or Wortmannin (1-100 µM) for 30 min at 37 °C. Cells were further incubated with approx. 1x10^9 PKH67-labelled SW480 EVs per well (113,000g, 70 min).
Extracellular vesicles derived from SW480 cancer colon cells are internalized by human primary monocytes but not by lymphocytes Beate Vestad 1,2, Lilly Alice Skaaraas1, Alicia Llorente2,3, Kari Bente Foss Haug1,2, Reidun Øvstebø1,2 1The
Blood Cell Research Group, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, 2Regional Research Network on Extracellular Vesicles, South-Eastern Norway Regional Health Authority, 3 Department of Molecular Cell Biology, Institute of Cancer Research, Oslo University Hospital, The Norwegian Radium Hospital, Oslo, Norway.
Aim:
Conclusion:
To investigate the endocytic pathways of extracellular vesicles (EVs) in human primary monocytes and lymphocytes.
Our findings show that SW480 EVs are internalized by human primary monocytes, but not by lymphocytes, thus suggesting that cells have different requirements for internalizing EVs. In addition, our results suggest that EVs are internalized by an actin- and PI3K-dependent endocytic pathway in primary monocytes.
Fluorescence microscopy of uptake of SW480 EVs in monocytes.
Results: 1:
SW480 EVs were internalized by human primary monocytes increasingly over time, in a dosedependent manner, but not by lymphocytes. a)
3:
c)
Imaging techniques showed that SW480 exosomes were internalized in varying amounts by human monocytes. Figure 3. Uptake of SW480 exosomes in monocytes were detected by different imaging techniques. Approx. 7x10^9 PKH67-labelled SW480 exosomes or a PKH67-PBS control (data not shown) were added per 1 mill monocytes from a healthy donor (containing approx. 90 % Mo and 10 % Ly) and incubated for 4 h. a) Fluorescent microscopy: Cells were trypsinized and washed before they were reattached to German glass slides for another 3 h. They were further fixed with 3.7 % PFA and a glycerol-containing mounting medium with 3 % 7-AAD solution was added. 7-AAD was used to stain the nucleus of the monocytes (red) and exosomes were labeled with PKH67 (green). b) Confocal microscopy: Monocytes were fixed in Mattek dishes (Poly-D-Lysin coating) with 4 % PFA, and mounting medium with DAPI was added to counter stain the nuclei (blue). PKH67 was used to label the exosomes (green). The image montage shows 15 slices of one confocal Z stack of a single monocyte.
b)
a)
Figure 1. Cells from healthy donors incubated with PKH67-labelled SW480 EVs were measured by flow cytometry, and uptake was measured as median fluorescence intensity (FI). a) Monocytes were incubated with 1x10^9 PKH67-labelled exosomes for different times. b) Monocytes were incubated with different concentrations of PKH67-labelled exosomes for 3 h. c) Monocytes and lymphocytes were incubated with 1x10^9 PKH67-labelled SW480 exosomes or microvesicles for 4 h. In a) and b), median FI values from a representative experiment are shown, whereas c) shows median FI values of monocytes (n=3) and lymphocytes (n=1). As controls, cells were incubated with PBS alone (a,b,c), or with exosomes at 4 °C (a). Mo: monocytes, Ly: lymphocytes, Exo: exosomes, MV: microvesicles.
2:
Internalization of SW480 exosomes was inhibited by an actin polymerization inhibitor (Cytochalasin D) and a PI3K-inhibitor (Wortmannin). a)
Figure 2a. Monocytes were incubated with 1x10^9 PKH67-labelled SW480 exosomes for 4 h in the presence of the inhibitors Cytochalasin D, Wortmannin and LY294402. Uptake was reported as median FI by flow cytometry. Optimal inhibitor concentrations were determined from dose-response experiments monitoring EV uptake inhibition (data not shown) combined with viability measurements (Figure 2b).
Methods: Elutriation-purified, cryopreserved human monocytes and lymphocytes from healthy donors were thawed and incubated (150,000 cells per well in 96-well plates, 1 mill/mL) with and without the actin polymerization inhibitor Cytochalasin D (1-10 µg/mL), or with the PI3K-inhibitors LY294002 (5-500 µM) or Wortmannin (1-100 µM) for 30 min at 37 °C. Cells were further incubated with approx. 1x10^9 PKH67-labelled SW480 EVs per well (113,000g, 70 min) (17,000g, 30 min) based on particle concentrations obtained by a Nanosight NS500 instrument, for 4 hours (3 hours in dose-response experiment) in 37 °C, 5 % CO2. Subsequently, the cells were carefully washed with PBS, trypsinized (0.25 % trypsin/EDTA), transferred to eppendorf tubes and further washed 3x with PBS (last wash with 1 % BSA). EV uptake was analyzed by flow cytometry (BD Accuri C6), reported as median fluorescence intensity (FI), as well as by fluorescence microscopy (Nikon Eclipse Ti, 60x1,5x objective), confocal microscopy and live imaging (Leica SP8, scan the barcodes to the right). Cell viability was assessed by flow cytometry, using anti-CD14, anti-Annexin V and propidium iodide, to determine the potential cellular toxicity of the inhibitors. In addition, lactate dehydrogenase released in cell supernatants was also measured as a marker of cell integrity (data not shown).
b)
b)
Figure 2b. Monocytes were incubated with four different concentrations of the inhibitors Cytochalasin D, Wortmannin and LY294402 for 4 h in the absence of EVs (data not shown), to determine inhibitor toxicity on cells. Per cent viable cells was determined by flow cytometry.
References: 1. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. The Journal of cell biology. 2013 Feb 18;200(4):373-83. PubMed PMID: 23420871. Pubmed Central PMCID: 3575529. 2. Mulcahy LA, Pink RC, Carter DR. Routes and mechanisms of extracellular vesicle uptake. Journal of extracellular vesicles. 2014;3. PubMed PMID: 25143819. Pubmed Central PMCID: 4122821.
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Background: Intercellular communication is an essential hallmark of multicellular organisms and can be mediated through direct cell-cell contact or transfer of secreted molecules. Extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer of membrane and cytosolic proteins, lipids, and RNA between cells (1). Functions of EVs in physiological and pathological processes depend on the ability of EVs to interact with recipient cells to deliver their contents. Cells have been shown to internalize EVs by a variety of endocytic pathways (2). Acknowledgements: Jens Eriksson and Stig Ove Bøe at the Advanced light microscopy core facility (Oslo University Hospital, Gaustad), and Hans Christian Aass and Anne-Marie Siebke Trøseid at The Blood Cell Research Group for contributing to interpreting of flow cytometry results and isolation of primary monocytes and lymphocytes.
