BSTRACTIt is unclear how changes in lipid droplet size and number are regulated -for example, it is not known whether this involves a signalling pathway or is directed by cellular lipid uptake. Here, we show that oleic acid stimulates lipid droplet formation by activating the long-chain fatty acid receptor FFAR4, which signals through a pertussis-toxin-sensitive G-protein signalling pathway involving phosphoinositide 3-kinase (PI3-kinase), AKT (also known as protein kinase B) and phospholipase D (PLD) activities. This initial lipid droplet formation is not dependent upon exogenous lipid, whereas the subsequent more sustained increase in the number of lipid droplets is dependent upon lipid uptake. These two mechanisms of lipid droplet formation point to distinct potential intervention points.

Sucrase-isomaltase (SI) is a highly N- and O-glycosylated intestinal brush border membrane protein. SI is sorted with high fidelity to the apical membrane via O-linked glycans that mediate its association with lipid rafts or detergent-resistant membranes (DRMs). Here, we show that N- and O-glycosylation and DRMs are implicated in the regulation of the function of SI in intestinal Caco-2 cells. The activities of sucrase (SUC) and isomaltase (IM) increase substantially in DRMs when N- and O-glycosylation patterns are intact. Disruption of DRMs by solubilization with Triton X-100 at 37 degrees C substantially reduces the activities of SUC and IM. Furthermore, modulation of O-glycosylation by benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside and N-glycosylation by deoxymannojirimycin is linked to a decreased capacity of SI to associate with DRMs, with a subsequent reduction of the enzymatic activities of SUC and IM. This is the first report that reveals a direct role of N- and O-glycans in association with DRMs in regulating the function of a membrane glycoprotein.

Background: Pancreatic ductal adenocarcinoma (PDAC), which ranks forth on the cancer-related death statistics still is both a diagnostic and a therapeutic challenge. Adenocarcinoma of the exocrine human pancreas originates in most instances from malignant transformation of ductal epithelial cells, alternatively by Acinar-Ductal Metaplasia (ADM). RA96 antibody targets to a mucin M1, according to the more recent nomenclature MUC5AC, an extracellular matrix component excreted by PDAC cells. In this study, we tested the usability of multimodal nanoparticle carrying covalently coupled RA96 Fab fragments for pancreatic tumor imaging. Methods: In order to make and evaluate a novel, better targeting, theranostic nanoparticle, iron nanoparticles and the optical dye indocyanin green (ICG) were encapsulated into the cationic sphingomyelin (SM) consisting liposomes. RA-96 Fab fragment was conjugated to the liposomal surface of the nanoparticle to increase tumor homing ability. ICG and iron nanoparticle-encapsulated liposomes were studied in vitro with cells and (i) their visibility in magnetic resonance imaging (MRI), (ii) optical, (iii) Magnetic particle spectroscopy (MPS) and (iv) photoacoustic settings was tested in vitro and also in in vivo models. The targeting ability and MRI and photoacoustic visibility of the RA-96-nanoparticles were first tested in vitro cell models where cell binding and internalization was studied. In in vivo experiments liposomal nanoparticles were injected into a tail vain using an orthotopic pancreatic tumor xenograft model and subcutaneous pancreas cancer cell xenografts bearing mice to determine in vivo targeting abilities of RA-96-conjugated liposomes. Results: Multimodal liposomes could be detected by MRI, MPS and by photoacoustic imaging in addition to optical imaging showing a wide range of imaging utility. The fluorescent imaging of ICG in pancreatic tumor cells Panc89 and Capan-2 revealed increased association of ICG-encapsulated liposomes carrying RA-96 Fab fragments in vitro compared to the control liposomes without covalently linked RA-96. Fluorescent molecular tomography (FMT) studies showed increased accumulation of the RA96-targeted nanoparticles in the tumor area compared to non-targeted controls in vivo. Similar accumulation in the tumor sites could be seen with liposomal ferric particles in MRI. Fluorescent tumor signal was confirmed by using an intraoperative fluorescent imaging system which showed fluorescent labeling of pancreatic tumors. Conclusion: These results suggest that RA-96-targeted liposomes encapsulating ICG and iron nanoparticles can be used to image pancreatic tumors with a variety of optical and magnetic imaging techniques. Additionally, they might be a suitable drug delivery tool to improve treatment of PDAC patients.

One contribution of 7 to a theme issue '3D biological cultures and organoids'.The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal-amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

Cancer cell invasion is a precondition for tumour metastasis and represents one of the most devastating characteristics of cancer. The development of drugs targeting cell migration, known as migrastatics, may improve the treatment of highly invasive tumours such as glioblastoma (GBM). In this study, investigations into the role of the cell adhesion protein Cellular communication network factor 1 (CCN1, also known as CYR61) in GBM cell migration uncovered a drug resistance mechanism adopted by cells when treated with the small molecule inhibitor CCG-1423. This inhibitor binds to importin α/β inhibiting the nuclear translocation of the transcriptional co-activator MKL1, thus preventing downstream effects including migration. Despite this reported role as an inhibitor of cell migration, we found that CCG-1423 treatment did not inhibit GBM cell migration. However, we could observe cells now migrating by mesenchymal–amoeboid transition (MAT). Furthermore, we present evidence that CCN1 plays a critical role in the progression of GBM with increased expression in higher-grade tumours and matched blood samples. These findings support a potential role for CCN1 as a biomarker for the monitoring and potentially early prediction of GBM recurrence, therefore as such could help to improve treatment of and increase survival rates of this devastating disease.

Background: Nanoparticle imaging and the imaging of the release of the loaded material from a nanoparticle system have attracted great attention in recent years. If the release of loaded molecules from delivery vehicles could be monitored reliably in vivo, it would speed up the development of drug delivery systems remarkably. Methods: Here we test a system that uses indocyanine green (ICG) as a fluorescent agent for studying release kinetics in vitro and in vivo of a lipid iron nanoparticle delivery system. The ICG spectral properties like its concentration, sensitivity and its fluctuations of absorption and emission wavelengths of the fluorescence can be utilized for gathering information about the change of the ICG surroundings. Results: We have found that the absorption, fluorescence, and photoacoustic spectra of the ICG in lipid iron nanoparticles differ from the spectra of ICG in the pure water and in plasma. We followed the ICG containing liposomal nanoparticle uptake into the squamous carcinoma cells (SCC) under a fluorescence microscopy and into SCC tumor in the nude mice orthotopic xenografts model under a surgical microscope. Conclusion: Absorption and emission properties of ICG in different solvent environment, like in plasma and human serum albumin, differ from those in aqueous solution. Photoacoustic spectral imaging confirmed a peak shift towards longer wavelengths and an intensity increase of ICG when bound to lipid. The SCC cells showed that the ICG containing liposomes bind to cell surface but are not internalized in the SCC-9 cells in 30 minutes’ incubation. We also showed here that ICG containing liposomal nanoparticles can be traced under a surgical camera in vivo in orthotopic SCC xenografts in mice.

Three-dimensional (3D) spheroid cultures are generating increasing interest in cancer research, e.g. for the evaluation of pharmacological effects of novel small molecule inhibitors. This is mainly due to the fact that such 3D structures reflect physiological characteristics of tumours and the cellular microenvironments they reside in more faithfully than two-dimensional (2D) cell cultures; in addition, they allow the reduction of animal experiments while providing significantly relevant human-based models. Quantification of such organoid structures as well as the mainly slice-based acquisition and thus forced 2D representation of 3D spheroids provide a challenge for the interpretation of the associated generated data. Here, we provide a novel open-source workflow to reconstruct a 3D entity from slice-recorded microscopical images with or without treatment with anti-migratory small molecule inhibitors. This reconstruction produces distinct point clouds as basis for subsequent comparison of basic readout parameters using average computer processor, memory and graphics resources within an acceptable time frame. We were able to validate the usefulness of this workflow using 3D data generated by various imaging techniques, including z -stacks from confocal microscopy and histochemically labelled spheroid sectioning, and demonstrate the possibility to accurately characterize inhibitor effects in great detail.

Receptor tyrosine kinases (RTKs) are highly regulated, single pass transmembrane proteins, fundamental to cellular function and survival. Aberrancies in regulation lead to corruption of signal transduction and a range of pathological outcomes. Although control mechanisms associated with the receptors and their ligands are well understood, little is known with respect to the impact of lipid/lipid and lipid/protein interactions in the proximal plasma membrane environment. Given that the transmembrane regions of RTKs change in response to extracellular ligand binding, the lipid interactions have important consequences in influencing signal transduction. Fibroblast growth factor receptor 2 (FGFR2) is a highly regulated RTK, including under basal conditions. Binding of the adaptor protein, growth factor receptor-bound protein 2 (GRB2) to FGFR2 prevents full activation and recruitment of downstream signalling effector proteins in the absence of extracellular stimulation. Here we demonstrate that the FGFR2-GRB2 complex is sustained in a defined lipid environment. Dissociation of GRB2 from this complex due to ligand binding, or reduced GRB2 expression, facilitates the dispersion of FGFR2 into detergent-resistant membrane (DRM) micro-domains. This modification of the plasma membrane proximal to FGFR2 provides a further regulatory checkpoint which controls receptor degradation, recycling and recruitment of intracellular signalling proteins.