Tumor-derived extracellular vesicles (tEVs), which are essential mediators for cell-to-cell communication during tumorigenesis and tumor development, have demonstrated significant diagnostic potential in cancer liquid biopsy, particularly through biomarkers like membrane proteins and inner microRNAs. However, traditional detection methods such as ELISA and qRT-PCR encounter challenges with low sensitivity and specificity, complex procedures, and high costs. Although emerging biosensors have been developed, these methods are limited to detecting a single type of tEV biomarker, which may result in misdiagnoses due to false-positive or false-negative signals. Herein, we introduce a specific and serum-stable membrane-fusion approach (SSMFA) capable of simultaneously detecting tEV proteins and microRNAs via dual-color fluorescence analysis. In this strategy, the established epithelial cell adhesion molecule (EpCAM) aptamer-modified serum-stable membrane-fusion liposome (AptSMFL) is labeled with fluorescence resonance energy transfer (FRET) dye pairs, which can specifically recognize EpCAM-overexpressed tEVs and induce serum-stable membrane fusion, allowing the quantification of EpCAM protein levels through red fluorescence changes resulting from FRET alterations. Meanwhile, SSMFA facilitates efficient transfection of the CRISPR/Cas13a probe into tEVs to analyze the levels of microRNA-21 (miR-21) in EpCAM-positive tEVs via green fluorescence detection. When tested on serum samples from hepatocellular carcinoma models, the SSMFA exhibited minimal sample volume requirement and rapid assay time (2 h) to effectively achieve accurate quantification of both tEV EpCAM protein and miR-21 levels. Additionally, this dual-biomarker detection method showed a strong correlation with tumor burden and significantly improved cancer diagnostic accuracy (AUC = 0.98), underscoring the potential of SSMFA as a promising tEV-based liquid biopsy assay for cancer diagnosis.

Cells are diverse systems with unique molecular profiles that support vital functions, such as energy production and nutrient absorption. Advances in omics have provided valuable insights into these cellular processes, but many of these tools rely on cell lysis, limiting the ability to track dynamic changes over time. To overcome this, methods for longitudinal profiling of living cells have emerged; however, challenges such as low throughput and genetic manipulation still need to be addressed. Nanomaterials, particularly nanowires, offer a promising solution due to their size, high aspect ratios, low cost, simplicity, and potential for high-throughput manufacturing. Here, we present a nanowire-based platform for longitudinal mRNA profiling in living cells using vertically aligned nickel nanowire arrays for efficient mRNA extraction with minimal cellular disruption. We demonstrate its ability to track enhanced green fluorescent protein expression and transcriptomic changes from drug responses in the same cells over time, showcasing the platform's potential for dynamic cellular analysis.

We present a novel acoustofluidic chromatography platform for high-throughput nanoparticle trapping and enrichment, with a focus on extracellular vesicles (EVs) from blood plasma. The system features a packed bed of polystyrene beads inside a rectangular glass capillary, acoustically actuated by a piezoelectric element. Using fluorescent polystyrene nanoparticles as small as 25 nm, we characterized device performance across a frequency range of 0.45-4 MHz, demonstrating particle trapping at all tested frequencies. The platform achieved recoveries of up to 42.9 ± 3.2% at input powers as low as 55 mW and operated at high flow rates of up to 200 μL/min. Trapping capacity reached 6.7 × 109 ± 2.5 × 109 particles for 25 nm polystyrene beads. For EV isolation, processing just 4 μL of blood plasma yielded 2 × 108 washed EV-sized particles eluted in 100 μL within 8 min. Micro BCA analysis confirmed a plasma protein background below 2 μg/mL, enabling downstream mass spectrometry. This platform provides an efficient, high-throughput approach for nanoparticle trapping and EV enrichment with minimal sample volumes, offering potential applications in diagnostics and therapeutic development. Future work will focus on optimizing bead properties for EV subpopulation separation and scaling the system for clinical applications.

SERPINI1 is a protein-coding gene, which has been reported to be related to malignancies, and the encoding protein is a secreted protein. Nevertheless, the specific effect of SERPINI1 on Hepatocellular carcinoma (HCC) remains unclear.

The expression level of SERPINI1 in cancers was detected by the Gene Expression Omnibus (GEO) database, the Gene Expression Profiling Interactive Analysis (GEPIA) database and the collected serum of HCC patients. The receiver operating characteristic (ROC) curve and area under curve (AUC) were used to evaluate the diagnostic effectiveness of serum SERPINI1 and the combination of AFP and SERPINI1 for HCC. The Kaplan-Meier (KM) survival was used to evaluate the prognostic capacity of SERPINI1 for HCC in GEPIA database. Furthermore, the correlations between clinicopathological characteristics and the level of serum SERPINI1 were analyzed. Besides, we detected the expression of SERPINI1 in HepG2 by qPCR and western blot, and confirmed the biological function of SERPINI1 through MTT, EdU, wound healing and transwell invasion assay.

The results indicated that the level of SERPINI1 was significantly increased in tissue and serum of HCC patients. ROC analysis displayed that SERPINI1 had a significantly diagnostic value for HCC, the combination of AFP and SERPINI1 gained the higher specificity and sensitivity. The KM survival curves indicated that patients with SERPINI1 overexpression had worse overall survival. Furthermore, we found the positive correlations between serum SERPINI1 level and some clinicopathological characteristics, such as tumor size, differentiation degrees and so on. In addition, in vitro experiments revealed that SERPINI1 could promote the proliferation and invasion of HCC.

Taken together, our study demonstrates that SERPINI1, which is highly expressed in HCC and closely related to cell proliferation and invasion, may serve as a novel biomarker for diagnosis and prognosis of HCC.

Hepatocellular carcinoma is a leading and increasing contributor to cancer-related death worldwide. Recent advancements in both liver-directed therapies in the form of yttrium-90 (90Y) radioembolization (RE) and systemic therapy in the form of immune checkpoint inhibitors (ICI) have expanded treatment options for patients with an otherwise poor prognosis. Despite these gains, ICIs and 90Y-RE each have key limitations with low objective response rates and persistent hazard of out-of-field recurrence, respectively, and overall survival remains low. However, each therapy's strength may mitigate the other's weakness, making them potentially ideal partners for combination treatment strategies. This review discusses the scientific and clinical rationale for combining 90Y-RE with ICIs, highlights early clinical trial data on its safety and effectiveness, and proposes key issues to be addressed in this emerging field. With optimal strategies, combination therapies can potentially result in increasing likelihood of durable and curative outcomes in later stage patients.

B7-H3 is a promising target for cancer therapy, notably in prostate cancer (PCa), particularly in metastatic, castration-resistant PCa (mCRPC). With the development of B7-H3-targeted therapies, there is a need for a rapid, reliable, and cost-effective method to detect and monitor B7-H3 expression. Leveraging their abundance and stability, we developed a liquid biopsy assay using extracellular vesicles (EVs) for this purpose.

B7-H3+ EVs were isolated using a B7-H3 antibody-mediated, click chemistry-based enrichment method. Antibodies were conjugated to methyltetrazine-grafted microbeads. EVs were isolated from 100 µL of plasma from metastatic, castration-sensitive PCa (mCSPC) (n = 43) and mCRPC (n = 103) patients and quantified using RT-qPCR of ACTB. Measurements were compared with the patient's disease status over time.

The assay detected higher B7-H3+ EVs in mCRPC than mCSPC and increased when mCSPC transitioned to mCRPC. Elevated B7-H3+ EVs were associated with lower overall survival (Hazard ratio (HR) 2.19, p = 0.01). In patients with serial plasma samples, B7-H3+ EV levels reflected treatment response and disease progression.

This B7-H3+ EV assay represents a significant advancement in utilizing tumor-derived EVs for a non-invasive, quantitative, and consistent real-time measurement of B7-H3. This assay warrants further development as a companion diagnostic for B7-H3 targeted therapies in PCa and other conditions.

Extracellular vesicles (EVs) are heterogeneous lipid containers carrying complex molecular cargoes, including proteins, nucleic acids, glycans, etc. These vesicles are closely associated with specific physiological characteristics, which makes them invaluable in the detection and monitoring of various diseases. However, traditional isolation methods are often labour-intensive, inefficient, and time-consuming. In addition, single biomarker analyses are no longer accurate enough to meet diagnostic needs. Routine isolation and molecular analysis of high-purity EVs in clinical applications is even more challenging. In this review, we discuss a promising solution, microfluidic-based techniques, that combine efficient isolation and multiplex detection of EVs, to further demystify EV heterogeneity. These microfluidic-based EV multiplexing platforms will hopefully facilitate development of liquid biopsies and offer promising opportunities for personalised therapy.

Hepatocellular carcinoma (HCC) is an aggressive primary liver malignancy often diagnosed at an advanced stage, resulting in a poor prognosis. Accurate risk stratification and early detection of HCC are critical unmet needs for improving outcomes. Several blood-based biomarkers and imaging tests are available for early detection, prediction, and monitoring of HCC. However, serum protein biomarkers such as alpha-fetoprotein have shown relatively low sensitivity, leading to inaccurate performance. Imaging studies also face limitations related to suboptimal accuracy, high cost, and limited implementation. Recently, liquid biopsy techniques have gained attention for addressing these unmet needs. Liquid biopsy is non-invasive and provides more objective readouts, requiring less reliance on healthcare professional's skills compared to imaging. Circulating tumor cells, cell-free DNA, and extracellular vesicles are targeted in liquid biopsies as novel biomarkers for HCC. Despite their potential, there are debates regarding the role of these novel biomarkers in the HCC care continuum. This review article aims to discuss the technical challenges, recent technical advancements, advantages and disadvantages of these liquid biopsies, as well as their current clinical application and future directions of liquid biopsy in HCC.

The complex and continuously evolving features of the tumor microenvironment, varying between tumor histotypes, are characterized by the presence of host cells and tumor cells embedded in a milieu shaped by hypoxia and low pH, resulting from the frequent imbalance between vascularity and tumor cell proliferation. These microenvironmental metabolic stressors play a crucial role in remodeling host cells and tumor cells, contributing to the stimulation of cancer cell heterogeneity, clonal evolution, and multidrug resistance, ultimately leading to progression and metastasis. The extracellular vesicles (EVs), membrane-enclosed structures released into the extracellular milieu by tumor/host cells, are now recognized as critical drivers in the complex intercellular communication between tumor cells and the local cellular components in a hypoxic/acidic microenvironment. Understanding the intricate molecular mechanisms governing the interactions between tumor and host cells within a hypoxic and acidic microenvironment, triggered by the release of EVs, could pave the way for innovative strategies to disrupt the complex interplay of cancer cells with their microenvironment. This approach may contribute to the development of an efficient and safe therapeutic strategy to combat cancer progression. Therefore, we review the major findings on the release of EVs in a hypoxic/acidic tumor microenvironment to appreciate their role in tumor progression toward metastatic disease.

Hepatocellular carcinoma (HCC) is characterized by a complex pathogenesis that confers aggressive malignancy, leading to a lack of dependable biomarkers for predicting invasion and metastasis, which results in poor prognoses in patients with HCC. Glycogen storage disease (GSD) is an uncommon metabolic disorder marked by hepatomegaly and liver fibrosis. Notably, hepatic adenomas in GSD patients present a heightened risk of malignancy compared to those in individuals without the disorder. In this investigation, PON1 emerged as a potential pivotal gene for HCC through bioinformatics analysis.

Transcriptomic profiling data of liver cancer were collected and integrated from TCGA and GEO databases. Bioinformatics analysis was conducted to identify mutated mRNAs associated with GSD, and the PON1 gene was selected as a key gene. Patients were grouped based on the expression levels of PON1, and differences in clinical characteristics, biological pathways, immune infiltration, and expression of immune checkpoints were compared.

The expression levels of the PON1 gene showed significant differences between the high-expression group and the low-expression group in HCC patients. Further analysis indicated that the PON1 gene at different expression levels might influence the clinical manifestations, biological processes, immune infiltration, and expression of immune checkpoints in HCC. Additionally, immunohistochemistry (IHC) results revealed high expression of PON1 in normal tissues and low expression in HCC tissues. These findings provide important clues and future research directions for the early diagnosis, prognosis, immunotherapy, and potential molecular interactions of HCC.

Our investigation underscores the noteworthy prognostic significance of PON1 in HCC, suggesting its potential pivotal role in modulating tumor progression and immune cell infiltration. These findings establish PON1 as a novel tumor biomarker with significant implications for the prognosis, targeted therapy, and immunotherapy of patients with HCC.

Extracellular vesicles (EVs) have garnered significant attention in biomedical applications. However, the rapid, efficient, and unbiased separation of EVs from complex biological fluids remains a challenge due to their heterogeneity and low abundance in biofluids. Herein, we report a novel approach to reconfigure and modify an artificial insertion peptide for the unbiased and rapid isolation of EVs in 20 min with ∼80% recovery in neutral conditions. Moreover, the approach demonstrates exceptional anti-interference capability and achieves a high purity of EVs comparable to standard ultracentrifugation and other methods. Importantly, the isolated EVs could be directly applied for downstream protein and nucleic acid analyses, including proteomics analysis, exome sequencing analysis, as well as the detection of both epidermal growth factor receptor (EGFR) and V-Ki-ras2 Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) gene mutation in clinical plasma samples. Our approach offers great possibilities for utilizing EVs in liquid biopsy, as well as in various other biomedical applications.

Hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) exhibit significant phenotypic heterogeneity and diverse gene expression profiles due to epithelial-mesenchymal transition (EMT). However, current detection methods lack the capacity for simultaneous quantification of multidimensional biomarkers, impeding a comprehensive understanding of tumor biology and dynamic changes. Here, the CTC Digital Simultaneous Cross-dimensional Output and Unified Tracking (d-SCOUT) technology is introduced, which enables simultaneous quantification and detailed interpretation of HCC transcriptional and phenotypic biomarkers. Based on self-developed multi-real-time digital PCR (MRT-dPCR) and algorithms, d-SCOUT allows for the unified quantification of Asialoglycoprotein Receptor (ASGPR), Glypican-3 (GPC-3), and Epithelial Cell Adhesion Molecule (EpCAM) proteins, as well as Programmed Death Ligand 1 (PD-L1), GPC-3, and EpCAM mRNA in HCC CTCs, with good sensitivity (LOD of 3.2 CTCs per mL of blood) and reproducibility (mean %CV = 1.80-6.05%). In a study of 99 clinical samples, molecular signatures derived from HCC CTCs demonstrated strong diagnostic potential (AUC = 0.950, sensitivity = 90.6%, specificity = 87.5%). Importantly, by integrating machine learning, d-SCOUT allows clustering of CTC characteristics at the mRNA and protein levels, mapping normalized heterogeneous 2D molecular profiles to assess HCC metastatic risk. Dynamic digital tracking of eight HCC patients undergoing different treatments visually illustrated the therapeutic effects, validating this technology's capability to quantify the treatment efficacy. CTC d-SCOUT enhances understanding of tumor biology and HCC management.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. HCC is highly heterogenous with diverse etiologies leading to different driver mutations potentiating unique tumor immune microenvironments. Current therapeutic options, including immune checkpoint inhibitors and combinations, have achieved limited objective response rates for the majority of patients. Thus, a precision medicine approach is needed to tailor specific treatment options for molecular subsets of HCC patients. Lipid nanovesicle platforms, either liposome- (synthetic) or extracellular vesicle (natural)-derived present are improved drug delivery vehicles which may be modified to contain specific cargos for targeting specific tumor sites, with a natural affinity for liver with limited toxicity. This mini-review provides updates on the applications of novel lipid nanovesicle-based therapeutics for HCC precision medicine and the challenges associated with translating this therapeutic subclass from preclinical models to the clinic.

Exosomes are small extracellular vesicles (EVs) derived from various cellular sources and have emerged as favorable biomarkers for cancer diagnosis and prognosis. These vesicles contain a variety of molecular components, including nucleic acids, proteins, and lipids, which can provide valuable information for cancer detection, classification, and monitoring. However, the clinical application of exosomes faces significant challenges, primarily related to the standardization and scalability of their use. In order to overcome these challenges, sophisticated methods such as liquid biopsy and imaging are being combined to augment the diagnostic capabilities of exosomes. Additionally, a deeper understanding of the interaction between exosomes and immune system components within the tumor microenvironment (TME) is essential. This review discusses the biogenesis and composition of exosomes, addresses the current challenges in their clinical translation, and highlights recent technological advancements and integrative approaches that support the role of exosomes in cancer diagnosis and prognosis.

The Scottish Inflammatory Prognostic Score (SIPS), an innovative scoring system, has emerged as a promising biomarker for predicting patient outcomes following cancer therapy. This study aimed to evaluate the value of SIPS as a prognostic indicator following hepatectomy in patients with hepatocellular carcinoma (HCC).

This retrospective study included 693 HCC patients who underwent hepatectomy. Survival outcomes were compared between propensity score-matched groups. Independent prognostic factors were identified through Cox regression analysis. Additionally, both traditional Cox proportional hazards models and machine learning models based on the SIPS were developed and validated.

A total of 693 HCC patients who underwent hepatectomy were included, with 102 in the high SIPS group and 591 in the low SIPS group. Following propensity score matching (1:3 ratio), both groups achieved balance, with 82 patients in the high SIPS group and 240 patients in the low SIPS group. The low SIPS group demonstrated significantly superior recurrence-free survival (RFS) (25 months vs. 21 months; P < 0.001) and overall survival (OS) (69 months vs. 58 months; P < 0.001) compared to the high SIPS group. Multivariable analysis identified SIPS as an independent adverse factor affecting both RFS and OS. The calibration curve for overall patient survival diagnosis displayed excellent predictive accuracy. Traditional COX prognostic models and machine learning models incorporating SIPS demonstrated excellent performance both the training and validation set.

This study confirms the prognostic significance of SIPS in post-hepatectomy HCC patients, providing a practical tool for risk stratification and clinical decision-making. Further research and validation are needed to consolidate its role in prognostic assessment.

In recent years, cancer, especially primary liver cancer (including hepatocellular carcinoma and intrahepatic cholangiocarcinoma), has posed a serious threat to human health. In the field of liver cancer, exosomes play an important role in liver cancer initiation, metastasis and interaction with the tumor microenvironment. Exosomes are a class of nanoscale extracellular vesicles (EVs)secreted by most cells and rich in bioactive molecules, including RNA, proteins and lipids, that mediate intercellular communication during physiological and pathological processes. This review reviews the multiple roles of exosomes in liver cancer, including the initiation, progression, and metastasis of liver cancer, as well as their effects on angiogenesis, epithelial-mesenchymal transformation (EMT), immune evasion, and drug resistance. Exosomes have great potential as biomarkers for liver cancer diagnosis and prognosis because they carry specific molecular markers that facilitate early detection and evaluation of treatment outcomes. In addition, exosomes, as a new type of drug delivery vector, have unique advantages in the targeted therapy of liver cancer and provide a new strategy for the treatment of liver cancer. The challenges and prospects of exosome-based immunotherapy in the treatment of liver cancer were also discussed. However, challenges such as the standardization of isolation techniques and the scalability of therapeutic applications remain significant hurdles.

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality globally. Most patients present with late diagnosis, leading to poor prognosis. This narrative review explores novel biomarkers for early HCC detection. We conducted a comprehensive literature review analyzing protein, circulating nucleic acid, metabolite, and quantitative proteomics-based biomarkers, evaluating the advantages and limitations of each approach. While established markers like alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin, and AFP-L3 remain relevant, promising candidates include circulating tumor DNA, microRNAs, long noncoding RNAs, extracellular vesicle, and metabolomic biomarkers. Multi-biomarker panels like the GALAD score, Oncoguard, and Helio liver test show promise for improved diagnostic accuracy. Non-invasive approaches like urine and gut microbiome analysis are also emerging possibilities. Integrating these novel biomarkers with current screening protocols holds significant potential for earlier HCC detection and improved patient outcomes. Future research should explore multi-biomarker panels, omics technologies, and artificial intelligence to further enhance early HCC diagnosis and management.

Liquid biopsy is a minimally invasive method that uses biofluid samples instead of tissue samples for cancer diagnosis. Exosomes are small extracellular vesicles secreted by donor cells and act as mediators of intercellular communication in human health and disease. Due to their important roles, exosomes have been considered as promising biomarkers for liquid biopsy. However, traditional methods for exosome isolation and cargo detection methods are time-consuming and inefficient, limiting their practical application. In the past decades, many new strategies, such as microfluidic chips, nanowire arrays and electrochemical biosensors, have been proposed to achieve rapid, accurate and high-throughput detection and analysis of exosomes. In this review, we discussed about the new advance in exosome-based liquid biopsy technology, including isolation, enrichment, cargo detection and analysis approaches. The comparison of currently available methods is also included. Finally, we summarized the advantages and limitations of the present strategies and further gave a perspective to their future translational use.

Hepatocellular carcinoma (HCC) seriously threatens the human health. Previous investigations revealed that γ-glutamyltranspeptidase (GGT) was tightly associated with the chronic injury, hepatic fibrosis, and the development of HCC, therefore might act as a potential indicator for monitoring the HCC-related processes. Herein, with the contribution of a structurally optimized probe ETYZE-GGT, the bimodal imaging in both far red fluorescence (FL) and photoacoustic (PA) modes has been achieved in multiple HCC-related models. To our knowledge, this work covered the most comprehensive models including the fibrosis and developed HCC processes as well as the premonitory induction stages (autoimmune hepatitis, drug-induced liver injury, non-alcoholic fatty liver disease). ETYZE-GGT exhibited steady and practical monitoring performances on reporting the HCC stages via visualizing the GGT dynamics. The two modes exhibited working consistency and complementarity with high spatial resolution, precise apparatus and desirable biocompatibility. In cooperation with the existing techniques including testing serum indexes and conducting pathological staining, ETYZE-GGT basically realized the universal application for the accurate pre-clinical diagnosis of as many HCC stages as possible. By deeply exploring the mechanically correlation between GGT and the HCC process, especially during the premonitory induction stages, we may further raise the efficacy for the early diagnosis and treatment of HCC.

Extracellular vesicle (EV)-based therapeutics have gained substantial interest in the areas of drug delivery, immunotherapy, and regenerative medicine. However, the clinical translation of EVs has been slowed due to limited yields and functional heterogeneity, as well as inadequate targeting. Engineering EVs to modify their inherent function and endow them with additional functions has the potential to advance the clinical translation of EV applications. Bio-orthogonal click chemistry is an engineering approach that modifies EVs in a controlled, specific, and targeted way without compromising their intrinsic structure. Here, we provide an overview of bio-orthogonal labeling approaches involved in EV engineering. We also present the isolation methods of bio-orthogonally labeled vesicles using magnetic beads, microfluidics, and microarray chip technologies. We highlight the in vivo applications of bio-orthogonal labeling EVs for diagnosis and therapy, especially the exciting potential of bio-orthogonal glycometabolic engineered EVs for targeted therapies.

There is a pressing need for non-invasive preoperative prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC). This study investigates the potential of exosome-derived mRNA in plasma as a biomarker for diagnosing MVI.

Patients with suspected HCC undergoing hepatectomy were prospectively recruited for preoperative peripheral blood collection. Exosomal RNA profiling was conducted using RNA sequencing in the discovery cohort, followed by differential expression analysis to identify candidate targets. We employed multiplexed droplet digital PCR technology to efficiently validate them in a larger sample size cohort.

A total of 131 HCC patients were ultimately enrolled, with 37 in the discovery cohort and 94 in the validation cohort. In the validation cohort, the expression levels of RSAD2, PRPSAP1, and HOXA2 were slightly elevated while CHMP4A showed a slight decrease in patients with MVI compared with those without MVI. These trends were consistent with the findings in the discovery cohort, although they did not reach statistical significance (P > 0.05). Notably, the expression level of exosomal PRPSAP1 in plasma was significantly higher in patients with more than 5 MVI than in those without MVI (0.147 vs 0.070, P = 0.035).

This study unveils the potential of exosome-derived PRPSAP1 in plasma as a promising indicator for predicting MVI status preoperatively.

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer often associated with chronic hepatitis B virus infection (CHB) and liver cirrhosis (LC), underscoring the critical need for biomarker discovery to improve patient outcomes. Emerging as a promising avenue for biomarker development, proteomic technology leveraging liquid biopsy from small extracellular vesicles (sEV) offers new insights. Here, we evaluated various methods for sEV isolation and identified polysaccharide chitosan (CS) as an optimal approach. Subsequently, we employed optimized CS-based magnetic beads (Mag-CS) for sEV separation from serum samples of healthy controls, CHB, LC, and HBV-HCC patients. Leveraging data-independent acquisition mass spectrometry coupled with machine learning, we uncovered potential vesicular protein biomarker signatures (KNG1, F11, KLKB1, CAPNS1, CDH1, CPN2, NME2) capable of distinguishing HBV-HCC from CHB, LC, and non-HCC conditions. Collectively, our findings highlight the utility of Mag-CS-based sEV isolation for identifying early detection biomarkers in HBV-HCC.

Because of the challenges posed by anatomical uncertainties and the low resolution of plain computed tomography (CT) scans, implementing adaptive radiotherapy (ART) for small hepatocellular carcinoma (sHCC) using artificial intelligence (AI) faces obstacles in tumor identification-alignment and automatic segmentation. The current study aims to improve sHCC imaging for ART using a gold nanoparticle (Au NP)-based CT contrast agent to enhance AI-driven automated image processing. The synthesized charged Au NPs demonstrated notable in vitro aggregation, low cytotoxicity, and minimal organ toxicity. Over time, an in situ sHCC mouse model was established for in vivo CT imaging at multiple time points. The enhanced CT images processed using 3D U-Net and 3D Trans U-Net AI models demonstrated high geometric and dosimetric accuracy. Therefore, charged Au NPs enable accurate and automatic sHCC segmentation in CT images using classical AI models, potentially addressing the technical challenges related to tumor identification, alignment, and automatic segmentation in CT-guided online ART.

Single-cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing the polyA tails of mRNAs, which exclude many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single-cell level. In this study, we developed a measurement protocol for nc886 as a model case and initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5'scRNA-seq. We then produced sequencing libraries for standard 5' gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines that express high, low, and zero levels of nc886. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows combined analysis of ncRNAs and gene expression phenotype.

The early detection of gene mutations in physiological and pathological processes is a powerful approach to guide decisions in precision medicine. However, detecting low-copy mutant DNA from clinical samples poses a challenge due to the enrichment of wild-type DNA backgrounds. In this study, we devised a novel strategy, named Mutation-Selected Amplification droplet digital PCR (MSA-ddPCR), to quantitatively analyze single nucleotide variants (SNVs) at low variant allele frequencies (VAFs). Using TP53R249S (a hotspot mutation associated with hepatocellular carcinoma) as a model, we optimized the concentration ratio of primers, the annealing temperature and nucleic acid amplification modifiers. Subsequently, we evaluated the linear range and precision of MSA-ddPCR by detecting TP53R249S and TP53wild-type (TP53WT) plasmid DNA, respectively. MSA-ddPCR demonstrated superior ability to discriminate between mutant DNA and wild-type DNA compared to traditional TaqMan-MGB PCR. We further applied MSA-ddPCR to analyze the TP53R249S mutation in 20 plasma samples and 15 formalin-fixed paraffin-embedded (FFPE) tissue samples, and assessed the agreement rates between MSA-ddPCR and amplicon high-throughput sequencing. The results showed that the limit of blanks of MSA-ddPCR are 0.449 copies μL-1 in the FAM channel and 0.452 copies μL-1 in the VIC channel. MSA-ddPCR could accurately quantify VAFs as low as 0.01 %, surpassing existing PCR and next-generation sequencing (NGS) methods. In the detection of clinical samples, a high correlation was found between MSA-ddPCR and amplicon high-throughput sequencing. Additionally, MSA-ddPCR outperformed sequencing methods in terms of detection time and simplicity of data analysis. MSA-ddPCR can be easily implemented into clinical practice and serve as a robust tool for detecting mutant genes due to its high sensitivity and accuracy.

This review comprehensively explores the complex interplay between extracellular vesicles (ECVs)/exosomes and circadian rhythms, with a focus on the role of this interaction in hepatocellular carcinoma (HCC). Exosomes are nanovesicles derived from cells that facilitate intercellular communication by transporting bioactive molecules such as proteins, lipids, and RNA/DNA species. ECVs are implicated in a range of diseases, where they play crucial roles in signaling between cells and their surrounding environment. In the setting of cancer, ECVs are known to influence cancer initiation and progression. The scope of this review extends to all cancer types, synthesizing existing knowledge on the various roles of ECVs. A unique aspect of this review is the emphasis on the circadian-controlled release and composition of exosomes, highlighting their potential as biomarkers for early cancer detection and monitoring metastasis. We also discuss how circadian rhythms affect multiple cancer-related pathways, proposing that disruptions in the circadian clock can alter tumor development and treatment response. Additionally, this review delves into the influence of circadian clock components on ECV biogenesis and their impact on reshaping the tumor microenvironment, a key component driving HCC progression. Finally, we address the potential clinical applications of ECVs, particularly their use as diagnostic tools and drug delivery vehicles, while considering the challenges associated with clinical implementation.

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells that contain various cellular components such as proteins, nucleic acids, and lipids from the parent cell. EVs are abundant in body fluids and can serve as circulating biomarkers for a variety of diseases or as a regulator of various biological processes. Considering these characteristics of EVs, analysis of the EV cargo has been spotlighted for disease diagnosis or to understand biological processes in biomedical research. Over the past decade, technologies for rapid and sensitive analysis of EVs in biofluids have evolved, but detection and isolation of targeted EVs in complex body fluids is still challenging due to the unique physical and biological properties of EVs. Recent advances in chemical biology provide new opportunities for efficient profiling of the molecular contents of EVs. A myriad of chemical biology tools have been harnessed to enhance the analytical performance of conventional assays for better understanding of EV biology. In this review, we will discuss the improvements that have been achieved using chemical biology tools.

Extracellular vesicles (EVs) have emerged as a novel cell-free strategy for the treatment of many diseases including cancer as they play important roles in cancer development and progression. Considering their natural capacity to facilitate cell-to-cell communication as well as their high physiochemical stability and biocompatibility, EVs serve as superior delivery systems for a wide range of therapeutic agents, including medicines, nanomaterials, nucleic acids, and proteins. Therefore, EVs-based cancer therapy is of greater interest to researchers. Mounting studies indicate that EVs can be improved in efficiency, specificity, and safety for cancer therapy. However, their heterogeneity of physicochemical properties and functions is not fully understood, hindering the achievement of bioactive EVs with high yield and purity. Herein, we paid more attention to the EVs applications and their significance in cancer therapy.

The hepatitis C virus (HCV) infection affects 58 million people worldwide. In the United States, the incidence rate of acute hepatitis C has doubled since 2014; during 2021, this increased to 5% from 2020. Acute hepatitis C is defined by any symptom of acute viral hepatitis plus either jaundice or elevated serum alanine aminotransferase (ALT) activity with the detection of HCV RNA, the anti-HCV antibody, or hepatitis C virus antigen(s). However, most patients with acute infection are asymptomatic. In addition, ALT activity and HCV RNA levels can fluctuate, and a delayed detection of the anti-HCV antibody can occur among some immunocompromised persons with HCV infection. The detection of specific biomarkers can be of great value in the early detection of HCV infection at an asymptomatic stage. The high rate of HCV replication (which is approximately 1010 to 1012 virions per day) and the lack of proofreading by the viral RNA polymerase leads to enormous genetic diversity, creating a major challenge for the host immune response. This broad genetic diversity contributes to the likelihood of developing chronic infection, thus leading to the development of cirrhosis and liver cancer. Direct-acting antiviral (DAA) therapies for HCV infection are highly effective with a cure rate of up to 99%. At the same time, many patients with HCV infection are unaware of their infection status because of the mostly asymptomatic nature of hepatitis C, so they remain undiagnosed until the liver damage has advanced. Molecular mechanisms induced by HCV have been intensely investigated to find biomarkers for diagnosing the acute and chronic phases of the infection. However, there are no clinically verified biomarkers for patients with hepatitis C. In this review, we discuss the biomarkers that can differentiate acute from chronic hepatitis C, and we summarize the current state of the literature on the useful biomarkers that are detectable during acute and chronic HCV infection, liver fibrosis/cirrhosis, and hepatocellular carcinoma (HCC).

The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization are proposed to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of EVs can enhance basic and clinical research in this field.

Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and a leading cause of cancer-related deaths worldwide. However, current diagnostic tools are often invasive and technically limited. In the last decade, non-invasive liquid biopsies have transformed the field of clinical oncology, showcasing the potential of various liquid-biopsy derived analytes, including extracellular vesicles (EVs), to diagnose and monitor HCC progression and metastatic spreading, serving as promising novel biomarkers. A prospective single-center cohort study including 37 HCC patients and 20 patients with non-malignant liver disease (NMLD), as a control group, was conducted. Serum EVs of both groups were analyzed before and after liver surgery. The study utilized microbead-based magnetic particle sorting and flow cytometry to detect 37 characteristic surface proteins of EVs. Furthermore, HCC patients who experienced tumor recurrence (R-HCC) within 12 months after surgery were compared to HCC patients without recurrence (NR-HCC). EVs of R-HCC patients (n = 12/20) showed significantly lower levels of CD31 compared to EVs of NR-HCC patients (p = 0.0033). EVs of NMLD-group showed significantly higher expressions of CD41b than EVs of HCC group (p = 0.0286). The study determined significant short-term changes in CD19 dynamics in EVs of the NMLD-group, with preoperative values being significantly higher than postoperative values (p = 0.0065). This finding of our pilot study suggests EVs could play a role as potential targets for the development of diagnostic and therapeutic approaches for the early and non-invasive detection of HCC recurrence. Further, more in-depth analysis of the specific EV markers are needed to corroborate their potential role as diagnostic and therapeutic targets for HCC.

The use of liquid biopsy in cancer research has grown exponentially, offering potential for early detection, treatment stratification, and monitoring residual disease and recurrence. Exosomes, released by cancer cells, contain tumor-derived materials and are stable in biofluids, making them valuable biomarkers for clinical evaluation. Bibliometric research on osteosarcoma (OS) and exosome-derived diagnostic biomarkers is scarce. Therefore, we aimed to conduct a bibliometric evaluation of studies on OS and exosome-derived biomarkers. Using the Web of Science Core Collection database, Microsoft Excel, the R "Bibliometrix" package, CiteSpace, and VOSviewer software, quantitative analyses of the country, author, annual publications, journals, institutions, and keywords of studies on exosome-derived biomarkers for OS from 1995 to 2023 were performed. High-quality records (average citation rate ≥ 10/year) were filtered. The corresponding authors were mainly from China, the USA, Australia, and Canada. The University of Kansas Medical Center, National Cancer Center, Japan, and University of Kansas were major institutions, with limited cooperation reported by the University of Kansas Medical Center. Keyword analysis revealed a shift from cancer progression to mesenchymal stem cells, exosome expression, biogenesis, and prognostic biomarkers. Qualitative analysis highlighted exosome cargo, including miRNAs, circRNAs, lncRNAs, and proteins, as potential diagnostic OS biomarkers. This research emphasizes the rapid enhancement of exosomes as a diagnostic frontier, offering guidance for the clinical application of exosome-based liquid biopsy in OS, contributing to the evolving landscape of cancer diagnosis.

The optimal treatment strategy for early-stage hepatocellular carcinoma (HCC) remains controversial, specifically in regard to surgical resection (SR) and ablation. The aim of this study was to investigate the impact of SR and ablation on recurrence and prognosis in early-stage HCC patients, to optimize treatment strategies and improve long-term survival.

A retrospective analysis was conducted on 801 patients diagnosed with Barcelona Clinic Liver Cancer (BCLC) stage 0/A HCC and treated with SR or ablation between January 2015 and December 2019. The effectiveness and complications of both treatments were analyzed, and patients were followed up to measure recurrence and survival. Propensity score matching (PSM) was employed to increase comparability between the two groups. The Kaplan-Meier method was used to analyze recurrence and survival, and a Cox risk proportional hazard model was used to identify risk factors that affect recurrence and surviva.

Before PSM, the overall survival (OS) rates were similar in both groups, with recurrence-free survival (RFS) rates better in the SR group than in the ablation group. After PSM, there was no significant difference in OS between the two groups. However, the RFS rates were significantly better in the SR group than in the ablation group. The ablation group exhibited superior outcomes compared to the SR group, with shorter treatment times, reduced bleeding, shorter hospital stays, and lower hospital costs. Concerning the location of the HCC within the liver, comparable efficacy was observed between SR and ablation for disease located in the noncentral region or left lobe. However, for HCCs located in the central region or right lobe of the liver, SR was more effective than ablation.

This study revealed no significant difference in OS between SR and ablation for early-stage HCC, with SR providing better RFS and ablation demonstrating better safety profiles and lower hospital costs. These findings offer valuable insights for clinicians in determining optimal treatment strategies for early-stage HCC patients, particularly in terms of balancing efficacy, safety, and cost considerations.

The diagnosis and management of hepatocellular carcinoma (HCC) have improved significantly in recent years. With the introduction of immunotherapy-based combination therapy, there has been a notable expansion in treatment options for patients with unresectable HCC. Simultaneously, innovative molecular tests for early detection and management of HCC are emerging. This progress prompts a key question: as liquid biopsy techniques rise in prominence, will they replace traditional tissue biopsies, or will both techniques remain relevant? Given the ongoing challenges of early HCC detection, including issues with ultrasound sensitivity, accessibility, and patient adherence to surveillance, the evolution of diagnostic techniques is more relevant than ever. Furthermore, the accurate stratification of HCC is limited by the absence of reliable biomarkers which can predict response to therapies. While the advantages of molecular diagnostics are evident, their potential has not yet been fully harnessed, largely because tissue biopsies are not routinely performed for HCC. Liquid biopsies, analysing components such as circulating tumour cells, DNA, and extracellular vesicles, provide a promising alternative, though they are still associated with challenges related to sensitivity, cost, and accessibility. The early results from multi-analyte liquid biopsy panels are promising and suggest they could play a transformative role in HCC detection and management; however, comprehensive clinical validation is still ongoing. In this review, we explore the challenges and potential of both tissue and liquid biopsy, highlighting that these diagnostic methods, while distinct in their approaches, are set to jointly reshape the future of HCC management.

Here we describe a cost-effective and simplified cell sorting method using tetrazine bioorthogonal chemistry. We successfully isolated SKOV3 cells from complex mixtures, demonstrating efficacy in separating mouse lymphocytes expressing interferon and HeLa cells expressing virally transduced green fluorescent protein post-infection.