elucidation of the 
 dynamic changes in secretomes , i.e. , the secreted 
 subset of the proteome , underlies a systems approach to understanding 
 the mechanisms controlling cell  cell and cell it is now clear that 
 changes in the cellular microenvironment determine disease progression , 
 and that these include two - way interactions between cancer cells and 
 surrounding stromal cells . several recent studies have 
 employed proteomic techniques to define 
 the secretomes of cancer cells . recent studies have also defined the secretome in breast and colon 
 cancer fibroblasts , but in general the interrogation 
 of stromal cell secretomes by proteomic methods has been limited . changes in secretomes may reflect alterations in secretory protein 
 abundance due to variations in gene expression , rates of exocytosis 
 or presecretory post - translational processing . but in addition , there 
 may also be extensive postsecretory proteolysis that defines the extracellular 
 tumor degradome . in cancer , alterations in extracellular proteolysis 
 through differential secretion of proteases or their inhibitors is 
 important , not least because it underlies disease progression and 
 sensitivity to protease targeted therapies . cancer - associated 
 fibroblasts , of which myofibroblasts are a subset , 
 are important stromal cells that exhibit an altered phenotype in many 
 cancers . myofibroblasts play important roles in defining the tissue microenvironment 
 through secretion of extracellular matrix components , growth factors , 
 proteases and their inhibitors . differences between 
 normal and cancer - associated myofibroblasts ( cams ) have been linked 
 to tumor progression by mechanisms including recruitment from circulating 
 mesenchymal stromal cells , global dna hypomethylation and changes 
 in gene expression profiles . since myofibroblasts stimulate 
 cancer cell invasion , in the present study we sought to define the 
 differences between gastric cams compared with adjacent tissue - derived 
 myofibroblasts ( atms ) with respect to proteolytic processing of their 
 secretomes . the data indicate both upregulation and activation of 
 matrix metalloproteinases ( mmps)-1 , -2 , and -3 are characteristic 
 of the cam secretome . myofibroblasts from gastric 
 cancers ( cams ) and adjacent tissue ( atms ) from two patients have been 
 described previously ( supporting information ) . the work was approved by the ethics 
 committee of the university of szeged , hungary , and subjects provided 
 informed consent . myofibroblasts were 
 cultured in dmem 
 silac media ( pierce , thermo scientific , rockford , il , usa ) for 6 population 
 doublings in the presence of either natural ( light ) or heavy c6-labeled l - arginine ( 0.94 mm ) and c6l - lysine ( 0.46 mm ) ( invitrogen , paisley , 
 renfrew , u.k . ) . media were further supplemented with 10% dialyzed 
 fetal bovine serum ( pierce ) , 2% antibiotic / antimycotic ( sigma - aldrich , 
 poole , u.k . ) and 1% penicillin / streptomycin ( sigma ) . media ( 10 ml , serum - free ) obtained 
 from 1  10 myofibroblasts plated in 10 cm diameter 
 dishes ( 8090% confluency ) were collected after 24 h. samples 
 were concentrated to approximately 0.5 ml using amicon ultra-15 3 
 kda centrifugal filter devices ( millipore , watford , u.k . ) , precipitated 
 with 20% tca and resuspended in 50 mm hepes , ph 7.4 , 100 mm nacl , 
 0.8% w / v chaps , 1% v / v protease inhibitor cocktail set iii , edta - free 
 ( calbiochem , merck biosciences , beeston , u.k . ) . equal amounts ( 35 
 g each ) of light and heavy silac - labeled secretome samples 
 from cams and atms were mixed following determination of protein concentration 
 by the bradford assay ( bio - rad lab , inc . , hemel hempstead , u.k . ) . methionyl - cofradic 
 was performed as described previously ( see supporting information methods ) . samples were reduced and s - alkylated , and following trypsinization 
 ( trypsin : protein 1:100 ) , peptides were fractionated by reversed - phase 
 hplc ( rp - hplc ) using an agilent 1100 hplc system with a zorbax 300sb - c18 column ( 2.1 mm ( internal diameter )  150 mm , agilent 
 technologies , wokingham , u.k . ) . the resulting hplc fractions were 
 further processed by incubating for 30 min with 0.1% w / v hydrogen 
 peroxide at 30 c . following oxidation of methionines , reaction 
 mixtures were immediately reinjected onto the rp - hplc column for secondary 
 rp - hplc separations under identical conditions . fractions with methionine 
 containing peptides displayed a hydrophilic shift and were collected 
 ( n = 90 ) and analyzed by lc n - terminal 
 cofradic was performed as described previously ( see supporting information methods ) . proteins were reduced and alkylated , and primary - and -amines 
 were blocked by trideutero - acetylation . samples were then trypsinized , 
 and n - terminal peptides were pre - enriched by strong cation exchange 
 chromatography at low ph . following a pyro - glutamate removal step , 
 peptides were separated by rp - hplc as described above . primary fractions 
 were incubated with 2,4,6-trinitrobenzenesulphonic acid ( tnbs ) to 
 modify internal tryptic peptides with free -n - termini . a series 
 of secondary rp - hplc runs was then performed on each individual primary 
 fraction , and n - terminal peptides ( which did not display a hydrophobic 
 shift ) were collected ( n = 36 ) for lc samples prepared for  shotgun  
 analysis of the secretomes were processed following the same method 
 as for met - cofradic up to the stage immediately before the first rp - hplc 
 run . at this point ms / ms analysis 
 ( see supporting information methods ) . for neo - n - terminal enrichment , the non - cofradic method employed a 
 scx - only enrichment of n - terminal peptides . samples were prepared 
 as for n - terminal cofradic up to the first rp - hplc run . at this stage 60 fractions of 1 min interval were collected , pooled to give a total 
 of 20 fractions , dried and prepared for lc  ms / ms analysis . peptides were analyzed using a ltq orbitrap xl mass spectrometer 
 ( thermo electron , bremen , germany ) as described previously . mascot generic files ( mgf ) were created using 
 the mascot distiller software ( version 2.2.1.0 , matrix science , ltd . , 
 london , u.k . ) . when generating peak lists , grouping of spectra was 
 performed with a maximum intermediate retention time of 30 s and maximum 
 intermediate scan count of 5 . a peak list was only generated when the spectrum 
 contained more than 10 peaks . there was no deisotoping , and the relative 
 signal - to - noise limit for both precursor and fragment ions was set 
 to 2 . the peak lists were then searched with mascot using the mascot 
 daemon interface ( version 2.2.0 , matrix science , ltd . ) against human 
 proteins in the swiss - prot database ( uniprot release 15.0 , containing 
 20  333 human protein sequences ) . spectra were searched with 
 semiargc / p enzyme settings , allowing no missed cleavages for the n - terminal 
 peptide experiments , and with trypsin / p settings allowing no missed 
 cleavages for the met - cofradic / shotgun experiments . mass tolerance 
 of the precursor ions was set to 10 ppm ( with mascot s c13 
 option set to 1 ) and of fragment ions to 0.5 da . variable modifications were acetylation of alpha - n - termini 
 and pyroglutamate formation of n - terminal glutamine residues ; fixed 
 modification was oxidation of methionine ( sulfoxide ) . additionally , 
 for n - terminal peptide experiments , trideutero - acetylation of the 
 n - terminus was set as variable peptide modification , and trideutero - acetylation 
 of lysine side chains was included as fixed modification . only peptides 
 that were ranked one and scored above the identity threshold score the fdr was calculated for every 
 search as described previously ( see supporting 
 information methods , table sm3 ) . identified peptides were quantified using the mascot distiller quantitation 
 toolbox ( www.matrixscience.com ) in the  precursor  
 mode as described previously . ratios 
 for all peptides of interest were validated by manual inspection of 
 spectra . for processing of all ms data , protein ratios were inferred 
 using the mean of the peptide group ratios for each protein . a peptide 
 group represents all quantifications of a single peptide sequence 
 in an experiment . the distribution of protein ratios as determined 
 by the met - cofradic and shotgun experiments was plotted using rover . this was used to define thresholds to give the 
 5% of proteins with the largest fold changes in cams relative to atms . all spectra have been stored in the pride database ( http://www.ebi.ac.uk/pride/ , accession numbers 2715727161 ) using pride converter . protein subcellular localizations and functional 
 classifications were manually curated , using the uniprot and hprd 
 online databases . immunocompromised 
 mice ( 6 weeks old , 
 balb / c nu / nu , charles river , wilmington , ma ) with xenogafts of mkn45 
 cells with or without cams ( supporting information methods ) were used for imaging mmp - activity using mmpsense 750 fast . these experiments were approved by the university of liverpool animal 
 welfare committee and were conducted in compliance with the u.k . myofibroblast 
 cell extracts were prepared 
 in ripa buffer containing protease and phosphatase inhibitors . cell 
 extracts or media were resolved by sds - page and processed for western 
 blotting as previously described . blots 
 were probed with antibodies against mmp-1 ( baf901 , r&d systems , 
 minneapolis , mn , usa ) , mmp-2 ( baf902 , r&d systems ) and mmp-3 ( baf513 , 
 r&d systems ) . membranes were reprobed with anti - gapdh antibody 
 ( biodesign , saco , maine , usa ) for assessing equal loading where appropriate . fluorogenic assays for mmp enzyme activity 
 were performed using selective substrates : dnp - pro - leu - ala - leu - trp - ala - arg - oh 
 ( mmp-1 ; calbiochem , bedfont cross , u.k . ) , dnp - pro - leu - gly - met - trp - ser - arg - oh 
 ( mmp-2/9 ; calbiochem ) , mca - pro - leu - ala - nva - dpa - ala - arg - nh2 ( mmp-2 ; calbiochem ) , dnp - pro - tyr - ala - ty - trp - met - arg - oh ( mmp-3 ; anaspec , 
 fremont , ca , usa ) and 5-fam - arg - pro - lys - pro - val - glu - nva - trp - arg - lys(tq2w)-nh2 ( mmp-3 ; enzo life sciences , exeter , u.k . ) as previously described . briefly , equal volumes of assay buffer and media from 10 myofibroblasts were incubated with 12 m substrate as appropriate . gastric cancer cell ( ags ) migration 
 ( 10 cells per 8 m pore filter insert ) ( bd biosciences , 
 oxford , u.k . ) was studied as previously described . conditioned media were collected as described above and treated 
 with mmp-1 neutralizing antibody ( mab901 , r&d systems ) , or selective 
 mmp-2 ( inhibitor i , calbiochem ) and mmp-3 ( inhibitor iv , calbiochem ) 
 inhibitors as appropriate . results are expressed 
 as mean  standard 
 error of the mean ( sem ) , unless otherwise stated . student s t - test or anova ( systat software , inc . , hounslow , london , 
 u.k . ) as appropriate were used to determine statistical significance 
 of results and considered significant at p < 0.05 , 
 unless otherwise stated . when myofibroblasts 
 derived from two gastric cancers and corresponding 
 atms were silac labeled and the secretomes analyzed by lc ms / ms , 
 approximately 350 unique proteins ( 310 and 392 ) were identified in 
 each pair on the basis of one or more peptide unique assignments with 
 validated quantification ( supporting information tables s1 , s2 ) . of these , 42 and 48% were characterized as extracellular 
 proteins on the basis of annotations in the uniprot and hprd databases . one of the paired samples was further analyzed using the cofradic 
 technology that enriches for methionine - containing peptides in an 
 attempt to increase overall proteome coverage by reducing sample complexity . this approach more than doubled the number of unique peptides and 
 proteins identified in the secretome , although a comparable proportion 
 ( 31% ) of the identifications were attributable to extracellular proteins 
 ( figure 1a ; supporting 
 information table s3 ) . the met - cofradic analysis identified 
 the majority ( 72% ) of proteins identified in the initial experiments 
 ( figure 1a ) . in total across the three experiments , 
 1460 unique proteins were identified , of which 364 were annotated 
 to be extracellular proteins . ( a ) venn diagram showing , 
 left , identification 
 of unique proteins , on the basis of one or more unique peptides with 
 a validated quantification , in media from pairs of cams / atms from 
 two patients , and overlap with met - cofradic identifications in patient 
 1 based on the same criteria ; right , extracellular proteins in this 
 data set . ( b ) functional classification of extracellular proteins 
 identified by met - cofradic and classical  shotgun  proteomics , 
 alongside cleaved proteins and total cleavage products in myofibroblast 
 secretomes by n - terminal peptide enrichment ( restricted to identifications 
 with successful quantification ) . extracellular proteins making up the secretome were grouped 
 into 
 11 functional classifications ( figure 1b ) . there was a broadly similar distribution across these groups in both 
 pairs of cams and atms , with binding proteins and extracellular matrix 
 proteins the two largest groups ( figure 1b ) . when 95% confidence limits were determined and applied to individual 
 experiments , 947 proteins were identified that were above 
 or below these limits ( supporting information figure s1 ; supporting information table 
 s4 ) . interestingly , mmp-1 , mmp-3 , mmp-10 and upa were identified as 
 upregulated in cams in both patients ( supporting 
 information table s4 ) . the largest groups showing differential 
 abundance were  binding  proteins , e.g. , the insulin - like 
 growth factor binding proteins ( igfbps ) ,  receptor  
 proteins , e.g. , epidermal growth factor receptor , and proteases , e.g. , 
 mmp-1 ( supporting information table s4 ) . we then extended 
 the analysis to the identification of neo - n - termini generated as a 
 consequence of proteolytic cleavage . thus , silac - labeled media samples 
 were enriched for n - terminal peptides using a strong cation exchange 
 ( scx ) step to remove nonterminal peptides . as part of this procedure , 
 neo - n - terminal peptides had been trideutero - acetylated prior to trypsinisation 
 to facilitate discrimination from other peptides by mass spectrometry , 
 and the results were filtered so that only peptides that were trideutero - acetylated 
 and had a valid mascot identification were used in subsequent analyses . 
 of unique quantified peptides , this 
 process identified peptides starting at residues 1 or 2 of the protein 
 or immediately after removal of the signal sequence , which were excluded 
 from further analysis as they were considered uninformative for present 
 purposes . for each of the remaining peptides , the ratio of relative 
 abundance between the cam and atm samples was manually validated by 
 inspecting the spectra and calculating the area under the peaks of 
 the heavy and light isotopes . one of the paired samples was 
 further analyzed using n - terminal cofradic in addition to the scx 
 enrichment step in an attempt to increase coverage of n - terminal peptides 
 by reducing sample complexity . similar numbers of peptides were identified 
 by n - terminal cofradic and scx - only enrichment , but over 2-fold more 
 of these identifications corresponded to neo - n - termini in the cofradic 
 data set ( supporting information table 
 s5 ) . approximately 50% of all unique proteins identified corresponded 
 to putative secreted proteins ( supporting information table s6 ) . in the functional classification , furthermore , 41 proteins were identified that 
 were not seen in the first set of silac experiments , of which 24 were 
 known extracellular proteins . in the data set as 
 a whole , neo - n - termini corresponding to putative proteolytic cleavage 
 sites were identified in a total of 94 unique proteins , of which collagens 
 alpha1(i ) and alpha-2(i ) and igfbp-5 had the most cleavage sites ( supporting information table s7 ) . in order to 
 refine cancer - related changes , we then sought those proteins for which 
 unique neo - n - termini were identified in cam secretomes relative to 
 their atm counterparts . applying this criterion , we identified 13 
 proteins that exhibited cam - restricted proteolytic cleavage ( table 1 ) : strikingly , these included cleavages in the propeptide 
 domains of mmp-1 ( interstitial collagenase ) and mmp-3 ( stromelysin-1 ) 
 ( table 1 and supporting 
 information figure s2 ) ; moreover , n - terminal cofradic also 
 identified increased prodomain cleavage in mmp-2 ( 72 kda type iv collagenase ) 
 ( supporting information table s7 ) . functional 
 classification and 
 cleavage sites start 
 and end position number of the n - terminally labeled peptide are relative 
 to unprocessed forms of the protein . to determine the in vivo relevance 
 of myofibroblasts for mmp activity in tumors , we made use of fmt imaging 
 of a mmp fluorescent substrate in a xenograft model consisting of 
 mkn45 gastric cancer cells alone or with human cams . in matched tumors 
 of similar size generated with and without coadministration of cams , 
 the mmpsense substrate revealed significantly increased activity when 
 human cams were present ( figure 2 ) . increased mmp 
 activity in vivo in xenografts containing cams . ( a ) 
 in balb / c nu / nu mice , mmp activity revealed by fmt imaging of mmpsense 
 750 fast is increased in xenografts of gastric cancer mkn45 cells 
 containing two different cams compared with mkn45 cells alone . ( b ) 
 representative fmt images of mice with xenografts of mkn45 cells alone 
 ( top row ) , mkn45 cells + cam2 ( middle row ) , and mkn45 cells + cam1 
 ( bottom row ) . the substrate 
 used for in vivo imaging does not distinguish individual mmps , and 
 subsequent studies therefore made use of in vitro techniques to assess 
 the relative contribution of mmps13 in cam media . western 
 blots of mmp-1 revealed increased abundance of a 42 kda form corresponding 
 to the active enzyme in cam compared with atm media ; in contrast , 
 in cell extracts of the two cell types there was similar abundance 
 of the precursor proteins of 57 and 52 kda ( figure 3a ) . for mmp-2 , we identified a precursor form of 72 kd in 
 cells and media of both cams and atms , again in similar abundance 
 in the two cell types . when blots of media were overexposed it was 
 possible to identify a minor band of 63 kda corresponding to the active 
 enzyme in cam but not atm media ( figure 3b ) . finally , for mmp-3 we identified precursor proteins of 59 and 54 kda 
 in cells and media of both cams and atms ; a band of 45 kda corresponding 
 to the active enzyme was found in media , and the abundance was greater 
 in cams compared with atms ( figure 3c ) . western blots show the active forms of mmp-1 , mmp-2 , and 
 mmp-3 
 in cam media . ( a ) representative western blots of mmp-1 in cam and 
 atm media ( left ) and cell extracts ( right ) . ( b ) representative western 
 blots of mmp-2 in cam and atm media ( left ) and cell extracts ( right ) . ( c ) representative western blots of mmp-3 in cam and atm media at 
 two different exposures ( left , center ) and cell extracts ( right ) . to establish the functional significance 
 of these results , we then 
 studied mmp enzyme activity in cam and atm media . using a fluorogenic 
 substrate for mmp-1 , we found significantly greater activity in cam 
 compared with the corresponding atm media ( figure 4a ) . similarly , substrates selective for mmp-2 , or for mmp-2/mmp-9 
 ( figure 4a ) , revealed greater activity in cam 
 than atm media , and the same pattern was observed with two different 
 mmp-3 substrates ( figure 4a ) . increased mmp-1 , mmp-2 , 
 and mmp-3 activity in cam media . ( a ) mmp-1 , 
 mmp-2 , and mmp-3 enzyme activities ( using the selective substrates 
 indicated in brackets ) are increased in cam compared with atm media ; 
 left , assays based on trp fluorescence and right on fret . ( b ) cam - cm 
 media stimulates ags cell migration and is inhibited by neutralizing 
 antibody to mmp-1 ( 2.5 g ml ) or inhibitors 
 ( see text ) of mmp-2 ( 6 m ) and mmp-3 ( 3 m ) . there 
 is mmp - stimulation of migration of ags gastric cancer cells in boyden 
 chambers , and this was then used to test 
 the functional significance of the changes in mmp abundance and activity 
 in cam media . conditioned media ( cm ) from both cams and atms stimulated 
 ags cell migration , but the response to the former was significantly 
 greater than to the latter ( cam - cm : 207  11 cells per field ; 
 atm - cm : 145  11 , p < 0.05 ; control media : 
 15  2 ) . neutralizing antibody to mmp-1 ( 2.5 g ml ) significantly suppressed ags cell migration in response 
 to cam - cm ( figure 4b ) ; similarly , previously 
 characterized selective inhibitors of mmp-2 or mmp-3 at concentrations 
 approximately 3-fold above their reported ki in each case ( 6 and 3 m , respectively ) also inhibited ags cell migration in response to cam - cm , indicating 
 that active mmp-2 and mmp-3 released from myobfibroblasts play a role 
 in cancer cell migration ( figure 4b ) . the tumor microenvironment reflects the secretomes of both cancer 
 and stromal cells including myofibroblasts , fibroblasts , pericytes , 
 endothelial cells , inflammatory and immune cells . crucially , interactions 
 between different secretomes influence cancer cell migration , invasion 
 and metastasis by multiple mechanisms including the activation or 
 inhibition of proteases with consequences for the proteolytic cleavage 
 of ecm proteins , growth factors , cytokines and chemokines . the secretomes of cancer cells have attracted 
 increasing attention in recent years , but little is yet known of stromal cell secretomes . differences 
 in the secretomes of myofibroblasts recovered from gastric cancers 
 and those recovered from adjacent tissue have been reported recently 
 using itraq . we have now used silac and 
 cofradic to determine the extent to which proteolysis influenced myofibroblast 
 secretomes in gastric cancer . our study identified neo - n - termini derived 
 from 94 proteins in cam secretomes including evidence of cleavage 
 of the prodomains of mmp-1 , -2 , and -3 leading to increased extracellular 
 proteolytic activity . the data indicate that a distinguishing feature 
 of cancer myofibroblasts is increased expression and increased activation 
 of these mmps in an autonomous manner in gastric cancer with the potential 
 for promoting cancer progression . the identification of secreted 
 biomarkers by cancer cells has been 
 a focus of interest for several generations and has been stimulated 
 more recently by the development and refinement of proteomic methods 
 and the prospect of rigorously defining the cancer secretome . extracellular proteolysis presents additional 
 challenges in defining the relevant secretomes ; it is important not 
 least because it underlies multiple mechanisms implicated in cancer 
 progression including angiogenesis , tumor cell migration and invasion . a number of proteomic methods have recently been used to identify 
 neo - n - termini generated in complex samples including cofradic and terminal amine isotopic labeling of substrates 
 ( tails ) , which has been applied to the 
 identification of substrates of mmp-2 and mmp-9 in fibroblast secretomes . since silac has previously been used successfully for secretome 
 studies in a range of cell types including stromal cells from other 
 tissues , and since cofradic coupled with silac labeling 
 is considered to offer a rigorous approach to n - terminomics , these methods were selected for the present studies . golgi secretory pathway ; however , as 
 commonly found in secretome studies , there were also proteins likely 
 to be released by other mechanisms including cytoplasmic proteins 
 liberated through cell death , shedding of membrane proteins and release 
 of exosomes . previous studies have identified similar rates of apoptosis 
 in cams and atms suggesting that differential 
 cell death is unlikely to account for differences in the cam and atm 
 secretomes . for proteins released through classical secretory mechanisms , 
 we were able to identify many previously reported in the secretomes 
 of fibroblasts and of the stem cells that may give rise to them , including 
 extracellular matrix proteins , igfbps , mmps and timps . the differences between cam and atm secretomes may reflect alterations 
 in gene expression , post - translational processing , relative rates 
 of secretion and in proteolysis following release . we have now defined 
 the latter though identification of neo - n - termini in cams compared 
 with their corresponding atms . for example , we found multiple neo - n - termini 
 in collagens alpha-2 ( i ) and alpha-1(i ) , and igfbp5 , as well as at 
 limited sites in 91 other proteins . in a small subset of proteins 
 we identified neo - n - termini that were unique to cams , and these included 
 six neo - n - termini in the prodomains of mmp-1 and mmp-3 , suggesting 
 that activation of these mmps might be functionally important in cams . it is well established that mmp activity is increased in tumors 
 and promotes cancer cell migration and invasion ; the present in vivo imaging data indicate that cams contribute 
 to this increased activity in an animal model . it is likely that mmps 
 have multiple roles in different tumor functions accounting for the 
 fact that mmp inhibitors have not yet led to successful anticancer 
 therapies . the present demonstration 
 of increased expression and activation of mmp-1 , mmp-2 and mmp-3 in 
 cam secretomes nevertheless suggests a novel dimension to the role 
 of these enzymes . in vivo there may be activation of myofibroblast 
 mmps by epithelial or cancer - derived proteases , e.g. , mmp-7 . importantly , however , the present data indicate 
 that increased mmp activity in cam media occurs independently of a 
 cancer or epithelial cell stimulus . this is nevertheless relevant 
 to cancer cell function not least because mmp-1 , mmp-2 , and mmp-3 
 stimulate cancer cell migration and make a substantial contribution 
 to the chemotactic properties of cam conditioned media . the prodomain 
 cleavages of mmp-1 , -2 , and -3 identified here are all on the n - terminal 
 side of the conserved cysteine switch sequence , and we think it is 
 possible that these facilitate activation by exposing the site for 
 autolysis much in the same way that trypsin activates mmps . however , the precise protease(s ) responsible 
 for cam - autonomous prodomain cleavages is presently unclear and should 
 now be investigated . in this context it is worth noting that there 
 was decreased abundance of protease inhibitors , including timps-1 , 
 -2 , and -3 in one patient ( supporting information table s4 ) , which may contribute to increased protease activity . cofradic has provided the 
 most detailed analysis of gastric cancer myofibroblast secretomes 
 to date . it extends previous studies , but in addition , it defines differences in the extracellular degradomes 
 of cams and atms . at least some of the differences between cafs or 
 cams and their normal tissue counterparts are thought to reflect interactions 
 that occur in the presence of cancer cells . the present data indicate that selective mmp - activation occurs in 
 the cam secretome even when these cells are cultured in the absence 
 of cancer cells . this property reflects a cell - autonomous mechanism 
 by which cams might contribute to a cancer - promoting microenvironment . the possibility of targeting anticancer therapies to stromal cells 
 has emerged recently , and the present 
 data indicate how these could be refined to include the stromal degradome .
cancer 
 progression involves changes in extracellular proteolysis , 
 but the contribution of stromal cell secretomes to the cancer degradome 
 remains uncertain . 
 we have now defined the secretome of a specific 
 stromal cell type , the myofibroblast , in gastric cancer and its modification 
 by proteolysis . 
 silac labeling and cofradic isolation of methionine 
 containing peptides allowed us to quantify differences in gastric 
 cancer - derived myofibroblasts compared with myofibroblasts from adjacent 
 tissue , revealing increased abundance of several proteases in cancer 
 myofibroblasts including matrix metalloproteinases ( mmp)-1 and -3 . 
 moreover , n - terminal cofradic analysis identified cancer - restricted 
 proteolytic cleavages , including liberation of the active forms of 
 mmp-1 , -2 , and -3 from their inactive precursors . in vivo imaging 
 confirmed increased mmp activity when gastric cancer cells were xenografted 
 in mice together with gastric cancer myofibroblasts . 
 western blot 
 and enzyme activity assays confirmed increased mmp-1 , -2 , and -3 activity 
 in cancer myofibroblasts , and cancer cell migration assays indicated 
 stimulation by mmp-1 , -2 , and -3 in cancer - associated myofibroblast 
 media . 
 thus , cancer - derived myofibroblasts differ from their normal 
 counterparts by increased production and activation of mmp-1 , -2 , 
 and -3 , and this may contribute to the remodelling of the cancer cell 
 microenvironment .