De novo formation of early endosomes during Rab5-to-Rab7a transition.

Rab5 and Rab7a are the main determinants of early and late endosomes and are important regulators of endosomal progression. The transport from early endosomes to late endosome seems to be regulated through an endosomal maturation switch, where Rab5 is gradually exchanged by Rab7a on the same endosome. Here, we provide new insight into the mechanism of endosomal maturation, for which we have discovered a stepwise Rab5 detachment, sequentially regulated by Rab7a. The initial detachment of Rab5 is Rab7a independent and demonstrates a diffusion-like first-phase exchange between the cytosol and the endosomal membrane, and a second phase, in which Rab5 converges into specific domains that detach as a Rab5 indigenous endosome. Consequently, we show that early endosomal maturation regulated through the Rab5-to-Rab7a switch induces the formation of new fully functional Rab5-positive early endosomes. Progression through stepwise early endosomal maturation regulates the direction of transport and, concomitantly, the homeostasis of early endosomes.

Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis.

BACKGROUND: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. METHODS: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. FINDINGS: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. INTERPRETATION: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. FUNDING: We received funding from the Norwegian research council and the Norwegian cancer society.

Tumor Killing by CD4+ T Cells Is Mediated via Induction of Inducible Nitric Oxide Synthase-Dependent Macrophage Cytotoxicity.

CD4+ T cells can induce potent anti-tumor immune responses. Due to the lack of MHC class II expression in most cancer cells, antigen recognition occurs indirectly via uptake and presentation on tumor-infiltrating antigen-presenting cells (APCs). Activation of the APCs can induce tumor rejection, but the mechanisms underlying tumor killing by such cells have not been established. To elucidate the molecular basis of CD4+ T-cell-mediated tumor rejection, we utilized a murine model of multiple myeloma, in which the T cells recognize a secreted tumor neoantigen. Our findings demonstrate that T cell recognition triggers inducible nitric oxide synthase activity within tumor-infiltrating macrophages. Diffusion of nitric oxide into surrounding tumor cells results in intracellular accumulation of toxic secondary oxidants, notably peroxynitrite. This results in tumor cell apoptosis through activation of the mitochondrial pathway. We find that this mode of cytotoxicity has strict spatial limitations, and is restricted to the immediate surroundings of the activated macrophage, thus limiting bystander killing. These findings provide a molecular basis for macrophage-mediated anti-tumor immune responses orchestrated by CD4+ T cells. Since macrophages are abundant in most solid tumors, evoking the secretion of nitric oxide by such cells may represent a potent therapeutic strategy.

Endosomal binding kinetics of Eps15 and Hrs specifically regulate the degradation of RTKs.

Activation of EGF-R and PDGF-R triggers autophosphorylation and the recruitment of Eps15 and Hrs. These two endosomal proteins are important for specific receptor sorting. Hrs is recruiting ubiquitinated receptors to early endosomes to further facilitate degradation through the ESCRT complex. Upon receptor activation Hrs becomes phosphorylated and is relocated to the cytosol, important for receptor degradation. In this work we have studied the endosomal binding dynamics of Eps15 and Hrs upon EGF-R and PDGF-R stimulation. By analysing the fluorescence intensity on single endosomes after ligand stimulation we measured a time-specific decrease in the endosomal fluorescence level of Eps15-GFP and Hrs-YFP. Through FRAP experiments we could further register a specific change in the endosomal-membrane to cytosol binding properties of Eps15-GFP and Hrs-YFP. This specific change in membrane fractions proved to be a redistribution of the immobile fraction, which was not shown for the phosphorylation deficient mutants. We here describe a mechanism that can explain the previously observed relocation of Hrs from the endosomes to cytosol after EGF stimulation and show that Eps15 follows a similar mechanism. Moreover, this specific redistribution of the endosomal protein binding dynamics proved to be of major importance for receptor degradation.

pH-responsive nano carriers for doxorubicin delivery.

PURPOSE: The aim of this study was to design stimuli-responsive nanocarriers for anti-cancer drug delivery. For this purpose, doxorubicin (DOX)-loaded, polysebacic anhydride (PSA) based nanocapsules (NC) were combined with pH-sensitive poly (L-histidine) (PLH). METHOD: PSA nano-carriers were first loaded with DOX and were coated with poly L-histidine to introduce pH sensitivity. The PLH-coated NCs were then covered with polyethylene glycol (PEG) to reduce macrophage uptake. The drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH5) and physiological (pH 7.4) media. The physical and chemical properties of the nanocapsules were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were conducted in MDA-MB-231 breast cancer cells. RESULTS: The results obtained by SEM and DLS revealed that nanocapsules have spherical morphology with an average size of 230 nm. Prepared pH sensitive nanocapsules exhibited pH-dependent drug release profile and promising intracellular release of drug. PEGylation of nanoparticles significantly prevented macrophage uptake compared to non-PEGylated particles.

Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment.

Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.