Metabolomics Analysis Reveals Altered Metabolic Pathways and Response to Doxorubicin in Drug-Resistant Triple-Negative Breast Cancer Cells

This study aimed to investigate metabolic changes following the acquisition of resistance to doxorubicin in the triple-negative breast cancer (TNBC) cell line MDA-MB-231. Two drug-resistant cell lines, DOX-RES-50 and DOX-RES-100, were generated by treating MDA-MB-231 cells with doxorubicin for 24 h and allowing them to recover for six weeks. Both drug-resistant cell lines demonstrated an increase in doxorubicin IC50 values, indicating acquired drug resistance. Metabolomics analysis showed clear separation between the parental MDA-MB-231 cell line and the drug-resistant cell lines. Pathway analysis revealed that arginine and proline metabolism, glutathione metabolism, and beta-alanine metabolism were significantly perturbed in the drug-resistant cell lines compared to the parental cell line. After matching signals to an in-house library of reference standards, significant decreases in short- and medium-chain acylcarnitines and significant increases in long-chain acylcarnitines, 5-oxoproline, and 7-ketodeoxycholic acid were observed in the resistant cell lines as compared to the parental MDA-MB-231 cell line. In addition to baseline metabolic differences, we also investigated differences in metabolic responses in resistant cell lines upon a second exposure at multiple concentrations. Results indicate that whereas the parental MDA-MB-231 cell line had many metabolites that responded to doxorubicin in a dose-dependent manner, the two resistant cell lines lost a dose-dependent response for the majority of these metabolites. The study’s findings provide insight into how metabolism is altered during the acquisition of resistance in TNBC cells and how the metabolic response to doxorubicin changes upon repeated treatment. This information can potentially identify novel targets to prevent or reverse multi-drug resistance in TNBC, and also demonstrate the usefulness of metabolomics technology in identifying new mechanisms of drug resistance in cancer and potential drug targets

Commonalities in Metabolic Reprogramming between Tobacco Use and Oral Cancer

Tobacco use is a major public health concern and is linked to myriad diseases, including cancer. The link between tobacco use and oral cancer, specifically, is very strong, making tobacco use one of the primary risk factors for oral cancer. While this association is well known, the underlying biochemical changes that result from tobacco use, and how this links to metabolic phenotypes of oral cancer, is not well understood. To address this knowledge gap, a combination of literature reviews and metabolomics studies were performed to identify commonalities in metabolic perturbations between tobacco use and oral cancers. Metabolomics analysis was performed on pooled reference urine from smokers and non-smokers, healthy and malignant oral tissues, and cultured oral cells with or without treatment of the well-known tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Alterations in amino acid metabolism, carbohydrates/oxidative phosphorylation, fatty acid oxidation, nucleotide metabolism, steroid metabolism, and vitamin metabolism were found to be shared between tobacco use and oral cancer. These results support the conclusion that tobacco use metabolically reprograms oral cells to support malignant transformation through these pathways. These metabolic reprogramming events may be potential targets to prevent or treat oral cancers that arise from tobacco use

Metabolomics Analysis Reveals Novel Targets of Chemosensitizing Polyphenols and Omega-3 Polyunsaturated Fatty Acids in Triple Negative Breast Cancer Cells

Triple negative breast cancer (TNBC) is a subtype of breast cancer with typically poorer outcomes due to its aggressive clinical behavior and lack of targeted treatment options. Currently, treatment is limited to the administration of high-dose chemotherapeutics, which results in significant toxicities and drug resistance. As such, there is a need to de-escalate chemotherapeutic doses in TNBC while also retaining/improving treatment efficacy. Dietary polyphenols and omega-3 polyunsaturated fatty acids (PUFAs) have been demonstrated to have unique properties in experimental models of TNBC, improving the efficacy of doxorubicin and reversing multi-drug resistance. However, the pleiotropic nature of these compounds has caused their mechanisms to remain elusive, preventing the development of more potent mimetics to take advantage of their properties. Using untargeted metabolomics, we identify a diverse set of metabolites/metabolic pathways that are targeted by these compounds following treatment in MDA-MB-231 cells. Furthermore, we demonstrate that these chemosensitizers do not all target the same metabolic processes, but rather organize into distinct clusters based on similarities among metabolic targets. Common themes in metabolic targets included amino acid metabolism (particularly one-carbon and glutamine metabolism) and alterations in fatty acid oxidation. Moreover, doxorubicin treatment alone generally targeted different metabolites/pathways than chemosensitizers. This information provides novel insights into chemosensitization mechanisms in TNBC

Assessment of corrosive attack of Fe9Cr1Mo alloys in pressurised CO2 for prediction of breakaway oxidation

To provide clarity on the poorly-understood mechanism of breakaway oxidation, corrosion of Fe9Cr1Mo steel in pressurised CO is quantified and modelled. The temperature range 400–640 , relevant to nuclear power plants, is emphasised. Attack is in the form of combined oxide scale growth and internal carburisation of the metal. Carbon activity in the metal at its surface exhibits a strong time dependence consistent with the kinetically-limited transport of carbon due to the slow Boudouard reaction. Breakaway is associated with the approach to saturation of the steel with respect to carbon. Diffusion modelling agrees well with steel carbide precipitation observations

The United States COVID-19 Forecast Hub dataset

Academic researchers, government agencies, industry groups, and individuals have produced forecasts at an unprecedented scale during the COVID-19 pandemic. To leverage these forecasts, the United States Centers for Disease Control and Prevention (CDC) partnered with an academic research lab at the University of Massachusetts Amherst to create the US COVID-19 Forecast Hub. Launched in April 2020, the Forecast Hub is a dataset with point and probabilistic forecasts of incident cases, incident hospitalizations, incident deaths, and cumulative deaths due to COVID-19 at county, state, and national, levels in the United States. Included forecasts represent a variety of modeling approaches, data sources, and assumptions regarding the spread of COVID-19. The goal of this dataset is to establish a standardized and comparable set of short-term forecasts from modeling teams. These data can be used to develop ensemble models, communicate forecasts to the public, create visualizations, compare models, and inform policies regarding COVID-19 mitigation. These open-source data are available via download from GitHub, through an online API, and through R packages

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017

Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis:a randomised, double-blind, placebo-controlled, phase 2b trial

Background: Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral CFTR gene therapy in patients with cystic fibrosis. Methods: We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50–90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene–liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867. Findings: Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups. Interpretation: Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials

A Review of Metabolic Targets of Anticancer Nutrients and Nutraceuticals in Pre-Clinical Models of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is notoriously aggressive and has poorer outcomes as compared with other breast cancer subtypes. Due to a lack of targeted therapies, TNBC is often treated with chemotherapeutics as opposed to hormone therapy or other targeted therapies available to individuals with estrogen receptor positive (ER+) breast cancers. Because of the lack of treatment options for TNBC, other therapeutic avenues are being explored. Metabolic reprogramming, a hallmark of cancer, provides potential opportunities to target cancer cells more specifically, increasing efficacy and reducing side effects. Nutrients serve a significant role in metabolic processes involved in DNA transcription, protein folding, and function as co-factors in enzyme activity, and may provide novel strategies to target cancer cell metabolism in TNBC. This article reviews studies that have investigated how nutrients/nutraceuticals target metabolic processes in TNBC cells alone or in combination with existing drugs to exert anticancer effects. These agents have been shown to cause perturbations in many metabolic processes related to glucose metabolism, fatty acid metabolism, as well as autophagy and oxidative stress-related metabolism. With this information, we present the potential of nutrients as metabolism-directed anticancer agents and the potential for using these agents alone or in cocktails as a new direction for TNBC therapy

Commonalities in Metabolic Reprogramming between Tobacco Use and Oral Cancer

Tobacco use is a major public health concern and is linked to myriad diseases, including cancer. The link between tobacco use and oral cancer, specifically, is very strong, making tobacco use one of the primary risk factors for oral cancer. While this association is well known, the underlying biochemical changes that result from tobacco use, and how this links to metabolic phenotypes of oral cancer, is not well understood. To address this knowledge gap, a combination of literature reviews and metabolomics studies were performed to identify commonalities in metabolic perturbations between tobacco use and oral cancers. Metabolomics analysis was performed on pooled reference urine from smokers and non-smokers, healthy and malignant oral tissues, and cultured oral cells with or without treatment of the well-known tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Alterations in amino acid metabolism, carbohydrates/oxidative phosphorylation, fatty acid oxidation, nucleotide metabolism, steroid metabolism, and vitamin metabolism were found to be shared between tobacco use and oral cancer. These results support the conclusion that tobacco use metabolically reprograms oral cells to support malignant transformation through these pathways. These metabolic reprogramming events may be potential targets to prevent or treat oral cancers that arise from tobacco use