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. 2009 Dec 4;284(49):34211-22.
doi: 10.1074/jbc.M109.041152. Epub 2009 Sep 30.

Microenvironmental pH is a key factor for exosome traffic in tumor cells

Isabella Parolini  1 , Cristina Federici, Carla Raggi, Luana Lugini, Simonetta Palleschi, Angelo De Milito, Carolina Coscia, Elisabetta Iessi, Mariantonia Logozzi, Agnese Molinari, Marisa Colone, Massimo Tatti, Massimo Sargiacomo, Stefano Fais
Affiliations

VSports在线直播 - Microenvironmental pH is a key factor for exosome traffic in tumor cells

Isabella Parolini et al. J Biol Chem. .

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 VSports手机版. 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. .

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Figures

FIGURE 1.
FIGURE 1.
Exosome release in acidic and buffered pH conditions. A, exosomes (100 μg) isolated from Mel1 cells culture medium were NHS-biotin-labeled and loaded on a 10–60% continuous sucrose gradient. Eleven fractions were analyzed by Western blotting with horseradish peroxidase-streptavidin. Results indicate a molecules profile with molecular mass ranging between 97 and 21 kDa enriched in fractions 3–6 corresponding to density 1.11–1.17 g/ml. These fractions were also found positive for exosome markers such as Lamp-2, CD81, and Rab 5B by Western blotting. B, Mel1 cells (3 × 106 cell/ml) were cultured in buffered (pH 7.4) or acidic (pH 6.0) media. At the indicated days exosomes were isolated, quantified by protein assay, and normalized on 1 × 106 viable cells. Points, means (n = 4); bars, S.D. *, p < 0.05. C, Western blotting of Lamp-2 and Rab 5B in exoMel1ac and exoMel1 released from parental cells at 2, 3, and 4 days. Note that Lamp-2 and Rab 5B expressions increased with time in the acidic exosomes. A representative Western blotting of three independent experiments is shown.
FIGURE 2.
FIGURE 2.
Exosome fusion with parental cells. A, a panel of confocal laser-scanning microscopy images of a human metastatic melanoma is shown. NHS-rhodamine-labeled exosomes (red) were incubated with cells for 4 h followed by fixation and labeling with green fluorescent antibodies directed to Rab 5B (early endosomes), Lamp-1 (lysosomes), and Golgina (Golgi apparatus). Arrows indicate the events of colocalization (yellow) in all samples, with the exception of Golgi compartment. Images in the right column represent magnification of the images on the left column. Bars: left panels, 10 μm; right panels, 4 μm. B, R18-exoMel1 were left untreated or mixed with 1 × 106 Mel1 cells. Note that a fluorescence dequenching (FD) curve was observed only after the addition of the cells. Tx 100, Triton X-100. C, R18-exoMel1 were left untreated or pretreated with 0.5% PAF before the addition to Mel1 cells, and fusion activity was tested. A representative fluorescence dequenching curve is shown. Inset, statistical analysis obtained by 20 min of kinetic experiments is shown. Values are the means ± S.D. a = p < 0.05 versus control (n = 3). D, Mel1 cells (1 × 106) were left untreated or treated with filipin, then subjected to a fusion test with R18-exoMel1. A representative fluorescence dequenching curve is shown. Inset, statistical analysis obtained by 30 min kinetic experiments is shown. Values are the means ± S.D. a = p < 0.01 versus control (n = 3). E, R18-exoMel1ac and R18-exoMel1 (10 μg) were mixed with parental cells at the corresponding pH, and fusion was monitored. A representative fluorescence dequenching curve is shown. Statistical analysis on 5-, 20-, and 30-min kinetic experiments is represented. Values are the means ± S.D. a, p < 0.05 (n = 3). F, streptavidin blotting is shown. 20 μg of biotinylated exosomes were incubated with parental cells (1 × 106) at the corresponding pH (lane 1, exoMel1 on Mel1; lane 2, exoMel1ac on Mel1ac) or with filipin-treated cells (lane 3, exoMel1 on Mel1; lane 4, exoMel1ac on Mel1ac) for 1 h at 37 °C. Immunoblotting of nucleolin protein expression represents a control for protein equal loading. A representative Western blot of three independent experiments is shown. Numbers expressed in arbitrary units (a.u.) represent streptavidin densitometry analysis.
FIGURE 3.
FIGURE 3.
In vivo exosome/melanoma lipids colocalization analysis through confocal laser-scanning microscopy. The panel shows three different fields (A, B, and C) of unfixed cell cultures in which co-cultivation of R18-labeled exosomes (red, magnifications in A1, B1, C1) and PKH67-labeled cells (green, magnifications in A2, B2, C2) is analyzed. In particular, arrows in A1, B1, and C1 images indicate free exosomes in cell culture medium not yet interacting with the cells. The yellow points (A, B, and C and arrows in magnification A3, B3, and C3) correspond to cell/exosome lipid mixing events, both at plasma membrane and intracellular levels. Bars: A, 48 μm; B, 46 μm; C, 42 μm; A3, 20 μm; B3, 24 μm; C3, 13 μm.
FIGURE 4.
FIGURE 4.
Exosomes and parental cells membrane fluidity and lipid phase state at different pH conditions. A, shown are Laurdan GP values (340-nm excitation wavelength; 37 °C) of buffered and acidic Mel1 cells (open bars) and exoMel1 and exoMel1ac (filled bars). The higher GP values of exosomes indicate a higher rigidity of such membranes with respect to bulk of the cellular membranes. ExoMel1ac are significantly more rigid than exoMel1. Data are the means ± S.E. of at least three independent experiments. *, p < 0.05 versus the corresponding bulk cellular membranes. B, Laurdan excitation GP spectra of Mel1 (a, filled triangles), Mel1ac cells (b, filled circles), exoMel1 (c, open triangles), and exoMel1ac (d, open circles) at 37 °C. Laurdan GP values decrease by increasing excitation wavelength, indicating that both cell and exosome membranes are in a liquid-crystalline lipid phase in the absence of coexisting gel-phase lipid domains. The curves are representatives of three consistent experiments.
FIGURE 5.
FIGURE 5.
Role of microenvironmental pH in exosome fusion. A, R18-exoMel1 and R18-exoMel1ac were mixed with both acidic and buffered Mel1 cells (1 × 106), and fusion efficiency was tested for 30 min. Points, means ± S.D. FD, fluorescence dequenching. B, NHS-biotin-buffered (pH 7.4) or acidic (pH 6.0) exosomes (25 μg) were incubated for 1 h with untreated or PPI-treated parental cells, then cells were analyzed by streptavidin blotting. Western blotting of nucleolin represents a control for cell protein equal loading. Numbers represent the whole lane densitometry analysis expressed in arbitrary units (a.u.). Points, means ± S.D. a p < 0.01 versus control (pH 6.0) (n = 3). C, R18-exoMel1 were added to metastatic melanoma (Mel1–3), primary melanoma (MelP1–3), and normal donor-derived PBMC, and fusion efficiency was tested for 30 min. Points, means ± S.D., (n = 3). D, R18-exoMel1 (red) were incubated for 3 h with PKH67-PBMC (green) and analyzed by confocal laser-scanning microscopy. The image clearly shows the absence of lipid colocalization areas. The rare event of colocalization observed in PBMC cells is likely due to the presence of monocytes, as recognizable from nuclear morphology. Bar, 20 μm.
FIGURE 6.
FIGURE 6.
Exosomes delivery of cav-1. A, R18-exoMe665/1ac and R18-exoMe665/1 (10 μg) were mixed with parental cells at the corresponding pH and fusion monitored. A representative fluorescence dequenching (FD) curve is shown. B, cav-1 and Lamp-2 immunoblotting on WM983A cell membranes after incubation with exoMe665/1 and exoMe665/1ac. Control represents WM983A membranes in the absence of exoMe665/1 incubation. A representative Western blot of two independent experiments is shown.
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