Joshua L. Hood

Exosomes Released by Melanoma Cells Prepare Sentinel Lymph Nodes for Tumor Metastasis

Exosomes are naturally occurring biological nanovesicles utilized by tumors to communicate signals to local and remote cells and tissues. Melanoma exosomes can incite a proangiogenic signaling program capable of remodeling tissue matrices. In this study, we show exosome-mediated conditioning of lymph nodes and define microanatomic responses that license metastasis of melanoma cells. Homing of melanoma exosomes to sentinel lymph nodes imposes synchronized molecular signals that effect melanoma cell recruitment, extracellular matrix deposition, and vascular proliferation in the lymph nodes. Our findings highlight the pathophysiologic role and mechanisms of an exosome-mediated process of microanatomic niche preparation that facilitates lymphatic metastasis by cancer cells.

Paracrine induction of endothelium by tumor exosomes

Cancers use a nanoscale messenger system known as exosomes to communicate with surrounding tissues and immune cells. However, the functional relationship between tumor exosomes, endothelial signaling, angiogenesis, and metastasis is poorly understood. Herein, we describe a standardized approach for defining the angiogenic potential of isolated exosomes. We created a powerful technique to rapidly and efficiently isolate and track exosomes for study using dynamic light scattering in conjunction with fluorescent exosome labeling. With these methods, melanoma exosomes were observed to interact with and influence endothelial tubule morphology as well as move between endothelial tubule cells by means of tunneling nanotube structures. Melanoma exosomes also were observed to rapidly stimulate the production of endothelial spheroids and endothelial sprouts in a dose-dependent manner. In concert, tumor exosomes simultaneously elicited paracrine endothelial signaling by regulation of certain inflammatory cytokines. These data suggest that, tumor exosomes can promote endothelial angiogenic responses, which could contribute to tumor metastatic potential.

Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and Golgi apparatus

Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na2CO3 or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are “embedded” within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell.

Maximizing exosome colloidal stability following electroporation.

Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum-derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label-free means of enriching exogenously modified exosomes and introduces the potential for MRI-driven theranostic exosome investigations in vivo.

Cytolytic nanoparticles attenuate HIV-1 infectivity.

BACKGROUND We investigated whether cytolytic melittin peptides could inhibit HIV-1 infectivity when carried in a nanoparticle construct that might be used as a topical vaginal virucide. Free melittin and melittin-loaded nanoparticles were prepared and compared for cytotoxicity and their ability to inhibit infectivity by CXCR4 and CCR5 tropic HIV-1 strains. METHODS TZM-bl reporter cells expressing luciferase under the control of the HIV-1 promoter were incubated with HIV-1 NLHX (CXCR4) or HIV-1 NLYU2 (CCR5) viral strains and different doses of soluble CD4 (positive control) or free melittin to determine infectivity and viability. Melittin-loaded nanoparticles were formulated and different doses tested against VK2 vaginal epithelial cells to determine cell viability. Based on VK2 viability, melittin nanoparticles were tested for prevention of CXCR4 and CCR5 tropic HIV-1 infectivity and viability of TZM-bl reporter cells. Low-speed centrifugation was used to compare the ability of blank non-melittin nanoparticles and melittin nanoparticles to capture CCR5 tropic HIV-1. RESULTS As expected, the soluble CD4 positive control inhibited CXCR4 (50% inhibitory concentration [IC₅₀] 3.7 μg/ml) and CCR5 (IC₅₀ 0.03 μg/ml) tropic HIV-1 infectivity. Free melittin doses <2 μM were not cytotoxic and were highly effective in reducing HIV-1 infectivity for both CXCR4 and CCR5 strains in TZM-bl reporter cells, while VK2 vaginal cell viability was adversely affected at all free melittin doses tested. However, VK2 cell viability was not affected at any dose of melittin-loaded nanoparticles. Melittin nanoparticles safely and significantly decreased CXCR4 (IC₅₀ 2.4 μM and IC₉₀ 6.9 μM) and CCR5 (IC₅₀ 3.6 μM and IC₉₀ 11.4 μM) strain infectivity of TZM-bl reporter cells. Furthermore, melittin nanoparticles captured more HIV-1 than blank nanoparticles. CONCLUSIONS These data illustrate the first proof-of-concept for therapeutic and safe nanoparticle-mediated inhibition of HIV-1 infectivity. Future investigations appear warranted to explore the antiviral prophylactic potential of melittin nanoparticles to capture, disrupt and prevent initial infection with HIV-1 or potentially other enveloped viruses.

A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM

AbstractExosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for “label-free” isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics. FigureFlow path of exosome sample through the asymmetrical field flow fractionation instrument, detectors, and transmission electron microscope.

Magnetic resonance imaging of melanoma exosomes in lymph nodes.

Exosomes are cell derived extracellular nanovesicles that relay molecular signals pertinent to both normal physiologic and disease processes. The ability to modify and track exosomes in vivo is essential to understanding exosome pathogenesis, and for utilizing exosomes as effective diagnostic and therapeutic nanocarriers to treat diseases.

A systematic approach to exosome-based translational nanomedicine.

Exosomes are a type of cell-derived extracellular nanovesicle. They relay information between cells. Some known exosome functions include immune modulation, promotion of angiogenesis, and tumor metastasis. To date, clinical use of exosomes has focused predominantly on evaluating their efficacy as cancer vaccines or diagnostically as biomarker containers. However, few investigations have explored their potential to serve as a platform for the development of semi-synthetic nanovesicles. Given their nanoscale size, potential to express targeting ligands in native conformations and deformable structure, exosomes offer a logical biological vesicle platform for adapting and producing semi-synthetic vesicles with excellent potential for nanomedicine applications. However, there are obstacles associated with realizing this potential that must be addressed. Thus, a systematic approach to isolating, modifying, and testing exosomes is presented to facilitate the introduction of exosome-based translational nanomedicine.

Lipid membrane editing with peptide cargo linkers in cells and synthetic nanostructures

Current strategies for deploying synthetic nanocarriers involve the creation of agents that incorporate targeting ligands, imaging agents, and/or therapeutic drugs into particles as an integral part of the formulation process. Here we report the development of an amphipathic peptide linker that enables postformulation editing of payloads without the need for reformulation to achieve multiplexing capability for lipidic nanocarriers. To exemplify the flexibility of this peptide linker strategy, 3 applications were demonstrated: converting nontargeted nanoparticles into targeting vehicles;adding cargo to preformulated targeted nanoparticles for in vivo site‐specific delivery;and labeling living cells for in vivo tracking. This strategy is expected to enhance the clinical application of molecular imaging and/or targeted therapeutic agents by offering extended flexibility for multiplexing targeting ligands and/or drug payloads that can be selected after base nanocarrier formulation.—Pan, H., Myerson, J. W., Ivashyna, O., Soman, N. R., Marsh, J. N., Hood, J. L., Lanza, G. M., Schlesinger, P. H., Wickline, S. A.. Lipid membrane editing with peptide cargo linkers in cells and synthetic nanostructures. FASEB J. 24, 2928–2937 (2010). www.fasebj.org

Nanoparticle incorporation of melittin reduces sperm and vaginal epithelium cytotoxicity

Melittin is a cytolytic peptide component of bee venom which rapidly integrates into lipid bilayers and forms pores resulting in osmotic lysis. While the therapeutic utility of free melittin is limited by its cytotoxicity, incorporation of melittin into the lipid shell of a perfluorocarbon nanoparticle has been shown to reduce its toxicity in vivo. Our group has previously demonstrated that perfluorocarbon nanoparticles containing melittin at concentrations <10 µM inhibit HIV infectivity in vitro. In the current study, we assessed the impact of blank and melittin-containing perfluorocarbon nanoparticles on sperm motility and the viability of both sperm and vaginal epithelial cells. We found that free melittin was toxic to sperm and vaginal epithelium at concentrations greater than 2 µM (p<0.001). However, melittin nanoparticles were not cytotoxic to sperm (p = 0.42) or vaginal epithelium (p = 0.48) at an equivalent melittin concentration of 10 µM. Thus, nanoparticle formulation of melittin reduced melittin cytotoxicity fivefold and prevented melittin toxicity at concentrations previously shown to inhibit HIV infectivity. Melittin nanoparticles were toxic to vaginal epithelium at equivalent melittin concentrations ≥20 µM (p<0.001) and were toxic to sperm at equivalent melittin concentrations ≥40 µM (p<0.001). Sperm cytotoxicity was enhanced by targeting of the nanoparticles to the sperm surface antigen sperm adhesion molecule 1. While further testing is needed to determine the extent of cytotoxicity in a more physiologically relevant model system, these results suggest that melittin-containing nanoparticles could form the basis of a virucide that is not toxic to sperm and vaginal epithelium. This virucide would be beneficial for HIV serodiscordant couples seeking to achieve natural pregnancy.