Extracellular Vesicles: A Mechanism to Reverse Metastatic Behaviour as a New Approach to Cancer Therapy

Extracellular vesicles (EVs) are membrane-bound particles shed from nearly all cell types into the extracellular environment. This collective term includes vesicles ranging in size from 30 nm to 5 μm in diameter. Various isolation techniques are used in different studies to separate EVs with no consensus protocol. EVs are released from cells under normal physiological conditions as well as in stressful and pathological conditions. In malignancies, they have been shown to be useful circulating markers for risk assessment, early diagnosis, monitoring of therapeutic effectiveness and prognosis. In addition, they appear to influence cell death and growth, angiogenesis, immune surveillance, extracellular matrix degradation and metastasis. In this respect, EVs have generated considerable interest for their potential use in cancer therapeutics. Since they appear to be responsible for transference of cellular components between cells and thereby transfer of functional characteristics of the donor to the recipient, two strategies for their role in cancer therapeutics may be envisaged. The first would be to prevent formation and/or shedding of EVs to prevent communication to or from cancer cells. The second would be to utilize them as carriers to deliver inhibitory/toxic components into cancer cells to destroy or neutralize them. In this review, we discuss the current state of research on characterization of EVs and highlight possible strategies for their use in cancer therapy

Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: Implications for Alzheimer's disease

© 2016 The Author(s). Background: Polymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer's disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME). Results: Following depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression. Conclusions: The depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD

Receptor crosslinking: a general method to trigger internalization and lysosomal targeting of therapeutic receptor: ligand complexes

A major unmet clinical need is a universal method for subcellular targeting of bioactive molecules to lysosomes. Delivery to this organelle enables either degradation of oncogenic receptors that are overexpressed in cancers, or release of prodrugs from antibody–drug conjugates. Here, we describe a general method that uses receptor crosslinking to trigger endocytosis and subsequently redirect trafficking of receptor:cargo complexes from their expected route, to lysosomes. By incubation of plasma membrane receptors with biotinylated cargo and subsequent addition of streptavidin to crosslink receptor:cargo–biotin complexes, we achieved rapid and selective lysosomal targeting of transferrin, an anti-MHC class I antibody, and the clinically approved anti-Her2 antibody trastuzumab. These three protein ligands each target a receptor with a distinct cellular function and intracellular trafficking profile. Importantly, we confirmed that crosslinking of trastuzumab increased lysosomal degradation of its cognate oncogenic receptor Her2 in breast cancer cell lines SKBR3 and BT474. These data suggest that crosslinking could be exploited for a wide range of target receptors, for navigating therapeutics through the endolysosomal pathway, for significant therapeutic benefit

Unraveling the cellular uptake of bioreducible poly(amido amine) — Gene complexes in cells of the retinal pigment epithelium

In vitro endocytosis of gene complexes composed of a bioreducible polyamidoamine CBA ABOL and plasmid DNA, in cells of the retinal pigment epithelium (RPE) was studied, the latter being an interesting target for ocular gene therapy. We found that cationic CBA ABOL DNA polyplexes attach to cell surface proteoglycans of these RPE cells and get subsequently internalized via a phagocytosis-like mechanism, as well as Flotillin dependent endocytosis.\ud \ud Introduction\ud \ud Proper delivery of therapeutic genes to designated cells and their availability at the intracellular site of action are crucial requirements for successful gene therapy. To this end, typically sub-micron sized particles are made by combining the therapeutic genes with a carrier material, such as cationic polymers, that aid in delivering the genes to the target site. In this work, for the first time, we have evaluated the ability of the highly promising bioreducible polymer carrier cystamin bisacrylamid aminobutanol (CBA ABOL) [1] (Fig. 1) to deliver plasmid DNA in cultured cells of the retinal pigment epithelium (ARPE-19) and characterized in vitro the cellular interactions with these target cells. These studies are of crucial importance since the further design and functionalization of polymeric gene carriers depend strongly on our understanding of the mechanisms involved in cellular adhesion, intracellular uptake and intracellular processing of the polyplexes.\ud \ud Experimental methods\ud \ud ARPE-19 cells (retinal pigment epithelial cell line; ATCC number CRL-2302) were cultured in DMEM:F12 supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and 2% penicillin-streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. CBA ABOL gene complexes with an average hydrodynamic diameter of 130 nm and an average zeta potential of + 45 mV in 20 mM HEPES buffer were obtained by adding the polymer in a mass ratio of 48/1 in 20 mM HEPES to the plasmid and vortexing the mixture for 10 min. For every transfection, fresh polyplexes were prepared and applied to the cells within 30 min after complexation. For all uptake studies, YOYO-1™ (λex = 491 nm, λem = 509 nm, Molecular Probes, Merelbeke, Belgium) labeled pGL4.13 plasmid (Promega, Leiden, The Netherlands) was used. For all transfection studies, gWiz™GFP plasmid (Aldevron, Freiburg, Germany) was used. siRNAs were all purchased from Dharmacon and transfected in cells with the help of LipofectaminRNAiMAX (Invitrogen, Merelbeke, Belgium). Protein knockdown was assessed on Western Blot. Uptake of polyplexes or endocytic markers or GFP expression was measured on a FACS Calibur Flow Cytometer (Beckton Dickinson, Erembodegem, Belgium). For genetic labeling of endosomes, cells were transfected with GFP-fusion proteins.\ud \ud For fluorescence colocalization studies with GFP labeled cellular structures, cells were transfected with GFP-fusion proteins using Lipofectamin2000 (Invitrogen, Merelbeke, Belgium) and 24 h later exposed to red fluorescent labeled polyplexes. For this, CBA ABOL was complexed with pGL4.13 plasmid, covalently labeled with Cy5 (Label IT Nucleic Acid Labeling Kit, Mirus Bio Corporation, WI, USA). Live cell fluorescence colocalization was then performed on a custom built laser epi-fluorescence microscope set-up. A Nikon Plan Apo VC 100× 1.4 NA oil immersion objective lens (Nikon Belux, Brussels, Belgium) was used for imaging. GFP and Cy5 were excited with 491 nm and 636 nm laser light and emission was detected on an EMCCD camera (Roper Scientific, Nieuwegein, The Netherlands). For live cell imaging the cells were placed in a stage top incubation chamber (Tokai Hit, Shizuoka, Japan), set at 37 °C, 5% CO2 and 100% humidity.\ud \ud Result and discussion\ud \ud First, we found evidence that these net positively charged CBA ABOL polyplexes adhere to the negatively charged heparan sulfate proteoglycans (HSPGs) at the cell surface and that polyplex internalization is blocked by antibodies against Toll-like receptor 9\u

siRNA versus pharmacological inhibition of endocytic pathways for studying cellular uptake of cell penetrating peptides and other drug delivery vectors

Cell-penetrating peptides (CPPs) have the potential to deliver numerous therapeutic macromolecules into cells including peptides, proteins, and nucleic acids. Under defined conditions endocytosis is thought to be of significant importance for CPP entry but identifying the exact uptake mechanism and pathway(s) involved has been difficult. Multiple pathways have been reported to contribute to uptake, including macropinocytosis and those regulated by clathrin and cavaeolin-1. This project aims enhance the use of cell penetrating peptides as drug delivery vectors by developing new technologies to study their mechanisms of uptake. Traditionally studies investigating the uptake of these molecules, and other drug delivery vectors, have been performed using chemical inhibitors but these are often toxic and lack specificity [1]. We have developed siRNA-based assays to silence endocytic proteins that have previously been shown to regulate distinct endocytic pathways. The effect of depleting these proteins was then assessed to investigate their roles in mediating the uptake of well characterised endocytic probes and CPPs. Two cell lines were predominantly used, HeLa (cervical cancer epithelial) and A431 (human epithelial carcinoma). Endocytic proteins clathrin heavy chain, flotillin-1, dynamin II, caveolin-1 and P21-activated kinase (PAK-1) were depleted using single siRNA sequences; siRNA against GFP was used as a control. In siRNA treated cells the uptake of fluorescent endocytic markers including; Alexa488-transferrin (clathrin mediated endocytosis), 40 kDa FITC Dextran (fluid phase uptake and macropinocytosis), FITC conjugated anti-CD59 antibody (flotillin-1 dependent uptake) and the uptake of Alexa488 CPPs (RRRRRRRRGC-Alexa488-R8, and GRKKRRQRRRPPQ-Alexa488-HIV-TAT) were measured by flow cytometry. Protein depletion was assessed from protein lysates using SDS PAGE and Western blotting. Overall, the data shows that siRNA transfection method could effectively reduce expression of clathrin heavy chain, caveolin-1 and flotillin-1 from HeLa cells and this then allowed for us to study effects on endocytosis of various probes. PAK-1 has been shown to regulate macropinocytosis and we show that the induction of macropinocytosis and PAK-1 expression are highly cell line dependent. Paralleled with this was our findings that cationic CPPs induce an increase in fluid phase uptake of dextran and the extent of this was cell line dependent. Comparative analysis of these experiments with those performed using pharmacological inhibitors, allowed us to determine the usefulness of this approach for drug delivery research

siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors

Flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/DNA polyplexes

Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway