAD.

Association of Tumor Cell Metabolic Subtype and Immune Response With the Clinical Course of Hepatocellular Carcinoma.

AIM: Tumor metabolism plays an important role in tumorigenesis and tumor progression. This study evaluated the potential association of tumor cell metabolism and immune cell tumor infiltration with the clinical course of hepatocellular carcinoma (HCC). METHODS: Gene-wise normalization and principal component analysis were performed to evaluate the metabolic system. A tumor microenvironment score system of tumor immune cell infiltration was constructed to evaluate its association with metabolic subtypes. Finally, we analyzed the impact of metabolism and immune cell infiltration on the clinical course of HCC. RESULTS: A total of 673 HCC patients were categorized into cholesterogenic (25.3%), glycolytic (14.6%), mixed (10.4%), and quiescent (49.8%) types based on glycolysis and cholesterol biosynthesis gene expression. The subgroups including the glycolytic genotyping expression (glycolytic and mixed types) showed a higher mortality rate. The glycolytic, cholesterogenic, and mixed types were positively correlated with M0 macrophage, resting mast cell, and naïve B-cell infiltration (P = .013, P = .019, and P = .006, respectively). In TCGA database, high CD8+ T cell and low M0 macrophage infiltration were associated with prolonged overall survival (OS, P = .0017 and P < .0001, respectively). Furthermore, in glycolytic and mixed types, patients with high M0 macrophage infiltration had a shorter OS (P = .03 and P = .013, respectively), and in quiescent type, patients with low naïve B-cell infiltration had a longer OS (P = .007). CONCLUSIONS: Tumor metabolism plays a prognostic role and correlates with immune cell infiltration in HCC. M0 macrophage and CD8+ T cell appear to be promising prognostic biomarker for HCC. Finally, M0 macrophages may represent a useful immunotherapeutic target in patients with HCC.

NFL-TBS.40-63 Peptide Gold Complex Nanovector: A Novel Therapeutic Approach to Increase Anticancer Activity by Breakdown of Microtubules in Pancreatic Adenocarcinoma (PDAC).

The role of the NFL-TBS.40-63 peptide is to destroy the microtubule network of target glioma cancer cells. Recently, we have conceived a gold-complex biotinylated NFL-TBS.40-63 (BIOT-NFL) to form a hybrid gold nanovector (BIOT-NFL-PEG-AuNPs). This methodology showed, for the first time, the ability of the BIOT-NFL-PEG-AuNPs to target the destruction of pancreatic cancer cells (PDAC) under experimental conditions, as well as detoxification and preclinical therapeutic efficacy regulated by the steric and chemical configuration of the peptide. For this aim, a mouse transplantation tumor model induced by MIA-PACA-2 cells was applied to estimate the therapeutic efficacy of BIOT-NFL-PEG-AuNPs as a nanoformulation. Our relevant results display that BIOT-NFL-PEG-AuNPs slowed the tumor growth and decreased the tumor index without effects on the body weight of mice with an excellent antiangiogenic effect, mediated by the ability of BIOT-NFL-PEG-AuNPs to alter the metabolic profiles of these MIA-PACA-2 cells. The cytokine levels were detected to evaluate the behavior of serum inflammatory factors and the power of BIOT-NFL-PEG-AuNPs to boost the immune system.

Correction: CTL-doxorubicin (DOX)-gold complex nanoparticles (DOX-AuGCs): from synthesis to enhancement of therapeutic effect on liver cancer model.

[This corrects the article DOI: 10.1039/D0NA00758G.].

Neoadjuvant Therapy for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is characterized by low resection and high postoperative recurrence rates, and conventional treatment strategies have failed to meet clinical needs. Neoadjuvant therapy (NAT) is widely employed in the routine management of several solid tumors because it increases resectability and reduces the rate of postoperative recurrence. However, a consensus has not been reached regarding the effects of NAT on HCC. As systemic therapy, particularly targeted therapy and immunotherapy, is given for HCC treatment, accumulating evidence shows that the "spring" of NAT for HCC is imminent. In the future, HCC researchers should focus on identifying biomarkers for treatment response, explore the mechanisms of resistance, and standardize the endpoints of NAT.

DOTAREM (DOTA)-Gold-Nanoparticles: Design, Spectroscopic Evaluation to Build Hybrid Contrast Agents to Applications in Nanomedecine.

Introduction: The realization of MRI contrast agents through chemical protocols of functionalization is a strong domain of research. In this work, we developed and formulated a novel hybrid gold nanoparticle system in which a gold salt (HAuCl4) is combined with dotarem, an MRI contrast agent (DOTA) by chelation (Method IN) and stabilized by a lactose-modified chitosan polymer (CTL; Chitlac) to form DOTA IN-CTL AuNPs. Result and Discussion: The authors demonstrate the biological efficiency of these nanoparticles in the case of three cell lines: Mia PaCa-2 (human pancreatic cancer cell line), TIB-75 (murine liver cell line) and KKU-M213 (cholangiocarcinoma cell line). DOTA IN-CTL AuNPs are stable under physiological conditions, are nontoxic, and are very efficient as PTT agents. The highlights, such as high stability and preliminary MRI in vitro and in vivo models, may be suitable for diagnosis and therapy. Conclusion: We proved that DOTA IN-CTL AuNPs have several advantages: i) Biological efficacy on three cell lines: MIA PaCa-2 (human pancreatic cancer cell line), TIB-75 (murine liver cell line) and KKU-M213 (cholangiocarcinoma cell line); ii) high stability, and no-toxicity; iii) high efficiency as a PPT agent. The study conducted on MRI in vitro and in vivo models will be suitable for diagnosis and therapy.

Autophagy-associated circRNA circATG7 facilitates autophagy and promotes pancreatic cancer progression.

Dysregulation of autophagy and circular RNAs (circRNAs) are involved in the pancreatic cancer (PC) progression. However, the regulatory network between circRNAs, autophagy, and PC progression remains unknown. Herein, we demonstrated that autophagy-associated circRNA circ-autophagy related 7 (circATG7) was elevated in PC tissues compared to adjacent tissues, and in PC cells treated with EBSS and hypoxia. circATG7 expression was positively associated with tumor diameter and lymph node invasion in patients with PC. circATG7 overexpression promoted PC cell proliferation, mobility, and autophagy in vitro, while circATG7 knockdown induced the opposite effects. ATG7 inhibition attenuated the effects of circATG7 on the biological functions of PC cells. CircATG7 is located in the cell cytoplasm and nucleus. Cytoplasmic circATG7 sponged miR-766-5p and decreased its expression, and increased the expression of ATG7, a target gene of miR-766-5p. Nuclear circATG7 acted as a scaffold to increase the interaction between the human antigen R protein and ATG7 mRNA and enhanced ATG mRNA stability. Furthermore, we demonstrated that circATG7 regulates PC cell proliferation and metastasis in vivo via ATG7-dependent autophagy. In conclusion, our results demonstrated that circATG7 accelerates PC progression via miR-766-5p/ATG7 and that HUR/ATG7 depends on autophagic flux. Thus, circATG7 may be a potential therapeutic target for PC.

Establishment of a 4-miRNA Prognostic Model for Risk Stratification of Patients With Pancreatic Adenocarcinoma.

Pancreatic adenocarcinomas (PAADs) often remain undiagnosed until later stages, limiting treatment options and leading to poor survival. The lack of robust biomarkers complicates PAAD prognosis, and patient risk stratification remains a major challenge. To address this issue, we established a panel constructed by four miRNAs (miR-4444-2, miR-934, miR-1301 and miR-3655) based on The Cancer Genome Atlas (TCGA) and Human Cancer Metastasis Database (HCMDB) to predicted the prognosis of PAAD patients. Then, a risk prediction model of these four miRNAs was constructed by using Cox regression analysis with the least absolute shrinkage and selection operator (LASSO) regression analysis. This model stratified TCGA PAAD cohort into the low-risk and high-risk groups based on the panel-based risk score, which was significantly associated with 1-, 2-, 3-year OS (AUC=0.836, AUC=0.844, AUC=0.952, respectively). The nomogram was then established with a robust performance signature for predicting prognosis compared to clinical characteristics of pancreatic cancer (PC) patients, including age, gender and clinical stage. Moreover, two GSE data were validated the expressions of 4 miRNAs with prognosis/survival outcome in PC. In the external clinical sample validation, the high-risk group with the upregulated expressions of miR-934/miR-4444-2 and downregulated expressions of miR-1301/miR-3655 were indicated a poor prognosis. Furthermore, the cell counting kit-8 (CCK-8) assay, clone formation, transwell and wound healing assay also confirmed the promoting effect of miR-934/miR-4444-2 and the inhibiting effect of miR-1301/miR-3655 in PC cell proliferation and migration. Taken together, we identified a new 4-miRNA risk stratification model could be used in predicting prognosis in PAAD.

Flavin Adenine Dinucleotide (FAD) Pegylated (PEG)-Complexes: Proof of Concept (PoC) of theranostic tool on a Murine Breast Cancer Model.

Flavin adenine dinucleotide (FAD) plays a key role in an extensive range of cellular oxidation-reduction reactions, which is engaged in metabolic pathways. The purpose of this study was to realize pegylated flavins formulation, named FAD and FAD-PEG diacid complex as theranostic tool in cancer therapy. For this objective, a murine breast cancer model, which was induced by mouse-derived4T1 breast cancer cells was studied to assess the therapeutic efficacy of FAD (named NP1) and FAD-PEG diacid complex (named NP2). The cytokines were monitored to evaluate the serum inflammatory factors to develop the blood cell content of different groups of nude mice. The experimental model shows that an intravenous injection of FAD (NP1) can significantly reduce tumour volume, tumour index and thymus index, and decrease neutrophils (NE), monocytes (MO), eosinophils (EO), and basophils (BA). At the same time, the content of IL-1α, IL-12P70, TNF α, IL-1ß and IL-6 was significantly reduced, and the content of IL-10 was significantly increased. These results provide the proof-of-concept for FAD as a smart adjuvant for cancer therapy and encourages their further development in the field of Nanomedicine.

A Pegylated Flavin Adenine Dinucleotide PEG Complex to Boost Immunogenic and Therapeutic Effects in a Liver Cancer Model.

Flavin adenine dinucleotide (FAD) is engaged in several metabolic diseases. Its main role is being a cofactor essential for the activity of many flavoproteins, which play a crucial role in electron transport pathways in living systems. The aim of this study was to apply a pegylated flavins formulation named FAD-PEG diacide complex as theranostic pathway in cancer therapy. For this purpose, a mouse liver cancer model induced by Hepa1-6 cells was used to evaluate the therapeutic efficacy of FAD (named NP1) and FAD-PEG diacide complex (named NP2). The cytokines were applied to screen the serum inflammatory factors, to establish the blood cell content of different groups of nude mice. The highlights follows that FAD formulations (NP1; NP2) significantly suppressed the tumor growth and reduced the tumor index without effects on the body weight of mice. Furthermore, NP2 significantly reduced the serum levels of cytokines IL-6, TNF-α and IL-12 (P70). The reported results provide the proof-of-concept for the synthesis of a smart adjuvant for liver cancer therapy and support their further development in the field of nanomedicine.

Tunable formaldehyde sensing properties of palladium cluster decorated graphene.

The ability to tune the adsorption strength of the targeted gas on sensing materials is crucial for sensing applications. By employing first-principles calculations the adsorption and sensing properties of HCHO on small Pd n (n = 1-6) cluster decorated graphene have been systematically investigated. The adsorption energy is found to depend on the size of the Pd n cluster and can be tuned in a wide range from -0.68 eV on Pd(111) to -1.98 eV on the Pd3/graphene system. We also find that the Pd n /graphene (n = 5 and 6) systems have an appropriate adsorption energy for HCHO gas sensing. The current-voltage curves are calculated by the non-equilibrium Green's function method for the two-probe nano-sensor devices along both the armchair and zigzag directions. The devices constructed with Pd n /graphene (n = 5 and 6), having the highest absolute response over 20% at small voltages, should be applicable for HCHO detection. This work provides a theoretical basis for exploring potential applications of metal cluster decorated graphene for gas sensing.

miR-375 Inhibits the Proliferation, Migration and Invasion of Esophageal Squamous Cell Carcinoma by Targeting XPR1.

OBJECTIVE: The purpose of this study was to explore the mechanism of the miR-375/XPR1 axis in esophageal squamous cell carcinoma (ESCC) and provide a new idea for targeted therapy of ESCC. METHODS: Differentially expressed genes in GEO and TCGA databases were analyzed by bioinformatics. The expression levels of miR-375 and XPR1 mRNA were detected by qRT-PCR. Protein expression of XPR1 was detected by western blot. Bioinformatics analysis and dual luciferase assay were conducted to confirm the target relationship between miR-375 and XPR1. The viability, proliferation, migration and invasion of cells in each treatment group were detected by CCK-8, colony formation, wound healing and Transwell assays. RESULTS: Significantly down-regulated miR-375 and remarkably up-regulated XPR1 were observed in ESCC tissue and cells. Overexpression of miR-375 inhibited proliferation, invasion and migration of ESCC cells, and greatly reduced the promoting effect of XPR1 overexpression on cell proliferation, migration and invasion. Dual luciferase assay confirmed that miR-375 targeted and inhibited XPR1 expression in ESCC. CONCLUSION: These results demonstrate the regulatory role of the miR-375/XPR1 axis in ESCC cells and provide a new potential target for the precise treatment of patients with ESCC.

miR-876 Inhibits EMT and Liver Fibrosis via POSTN to Suppress Metastasis in Hepatocellular Carcinoma.

BACKGROUND: The asymptomatic onset, frequent recurrence, and poor prognosis of hepatocellular carcinoma (HCC) prompted us to identify new therapeutic targets or predictive markers of HCC diagnosis or prognosis. METHODS: In this study, bioinformatics analysis was used to screen for target miRNAs from the open-access TCGA database. Transwell assays, Western blotting, and qRT-PCR analyses were used to detect cellular functions and gene expression in HCC cells and samples. A nude mouse tumorigenesis model was established to facilitate the observation of HCC progression. Other assays included luciferase reporter assays, IHC, and survival analysis. RESULTS: We found that the identified miR-876 from TCGA was expressed at low levels in HCC cell lines and that low miR-876 expression was corrected with liver cirrhosis, tumor thrombus, and TNM stage. Further research revealed that miR-876 regulated cell invasion, EMT, and collagen expression by targeting POSTN expression. miR-876 and POSTN were inversely correlated in HCC samples and associated with EMT status and liver fibrosis in clinical HCC tissues. miR-876 inhibited the liver cancer progression in in vivo animal assays. Finally, both miR-876 and POSTN were risk factors for HCC survival, and HCC patients with combined low miR-876 and high POSTN expression had worse prognosis. CONCLUSIONS: miR-876 inhibited HCC EMT and fibrosis by targeting POSTN, thus affecting HCC progression and prognosis. miR-876 and POSTN may be useful therapeutic targets or prognostic markers of HCC.

Administration of glycyrrhetinic acid reinforces therapeutic effects of mesenchymal stem cell-derived exosome against acute liver ischemia-reperfusion injury.

Recent studies have shown that mesenchymal stem cell-derived exosome could attenuate ischaemia-reperfusion (I/R) injury by suppressing inflammatory response in the liver. Glycyrrhetinic acid was also shown to be capable of repressing the TLR4 signalling pathway. However, it remains to be explored as whether the combined administration of mesenchyma stem cell (MSC)-derived exosome and glycyrrhetinic acid (GA) could increase their therapeutic effects on I/R injury. Western blot was performed to evaluate the expression of proteins associated with inflammatory response in THP-1 cells and I/R rat models treated under different conditions. Flow cytometry was carried out to analyse the proportions of different subtypes of peripheral blood cells in I/R rats. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess the liver injury in I/R rats. Combined treatment with MSC-derived exosome and GA effectively maintained the expression of key proteins involved in inflammatory response in LPS stimulated THP-1 cells and THP-1 cells treated under hypoxia conditions. In the established of I/R rat models, GA administration reinforced the therapeutic efficiency of MSC-derived exosomes by maintaining the proportion of different subgroups of peripheral blood cells, decreasing the concentration of ALT and AST, and restoring the expression of dysregulated proteins associated with inflammation. Our results demonstrated that treatment with exosomes derived from mesenchymal stem cells (MSCs) attenuated liver I/R injury, while the pre-treatment with GA may further promote the therapeutic effect of mesenchymal stem cell-derived exosome against acute liver ischaemia-reperfusion injury.

A practical new strategy to prevent bile duct injury during laparoscopic cholecystectomy. A single-center experience with 5539 cases.

Purpose Bile duct injury (BDI) is a catastrophic complication of cholecystectomy, and misidentification of the cystic anatomy is considered to be the main cause. Although several techniques have been developed to prevent BDI, such as the "critical view of safety", the infundibular technique, the rates remain higher during laparoscopic cholecystectomy (LC) than during open surgery. We, here, propose a practical new strategy for ductal identification, that can help to prevent laparoscopic bile duct injury. Methods A retrospective study of 5539 patients who underwent LC from March 2007 to February 2019 at a single institution was conducted. The gallbladder infundibulum was classified by its position located on an imaginary clock with the gallbladder neck as the center point of the dial, 3-o'clock position as cranial, 6-o'clock as dorsal, 9-o'clock as caudal, and 12-o'clock as ventral, as well as the axial position. Patient demographics, pathologic variables and infundibulum classification were evaluated. Detailed analysis of ductal identification based on gallbladder infundibulum position was performed in this study. All infundibulum positions were recorded by intraoperative laparoscopic video or photographic images. Results All the patients successfully underwent LC during the study period. No conversion or serious complications such as biliary injury occurred. Gallbladders with infundibulum of 3-o'clock position, 6-o'clock position, 9-o'clock position, 12-o'clock position, axial position were 12.3%, 23.4%, 28.0%, 4.2%, and 32.1%, respectively. The 3-o'clock and 12-o'clock position were pitfalls that might cause biliary injury. Conclusion The gallbladder infundibulum as a navigator is useful for ductal identification to reduce BDI and improve the safety of LC.

Aptamer-Gold(III) Complex Nanoparticles: A New Way to Detect Cu, Zn SOD Glycoprotein.

Aptamers are small biomolecules composed of 20-100 nucleotides that recognize target molecules in three-dimensional structures. These natural targeting molecules have attracted interest in the biomedical field as biomarkers for cancer diagnostics. In this study, we investigated the interaction of a characteristic aptamer with its target protein, Cu, Zn superoxide dismutase (SOD 4), on a gold nanoparticle (AuNP) surface under experimental conditions. For this purpose, we applied two protocols to coat SOD 4 aptamer (APT) on the nanoparticle surface: carbodiimide chemistry (EDC/NHS) (Method ON) and a complexation methodology (Method IN). The nano-aptamer's interactions with SOD 4 were detected by UV-vis absorption and Raman spectroscopy in a range of protein concentrations (from 1 µM to 50 nM). We believe that the interaction is heavily dependent on the nature of the biomarker (SOD 4) and also on the steric arrangement of the aptamer on the gold nanoparticle surface. The lowest detectable concentration (limit of detection, LOD) was about 2 nM for APT IN PEG-AuNPs and 8 nM for APT ON PEG-AuNPs. For the first time, we demonstrated a very sensitive detection of SOD 4 in the nanomolar concentration range with new ways of biosensor synthesis (APT IN and ON), providing a very strong tool to understand the effect of aptamer conformation to detect SOD 4.

Oncogenic hsa_circ_0091581 promotes the malignancy of HCC cell through blocking miR-526b from degrading c-MYC mRNA.

Circular RNAs (circRNAs) are new types of endogenous non-coding RNAs, which are identified to have critical regulatory roles in cancer biology. In this study, we aimed to find the abnormally expressed circRNAs in hepatocellular carcinoma (HCC) and investigate the function and mechanism of circRNAs in HCC progression. Upregulation of hsa_circ_0091581 was identified by RNA-sequencing and validated by quantitative real-time polymerase chain reaction (qRT-PCR). Hsa_circ_0091581 expression was correlated with tumor size, disease-free survial and overall survival of HCC patients. Functionally, hsa_circ_0091581 could promote the proliferation of HCC cells in vitro. Mechanism research showed that hsa_circ_0091581 promoted cell proliferation via hsa_circ_0091581/miR-526b/c-Myc axis in HCC cells. Also, the expression of hsa_circ_0091581 in HCC could be regulated by c-Myc. These results revealed that hsa_circ_0091581/miR-526b/c-Myc/hsa_circ_0091581 positive feedback loop plays a vital role in HCC progression and hsa_circ_0091581 may be a potential prognostic biomarker and therapeutic target for HCC.