Microenvironmental pH Is a Key Factor for Exosome Traffic in Tumor Cells

Abstract

Exosomes secreted by normal and cancer cells carry and deliver a variety of molecules. To date, mechanisms referring to tumor exosome trafficking, including release and cell-cell transmission, have not been described. To gain insight into this, exosomes purified from metastatic melanoma cell medium were labeled with a lipid fluorescent probe, R18, and analyzed by spectrofluorometry and confocal microscopy. A low pH condition is a hallmark of tumor malignancy, potentially influencing exosome release and uptake by cancer cells. Using different pH conditions as a modifier of exosome traffic, we showed (i) an increased exosome release and uptake at low pH when compared with a buffered condition and (ii) exosome uptake by melanoma cells occurred by fusion. Membrane biophysical analysis, such as fluidity and lipid composition, indicated a high rigidity and sphingomyelin/ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) content in exosomes released at low pH. This was likely responsible for the increased fusion efficiency. Consistent with these results, pretreatment with proton pump inhibitors led to an inhibition of exosome uptake by melanoma cells. Fusion efficiency of tumor exosomes resulted in being higher in cells of metastatic origin than in those derived from primary tumors or normal cells. Furthermore, we found that caveolin-1, a protein involved in melanoma progression, is highly delivered through exosomes released in an acidic condition. The results of our study provide the evidence that exosomes may be used as a delivery system for paracrine diffusion of tumor malignancy, in turn supporting the importance of both exosomes and tumor pH as key targets for future anti-cancer strategies.

Exosomes as intercellular signalosomes and pharmacological effectors

“…In any cases, since miRNA vehiculated by exosomes are able to modulate genes in a target cell [23], this indicates that exosomes are able to deliver their content into the recipient cells. Delivery of exosome content can occur either by fusion between exosome and the plasma membrane [77], internalization of exosome with subsequent opening of the vesicule inside target cell [78], or internalization and degradation of the exosome membrane inside the cell [35].…”

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8

“…EV uptake is considered to depend on the EV donor and recipient cell type, their physiological state, and the presence or absence of interfering molecules in the extracellular microenvironment [20–22]. Although direct fusion of EVs and plasma membranes has been reported for melanoma cells [23], multiple studies point to interactions involving endocytic pathways. EV uptake is inhibited at 4°C, indicating an active, energy-dependent process [24–26].…”

Therapeutic and diagnostic applications of extracellular vesicles

“…For instance, hypoxia triggers cancer cells to release more CD63-positive vesicles [49] with a modified molecular composition and distinct effect on recipient cells [50]. Comparable observations were made for an altered extracellular pH [51] and the presence of stress-inducing molecules (e.g. lipopolysaccharide, H2O2, etc.)…”

“…Indeed, EVs are believed to encompass many interesting features for drug delivery: (1) a proteo-lipid architecture that protects the encapsulated cargo, (2) their nanosize and specific composition minimizes recognition by the mononuclear phagocyte system (MPS) [110], (3) their patient self-derived nature mitigates activation of the adaptive immune system, (4) they contain specific lipids that help stabilize the vesicles in the blood circulation (e.g. GM3, sphingomyelin and cholesterol) and stimulate membrane fusion [51,111] as well as surface proteins that have likewise been linked to membrane fusion in cell-cell and viruscell interactions (e.g. CD9, CD81) [112,113] and finally (5) EVs seem to possess intrinsic cell and tissue targeting properties [114].…”

“…Some studies labeled EVs with a self-quenching dye after which they were incubated with cells. An enhancement of fluorescence was indicative of dye dequenching and hence fusion of (a fraction of) EVs with cellular membranes [51,91]. Alternatively, luciferin containing EVs were able to evoke a luminescent signal after interaction with luciferase expressing cells, suggesting cytosolic delivery of the EV luminal cargo [91].…”