本周三请大家随小编一起看看这篇文章：
Cisplatininduces the release of
extracellularvesicles from ovarian cancer
cells that can induce invasiveness and
drug resistance in bystander cells
（顺铂诱导卵巢癌细胞释放胞外囊泡，诱导细胞侵袭和耐药。）
摘要：
Ovariancancer has a poor overall survival that is partly caused by resistance to drugssuch as cisplatin. sReistance can be acquired as a result of changes to thetumour or due to altered interactions within the tumour microenvironment.Extracellular vesicles (EVs), small lipid-bound vesicles that are loaded withmacromolecular cargo and released by cells, are emerging as mediators ofcommunication in the tumour microenvironment. We previously showed that EVsmediate the bystander effect, a phenomenon in which stressed cells cancommunicate with neighbouring naive cells leading to various effects includingDNA damage; however, the role of EVs released following cisplatin treatment hasnot been tested. Here we show that treatment of cells with cisplatin led to therelease of EVs that could induce invasion and increased resistance when takenup by bystander cells. This coincided with changes in p38 and JNK signalling,suggesting that these pathways may be involved in mediating the effects. Wealso show that EV uptake inhibitors could prevent this EV-mediated adaptiveresponse and thus sensitize cells in vitro to the effects of cisplatin. Ourresults suggest that preventing pro-tumourigenic EV cross-talk duringchemotherapy is a potential therapeutic target for improving outcome in ovariancancer patients. This article is part of the discussion meeting issue‘Extracellular vesicles and the tumour microenvironment’.
结果：
1.Characteristics of extracellular vesiclesfrom control and cisplatin-treated cells.
Figure 1. Characterizationof cisplatin and control EVs derived from A2780 cells. (a) Control andcisplatin-treated cell and EV protein lysates were characterized by Westernblotting; samples were probed for GAPDH, calnexin, HSP70 and cytochrome coxidase. (b) Quantification of EVs secreted by control and cisplatin-treated A2780cells by nanoparticle tracking analysis (at least two replicates). (c) Imagesof electron microscopy grids of control and cisplatin EVs visualized by transmissionelectron microscopy. (d) Average diameter of EVs secreted by cisplatin-treatedand control A2780 cells measured on electron microscopy grids (c). (Onlineversion in colour.)
2.
Extracellular vesicles released by cisplatin-treated cells have thecapacity to induce invasion.
Figure 2. The effect ofcisplatin-treated cell-derived EVs upon the invasive capacity of ovarian cancercell lines. The Matrigel transwell invasion assay was used to determine theeffect of cisplatin-treated cell-derived EVs on invasive potential of twoovarian cancer cell lines, A2780 (a) and IGROV-1 (b). Extracted EVs wereadministered to approximately 1 million cells and after 24 h, 100 000 cellswere distributed into each insert of the transwell assay and another dose ofEVs was added. After 24 h, the Matrigel membranes were cleared of noninvasivecells and invasive cells were stained with crystal violet. The number ofinvasive cells on each membrane was counted. The graphs represent fold changein terms of the total number of cells that invaded the Matrigel membranefollowing treatment with either control or cisplatin-treated cell-derived EVs.Each sample group contained six biological replicates. Error bars representstandard error of the mean of the biological replicates. p-Values werecalculated using Student’s t-test. Representative images are shown below eachgroup. (Online version in colour.)
3.
Cisplatin extracellular vesicles can cause bystander effect and anadaptive response to cisplatin
Figure 3. Cisplatin-derivedEVs cause bystander effect and an adaptive response to cisplatin. (a) A2780cells were seeded in 96-well plates. They were treated with PBS (control), EVsfrom cisplatin-treated cells (cis EV) or EVs from control cells (control EV)with or without 30 min pre-treatment with 10 mg ml21 heparin to inhibit EVuptake; 4 d later overall viability was measured using the MTT assay. cis EVscaused a significant decrease in viability; this effect was not present in thegroup treated with heparin. (b) A2780 cells were treated with PBS, cis EVs orcontrol EV (as in a) with or without pre-treatment with heparin (10 mg ml21);after 24 h, cells were further treated with cisplatin and survival was assayedusing the MTT assay (results are normalized to control). Cells pre-treated withcis EVs are more resistant to cisplatin; this effect is decreased with heparin.Each column shows the mean of at least six replicates; error bars show standarderror of mean. (Online version in colour.)
4.
Pathways differentially modulated by cisplatin extracellularvesicles in cells.
Figure 4. Relativephosphorylation levels of 26 proteins in A2780 cells following treatment witheither control or cisplatin EVs determined using the proteome profiler humanPhosphoMAPK array. (a) Blots showing intensity for each kinase on duplicatespots for each EV treatment. (b) Intensity levels of each kinase in A2780 cellstreated with either control or cisplatin EVs. Differences in kinasephosphorylation were calculated using the Student’s two-tailed t-test. (Onlineversion in colour.)
