h1650 ( nci - h1650 ; atcc ) and h3255 ( nci - h3255 ; atcc ) non - small - cell lung cancer ( nsclc ) cells were maintained in rpmi-1640 ( lonza biowhittaker , verviers , belgium ) . the glioma cell lines u87 all cell culture media were supplemented with 10% foetal bovine serum ( fbs ) , 100 u / ml penicillin and 0.1 mg / ml streptomycin , referred to as complete dmem . ev - depleted medium was obtained by 2 h ultracentrifugation of serum at 100,000g before use as a medium supplement . drug treatments were performed with erlotinib ( lc laboratories , woburn , ma , usa ) in dmem containing ev - depleted serum . cell viability was assessed by 2 h incubation with mtt 3-(4,5-dimethylthiazolyl-2)-2,5-diophenyl tetrazolium bromide ( sigma - aldrich , zwijndrecht , the netherlands ) . supernatants from cells , cultured for 32 h in serum - free or with 10% ev - depleted serum , were centrifuged at 500g for 10 min to remove cells . after another centrifugation step at 2,000g for 15 min , the samples were concentrated by retention and collection using 100 kda cut - off filters ( amicon ultra-15 , millipore , amsterdam , the netherlands ) and stored at 20c . centrifugation at 10,000g for 45 min was applied , before evs were pelleted by ultracentrifugation at 100,000g for 90 min , using a sw32ti rotor ( beckmann instruments , woerden , the netherlands ) . the ev pellet was treated with proteases ( 0.5 g / l , trypsin , lonza biowhittaker ) or erlotinib ( 10 m ) at 37c for 1 h when indicated . subsequently , evs were washed in pbs followed by centrifugation at 100,000g for 90 min . pellets were solubilized in m - per mammalian protein extraction reagent , supplemented with phosphatase and protease inhibitor cocktails ( pierce , thermo scientific , breda , the netherlands ) . evs were isolated from 2 independent cell cultures ( 10 t175 ) for each cell line and isolated as described above . full sample yield ( corresponding to 1015 g of protein ) was loaded on nupage precast gradient gels , and proteins were separated at 125 v. after electrophoresis , gels were fixed in 50% ethanol containing 3% phosphoric acid and stained with coomassie r-250 and washed in milli - q water ( supplementary fig . gel lanes were cut in 5 slices and each slice was processed by in - gel - digestion according to the protocol previously published by shevchenko ( 15 ) . briefly , samples were treated with dithiothreitol ( 10 mm ) for 1 h at 56c and iodoacetamide ( 50 mm ) for 45 min at rt . peptides were extracted first in 1% formic acid and then in 50% acn/5% formic acid . subsequently , peptides were separated by an ultimate 3,000 nanolc - ms / ms system ( dionex lc - packings , amsterdam , the netherlands ) . after injection , peptides were trapped on a 10 mm100 m i d trap column and separated at 300 nl / min in a 1040% buffer gradient ( 80% acn+0.05% formic acid ) in 60 min . peptides were measured on - line after nanospray ionization , using a q exactive mass spectrometer ( thermofisher , bremen , germany ) . intact masses were measured at resolution 70,000 ( at m / z 200 ) in the orbitrap . ms / ms spectra were acquired at resolution 17,500 ( at m / z 200 ) in the orbitrap using an agc target value of 210 charges and an underfill ratio of 0.1% . ms / ms spectra were searched against the uniprot human reference proteome fasta file ( release sept 2012 ; 147,951 entries ) using maxquant 1.3.0.5 ( default settings ) ( 16 ) . cysteine carboxamidomethylation was treated as fixed modification and methionine oxidation and n - terminal acetylation as variable modifications . peptide and protein identifications were filtered at an fdr of 1% using the decoy database strategy . proteins were quantified by spectral counting ( 17,18 ) , that is , the sum of all ms / ms spectra for each identified protein . these counts were normalized by dividing the spectral counts per protein by the sum of all counts per sample and multiplying by the average sum across all samples ( 18 ) . permeabilization was performed with ice - cold methanol ( 10 min ) , followed by 1 h incubation with 5% bovine serum albumin ( bsa ) . staining with primary antibodies against egfr ( cell signaling # 2232 , 1:200 ) or phospho - y ( 4g10 , millipore , usa , 1:200 ) protein content of samples was determined with the micro bca protein assay ( pierce , thermo scientific , the netherlands ) . for western electrophoresis was performed on precast 412% sds polyacrylamide gels ( nupage , life technologies , bleiswijk , the netherlands ) and transferred to pvdf membranes for immunoblotting of proteins . membranes were probed with antibodies against phospho - egfr - y1068 ( cell signaling , leiden , the netherlands # 2236s ) , egfr ( cs # 2232 ) , phospho - akt - s473 ( cs # 4051s ) , akt ( cs # 9272 ) , phospho - erk1/2-t202/204 ( cs # 9101s ) , erk1/2 ( cs # 9107s ) , -tubulin ( cs # 2144s ) , alix ( cs # 2171s ) , cd63 ( bd bioscience , etten - leur , the netherlands 556,019 ) , cytochrome - c ( bd bioscience 556,433 ) and gapdh ( cs # 2118 ) . irdye infrared dye ( li - cor biosciences , bad homburg , germany ) labelled secondary antibodies were used for detection . membranes were scanned using an odyssey infrared imaging system and accompanying software ( version 3.0 , licor biosciences ) . signal intensity was quantified using image j imaging analysis software ( nih , bethesda , md ) . all protein kinases were identified by a cross search using protein ids of the human kinome . all patients and/or their guardians gave written and oral consent prior to providing blood for isolation of circulating evs . the protocol was approved by the dutch competent authority , centrale commissie mensgebonden onderzoek ( ccmo ) and the ethics committee , medisch etische toesting commissie ( metc ) vumc ( protocol number : nl38229.029.11 ) . we collected 12 ml of edta blood ( in 2 bd vacutainer # 368661 )  yielding 36.5 ml of plasma  from patients with various types of cancer ( n=32 ) and healthy donors ( n=24 ) . samples were centrifuged twice at 2,000g for 10 min to remove cells and frozen at 80c until further processing . statistical analysis was performed using the 2-tailed ( un)paired student 's t - test embedded in graphpad prism software ( version 5.0 for windows , graphpad software , ca , usa ) . for the survival h1650 ( nci - h1650 ; atcc ) and h3255 ( nci - h3255 ; atcc ) non - small - cell lung cancer ( nsclc ) cells were maintained in rpmi-1640 ( lonza biowhittaker , verviers , belgium ) . the glioma cell lines u87 all cell culture media were supplemented with 10% foetal bovine serum ( fbs ) , 100 u / ml penicillin and 0.1 mg / ml streptomycin , referred to as complete dmem . ev - depleted medium was obtained by 2 h ultracentrifugation of serum at 100,000g before use as a medium supplement . drug treatments were performed with erlotinib ( lc laboratories , woburn , ma , usa ) in dmem containing ev - depleted serum . cell viability was assessed by 2 h incubation with mtt 3-(4,5-dimethylthiazolyl-2)-2,5-diophenyl tetrazolium bromide ( sigma - aldrich , zwijndrecht , the netherlands ) . supernatants from cells , cultured for 32 h in serum - free or with 10% ev - depleted serum , were centrifuged at 500g for 10 min to remove cells . after another centrifugation step at 2,000g for 15 min , the samples were concentrated by retention and collection using 100 kda cut - off filters ( amicon ultra-15 , millipore , amsterdam , the netherlands ) and stored at 20c . centrifugation at 10,000g for 45 min was applied , before evs were pelleted by ultracentrifugation at 100,000g for 90 min , using a sw32ti rotor ( beckmann instruments , woerden , the netherlands ) . the ev pellet was treated with proteases ( 0.5 g / l , trypsin , lonza biowhittaker ) or erlotinib ( 10 m ) at 37c for 1 h when indicated . subsequently , evs were washed in pbs followed by centrifugation at 100,000g for 90 min . pellets were solubilized in m - per mammalian protein extraction reagent , supplemented with phosphatase and protease inhibitor cocktails ( pierce , thermo scientific , breda , the netherlands ) . evs were isolated from 2 independent cell cultures ( 10 t175 ) for each cell line and isolated as described above . full sample yield ( corresponding to 1015 g of protein ) was loaded on nupage precast gradient gels , and proteins were separated at 125 v. after electrophoresis , gels were fixed in 50% ethanol containing 3% phosphoric acid and stained with coomassie r-250 and washed in milli - q water ( supplementary fig . gel lanes were cut in 5 slices and each slice was processed by in - gel - digestion according to the protocol previously published by shevchenko ( 15 ) . briefly , samples were treated with dithiothreitol ( 10 mm ) for 1 h at 56c and iodoacetamide ( 50 mm ) for 45 min at rt . peptides were extracted first in 1% formic acid and then in 50% acn/5% formic acid . subsequently , peptides were separated by an ultimate 3,000 nanolc - ms / ms system ( dionex lc - packings , amsterdam , the netherlands ) . after injection , peptides were trapped on a 10 mm100 m i d trap column and separated at 300 nl / min in a 1040% buffer gradient ( 80% acn+0.05% formic acid ) in 60 min . peptides were measured on - line after nanospray ionization , using a q exactive mass spectrometer ( thermofisher , bremen , germany ) . intact masses were measured at resolution 70,000 ( at m / z 200 ) in the orbitrap . ms / ms spectra were acquired at resolution 17,500 ( at m / z 200 ) in the orbitrap using an agc target value of 210 charges and an underfill ratio of 0.1% . ms / ms spectra were searched against the uniprot human reference proteome fasta file ( release sept 2012 ; 147,951 entries ) using maxquant 1.3.0.5 ( default settings ) ( 16 ) . cysteine carboxamidomethylation was treated as fixed modification and methionine oxidation and n - terminal acetylation as variable modifications . peptide and protein identifications were filtered at an fdr of 1% using the decoy database strategy . proteins were quantified by spectral counting ( 17,18 ) , that is , the sum of all ms / ms spectra for each identified protein . these counts were normalized by dividing the spectral counts per protein by the sum of all counts per sample and multiplying by the average sum across all samples ( 18 ) . permeabilization was performed with ice - cold methanol ( 10 min ) , followed by 1 h incubation with 5% bovine serum albumin ( bsa ) . staining with primary antibodies against egfr ( cell signaling # 2232 , 1:200 ) or phospho - y ( 4g10 , millipore , usa , 1:200 ) protein content of samples was determined with the micro bca protein assay ( pierce , thermo scientific , the netherlands ) . for western electrophoresis was performed on precast 412% sds polyacrylamide gels ( nupage , life technologies , bleiswijk , the netherlands ) and transferred to pvdf membranes for immunoblotting of proteins . membranes were probed with antibodies against phospho - egfr - y1068 ( cell signaling , leiden , the netherlands # 2236s ) , egfr ( cs # 2232 ) , phospho - akt - s473 ( cs # 4051s ) , akt ( cs # 9272 ) , phospho - erk1/2-t202/204 ( cs # 9101s ) , erk1/2 ( cs # 9107s ) , -tubulin ( cs # 2144s ) , alix ( cs # 2171s ) , cd63 ( bd bioscience , etten - leur , the netherlands 556,019 ) , cytochrome - c ( bd bioscience 556,433 ) and gapdh ( cs # 2118 ) . irdye infrared dye ( li - cor biosciences , bad homburg , germany ) labelled secondary antibodies were used for detection . membranes were scanned using an odyssey infrared imaging system and accompanying software ( version 3.0 , licor biosciences ) . signal intensity was quantified using image j imaging analysis software ( nih , bethesda , md ) . all protein kinases were identified by a cross search using protein ids of the human kinome . all patients and/or their guardians gave written and oral consent prior to providing blood for isolation of circulating evs . the protocol was approved by the dutch competent authority , centrale commissie mensgebonden onderzoek ( ccmo ) and the ethics committee , medisch etische toesting commissie ( metc ) vumc ( protocol number : nl38229.029.11 ) . we collected 12 ml of edta blood ( in 2 bd vacutainer # 368661 )  yielding 36.5 ml of plasma  from patients with various types of cancer ( n=32 ) and healthy donors ( n=24 ) . samples were centrifuged twice at 2,000g for 10 min to remove cells and frozen at 80c until further processing . statistical analysis was performed using the 2-tailed ( un)paired student 's t - test embedded in graphpad prism software ( version 5.0 for windows , graphpad software , ca , usa ) . for the survival we first investigated the variety and abundance of protein kinases in evs from different cell lines . evs were isolated from conditioned media of 2 nsclc cell lines ( h1650 and h3255 ) and u87 glioma cells , lysed and subjected to tandem mass spectrometry . in total , 3,773 different proteins were identified in at least 2 independent samples . marker proteins for exosomes such as tsg101 , cd63 , flot1 and alix were abundantly present in the samples ( see supplementary table i for full proteome results ) . we next searched for protein kinases in the data set by overlaying all protein ids with publicly available protein kinase ids that were previously published ( 19 ) . kinases were found from all 7 designated kinase groups ( tyrosine kinase , tyrosine kinase like , agc , serine / threonine ( ste ) , cmcg , casein kinase 1 , calcium and calmodulin  dependent kinases ) , but also several kinases from atypical classes ( rgc , other ) were present . evs from u87 cells contained 92 different kinases , while evs from h1650 and h3255 nsclc cells yielded , respectively , 85 and 106 protein kinases ( fig . this analysis shows that the majority of the kinases in evs are present in evs from multiple cell lines . overview of average spectral counts for the tyrosine kinases detected in extracellular vesicle samples from different cell lines as most clinically approved drugs , target kinases from the tyrosine kinase group , we focused on this subset of the kinome . multiple kinases from the tyrosine kinase group were found , with comparable representation ( 32 from a total of 130 kinases ) as in the human genome ( 90 from 518 ) , suggesting rather random inclusion of protein kinases during release of evs . both receptor tyrosine kinases ( egfr , erbb2 , epha2 , axl , met , pdgfr - b ) as well as non - receptor tyrosine kinases were present ( src , jak1 , fak , yes1 ) in evs ( table i ) . in addition multiple cell cycle kinases ( cdk2 , plk1 and aurkb ) and well - known drug targets were identified such as mtor , egfr and met . for quantitation of protein levels in the different samples , egfr and epha2 were among the most prominent receptor tyrosine kinases in evs , while jak1 and yes1 non - receptor tyrosine kinases were released with particular high levels . h3255 evs uniquely showed high levels of erbb2 , while only u87 evs contained pdgfrb . for example , akt was found in evs from u87 and h3255 cells , while erk1/2 was present in all samples . different receptor tyrosine kinases were found to engage similar signalling components , ultimately influencing a relatively small number of downstream kinases ( 20,21 ) . we reasoned that , in particular , the protein kinases akt and erk1/2 could be valuable biomarkers as they reside downstream of multiple oncogenic receptors that are currently being targeted by drugs . akt and erk1/2 could therefore provide information on activity of multiple receptor protein kinases . to study differential phosphorylation , an isogenic human cell line model was used , consisting of u87 glioma cells with and without overexpression of the constitutively active mutant kinase egfr ( egfrviii ) ( fig . introduction of egfrviii in these cells enhances the proliferation rate and results in increased sensitivity of these cells to the egfr inhibitor erlotinib ( fig . ic50 values as measured by the mtt cell viability assay were 8.5 m for the parental cells and 2.3 m for the egfrviii cells ( p<0.001 ) . increased phosphorylation of akt and erk1/2 was found in cells expressing egfrviii in comparison to u87 parental cells ( fig . 2c ) . to visualize the impact of erlotinib treatment on phosphorylation of akt and erk1/2 , quantification of the respective phospho - staining intensity treatment of egfrviii cells with low concentrations of erlotinib ( 1 m ) reduced phosphorylation of both akt and erk1/2 . in u87 parental cells in contrast , reductions in phosphorylation were only noted at a 10-fold higher concentration of erlotinib . no effect was found on egfr , akt and erk1/2 protein expression levels ( fig . 2c ) . u87 cells with or without overexpression of the constitutively active egfrviii receptor were used as model cell line . ( a ) sensitivity of cell lines to the egfr inhibitor erlotinib was determined in the mtt cell viability assay . ( b ) subsequently , u87 and u87-egfrviii cells were treated with increasing concentrations of erlotinib or dmso vehicle . ( c ) intensity of akt and erk1/2 phospho - stainings was quantified by image j. relative phosphorylation during erlotinib treatment is shown . ( d ) to assess statistical significance , the 2-tailed unpaired student 's t - test was applied . * * * p0.001 . to further subtype the evs released by u87 cells in vitro , relative abundance of the exosome markers alix , cd63 and apoptotic marker cytochrome - c these results suggest a significant enrichment of exosomes in the ev pool . to determine whether egfrviii expression influences ev production , protein yields from isolations were measured . total protein yield as measured by the micro bca protein assay was normalized to cell counts . no significant differences were observed in the yield from the isolations of u87 cells or egfrviii - containing cells ( 15.5 and 13.3 g , respectively ; fig . 3b ) . to investigate whether evs could provide a read - out of oncogenic signalling in cancer cells , evs were isolated from cells treated with erlotinib and subjected to western blot . total akt and erk1/2 protein levels were readily detected by western blot , while egfr western blot analysis of ev samples appeared less efficient . phosphorylation as well as total protein levels of protein kinases was lower in evs than in u87-egfrviii cells , used as positive control for immunostaining . on the other hand , phosphorylation of egfr , akt and erk1/2 kinases was elevated in evs from egfrviii cells in comparison to evs from parental u87 cells ( fig . similar to what was found in cells , treatment of the erlotinib - sensitive u87-egfrviii cells with 1 m erlotinib for 32 h resulted in a decrease in phosphorylation of akt and erk1/2 in evs . however , in the evs from the erlotinib - resistant parental u87 cells only a modest reduction in phosphorylation was observed at a 10-fold higher erlotinib dose ( fig . altogether these results indicate that the phosphorylation profiles of akt and erk1/2 kinases in evs may reflect the intracellular phosphorylation status during treatment with erlotinib . evs were isolated from u87 cells . to further characterize evs , relative abundance of exosome markers alix , cd63 and ( a ) ev protein yield from cells with or without egfrviii was determined by micro bca protein assay . ( b ) u87 cells with or without egfrviii were treated with erlotinib and evs were isolated . phosphorylation of egfr and downstream mediators akt and erk1/2 in evs was determined by western blot . ( c ) intensity of phosphostainings was quantified by image j. relative phosphorylation during treatment with erlotinib is shown . ( d ) to assess statistical significance , the 2-tailed unpaired student 's t - test was applied . ns = non - significant . in order to determine whether the double - layered membranes of evs protect their content from proteases in whole cells lysates in contrast , complete digestion was observed after 1 h of treatment ( fig . short - term exposure of the evs to high concentrations of erlotinib ( 10 m ) had no effect on either phosphorylation or total protein levels in the evs ( fig . we conclude that akt and erk1/2 protein kinases reside within evs , which protects them from potential detrimental influences of enzymes in the circulation . evs were isolated from culture medium of an equal number of u87 cells with or without egfrviii . g / l ) or erlotinib ( 10 m ) for 1 h at 37c . western blot analysis was performed using equal volumes to determine protein levels and their phosphorylation . we next investigated whether akt and erk1/2 kinase phosphorylation may be used as biomarker in patients with cancer . since limited information is available on the production of evs by different cancer cells , a cohort of 42 patients was recruited with a variety a cancer types ( table ii ) . three patients with oesophageal cancer , 3 patients with breast cancer , 21 patients with nsclc and 4 patients with glioma were included among others ( table ii ) . thirty - three patients ( 76% ) had metastatic disease , while 9 patients ( 21% ) had either glioma or localized disease . at the time , the first blood samples were obtained none of the patients received active systemic treatment . patient characteristics first , ev protein levels in blood of healthy volunteers ( n=22 ) and the patients with cancer were measured . ev protein yield from each sample was measured using the micro bca protein assay ( fig . ev samples derived from patients with cancer contained significantly more protein ( p=0.0067 ) compared to healthy donors . in 6 patients , increased ev protein levels were observed . two of these patients had nsclc , while the others had crc , sclc , oesophageal cancer and rcc . no significant difference was detected in the amount of ev protein between patients with metastatic disease and patients with localized disease ( p=0.78 ) ( fig . 5b ) . subsequently , akt and erk1/2 protein levels and their phosphorylation status in circulating evs in patients and healthy donors were determined . western blot analysis was performed using equal protein amounts from 21 randomly selected patients and 22 healthy donors . total akt protein was readily detected in all ev samples , whereas total erk1/2 levels showed more variation between samples ( fig . alix staining was not detectable in ev samples isolated from plasma , whereas gapdh staining was high ( supplementary fig . phosphorylation of akt and erk1/2 was measured in ev samples from both patients as well as healthy volunteers . as compared to phosphorylation levels of these proteins in cancer cells , phosphorylation of akt and erk1/2 was relatively low ( fig . semi - quantification of phosphorylation was performed by calculating the relative signal intensity from phosphorylated and total protein levels . no significant differences were observed in the phosphorylation status of akt and erk1/2 between patients with cancer and healthy volunteers , p=0.78 and p=0.76 , respectively ( fig . 5d and e ) . additionally , the subgroup of patients with high protein levels did not demonstrate increased akt ( p=0.56 ) or erk1/2 phosphorylation ( p=0.98 ) as compared to the healthy donors ( fig . 5d and e ) . to determine variability in total ev kinase levels in plasma , maximum protein amounts substantial variation in total akt and erk1/2 levels were noted in evs from these patients ( fig . ( a  b ) phosphorylation of akt and erk1/2 was measured by western blot using equal protein amounts from 21 patients and 22 healthy donors . ( c ) signal intensities were quantified by image j imaging analysis software and phosphorylation was measured by calculating the relative signal intensity from phosphorylated and total protein levels . subgroup analysis was performed with patients having high ev protein levels as shown in red . e ) to determine variability in total ev kinase levels in plasma , maximum protein amounts were loaded from a subgroup of patients with nsclc . ( f ) statistical significance was tested using the 2-tailed paired student 's t - test . ns = not significant , p0.05 , * * p0.01 , * * * p0.001 . a subgroup of 12 patients with kras mutated nsclc ( n=12 ) were included in an on - going single arm phase ii study ( more detailed information can be found in supplementary table ii ) . based on the kras mutation status , patients were selected for treatment with sorafenib and metformin . sorafenib is a small molecule tyrosine kinase inhibitor , which inhibits angiogenesis in tumours through blockade of vegfr2/3 and pdgf receptors . in addition , it is known to have affinity for craf and braf , both mediators downstream of kras . metformin , on the other hand , reduces systemic glucose and insulin levels , thereby interfering with insulin / insulin - like growth factor receptor ( igfr1 ) signalling in cancer cells . since akt and erk1/2 act downstream of igfr1 and ras / raf , respectively , ev protein levels were investigated as potential predictive biomarkers . consistent with previous results both before and during treatment , phosphorylation in evs was low ( data not shown ) . before and 3 weeks after onset of treatment , ct scans and blood plasma samples were collected . treatment with sorafenib / metformin resulted in partial response / stable disease or progressive disease ( pd ) at 3 weeks according to recist criteria ( fig . median progression free survival ( pfs ) of the favourable response group was 4.1 months ( 95% ci 0.67.5 ) , while the resistant group responded for only 0.8 months ( 95% ci 0.70.8 ) . overall survival of the favourable response group was 6.5 months ( 95% ci 3.19.9 ) , while the patients with pd survived for only 2.1 months ( 95% ci 0.33.9 ) . plasma evs were isolated and samples were subjected to western blot analysis of akt and erk1/2 protein levels ( fig . protein levels at baseline and during treatment were quantified and correlated to the treatment response ( fig . no reduction in the akt and erk1/2 protein levels in evs were observed during treatment with sorafenib / metformin in patients with pd at 3 weeks . however , in patients with partial response / stable disease ( n=7 ) , significant reductions were observed in akt protein levels in plasma evs ( p<0.005 , fig . 6b and c ) . similarly , erk1/2 levels in evs decreased during treatment in patients with a favourable treatment response ; however , this difference did not reach significance ( p=0.297 , fig . overall we conclude that substantial variation is observed in akt and erk1/2 protein levels in evs particularly during treatment with sorafenib / metformin . akt protein levels in evs may serve as potential predictive biomarker for the response of patients with nsclc to sorafenib and metformin . representative ct images of patient # 7 at baseline and with stable disease ( sd ) at 3 weeks on sorafenib / metformin and of patient # 9 at baseline and with progressive disease ( pd ) at 3 weeks on sorafenib / metformin . ( a ) western blot analysis was performed of plasma evs of nsclc patients ( n=12 ) . ( b ) total akt and erk1/2 levels in evs , 3 weeks after start of treatment , are shown . patients with pd and sd or partial response ( pr ) on sorafenib / metformin treatment are grouped . ( b ) samples are paired pre- and on - treatment samples depicted with  and + , respectively . ( c ) kaplan meier curves were generated for the treatment response ( pfs ) of patients with stable and decreased akt and erk1/2 levels in evs . ( c  d ) to assess statistical significance , the 2-tailed unpaired t - test was applied . * p<0.001 , * * p<0.0001 . the log - rank test was used for statistical analysis of survival analyses . we first investigated the variety and abundance of protein kinases in evs from different cell lines . evs were isolated from conditioned media of 2 nsclc cell lines ( h1650 and h3255 ) and u87 glioma cells , lysed and subjected to tandem mass spectrometry . in total , 3,773 different proteins were identified in at least 2 independent samples . marker proteins for exosomes such as tsg101 , cd63 , flot1 and alix were abundantly present in the samples ( see supplementary table i for full proteome results ) . we next searched for protein kinases in the data set by overlaying all protein ids with publicly available protein kinase ids that were previously published ( 19 ) . kinases were found from all 7 designated kinase groups ( tyrosine kinase , tyrosine kinase like , agc , serine / threonine ( ste ) , cmcg , casein kinase 1 , calcium and calmodulin  dependent kinases ) , but also several kinases from atypical classes ( rgc , other ) were present . evs from u87 cells contained 92 different kinases , while evs from h1650 and h3255 nsclc cells yielded , respectively , 85 and 106 protein kinases ( fig . this analysis shows that the majority of the kinases in evs are present in evs from multiple cell lines . overview of average spectral counts for the tyrosine kinases detected in extracellular vesicle samples from different cell lines as most clinically approved drugs , target kinases from the tyrosine kinase group , we focused on this subset of the kinome . multiple kinases from the tyrosine kinase group were found , with comparable representation ( 32 from a total of 130 kinases ) as in the human genome ( 90 from 518 ) , suggesting rather random inclusion of protein kinases during release of evs . both receptor tyrosine kinases ( egfr , erbb2 , epha2 , axl , met , pdgfr - b ) as well as non - receptor tyrosine kinases were present ( src , jak1 , fak , yes1 ) in evs ( table i ) . in addition multiple cell cycle kinases ( cdk2 , plk1 and aurkb ) and well - known drug targets were identified such as mtor , egfr and met . for quantitation of protein levels in the different samples , egfr and epha2 were among the most prominent receptor tyrosine kinases in evs , while jak1 and yes1 non - receptor tyrosine kinases were released with particular high levels . h3255 evs uniquely showed high levels of erbb2 , while only u87 evs contained pdgfrb . for example , akt was found in evs from u87 and h3255 cells , while erk1/2 was present in all samples . different receptor tyrosine kinases were found to engage similar signalling components , ultimately influencing a relatively small number of downstream kinases ( 20,21 ) . we reasoned that , in particular , the protein kinases akt and erk1/2 could be valuable biomarkers as they reside downstream of multiple oncogenic receptors that are currently being targeted by drugs . akt and erk1/2 could therefore provide information on activity of multiple receptor protein kinases . to study differential phosphorylation , an isogenic human cell line model was used , consisting of u87 glioma cells with and without overexpression of the constitutively active mutant kinase egfr ( egfrviii ) ( fig . introduction of egfrviii in these cells enhances the proliferation rate and results in increased sensitivity of these cells to the egfr inhibitor erlotinib ( fig . ic50 values as measured by the mtt cell viability assay were 8.5 m for the parental cells and 2.3 m for the egfrviii cells ( p<0.001 ) . increased phosphorylation of akt and erk1/2 was found in cells expressing egfrviii in comparison to u87 parental cells ( fig . 2c ) . to visualize the impact of erlotinib treatment on phosphorylation of akt and erk1/2 , quantification of the respective phospho - staining intensity treatment of egfrviii cells with low concentrations of erlotinib ( 1 m ) reduced phosphorylation of both akt and erk1/2 . in u87 parental cells in contrast , reductions in phosphorylation were only noted at a 10-fold higher concentration of erlotinib . no effect was found on egfr , akt and erk1/2 protein expression levels ( fig . u87 cells with or without overexpression of the constitutively active egfrviii receptor were used as model cell line . ( a ) sensitivity of cell lines to the egfr inhibitor erlotinib was determined in the mtt cell viability assay . ( b ) subsequently , u87 and u87-egfrviii cells were treated with increasing concentrations of erlotinib or dmso vehicle . ( c ) intensity of akt and erk1/2 phospho - stainings was quantified by image j. relative phosphorylation during erlotinib treatment is shown . ( d ) to assess statistical significance , the 2-tailed unpaired student 's t - test was applied . * * * p0.001 . to further subtype the evs released by u87 cells in vitro , relative abundance of the exosome markers alix , cd63 and apoptotic marker cytochrome - c 3a ) . increased levels of cd63 and alix were observed , while cytochrome - c was not detected in ev samples . these results suggest a significant enrichment of exosomes in the ev pool . to determine whether egfrviii expression influences ev production , protein yields from isolations were measured . total protein yield as measured by the micro bca protein assay was normalized to cell counts . no significant differences were observed in the yield from the isolations of u87 cells or egfrviii - containing cells ( 15.5 and 13.3 g , respectively ; fig . 3b ) . to investigate whether evs could provide a read - out of oncogenic signalling in cancer cells , evs were isolated from cells treated with erlotinib and subjected to western blot . total akt and erk1/2 protein levels were readily detected by western blot , while egfr western blot analysis of ev samples appeared less efficient . phosphorylation as well as total protein levels of protein kinases was lower in evs than in u87-egfrviii cells , used as positive control for immunostaining . on the other hand , phosphorylation of egfr , akt and erk1/2 kinases was elevated in evs from egfrviii cells in comparison to evs from parental u87 cells ( fig . similar to what was found in cells , treatment of the erlotinib - sensitive u87-egfrviii cells with 1 m erlotinib for 32 h resulted in a decrease in phosphorylation of akt and erk1/2 in evs . however , in the evs from the erlotinib - resistant parental u87 cells only a modest reduction in phosphorylation was observed at a 10-fold higher erlotinib dose ( fig . altogether these results indicate that the phosphorylation profiles of akt and erk1/2 kinases in evs may reflect the intracellular phosphorylation status during treatment with erlotinib . evs were isolated from u87 cells . to further characterize evs , relative abundance of exosome markers alix , cd63 and the apoptotic marker cytochrome - c was analyzed by western blot . ( a ) ev protein yield from cells with or without egfrviii was determined by micro bca protein assay . ( b ) u87 cells with or without egfrviii were treated with erlotinib and evs were isolated . phosphorylation of egfr and downstream mediators akt and erk1/2 in evs was determined by western blot . ( c ) intensity of phosphostainings was quantified by image j. relative phosphorylation during treatment with erlotinib is shown . ( d ) to assess statistical significance , the 2-tailed unpaired student 's t - test was applied . ns = non - significant . in order to determine whether the double - layered membranes of evs protect their content from proteases in whole cells lysates in contrast , complete digestion was observed after 1 h of treatment ( fig . simultaneously , a reduction of the alix levels was found in the protease treated samples . short - term exposure of the evs to high concentrations of erlotinib ( 10 m ) had no effect on either phosphorylation or total protein levels in the evs ( fig . we conclude that akt and erk1/2 protein kinases reside within evs , which protects them from potential detrimental influences of enzymes in the circulation . evs were isolated from culture medium of an equal number of u87 cells with or without egfrviii . ev pellets were treated with proteases ( trypsin , 0.5 g / l ) or erlotinib ( 10 m ) for 1 h at 37c . western blot analysis was performed using equal volumes to determine protein levels and their phosphorylation . we next investigated whether akt and erk1/2 kinase phosphorylation may be used as biomarker in patients with cancer . since limited information is available on the production of evs by different cancer cells , a cohort of 42 patients was recruited with a variety a cancer types ( table ii ) . three patients with oesophageal cancer , 3 patients with breast cancer , 21 patients with nsclc and 4 patients with glioma were included among others ( table ii ) . thirty - three patients ( 76% ) had metastatic disease , while 9 patients ( 21% ) had either glioma or localized disease . at the time , the first blood samples were obtained none of the patients received active systemic treatment . patient characteristics first , ev protein levels in blood of healthy volunteers ( n=22 ) and the patients with cancer were measured . ev protein yield from each sample was measured using the micro bca protein assay ( fig . ev samples derived from patients with cancer contained significantly more protein ( p=0.0067 ) compared to healthy donors . in 6 patients , increased ev protein levels were observed . two of these patients had nsclc , while the others had crc , sclc , oesophageal cancer and rcc . no significant difference was detected in the amount of ev protein between patients with metastatic disease and patients with localized disease ( p=0.78 ) ( fig . 5b ) . subsequently , akt and erk1/2 protein levels and their phosphorylation status in circulating evs in patients and healthy donors were determined . western blot analysis was performed using equal protein amounts from 21 randomly selected patients and 22 healthy donors . total akt protein was readily detected in all ev samples , whereas total erk1/2 levels showed more variation between samples ( fig . alix staining was not detectable in ev samples isolated from plasma , whereas gapdh staining was high ( supplementary fig . subsequently , phosphorylation of akt and erk1/2 was measured in ev samples from both patients as well as healthy volunteers . as compared to phosphorylation levels of these proteins in cancer cells , phosphorylation of akt and erk1/2 was relatively low ( fig . semi - quantification of phosphorylation was performed by calculating the relative signal intensity from phosphorylated and total protein levels . no significant differences were observed in the phosphorylation status of akt and erk1/2 between patients with cancer and healthy volunteers , p=0.78 and p=0.76 , respectively ( fig . 5d and e ) . additionally , the subgroup of patients with high protein levels did not demonstrate increased akt ( p=0.56 ) or erk1/2 phosphorylation ( p=0.98 ) as compared to the healthy donors ( fig . 5d and e ) . to determine variability in total ev kinase levels in plasma , maximum protein amounts substantial variation in total akt and erk1/2 levels were noted in evs from these patients ( fig . ( a  b ) phosphorylation of akt and erk1/2 was measured by western blot using equal protein amounts from 21 patients and 22 healthy donors . ( c ) signal intensities were quantified by image j imaging analysis software and phosphorylation was measured by calculating the relative signal intensity from phosphorylated and total protein levels . subgroup analysis was performed with patients having high ev protein levels as shown in red . e ) to determine variability in total ev kinase levels in plasma , maximum protein amounts were loaded from a subgroup of patients with nsclc . ( f ) statistical significance was tested using the 2-tailed paired student 's t - test . ns = not significant , p0.05 , * * p0.01 , * * * p0.001 . a subgroup of 12 patients with kras mutated nsclc ( n=12 ) were included in an on - going single arm phase ii study ( more detailed information can be found in supplementary table ii ) . based on the kras mutation status sorafenib is a small molecule tyrosine kinase inhibitor , which inhibits angiogenesis in tumours through blockade of vegfr2/3 and pdgf receptors . in addition , it is known to have affinity for craf and braf , both mediators downstream of kras . metformin , on the other hand , reduces systemic glucose and insulin levels , thereby interfering with insulin / insulin - like growth factor receptor ( igfr1 ) signalling in cancer cells . since akt and erk1/2 act downstream of igfr1 and ras / raf , respectively , ev protein levels were investigated as potential predictive biomarkers . consistent with previous results both before and during treatment , phosphorylation in evs was low ( data not shown ) . before and 3 weeks after onset of treatment , ct scans and blood plasma samples were collected . treatment with sorafenib / metformin resulted in partial response / stable disease or progressive disease ( pd ) at 3 weeks according to recist criteria ( fig . median progression free survival ( pfs ) of the favourable response group was 4.1 months ( 95% ci 0.67.5 ) , while the resistant group responded for only 0.8 months ( 95% ci 0.70.8 ) . overall survival of the favourable response group was 6.5 months ( 95% ci 3.19.9 ) , while the patients with pd survived for only 2.1 months ( 95% ci 0.33.9 ) . plasma evs were isolated and samples were subjected to western blot analysis of akt and erk1/2 protein levels ( fig . protein levels at baseline and during treatment were quantified and correlated to the treatment response ( fig . no reduction in the akt and erk1/2 protein levels in evs were observed during treatment with sorafenib / metformin in patients with pd at 3 weeks . however , in patients with partial response / stable disease ( n=7 ) , significant reductions were observed in akt protein levels in plasma evs ( p<0.005 , fig . similarly , erk1/2 levels in evs decreased during treatment in patients with a favourable treatment response ; however , this difference did not reach significance ( p=0.297 , fig . overall we conclude that substantial variation is observed in akt and erk1/2 protein levels in evs particularly during treatment with sorafenib / metformin . akt protein levels in evs may serve as potential predictive biomarker for the response of patients with nsclc to sorafenib and metformin . representative ct images of patient # 7 at baseline and with stable disease ( sd ) at 3 weeks on sorafenib / metformin and of patient # 9 at baseline and with progressive disease ( pd ) at 3 weeks on sorafenib / metformin . ( a ) western blot analysis was performed of plasma evs of nsclc patients ( n=12 ) . ( b ) total akt and erk1/2 levels in evs , 3 weeks after start of treatment , are shown . patients with pd and sd or partial response ( pr ) on sorafenib / metformin treatment are grouped . ( b ) samples are paired pre- and on - treatment samples depicted with  and + , respectively . ( c ) kaplan meier curves were generated for the treatment response ( pfs ) of patients with stable and decreased akt and erk1/2 levels in evs . ( c  d ) to assess statistical significance , the 2-tailed unpaired t - test was applied . * p<0.001 , * * p<0.0001 . the log - rank test was used for statistical analysis of survival analyses . we found that evs contain multiple protein kinases and that their phosphorylation corresponds to the profile in tumour cells in vitro . in addition , preliminary evaluation of ev associated akt protein levels in patients reveals a correlation with response to sorafenib / metformin treatment . presence of multiple tumour - derived drug targets in evs suggests potential as biomarker platform . mutational analysis of genes encoding kinase inhibitor targets , such as egfr and braf in tumour tissue biopsies , proved to be a powerful tool to predict treatment response in patients with nsclc and melanoma ( 22,23 ) . tumour mutations are also readily detectable in several blood compartments such as plasma , evs and blood platelets ( 8,24 ) 
 ( 25 ) , allowing for non - invasive diagnostics . important advantage of blood - based biomarkers is that they are easily available and also allow longitudinal monitoring during treatment . mutations in dna or rna are stable and tumour specific , but biological effects may be hampered by , for example methylation or rna interference . protein levels and protein phosphorylation , in contrast , directly correlate with biological effects in cells , yielding powerful biomarkers ( 26 ) . circulating peptide and protein signatures previous studies indicated that upon treatment with small molecule kinase inhibitors , multiple modulations of phosphorylation occur in tumours of patients ( 31,32 ) . for example , in tumour biopsies from patients with gastric cancer , a reduction in akt phosphorylation was found , with an interesting association with apoptosis ( 33 ) . another study , investigating evs as potential biomarker platform , indicated that analysis of protein levels in evs allows real - time monitoring of treatment effects induced in tumours in vivo 
 ( 7 ) . here , we show that proteins in evs are protected against external influences and also that post - translational modifications such as phosphorylation are conserved . moreover , our in vitro data suggest that circulating evs may provide a read - out of oncogenic signalling in tumour cells . however , despite very high phosphorylation in cancer cells , we found relatively low phosphorylation of akt and erk1/2 in plasma evs . since phosphorylation was low , but fairly detectable in cell culture samples , it is unlikely that phosphorylation is lost during the vesicle isolation . levels in healthy donors suggest that akt and erk1/2 studies may suffer from a high background of vesicles released by other cells . therefore , it should be evaluated whether more tumour - specific proteins may give lower background and therefore are more attractive as a biomarker . because of the role of akt and erk1/2 in signalling of multiple receptor tyrosine kinases , phosphorylation of these proteins in cells as well as their evs may still be of great interest . although we found that phosphorylation of akt and erk1/2 in evs is low , it remains to be investigated whether this event has any value as a biomarker . for such a study , it is important to develop a more sensitive and specific method for quantitative measurements of phosphorylated proteins in evs . these may be based on , for example , elisa , nmr , peptide array or mass spectrometry techniques ( 7,34 ) . current ev isolation methods typically yield relatively low protein levels , which limits protein biomarker studies . for clinical applications , the ev studies may benefit from a simple and robust isolation strategy that also allows specific isolation of tumour - derived evs from blood . flow cytometry methods have been described , but may be particularly beneficial for the larger vesicles ( e.g. apoptotic bodies ) ( 35 ) . capture - based isolation procedures are convenient but use surface markers to specifically bind subsets of evs ( exosomes , microvesicles ) , and this may also bias and limit biomarker studies . we did not detect alix in our plasma ev analysis , while in our in vitro analysis a pronounced exosome signature was noted in the ev samples with abundant alix , tsg101 and cd63 . to the best of our knowledge , future biomarker research may therefore also benefit from further subtyping of the different evs . combinations of antibodies against ev subtype markers ( e.g. cd63 , cytochrome - c ) with antibodies raised against epitopes exclusively present on mutant proteins ( 36 ) may help in isolation of tumour - specific evs . we found increased ev protein amounts in patients with cancer compared to healthy volunteers . in our sorafenib / metformin analysis , high akt levels during treatment were associated with treatment failure and poor survival . met protein levels in evs were previously found to be correlated with overall survival of patients with melanoma ( 5 ) . these results suggest that high kinase levels in evs are a poor prognostic feature in patients with cancer . it would be of interest to investigate the value of ev counts as well as ev protein amounts both at baseline as well as during treatment , in conjunction with protein kinase levels as biomarker . the biomarker analysis also showed downmodulation of total akt and to some extent erk1/2 protein levels during treatment with sorafenib / metformin . unfortunately , no tumour tissue samples were available for comparative analyses between tumour tissue and ev levels . whether these results can be related to direct biological effects of the treatment on tumour cells or reflect a mere systemic response remains enigmatic . however , this analysis warrants further investigation and proves potential of protein kinase levels as biomarker in patients with cancer . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .
backgroundextracellular vesicles ( evs ) are small nanometre - sized vesicles that are circulating in blood . 
 they are released by multiple cells , including tumour cells . we hypothesized that circulating evs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors.methodsevs released by u87 glioma cells , h3255 and h1650 non - small - cell lung cancer ( nsclc ) cells were profiled by tandem mass spectrometry . 
 total akt / protein kinase b and extracellular signal regulated kinase 1/2 ( erk1/2 ) levels as well as their relative phosphorylation were measured by western blot in isogenic u87 cells with or without mutant epidermal growth factor receptor ( egfrviii ) and their corresponding evs . to assess biomarker potential , 
 plasma samples from 24 healthy volunteers and 42 patients with cancer were used.resultsin total , 130 different protein kinases were found to be released in evs including multiple drug targets , such as mammalian target of rapamycin ( mtor ) , akt , erk1/2 , axl and egfr . 
 overexpression of egfrviii in u87 cells results in increased phosphorylation of egfr , akt and erk1/2 in cells and evs , whereas a decreased phosphorylation was noted upon treatment with the egfr inhibitor erlotinib . 
 ev samples derived from patients with cancer contained significantly more protein ( p=0.0067 ) compared to healthy donors . 
 phosphorylation of akt and erk1/2 in plasma evs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells . 
 preliminary analysis of total akt and erk1/2 levels in plasma evs from patients with nsclc before and after sorafenib / metformin treatment ( n=12 ) shows a significant decrease in akt levels among patients with a favourable treatment response ( p<0.005).conclusionphosphorylation of protein kinases in evs reflects their phosphorylation in tumour cells . 
 total akt protein levels may allow monitoring of kinase inhibitor responses in patients with cancer .