Establishing the capacity to monitor proteins relevant to the study of drug exposure and response using liver-derived extracellular vesicles.

AIMS: Drug exposure and response is determined by pharmacokinetic (PK) and pharmacodynamic (PD) profiles. Interindividual differences in abundance of drug metabolizing enzymes (DMEs) and drug target proteins underpin PK and PD variability and impact treatment efficacy and tolerability. Extracellular vesicles (EVs) carry protein cargo inherited from originating cells and may be useful for defining differences in key proteins related to hepatic drug metabolism and the treatment of metabolic-associated fatty liver disease (MAFLD). We sought to quantify these proteins in liver-derived EVs and establish the profile relative to paired tissue. METHODS: EVs were recovered from human liver tissue samples (LT-EV, n = 11). Targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays were employed for absolute quantification of proteins in EV isolates and matched liver tissue. RESULTS: DMEs and MAFLD drug targets were readily detected and quantified in LT-EVs. Twelve of 15 DMEs exhibited moderate to strong correlation (Spearman ⍴ = 0.618-0.973) between tissue and EVs. Correlation in protein abundance was influenced by the extent of extra-hepatic expression of the target. CONCLUSIONS: This study provides evidence that key proteins related to PK and PD profiles can be measured in liver-derived EVs and abundance of liver-enriched DMEs are robustly correlated between paired tissue and EVs. The robust detection of protein markers related to drug PD profile in MAFLD opens the possibility to track within-subject changes in MAFLD and lays the foundation for future development of a liver-derived EV liquid biopsy to assess markers of drug exposure and response in vivo.

Proteomic profiling of paired human liver homogenate and tissue derived extracellular vesicles.

Advances in technologies to isolate extracellular vesicles (EVs) and detect/quantify their cargo underpin the novel potential of these circulating particles as a liquid biopsy to understand physiology and disease. One organ of particular interest in terms of utilizing EVs as a liquid biopsy is the liver. The extent to which EVs originating from the liver reflect the functional status of this organ remains unknown. This is an important knowledge gap that underpins the utility of circulating liver derived EVs as a liquid biopsy. The primary objective of this study was to characterize the proteomic profile of EVs isolated from the extracellular space of liver tissue (LEV) and compare this profile to that of paired tissue (LH). LCMS analyses detected 2892 proteins in LEV and 2673 in LH. Of the 2673 proteins detected in LH, 1547 (58%) were also detected in LEV. Bioinformatic analyses demonstrated comparable representation of proteins in terms of biological functions and cellular compartments. Although, enriched representation of membrane proteins and associated functions was observed in LEV, while representation of nuclear proteins and associated functions was depleted in LEV. These data support the potential use of circulating liver derived EVs as a liquid biopsy for this organ.

Role of Extracellular Vesicle-Derived Biomarkers in Drug Metabolism and Disposition.

Extracellular vesicles (EVs) are small, nonreplicating, lipid-encapsulated particles that contain a myriad of protein and nucleic acid cargo derived from their tissue of origin. The potential role of EV-derived biomarkers to the study of drug metabolism and disposition (DMD) has gained attention in recent years. The key trait that makes EVs an attractive biomarker source is their capacity to provide comparable insights to solid organ biopsy through an appreciably less invasive collection procedure. Blood-derived EVs exist as a heterogenous milieu of biologically distinct particles originating from different sources through different biogenesis pathways. Furthermore, blood (plasma and serum) contains an array of vesicular and nonvesicular contaminants, such as apoptotic bodies, plasma proteins, and lipoproteins that are routinely coisolated with EVs, albeit to a different extent depending on the isolation technique. The following minireview summarizes current studies reporting DMD biomarkers and addresses elements of EV isolation and quantification relevant to the application of EV-derived DMD biomarkers. Evidence based-best practice guidance aligned to Minimum Information for the Study of Extracellular Vesicles and EV-TRACK reporting standards are summarized in the context of DMD studies. SIGNIFICANCE STATEMENT: Extracellular vesicle (EV)-derived protein and nucleic acid cargo represent a potentially game-changing source of novel DMD biomarkers with the capacity to define within- and between-individual variability in drug exposure irrespective of etiology. However, robust translation of EV-derived biomarkers requires the generation of transparent reproducible evidence. This review outlines the critical elements of data generation and reporting relevant to achieving this evidence in a drug metabolism and disposition context.

Increased extracellular vesicles mediate inflammatory signalling in cystic fibrosis.

RATIONALE: Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene form the basis of cystic fibrosis (CF). There remains an important knowledge gap in CF as to how diminished CFTR activity leads to the dominant inflammatory response within CF airways. OBJECTIVES: To investigate if extracellular vesicles (EVs) contribute to inflammatory signalling in CF. METHODS: EVs released from CFBE41o-, CuFi-5, 16HBE14o- and NuLi-1 cells were characterised by nanoparticle tracking analysis (NTA). EVs isolated from bronchoalveolar lavage fluid (BALF) from 30 people with CF (PWCF) were analysed by NTA and mass spectrometry and compared with controls. Neutrophils were isolated from the blood of 8 PWCF to examine neutrophil migration in the presence of CFBE41o- EVs. RESULTS: A significantly higher level of EVs were released from CFBE41o- (p<0.0001) and CuFi-5 (p=0.0209) relative to control cell lines. A significantly higher level of EVs were detected in BALF of PWCF, in three different age groups relative to controls (p=0.01, 0.001, 0.002). A significantly lower level of EVs were released from CFBE41o- (p<0.001) and CuFi-5 (p=0.0002) cell lines treated with CFTR modulators. Significant changes in the protein expression of 126 unique proteins was determined in EVs obtained from the BALF of PWCF of different age groups (p<0.001-0.05). A significant increase in chemotaxis of neutrophils derived from PWCF was observed in the presence of CFBE41o EVs (p=0.0024) compared with controls. CONCLUSION: This study demonstrates that EVs are produced in CF airway cells, have differential protein expression at different ages and drive neutrophil recruitment in CF.

Anti-cancer effects of baicalein in non-small cell lung cancer in-vitro and in-vivo.

BACKGROUND: Baicalein is a widely used Chinese herbal medicine derived from Scutellaria baicalenesis, which has been traditionally used as anti-inflammatory and anti-cancer therapy. In this study we examined the anti-tumour pathways activated following baicalein treatment in non-small cell lung cancer (NSCLC), both in-vitro and in-vivo. METHODS: The effect of baicalein treatment on H-460 cells in-vitro was assessed using both BrdU assay (cell proliferation) and High Content Screening (multi-parameter apoptosis assay). A xenograft nude mouse model was subsequently established using these cells and the effect of baicalein on tumour growth and survival assessed in-vivo. Tumours were harvested from these mice and histological tissue analysis carried out. VEGF, 12-lipoxygenase and microvessel density (CD-31) were assessed by immunohistochemistry (IHC), while H and E staining was carried out to assess mitotic index. Gene expression profiling was carried out on corresponding RNA samples using Human Cancer Pathway Finder Arrays and qRT-PCR, with further gene expression analysis carried out using qRT-PCR. RESULTS: Baicalein significantly decreased lung cancer proliferation in H-460 cells in a dose dependent manner. At the functional level, a dose-dependent induction in apoptosis associated with decreased cellular f-actin content, an increase in nuclear condensation and an increase in mitochondrial mass potential was observed. Orthotopic treatment of experimental H-460 tumours in athymic nude mice with baicalein significantly (p < 0.05) reduced tumour growth and prolonged survival. Histological analysis of resulting tumour xenografts demonstrated reduced expression of both 12-lipoxygenase and VEGF proteins in baicalein-treated tumours, relative to untreated. A significant (p < 0.01) reduction in both mitotic index and micro-vessel density was observed following baicalein treatment. Gene expression profiling revealed a reduction (p < 0.01) in both VEGF and FGFR-2 following baicalein treatment, with a corresponding increase (p < 0.001) in RB-1. CONCLUSION: This study is the first to demonstrate efficacy of baicalein both in-vitro and in-vivo in NSCLC. These effects may be mediated in part through a reduction in both cell cycle progression and angiogenesis. At the molecular level, alterations in expression of VEGF, FGFR-2, and RB-1 have been implicated, suggesting a molecular mechanism underlying this in-vivo effect.

Extracellular vesicles: A potential new way to assess cholestasis.

Extracellular vesicles (EVs) are small, nonreplicating, lipid-encapsulated nanoparticles that carry protein and nucleic acid cargo derived from their tissue of origin. Due to their capacity to provide comparable insights to solid organ biopsy through a minimally invasive collection procedure, EVs provide an attractive biomarker source. This review will provide an insight, how EVs in circulation may provide a novel way to assess cholestasis and will address the possibility of getting a better understanding of the mechanisms of cholestasis of pregnancy through the use of serial hepatic-specific EVs as a window.

Study of the ketohexokinase inhibitor PF-06835919 as a clinical cytochrome P450 3A inducer: Integrated use of oral midazolam and liquid biopsy.

PF-06835919, a ketohexokinase inhibitor, presented as an inducer of cytochrome P450 3A4 (CYP3A4) in vitro (human primary hepatocytes), and static mechanistic modeling exercises predicted significant induction in vivo (oral midazolam area under the plasma concentration-time curve [AUC] ratio [AUCR] = 0.23-0.79). Therefore, a drug-drug interaction study was conducted to evaluate the effect of multiple doses of PF-06835919 (300 mg once daily × 10 days; N = 10 healthy participants) on the pharmacokinetics of a single oral midazolam 7.5 mg dose. The adjusted geometric means for midazolam AUC and its maximal plasma concentration were similar following co-administration with PF-06835919 (vs. midazolam administration alone), with ratios of the adjusted geometric means (90% confidence interval [CI]) of 97.6% (90% CI: 79.9%-119%) and 98.9% (90% CI: 76.4%-128%), respectively, suggesting there was minimal effect of PF-06835919 on midazolam pharmacokinetics. Lack of CYP3A4 induction was confirmed after the preparation of subject plasma-derived small extracellular vesicles (sEVs) and conducting proteomic and activity (midazolam 1'-hydroxylase) analysis. Consistent with the midazolam AUCR observed, the CYP3A4 protein expression fold-induction (geometric mean, 90% CI) was low in liver (0.9, 90% CI: 0.7-1.2) and non-liver (0.9, 90% CI: 0.7-1.2) sEVs (predicted AUCR = 1.0, 90% CI: 0.9-1.2). Likewise, minimal induction of CYP3A4 activity (geometric mean, 90% CI) in both liver (1.1, 90% CI: 0.9-1.3) and non-liver (0.9, 90% CI: 0.5-1.5) sEVs was evident (predicted AUCR = 0.9, 90% CI: 0.6-1.4). The results showcase the integrated use of an oral CYP3A probe (midazolam) and plasma-derived sEVs to assess a drug candidate as inducer.

Exploring the role of sporadic BRAF and KRAS mutations during colorectal cancer pathogenesis: A spotlight on the contribution of the endosome-lysosome system.

The highly heterogenous nature of colorectal cancer can significantly hinder its early and accurate diagnosis, eventually contributing to high mortality rates. The adenoma-carcinoma sequence and serrated polyp-carcinoma sequence are the two most common sequences in sporadic colorectal cancer. Genetic alterations in adenomatous polyposis coli (APC), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and tumour protein 53 (TP53) genes are critical in adenoma-carcinoma sequence, whereas v-Raf murine sarcoma viral oncogene homolog B (BRAF) and MutL Homolog1 (MLH1) are driving oncogenes in the serrated polyp-carcinoma sequence. Sporadic mutations in these genes contribute differently to colorectal cancer pathogenesis by introducing distinct alterations in several signalling pathways that rely on the endosome-lysosome system. Unsurprisingly, the endosome-lysosome system plays a pivotal role in the hallmarks of cancer and contributes to specialised colon function. Thus, the endosome-lysosome system might be distinctively influenced by different mutations and these alterations may contribute to the heterogenous nature of sporadic colorectal cancer. This review highlights potential connections between major sporadic colorectal cancer mutations and the diverse pathogenic mechanisms driven by the endosome-lysosome system in colorectal carcinogenesis.

Analysis of Extracellular Vesicle and Contaminant Markers in Blood Derivatives Using Multiple Reaction Monitoring.

Extracellular vesicles (EVs) are naturally occurring membranous particles that can be isolated from blood and other biofluids. EVs have drawn considerable attention for their potential as a minimally invasive biomarker source for a range of conditions, based on tissue-specific expression of proteins and other molecular information. To promote robust characterization of EV isolates, the International Society for Extracellular Vesicles (ISEV) has established consensus minimal requirements for the study of extracellular vesicles (MISEV) reporting guidelines. A core element of MISEV guidance is the recommendation for the analysis of protein markers in samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the sample matrix. Furthermore, there is growing interest in circulating EVs enriched for tissue-specific origin, and in this context, the degree of nontarget EV "contamination" (e.g., EVs derived from blood cells) may inform assessment of sample purity. The increasing application of EVs as a liquid biopsy for clinical applications requires a high-throughput multiplexed approach that enables analysis of protein markers from small volumes of starting material, ideally utilizing the same platform for measuring biomarkers of interest. To this end, targeted liquid chromatography mass spectrometry using multiple reaction monitoring (LC-MRM-MS) is a key platform for the quantitative assessment of target proteins within EV samples. Here we describe a protocol for the isolation of EVs from blood and parallel analytical methods targeting general EV markers and blood cell-derived EV markers, along with guidance of best practice for sample collection and processing.

Addressing MISEV guidance using targeted LC-MS/MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood.

Extracellular vesicles (EVs) are membrane-bound nanosized particles released by cells into bodily fluids containing an array of molecular cargo. Several characteristics, including stability and accessibility in biofluids such as blood and urine, make EVs and associated cargo attractive biomarkers and therapeutic tools. To promote robust characterisation of EV isolates, the minimal requirements for the study of extracellular vesicles (MISEV) guidelines recommend the analysis of proteins in EV samples, including positive EV-associated markers and negative contaminant markers based on commonly co-isolated components of the starting material. Western blot is conventionally used to address the guidelines; however, this approach is limited in terms of quantitation and throughput and requires larger volumes than typically available for patient samples. The increasing application of EVs as liquid biopsy in clinical contexts requires a high-throughput multiplexed approach for analysis of protein markers from small volumes of starting material. Here, we document the development and validation of a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantification of markers associated with EVs and non-vesicle contaminants from human blood samples. The assay was highly sensitive, requiring only a fraction of the sample consumed for immunoblots, fully quantitative and high throughput. Application of the assay to EVs isolated by size exclusion chromatography (SEC) and precipitation revealed differences in yield, purity and recovery of subpopulations.

Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH.

Optimisation and comparison of orthogonal methods for separation and characterisation of extracellular vesicles to investigate how representative infant milk formula is of milk.

Many infants are fed infant milk formula (IMF). However, IMF production from skim milk (SM) involves harsh treatment. So, we hypothesised that the quantity and/or quality of extracellular vesicles (EVs) in IMF may be reduced. Thus, firstly, we aimed to optimise separation of EVs from IMF and SM and, secondly, we aimed to compare the EV isolates from these two sources. Prior to EV isolation, abundant casein micelles of similar sizes to EVs were removed by treating milk samples with either acetic acid or hydrochloric acid. Samples progressed to differential ultracentrifugation (DUC) or gradient ultracentrifugation (GUC). EV characterisation included BCA, SDS-PAGE, nanoparticle tracking (NTA), electron microscopy (TEM), immunoblotting, and imaging flow cytometry (IFCM). Reduced EV concentrations were found in IMF. SM-derived EVs were intact, while IMF contained disrupted EV-like structures. EV biomarkers were more abundant with isolates from SM, indicating EV proteins in IMF are compromised. Altogether, a suitable method combining acid pre-treatment with GUC for EV separation from milk products was developed. EVs appear to be substantially compromised in IMF compared to SM.

Importance of between and within Subject Variability in Extracellular Vesicle Abundance and Cargo when Performing Biomarker Analyses.

Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a 'liquid biopsy' to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.

Exploring the Use of Serum-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Hepatic Cytochromes P450 and Organic Anion Transporting Polypeptides.

Liver-derived small extracellular vesicles (sEVs), prepared from small sets of banked serum samples using a novel two-step protocol, were deployed as liquid biopsy to study the induction of cytochromes P450 (CYP3A4, CYP3A5, and CYP2D6) and organic anion transporting polypeptides (OATP1B1 and OATP1B3) during pregnancy (nonpregnant (T0), first, second, and third (T3) trimester women; N = 3 each) and after administration of rifampicin (RIF) to healthy male subjects. Proteomic analysis revealed induction (mean fold-increase, 90% confidence interval) of sEV CYP3A4 after RIF 300 mg × 7 days (3.5, 95% CI = 2.5-4.5, N = 4, P = 0.029) and 600 mg × 14 days (3.7, 95% CI = 2.1-6.0, N = 5, P = 0.018) consistent with the mean oral midazolam area under the plasma concentration time curve (AUC) ratio in the same subjects (0.28, 95% CI = 0.22-0.34, P < 0.0001; and 0.17, 95% CI = 0.13-0.22, P < 0.0001). Compared with CYP3A4, liver sEV CYP3A5 protein (subjects genotyped CYP3A5*1/*3) was weakly induced (≤ 1.5-fold). It was also possible to measure liver sEV-catalyzed dextromethorphan (DEX) O-demethylation to dextrorphan (DXO), correlated with sEV CYP2D6 expression (r = 0.917, P = 0.0001; N = 10) and 3-hour plasma DXO-to-DEX concentration ratio (r = 0.843, P = 0.002, N = 10), and show that CYP2D6 was not induced by RIF. Nonparametric analysis of liver sEV revealed significantly higher CYP3A4 (3.2-fold, P = 0.003) and CYP2D6 (3.7-fold, P = 0.03) protein expression in T3 vs. T0 women. In contrast, expression of both OATPs in liver sEV was unaltered by RIF administration and pregnancy.

Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation.

MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary.

Correction: BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer.

[This corrects the article DOI: 10.18632/oncotarget.24590.].

BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer.

Triple-negative breast cancer (TNBC), is a heterogeneous disease characterised by absence of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of human epidermal growth factor receptor 2 (HER2). TNBC patients can exhibit poor prognosis and high recurrence stages despite early response to chemotherapy treatment. In this study, we identified a pro-survival signalling protein BCL2- associated athanogene 3 (BAG3) to be highly expressed in a subset of TNBC cell lines and tumour tissues. High mRNA expression of BAG3 in TNBC patient cohorts significantly associated with a lower recurrence free survival. The epidermal growth factor receptor (EGFR) is amplified in TNBC and EGFR signalling dynamics impinge on cancer cell survival and disease recurrence. We found a correlation between BAG3 and EGFR expression in TNBC cell lines and determined that BAG3 can regulate tumour cell proliferation, migration and invasion in EGFR expressing TNBC cells lines. We identified an interaction between BAG3 and components of the EGFR signalling networks using mass spectrometry. Furthermore, BAG3 contributed to regulation of proliferation in TNBC cell lines by reducing the activation of components of the PI3K/AKT and FAK/Src signalling subnetworks. Finally, we found that combined targeting of BAG3 and EGFR was more effective than inhibition of EGFR with Cetuximab alone in TNBC cell lines. This study demonstrates a role for BAG3 in regulation of distinct EGFR modules and highlights the potential of BAG3 as a therapeutic target in TNBC.

The microenvironment of visceral adipose tissue and liver alter natural killer cell viability and function.

The role of NK cells in visceral adipose tissue (VAT) and liver inflammation in obesity is not fully understood. This study investigated the frequency, cytokine expression, chemokine receptor, and cytotoxicity receptor profile of NK cells in the blood, omentum, and liver of patients with the obesity-associated cancer, oesophageal adenocarcinoma (OAC). The effect of chronically inflamed tissue microenvironments on NK cell viability and function was also examined. We identified significantly lower NK cell frequencies in the liver of OAC patients compared with healthy controls and within the omentum and liver of OAC patients compared with blood, whereas IL-10-producing populations were significantly higher. Interestingly, our data suggest that reduced frequencies of NK cells in omentum and liver of OAC patients are not a result of impaired NK cell chemotaxis to these tissues. In fact, our functional data revealed that secreted factors from omentum and liver of OAC patients induce significant levels of NK cell death and lead to reduced percentages of TNF-α+ and NKP46+ NK cells and higher frequencies of IL-10-producing NK cells. Together, these data suggest that the omental and hepatic microenvironments of OAC patients alter the NK cell phenotype to a more anti-inflammatory homeostatic role.