Circulating tumour cells (CTCs) are attractive "liquid biopsy" candidates that could provide insights into the different phenotypes of tumours present within a patient. The epithelial-to-mesenchymal transition (EMT) of CTCs is considered a critical step in tumour metastasis; however, it may confound traditional epithelial feature-based CTC isolation and detection. We applied single-cell copy number alteration (CNA) analysis for the identification of genomic alterations to confirm the neoplastic nature of circulating cells with only mesenchymal phenotypes.We isolated CTCs from blood samples collected from 46 NSCLC patients using the Parsortix system. Enriched cells were subjected to immunofluorescent staining for CTC identification using a multi-marker panel comprising both epithelial and mesenchymal markers. A subset of isolated CTCs was subjected to whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for the analysis of copy number alterations (CNAs).CTCs were detected in 16/46 (34.8%) patients, inclusive of CK+/EpCAM+ CTCs (3/46, 6.5%) and Vim+ CTCs (13/46, 28.3%). Clusters of Vim+ cells were detected in 8 samples, which constitutes 50% of the total number of NSCLC patients with CTCs. No patients had detectable hybrid CK+/EpCAM+/Vim+ cells. All of the tested CK+/EpCAM+ CTCs and 7/8 Vim+ CTCs or CTC clusters carried CNAs confirming their neoplastic nature. Notably, the Vim+ cluster with no CNAs was characterised by spindle morphology and, therefore, defined as normal mesenchymal circulating cells.Our results revealed that CK-negative, vimentin-expressing cells represent a large proportion of CTCs detected in NSCLC patients, which are likely missed by standard epithelial-marker-dependent CTC categorisation.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and neuropathological features, including abnormal deposition of amyloid-β (Aβ) peptides, intracellular neurofibrillary tangles, and neuronal death. Identifying therapeutics which can reduce memo ry deficits at an early stage of the disease has the advantage of slowing or even reversing disease progression before irreversible brain damage has occurred. Consequently, in this study, we investigated the ability of the histone deacetylase inhibitor sodium butyrate (NaB) to attenuate memory deficits in the 5xFAD mouse model of AD following a 12-week feeding regimen. 5xFAD mice demonstrate a unique time course of Aβ pathology, developing Aβ plaques as early as 2 months. Male mice were assigned to either a control diet or a NaB-supplemented diet which was administered at either 5 mg/kg/day, or 15 mg/kg/day for 12 weeks (each group, N = 15). Supplementation commenced at an early disease stage (8–10 weeks of age). Behavioral testing (contextual and cued fear conditioning) was undertaken, and brain Aβ levels measured, at the end of the 12-week intervention. NaB had profound effects on Aβ levels and on associative learning and cognitive functioning. A 40% reduction in brain Aβ levels and a 25% increase in fear response in both the cued and contextual testing was observed in the NaB-treated animals compared to the control group. These findings suggest that NaB warrants further investigation as a potential therapeutic agent in the treatment of cognitive deficits associated with early stages of AD.
Latrepirdine (DimebonTM) is a promising drug for reducing the cognitive dysfunction of AD and is currently in phase III clinical trials. Although its mechanism of action is not completely understood, latrepirdine has recently been reported to modulate Aβ metabolism and to do so in an unexpected way: latrepirdine increases extracellular Aβ concentrations in the interstitial fluid of the living mouse brain and in the releasate from isolated intact nerve terminals. Further investigations into how latrepirdine modulates Aβ metabolism, and how the drug may otherwise be producing the observed clinical benefits in AD are essential for the full characterization of this promising treatment. Our aim in this study was to determine the effects of latrepirdine on Aβ oligomer formation, clearance, and associated toxicity. The ability of latrepirdine to reduce Aβ oligomer induced toxicity was assessed in neuroblastoma and yeast (Saccharomyces cerevisiae) cells. Effects on Aβ oligomer formation were assessed by SDS-PAGE and TEM analysis. Intracellular Aβ clearance was assessed in vitro in yeast cells and peripheral clearance of Aβ42 was assessed in APOE KO mice in the presence of a human APOE ∊4 transgene. Latrepirdine reduced toxicity in neuroblastoma and yeast cells. Oligomer formation was reduced at the lower concentrations of latrepirdine whereas at higher concentrations, the drug enhanced oligomers formation and failed to rescue cell death. Latrepirdine was also shown to enhance the clearance of GFP-labeled Aβ42 in yeast cells, apparently through enhancing autophagy. Latrepirdine did not improve viability of Aβ treated cells lacking essential genes for autophagy, suggesting that autophagy may be one mechanism by which latrepirdine improves cell viability. In vivo, peripheral clearance of Aβ42 was increased and uptake by the liver was increased in the presence of latrepirdine, providing evidence that latrepirdine may enhance Aβ clearance in an intact mammal. Our data suggest novel actions for latrepirdine in modulating Aβ oligomer formation, enhancing Aβ clearance, and attenuating Aβ induced toxicity. How these effects relate to the clinically apparent drug-related changes in cognition and behavior will need to be determined in order to optimize the development of additional latrepirdine-related molecules that specifically target AD.
Antibodies against the programmed death-1 (PD-1) receptor and its ligand (PD-L1) have been recently approved for small-cell lung cancer (SCLC) treatment. Circulating tumour cells (CTCs) have emerged as an appealing liquid biopsy candidate that could enhance treatment decision-making in systemic therapy for SCLC patients. Several current technologies enrich CTCs using specific surface epitopes, size, rigidity, or dielectric properties. However, they are hampered by the heterogeneity of the enriched cells from blood samples.We evaluated two CTC enrichment systems: EpCAM conjugated to magnetic beads and a microfluidic device (Parsortix, Angle plc). PD-L1 expression was evaluated on the isolated CTCs. Twenty-three blood samples were collected from 21 patients with SCLC. PD-L1 expression was determined on CTCs through immunofluorescent staining.CTCs were found in 14/23 (60.9%) of the samples, with 11/23 (47.8%) through EpCAM-coated magnetic beads (range, 4-1,611 CTCs/8 mL; median =5) and 11/20 (55.0%) using the Parsortix system (range, 1-165 CTCs/8 mL; median =4). Notably, a total of 17 EpCAM-negative CTCs were isolated using the Parsortix system. PD-L1 expression was detected on 268 of the 3,501 (7.7%) CTCs isolated with EpCAM-coated beads and in 33/366 (9.0%) of the CTCs isolated with the Parsortix system. No vimentin expression was observed in any of the detected CTCs.Overall, we identified a population of EpCAM-negative SCLC CTCs and showed that PD-L1 expression can be assessed on CTCs from SCLC patients. Comparison to tumour and treatment outcomes is needed to validate the potential of CTCs as an alternative sample for the assessment of PD-L1 expression in SCLC.
Abstract Baculoviral protein expression in insect cells has been previously used to generate large quantities of a protein of interest for subsequent use in biochemical and structural analyses. The MultiBac baculovirus protein expression system has enabled, the use of a single baculovirus to reconstitute a protein complex of interest, resulting in a larger protein yield. Using this system, we aimed to reconstruct the gamma (γ)-secretase complex, a multiprotein enzyme complex essential for the production of amyloid-β (Aβ) protein. A MultiBac vector containing all components of the γ-secretase complex was generated and expression was observed for all components. The complex was active in processing APP and Notch derived γ-secretase substrates and proteolysis could be inhibited with γ-secretase inhibitors, confirming specificity of the recombinant γ-secretase enzyme. Finally, affinity purification was used to purify an active recombinant γ-secretase complex. In this study we demonstrated that the MultiBac protein expression system can be used to generate an active γ-secretase complex and provides a new tool to study γ-secretase enzyme and its variants.
Latrepirdine (Dimebon™,) is the first drug to have produced both improvements in cognition in Alzheimer's disease (AD) while also showing evidence of stabilising disease progression; however, its mechanism(s) of action is not fully understood. Recent evidence has shown that acute dosing with latrepiridine causes an elevation in extracellular levels of Aβ42. The significance of this increase remains to be determined and may suggest that latrepirdine modulates Aβ metabolism, clearance, and/or aggregation. In the current study, we focused on whether latrepiridine modulated Aβ aggregation and whether this was associated with suppression in Aβ toxicity in neuronal and/or yeast (Saccharomyces cerevisiae) cells. Oligomeric Aβ42 (oAβ42) prepared in the presence or absence of various concentrations of latrepirdine were analysed by SDS-PAGE and immunoblotting. The oAβ42 preparations were also visualized by transmission electron microscopy (TEM). M17 neuroblastoma and S. cerevisiae cells were treated with oAβ42 in the presence or absence of latrepirdine (5 nM - 20 μM), and cell viability was assessed by MTS assay. SDS-PAGE analysis showed that oligomer formation was reduced at 10:1 and 5:1 molar ratios of Aβ: latrepirdine, whereas at 50 μM (2:1 ratio), Aβ aggregation was enhanced. At similar concentrations, TEM analysis of oAβ42-latrepirdine preparations showed that latrepirdine may promote conformational changes in oAβ42. Pretreatment of neuroblastoma cells with latrepirdine (0.5-5 μM) prior to the addition of oAβ42 suggested that latrepirdine significantly reduced oAβ42-associated toxicity, whilst higher concentrations (10 and 20 μM) showed no significant protective effects. Similar results were observed for S. cerevisiae cells treated with oAβ42 in the absence or presence of latrepirdine. These are the first data to suggest that latrepirdine can reduce Aβ oligomer formation and thereby attenuate oAβ42 toxicity in neuroblastoma and yeast cells. Anti-oligomerization properties of latrepirdine could underlie some of its cognitive benefits in AD.
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