Proteomic profiling of small extracellular vesicles derived from mouse pancreatic cancer and stellate cells: Role in pancreatic cancer.

Small extracellular vesicles (sEVs) are cell-derived vesicles evolving as important elements involved in all stages of cancers. sEVs bear unique protein signatures that may serve as biomarkers. Pancreatic cancer (PC) records a very poor survival rate owing to its late diagnosis and several cancer cell-derived proteins have been reported as candidate biomarkers. However, given the pivotal role played by stellate cells (PSCs, which produce the collagenous stroma in PC), it is essential to also assess PSC-sEV cargo in biomarker discovery. Thus, this study aimed to isolate and characterise sEVs from mouse PC cells and PSCs cultured alone or as co-cultures and performed proteomic profiling and pathway analysis. Proteomics confirmed the enrichment of specific markers in the sEVs compared to their cells of origin as well as the proteins that are known to express in each of the culture types. Most importantly, for the first time it was revealed that PSC-sEVs are enriched in proteins (including G6PI, PGAM1, ENO1, ENO3, and LDHA) that mediate pathways related to development of diabetes, such as glucose metabolism and gluconeogenesis revealing a potential role of PSCs in pancreatic cancer-related diabetes (PCRD). PCRD is now considered a harbinger of PC and further research will enable to identify the role of these components in PCRD and may develop as novel candidate biomarkers of PC.

Single-Circulating Tumor Cell Whole Genome Amplification to Unravel Cancer Heterogeneity and Actionable Biomarkers.

The field of single-cell analysis has advanced rapidly in the last decade and is providing new insights into the characterization of intercellular genetic heterogeneity and complexity, especially in human cancer. In this regard, analyzing single circulating tumor cells (CTCs) is becoming particularly attractive due to the easy access to CTCs from simple blood samples called "liquid biopsies". Analysis of multiple single CTCs has the potential to allow the identification and characterization of cancer heterogeneity to guide best therapy and predict therapeutic response. However, single-CTC analysis is restricted by the low amounts of DNA in a single cell genome. Whole genome amplification (WGA) techniques have emerged as a key step, enabling single-cell downstream molecular analysis. Here, we provide an overview of recent advances in WGA and their applications in the genetic analysis of single CTCs, along with prospective views towards clinical applications. First, we focus on the technical challenges of isolating and recovering single CTCs and then explore different WGA methodologies and recent developments which have been utilized to amplify single cell genomes for further downstream analysis. Lastly, we list a portfolio of CTC studies which employ WGA and single-cell analysis for genetic heterogeneity and biomarker detection.

Geospatial Variation of Real-World Tailpipe Emission Rates for Light-Duty Gasoline Vehicles.

Spatial variability in real-world on-road tailpipe light-duty gasoline vehicle nitrogen oxides, hydrocarbon, carbon monoxide, and carbon dioxide emission rates, the locations of emissions hotspots, and factors that explain spatial variability are quantified. A sample of 205 vehicles were measured on four predefined round-trip study routes using Portable Emission Measurement Systems. The trips on each route were divided into segments, averaging 1/4 mile in length. Segment-average emission rates were estimated based on measured 1 Hz emission rates. Emission hotspots are defined as segments with ≥90th percentile of segment-average emission rates. The hotspots have average emission rates 2-4 times greater, depending on the pollutant, than other segments. Hotspots are of heterogeneous characteristics including road attributes and vehicle activity metrics. For example, some hotspots were on arterial roads with an upstream signalized intersection and positive road grade, whereas some hotspots were on interstates with positive grade. Vehicle activity metrics, including average vehicle specific power and relative positive acceleration, help identify the hotspots. To reliably identify a fleet-average hotspot, data are needed for at least 36-130 vehicles, depending on the pollutant.

Air quality and climate impacts due to CNG conversion of motor vehicles in Dhaka, Bangladesh.

Dhaka had recently experienced rapid conversion of its motor vehicle fleet to run on compressed natural gas (CNG). This paper quantifies ex-post the air quality and climate benefits of the CNG conversion policy, including monetary valuations, through an impact pathway approach. Around 2045 (1665) avoided premature deaths in greater Dhaka (City Corporation) can be attributed to air quality improvements from the CNG conversion policy in 2010, resulting in a saving of around USD 400 million. Majority of these health benefits resulted from the conversion of high-emitting diesel vehicles. CNG conversion was clearly detrimental from climate change perspective using the changes in CO2 and CH4 only (CH4 emissions increased); however, after considering other global pollutants (especially black carbon), the climate impact was ambiguous. Uncertainty assessment using input distributions and Monte Carlo simulation along with a sensitivity analysis show that large uncertainties remain for climate impacts. For our most likely estimate, there were some climate costs, valued at USD 17.7 million, which is an order of magnitude smaller than the air quality benefits. This indicates that such policies can and should be undertaken on the grounds of improving local air pollution alone and that precautions should be taken to reduce the potentially unintended increases in GHG emissions or other unintended effects.

Prognostic and Predictive Value of Liquid Biopsy-Derived Androgen Receptor Variant 7 (AR-V7) in Prostate Cancer: A Systematic Review and Meta-Analysis.

In advanced prostate cancer, access to recent diagnostic tissue samples is restricted and this affects the analysis of the association of evolving biomarkers such as AR-V7 with metastatic castrate resistance. Liquid biopsies are emerging as alternative analytes. To clarify clinical value of AR-V7 detection from liquid biopsies, here we performed a meta-analysis on the prognostic and predictive value of androgen receptor variant 7 (AR-V7) detected from liquid biopsy for patients with prostate cancer (PC), three databases, the Embase, Medline, and Scopus were searched up to September 2021. A total of 37 studies were included. The effects of liquid biopsy AR-V7 status on overall survival (OS), radiographic progression-free survival (PFS), and prostate-specific antigen (PSA)-PFS were calculated with RevMan 5.3 software. AR-V7 positivity detected in liquid biopsy significantly associates with worse OS, PFS, and PSA-PFS (P <0.00001). A subgroup analysis of patients treated with androgen receptor signaling inhibitors (ARSi such as abiraterone and enzalutamide) showed a significant association of AR-V7 positivity with poorer OS, PFS, and PSA-PFS. A statistically significant association with OS was also found in taxane-treated patients (P = 0.04), but not for PFS (P = 0.21) or PSA-PFS (P = 0.93). For AR-V7 positive patients, taxane treatment has better OS outcomes than ARSi (P = 0.01). Study quality, publication bias and sensitivity analysis were integrated in the assessment. Our data show that liquid biopsy AR-V7 is a clinically useful biomarker that is associated with poor outcomes of ARSi-treated castrate resistant PC (CRPC) patients and thus has the potential to guide patient management and also to stratify patients for clinical trials. More studies on chemotherapy-treated patients are warranted. Systematic Review Registration: PROSPERO, CRD42021239353.

Choice of antibody is critical for specific and sensitive detection of androgen receptor splice variant-7 in circulating tumor cells.

Androgen receptor variant 7 (AR-V7) is an important biomarker to guide treatment options for castration-resistant prostate cancer (CRPC) patients. Its detectability in circulating tumour cells (CTCs) opens non-invasive diagnostic avenues. While detectable at the transcript level, AR-V7 protein detection in CTCs may add additional information and clinical relevance. The aim of this study was to compare commercially available anti-AR-V7 antibodies and establish reliable AR-V7 immunocytostaining applicable to CTCs from prostate cancer (PCa) patients. We compared seven AR-V7 antibodies by western blotting and immmunocytostaining using a set of PCa cell lines with known AR/AR-V7 status. The emerging best antibody was validated for detection of CRPC patient CTCs enriched by negative depletion of leucocytes. The anti-AR-V7 antibody, clone E308L emerged as the best antibody in regard to signal to noise ratio with a specific nuclear signal. Moreover, this antibody detects CRPC CTCs more efficiently compared to an antibody previously shown to detect AR-V7 CTCs. We have determined the best antibody for AR-V7 detection of CTCs, which will open future studies to correlate AR-V7 subcellular localization and potential co-localization with other proteins and cellular structures to patient outcomes.

The Prospect of Identifying Resistance Mechanisms for Castrate-Resistant Prostate Cancer Using Circulating Tumor Cells: Is Epithelial-to-Mesenchymal Transition a Key Player?

Prostate cancer (PCa) is initially driven by excessive androgen receptor (AR) signaling with androgen deprivation therapy (ADT) being a major therapeutic approach to its treatment. However, the development of drug resistance is a significant limitation on the effectiveness of both first-line and more recently developed second-line ADTs. There is a need then to study AR signaling within the context of other oncogenic signaling pathways that likely mediate this resistance. This review focuses on interactions between AR signaling, the well-known phosphatidylinositol-3-kinase/AKT pathway, and an emerging mediator of these pathways, the Hippo/YAP1 axis in metastatic castrate-resistant PCa, and their involvement in the regulation of epithelial-mesenchymal transition (EMT), a feature of disease progression and ADT resistance. Analysis of these pathways in circulating tumor cells (CTCs) may provide an opportunity to evaluate their utility as biomarkers and address their importance in the development of resistance to current ADT with potential to guide future therapies.

Detection of AR-V7 in Liquid Biopsies of Castrate Resistant Prostate Cancer Patients: A Comparison of AR-V7 Analysis in Circulating Tumor Cells, Circulating Tumor RNA and Exosomes.

Detection of androgen receptor (AR) variant 7 (AR-V7) is emerging as a clinically important biomarker in castrate resistant prostate cancer (CRPC). Detection is possible from tumor tissue, which is often inaccessible in the advanced disease setting. With recent progress in detecting AR-V7 in circulating tumor cells (CTCs), circulating tumor RNA (ctRNA) and exosomes from prostate cancer patients, liquid biopsies have emerged as an alternative to tumor biopsy. Therefore, it is important to clarify whether these approaches differ in sensitivity in order to achieve the best possible biomarker characterization for the patient. In this study, blood samples from 44 prostate cancer patients were processed for CTCs and ctRNA with subsequent AR-V7 testing, while exosomal RNA was isolated from 16 samples and tested. Detection of AR and AR-V7 was performed using a highly sensitive droplet digital PCR-based assay. AR and AR-V7 RNA were detectable in CTCs, ctRNA and exosome samples. AR-V7 detection from CTCs showed higher sensitivity and has proven specificity compared to detection from ctRNA and exosomes. Considering that CTCs are almost always present in the advanced prostate cancer setting, CTC samples should be considered the liquid biopsy of choice for the detection of this clinically important biomarker.

Comparison of real-world and certification emission rates for light duty gasoline vehicles.

U.S. light duty vehicles are subject to the U.S. Environmental Protection Agency (EPA) emission standards. Emission compliance is determined by certification testing of selected emissions from representative vehicles on standard driving cycles using chassis dynamometers. Test results are also used in many emission inventories. The dynamometer based emission rates are adjusted to provide the certification levels (CL), which must be lower than the standards for compliance. Although standard driving cycles are based on specific observations of real-world driving, they are not necessarily real-world representative. A systematic comparison of the real-world emission rates of U.S. light duty gasoline vehicles (LDGVs) versus CL, and emission standards has not been previously reported. The purpose of this work is to compare regulatory limits (both CLs and emission standards) and the real-world emissions of LDGVs. The sensitivity of the comparisons to cold start emission was assessed. Portable Emission Measurement Systems (PEMS) were used to measure hot stabilized exhaust emissions of 122 LDGVs on a specified 110 mile test route. Cold start emissions were measured with PEMS for a selected vehicle sample of 32 vehicles. Emissions were measured for carbon dioxide (CO2), carbon monoxide (CO), hydrocarbons (HC) and nitrogen oxides (NOx). For each vehicle, a Vehicle Specific Power (VSP) modal emission rate model was developed. The VSP modal rates were weighted by the standard driving cycles and real-world driving cycles to estimate the respective cycle average emission rates (CAERs). Measured vehicles were matched with certification test vehicles for comparison. For systematic trends in comparison, vehicles were classified into four groups based on the Tier 1 and Tier 2 emission regulation, and the vehicle type such as passenger car and passenger truck. Depending on the cycle-pollutant and the vehicle groups, hot stabilized CAERs are on average either statistically significantly higher than or significantly not different from the CLs, with the exception of CO on the US06 cycle, for which real-world rates are lower than CLs. Compared to the emission standards, hot stabilized CAERs are on average significantly lower. However, comparisons of CAERs and standards are sensitive to cold start emissions. For some combinations of pollutants and vehicle groups, cold start inclusive CAERs are higher than the corresponding CLs and as high as the standards. The CLs, which are based on standard driving cycles, tend to underestimate real-world emission rates. Therefore, emission inventory estimates using certification test results are potentially underestimated.