In vivo investigation of hyperpolarized [1,3-13C2]acetoacetate as a metabolic probe in normal brain and in glioma.

Dysregulation in NAD+/NADH levels is associated with increased cell division and elevated levels of reactive oxygen species in rapidly proliferating cancer cells. Conversion of the ketone body acetoacetate (AcAc) to β-hydroxybutyrate (β-HB) by the mitochondrial enzyme β-hydroxybutyrate dehydrogenase (BDH) depends upon NADH availability. The β-HB-to-AcAc ratio is therefore expected to reflect mitochondrial redox. Previous studies reported the potential of hyperpolarized 13C-AcAc to monitor mitochondrial redox in cells, perfused organs and in vivo. However, the ability of hyperpolarized 13C-AcAc to cross the blood brain barrier (BBB) and its potential to monitor brain metabolism remained unknown. Our goal was to assess the value of hyperpolarized [1,3-13C2]AcAc in healthy and tumor-bearing mice in vivo. Following hyperpolarized [1,3-13C2]AcAc injection, production of [1,3-13C2]β-HB was detected in normal and tumor-bearing mice. Significantly higher levels of [1-13C]AcAc and lower [1-13C]β-HB-to-[1-13C]AcAc ratios were observed in tumor-bearing mice. These results were consistent with decreased BDH activity in tumors and associated with increased total cellular NAD+/NADH. Our study confirmed that AcAc crosses the BBB and can be used for monitoring metabolism in the brain. It highlights the potential of AcAc for future clinical translation and its potential utility for monitoring metabolic changes associated with glioma, and other neurological disorders

Determination of epithelial growth factor receptor mutations in circulatory tumour cells from non-small cell lung cancer patients isolated using a novel microfluidic device

Patients with epidermal growth factor receptor (EGFR) sensitizing mutations in non small cell lung cancer (NSCLC) receive benefit from Tyrosine Kinase inhibitors. Accurate selection of patients before treatment is highly dependent on precise molecular diagnosis of EGFR mutations. Presently in the clinic, the diagnostic samples routinely used tumour biopsy and/or cell free DNA (cfDNA), are not sufficiently effective for precise diagnosis. Circulatory tumour cells (CTC) in blood have been explored successfully as alternative and complementary diagnostic markers to the current clinical tools. However, utility in the clinics has been hampered by the relatively low concentration of CTC in blood, and the lack of robust technologies that are adaptable for routine use. The present study describes the design and optimization of an immunomagnetic based microfluidic device (Lung card version II) that isolates CTC expressing the epithelial cell adhesion molecule (EpCAM) from blood with high capture efficiency and purity. The device is a 2-part system comprising a disposable chip that is simple in design and a reusable microfluidic unit that contains a mobile magnetic arm. The simple design and work-flow process of the device ensures cost efficiency for scalability and, ultimately, use in the clinic. The device was initially validated for its capability to isolate EpCAM positive cells. Results from spiking carboxylfluorescein succinimidyl ester stained EpCAM positive cells in media/blood showed a capture efficiency of ≥ 65% and a purity ≥ 97% from a 13ml sample in 50 minutes. The isolated CTC from NSCLC patients (n=38) were analysed for mRNA markers specific to malignant cells and were characterized for EGFR mutations following PCR and next generation sequencing. The mutational status of CTC was compared to that obtained from matched, tumour biopsy, samples. Significantly more mutations (P=0.0173) were detected in CTC enriched samples than the matched biopsy. Interestingly, mutations were detected in only 4 biopsy samples and the mutations detected in the biopsy were only concordant with results from CTC enriched samples for 1 patient. Exon 19 deletion was the most frequent mutation detected (86.7%) with rare mutations such as: L792P, C797S, H509R also been detected in CTC, and the present study reports the detection of K708R mutation in NSCLC for the first time. The clinical outcomes of patients who were positive for EGFR mutation from CTC, but had been placed on therapies based on mutation results from tissue biopsy were evaluated in this study. The results showed that no significant progression free survival (PFS) benefit was attained when comparing treatment response between patients whose CTC possessed an EGFR mutation and patients whose CTC possessed no EGFR mutation (10 months vs26 months p value-0.3420 HR- 0.76 95% CI- 0.2498-2.319). In summary the results from this study showed that the microfluidic device captured CTC with efficiency equal to other immuno-affinity based devices but had better purity rates and throughput and also that the device can be utilized for CTC processing for downstream analysis. Results from this current study further demonstrated the clinical potential of CTC+NGS matrix for the detection of EGFR mutations and the prospective impact it would have for precision oncology in NSCLC are discussed

Sugar Alcohols Have a Key Role in Pathogenesis of Chronic Liver Disease and Hepatocellular Carcinoma in Whole Blood and Liver Tissues.

The major risk factors for hepatocellular carcinoma (HCC) are hepatitis C and B viral infections that proceed to Chronic Liver Disease (CLD). Yet, the early diagnosis and treatment of HCC are challenging because the pathogenesis of HCC is not fully defined. To better understand the onset and development of HCC, untargeted GC-TOF MS metabolomics data were acquired from resected human HCC tissues and their paired non-tumor hepatic tissues (n = 46). Blood samples of the same HCC subjects (n = 23) were compared to CLD (n = 15) and healthy control (n = 15) blood samples. The participants were recruited from the National Liver Institute in Egypt. The GC-TOF MS data yielded 194 structurally annotated compounds. The most strikingly significant alteration was found for the class of sugar alcohols that were up-regulated in blood of HCC patients compared to CLD subjects (p < 2.4 × 10-12) and CLD compared to healthy controls (p = 4.1 × 10-7). In HCC tissues, sugar alcohols were the most significant (p < 1 × 10-6) class differentiating resected HCC tissues from non-malignant hepatic tissues for all HCC patients. Alteration of sugar alcohol levels in liver tissues also defined early-stage HCC from their paired non-malignant hepatic tissues (p = 2.7 × 10-6). In blood, sugar alcohols differentiated HCC from CLD subjects with an ROC-curve of 0.875 compared to 0.685 for the classic HCC biomarker alpha-fetoprotein. Blood sugar alcohol levels steadily increased from healthy controls to CLD to early stages of HCC and finally, to late-stage HCC patients. The increase in sugar alcohol levels indicates a role of aldo-keto reductases in the pathogenesis of HCC, possibly opening novel diagnostic and therapeutic options after in-depth validation

Losartan Treatment Attenuates Tumor-induced Myocardial Dysfunction

Fatigue and muscle wasting are common symptoms experienced by cancer patients. Data from animal models demonstrate that angiotensin is involved in tumor-induced muscle wasting, and that tumor growth can independently affect myocardial function, which could contribute to fatigue in cancer patients. In clinical studies, inhibitors of angiotensin converting enzyme (ACE) can prevent the development of chemotherapy-induced cardiovascular dysfunction, suggesting a mechanistic role for the renin–angiotensin–aldosterone system (RAAS). In the present study, we investigated whether an angiotensin (AT) 1-receptor antagonist could prevent the development of tumor-associated myocardial dysfunction. Methods and results: Colon26 adenocarcinoma (c26) cells were implanted into female CD2F1 mice at 8 weeks of age. Simultaneously, mice were administered Losartan (10 mg/kg) daily via their drinking water. In vivo echocardiography, blood pressure, in vitro cardiomyocyte function, cell proliferation assays, and measures of systemic inflammation and myocardial protein degradation were performed 19 days following tumor cell injection. Losartan treatment prevented tumor-induced loss of muscle mass and in vitro c26 cell proliferation, decreased tumor weight, and attenuated myocardial expression of interleukin-6. Furthermore, Losartan treatment mitigated tumor-associated alterations in calcium signaling in cardiomyocytes, which was associated with improved myocyte contraction velocity, systolic function, and blood pressures in the hearts of tumor-bearing mice. Conclusions: These data suggest that Losartan may mitigate tumor-induced myocardial dysfunction and inflammation

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Here, we present stable isotope cysteine labelling with iodoacetamide (SICyLIA), a mass spectrometry-based workflow to assess proteome-scale cysteine oxidation. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. Applying SICyLIA to diverse cellular models and primary tissues provides detailed insights into thiol oxidation proteomes. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys identifies oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies