Is the proteome of bronchoalveolar lavage extracellular vesicles a marker of advanced lung cancer?

cance s A icle Is he P o eome o B onchoal eola La age Ex acellula Vesicles a Ma ke o Ad anced Lung Cance ? Ana So ia Ca alho 1,* , Ma ia Ca olina S ano Mo aes 2, Chan Hyun Na 3, I o Fie o-Mon i 1, And eia Hen iques 1, Sa a Zahedi 1, C is ian Bodo 2, E in M T an ield 4, Ana Lau a Sousa 4, Ana Fa inho 5, Luís Vaz Rod igues 6, Paula Pin o 7, C is ina Bá ba a 8, Leono Mo a 7, Tiago Ta a es de Ab eu 7, Júlio Semedo 7, Susana Seixas 9, P ashan Kuma 10,11 , B uno Cos a-Sil a 2, Akhilesh Pandey 10,11,12 and Rune Ma hiesen 1,* 1 Compu a ional and Expe imen al Biology G oup, Ch onic Diseases Resea ch Cen e, NOVA Medical School, Faculdade de Ciencias Medicas, Uni e sidade NOVA de Lisboa, Campo dos Ma i es da Pa ia, 130, 1169-056 Lisboa, Po ugal; [email p o ec ed] (I.F.-M.); [email p o ec ed] (A.H.); [email p o ec ed] (S.Z.) 2 Sys ems Oncology G oup, Champalimaud Resea ch, Champalimaud Cen e o he Unknown, A . B asilia, Doca de Ped oucos, 1400-038 Lisbon, Po ugal; ca olina.s anomo aes@ esea ch. champalimaud.o g (M.C.S.M.); c is ian.bodo@ esea ch. champalimaud.o g (C.B.); b uno.cos adasil a@ esea ch. champalimaud.o g (B.C.-S.) 3Depa men o Neu ology, Ins i u e o Cell Enginee ing, Johns Hopkins Uni e si y School o Medicine, Bal imo e, MD 21205, USA; [email p o ec ed] 4Elec on Mic oscopy Facili y, Ins i u o Gulbenkian de Ciência—Rua da Quin a G ande, 6, 2780-156 Oei as, Po ugal; [email p o ec ed] (E.M.T.); [email p o ec ed] (A.L.S.) 5iNOVA4Heal h—Ad ancing P ecision Medicine, CEDOC—Ch onic Diseases Resea ch Cen e, NOVA Medical School/Faculdade de Ciências Médicas, Uni e sidade NOVA de Lisboa, Campo dos Ma i es da Pa ia, 130, 1169-056 Lisboa, Po ugal; [email p o ec ed] 6 Depa men o Pneumology, Unidade Local de Sa ú de da Gua da (USLGua da), 6300-659 Gua da, Po ugal; [email p o ec ed] 7Unidade de Técnicas In asi as Pneumológicas, Pneumologia II, Hospi al Pulido Valen e, Cen o Hospi ala Lisboa No e, 1649-028 Lisbon, Po ugal; [email p o ec ed] (P.P.); [email p o ec ed] (L.M.); a a esdeab [email p o ec ed] (T.T.d.A.); [email p o ec ed] (J.S.) 8Ins i u o de Saúde Ambien al (ISAMB), Faculdade de Medicina, Uni e sidade de Lisboa, Cen o Hospi ala Uni e si á io Lisboa No e, 1649-028 Lisbon, Po ugal; [email p o ec ed] 9Ins i u o de In es igação e Ino ação em Saúde (I3S), Uni e sidade do Po o, 4200-135 Po o, Po ugal; [email p o ec ed] 10 Ins i u e o Bioin o ma ics, Disco e e building, ITPL, Bangalo e 560066, India; [email p o ec ed] (P.K.); pandey[email p o ec ed] (A.P.) 11 Manipal Academy o Highe Educa ion (MAHE), Manipal 576104, India 12 Depa men o Labo a o y Medicine and Pa hology, Mayo Clinic, Roches e , MN 55905, USA *Co espondence: [email p o ec ed] (A.S.C.); [email p o ec ed] (R.M.) Recei ed: 15 Oc obe 2020; Accep ed: 19 No embe 2020; Published: 20 No embe 2020   Simple Summa y: B onchoal eola la age is ou inely collec ed du ing b onchoscopy o cy ology analysis in he diagnos ic o lung cance . Due o low sensi i i y o his me hod, ea ly-s age cance s a e unde ec ed, lowe ing he ea men success. In his s udy, we analyzed ex acellula esicles isola ed om b onchoal eola la age o lung cance suspec s by mass spec ome y-based p o eomics. The p o ein composi ion o b onchoal eola la age ex acellula esicles o la e-s age cance showed a highe p o eome complexi y associa ed wi h mo ali y wi hin he wo yea ollow-up pe iod. We iden i ied a po en ial he apeu ic a ge DNMT3B complex which was signi ican ly exp essed in b onchoal eola la age ex acellula esicles as well as in umo issue. B onchoal eola la age Cance s 2020,12, 3450; doi:10.3390/cance s12113450 www.mdpi.com/jou nal/cance s Cance s 2020,12, 3450 2 o 18 ex acellula esicles p o eome analysis o immune ma ke s indica es he p esence o ma ke s o inna e immune and ib oblas cells. Abs ac : Acellula b onchoal eola la age (BAL) p o eomics can pa ially sepa a e lung cance om non-lung cance pa ien s based on p incipal componen analysis and mul i a ia e analysis. Fu he mo e, he a iance in he p o eomics da a se s is co ela ed mainly wi h lung cance s a us and, o a lesse ex en , smoking s a us and gende . Despi e hese ad ances BAL small and la ge ex acellula ehicles (EVs) p o eomes e eal abe an p o ein exp ession in pa ac ine signaling mechanisms in cance ini ia ion and p og ession. We consequen ly p esen a case-con ol s udy o 24 b onchoal eola la age ex acellula esicle samples which we e analyzed by s a e-o - he-a liquid ch oma og aphy-mass spec ome y (LC-MS). We ob ained e idence ha BAL EVs p o eome complexi y co ela ed wi h lung cance s age 4 and mo ali y wi hin wo yea s ´ ollow-up (p alue =0.006). The po en ial he apeu ic a ge DNMT3B complex is signi ican ly up- egula ed in umo issue and BAL EVs. The compu a ional analysis o he immune and ib oblas cell ma ke s in EVs sugges s ha pa ien s who deceased wi hin he ollow-up pe iod display highe ma ke exp ession indica i e o inna e immune and ib oblas cells ( ou ou o i e cases). This s udy p o ides insigh s in o he p o eome con en o BAL EVs and hei co ela ion o clinical ou comes. Keywo ds: ex acellula esicles; lung cance ; p o eomics; immuno oncology; b onchoal eola la age 1. In oduc ion A p esen , lung cance pe sis ed as he mos p e alen oncological disease wi h an es ima ed incidence o mo e han 2 million new cases wo ldwide, and a mo ali y a e o 84% wi h a o al o 1.8 million dea hs yea ly [ 1 , 2 ]. The la e de ec ion o he disease is he main cause o such a dismal ou come as demons a ed by he i e-yea o e all su i al a es o non–small cell lung cance (NSCLC): 14–49% o s ages I o IIIA, and <5% o s age IIIB/IV [3]. The cha ac e iza ion o lung umo issue and he su ounding mic oen i onmen a he molecula le el con ibu ed o he inc eased knowledge on he physiology o he disease as well as he design o no el he apies. Ne e heless, he collec ion o issue biopsies o pe iphe al adenoca cinomas in dis al ai ways by minimally in asi e echniques is limi ed, and du ing he pas yea s, se e al s udies ha e a ge ed di e en ypes o biological specimens, which ange om umo issues o di e en ypes o liquid biopsies [ 4 ]. B onchoal eola la age (BAL) liquid biopsies a e ob ained om minimally in asi e p ocedu es, o example, ibe op ic b onchoscopy. B onchoscopy is ypically pe o med upon suspicion o lung cance , o example, de i ed om p e iously ob ained imaging o he lungs. Due o he close ana omical p oximi y o BAL compa ed o sali a, mo e accu a e bioma ke signa u es a e expec ed om BAL han om bio luids such as blood, sali a, o spu um. Blood and sali a, on he o he hand, migh se e as po en ial ma ke s o ea ly sc eening. In he pas , se e al molecula echnologies we e applied o p o ile he molecula con en o BAL such as miRNA [ 5 ], mRNA [ 6 ], DNA [ 7 ], DNA me hyla ion [ 8 ], me aboli es [ 9 ], mic obio a [ 10 ], and p o eomics. O e he yea s, p o eomics echnologies ha e e ol ed and consequen ly, BAL p o eome p o iling has been a emp ed mul iple imes. The i s BAL p o eomics p o iling applied MALDI TOF-MS [ 11 ]. La e , 2D-PAGE and MALDI-TOF we e combined [ 12 ]. Nowadays, liquid ch oma og aphy-mass spec ome y (LC-MS) app oaches a e ypically applied o BAL p o eome p o iling [ 13 – 15 ]. Abundan p o eins like albumin pa ially hampe he p o ein co e age ob ainable by di ec LC-MS analysis o acellula BAL samples. The e o e, Sim e al. [ 16 ] es ablished a no el me hodology based on combining an ibody-based deple ion o high abundan BALF p o eins, high pH pep ide ac iona ion, and label- ee quan i a ion on a high- esolu ion O bi ap Fusion Lumos ins umen . Howe e , an ibody deple ion o abundan Cance s 2020,12, 3450 3 o 18 p o eins isks he emo al o clinically ele an a ge p o eins. Consequen ly, i is impe a i e o explo e addi ional sample p epa a ion me hodologies. The en ichmen o small (sEVs) and la ge (lEVs) EVs ep esen s an al e na i e me hodology o he deple ion o abundan p o eins in bioma ke s udies based on liquid biopsies [ 17 ]. Cu en ly, no s udy has a ge ed BAL sEVs and lEVs om suspec ed lung cance pa ien s. We, he e o e, pe o med an explo a o y case-con ol s udy o e alua e he p o ein complexi y om p o eomics p o iling o BAL EV ac ions. The sEVs p o eins cons i u ed he mos complex p o eome when compa ed o acellula BAL, esicle-deple ed BAL, and lEVs. We iden i ied and quan i ied 7484 p o ein iso o ms om 3158 encoding genes om sEVs. The lEVs and sEVs we e en iched by sequen ial cen i uga ion ollowed by ul acen i uga ion and loa a ion on suc ose g adien cushion, which cap u es all he subpopula ions o sEVs while deple ing om non- esicula p o ein agg ega es and complexes. Besides, he po en ial o BAL sEV p o eome as a sou ce o bioma ke s was explo ed. The da a ob ained sugges ed ha BAL sEV p o eome complexi y co ela es wi h cance s age IV and dea h (Wilcoxon ank-sum es p alue =0.006). The p o eomes we e compa ed, by sys em biology app oaches, o p e iously ob ained p o eomes om lung umo issues and acellula BAL p o eome [ 13 , 18 ]. sEVs and umo issue displayed a common signi ican egula ion o se e al en iched unc ional ca ego ies wi h po en ial he apeu ic alue. Finally, sEV p o ein ma ke s o inna e immune and ib oblas cells co ela ed wi h poo p ognosis (mo ali y wi hin wo yea s ollow-up in ou ou o i e cases). 2. Resul s 2.1. Ou line o S udy As a p oo o concep , we compa ed di e en BAL ac ions by high- h oughpu mass spec ome y analysis such as acellula BAL, esicle deple ed BAL, lEVs and sEVs om con ol, and lung cance pa ien s. The p o eome con en was compa ed by LC-MS (Figu e S1). We obse ed ha sEVs con ained he la ge p o eome complexi y wi h he mos signi ican po en ial o bioma ke disco e y (Sec ion 2.2 and Figu e S2). The e o e, sEVs we e isola ed om 24 acellula BAL samples and analyzed by mass spec ome y and u he cha ac e ized by ansmission elec on mic oscopy (TEM), wes e n blo , and nanopa icle acking analysis. The quan i a i e MS da a we e compa ed wi h iBAQ alues ob ained om eanalyzed da a o p e ious s udies on acellula BAL (PXD004700) [ 13 ] and pa ien -ma ched umo e sus no mal issue (PXD000853) [18]. 2.2. P o eome Con en o lEVs, sEVs, Acellula BAL, and EV Deple ed BAL (DB) BAL luids (acellula BAL, aBAL) om wo di e en cance s a us samples (NO and YES) we e ac iona ed by sequen ial cen i uga ion and ul acen i uga ion in o h ee ac ions: (1) deple ed BAL (DB), (2) lEVs and (3) sEVs (Figu e S1). To add ess which BAL ac ion holds he g ea es p omise in e ms o signi ican cance s a us disc imina ion based on p o ein iden i ica ion and quan i a ion, each ac ion was analyzed by LC-MS in pa allel wi h he espec i e aBAL luid. Figu e 1compa es he p o ein iden i ica ion in cance e sus con ol o (a) sEVs, (b) BAL, (c) lEVs, and (d) DB. The sEVs ac ion esul ed in he highes numbe o p o ein iden i ica ions and he highes numbe o p o eins unique o cance (Figu e 1a). KEGG unc ional en ichmen o all he iden i ica ions om each o he ac ions was compa ed o add ess he ques ion o which ac ion has he g ea es po en ial o sepa a ing cance s a us (Figu e 1e). Figu e S2 displays he o e lap be ween he iden i ied p o eins in each ac ion ype ob ained om LC-MS analysis. The highes numbe s o quan i ied p o eins we e ob ained in BAL sEVs om YES and NO cance s a us samples. On a e age he numbe o iden i ied p o eins om sEVs was almos double compa ed o aBAL, while in BAL esicle-deple ed and lEVs ac ions he numbe o iden i ied p o eins was lowe , wi h he lEVs p o eome being he simples p o eome (Figu e S2). KEGG unc ional en ichmen analysis o all iden i ied p o eins e ealed unc ional di e si y be ween he BAL ac ions analyzed (Figu e 1e). The samples clus e ed acco ding o BAL ac ions based on he unc ional KEGG anno a ion, s ongly sugges ing ha each ac ion con ains dis inc p o ein unc ional g oups. The iden i ied p o eins o e all Cance s 2020,12, 3450 4 o 18 exhibi ed signi ican en ichmen in pa hways ela ed o in ec ion, in lamma ion, and complemen and coagula ion pa hways. sEVs uniquely displayed signi ican en ichmen in p o easome and ibosome ac o s while no en ichmen in glycolysis and glycogenesis ac o s was obse ed in con as o he o he BAL ac ions (Figu e 1). P o easome and ibosome ac o s a e known as abundan ac o s in cance due o inc eased p o ein u no e . Al hough lEV ac ions esul ed in he lowes amoun o iden i ica ions, i p esen ed unique unc ional en ichmen ela ed o, e.g., chemokine and synapse signaling in cance . In u n, he wo esicles con aining ac ions displayed he maximum sepa a ion in e ms o sample clus e ing. None o he ac ions ully encapsula ed all en iched unc ional ca ego ies. P o eomics analysis o sEV ac ions showed he highes numbe o iden i ied p o eins and he highes di e si y o unc ional en iched KEGG pa hways. In his iew, we ha e analyzed he p o eome o sEVs o bioma ke s disco e y in a case-con ol s udy o 12 con ols and 12 lung cance cases. Cance s 2020, 12, x FOR PEER REVIEW 4 o 20 sugges ing ha each ac ion con ains dis inc p o ein unc ional g oups. The iden i ied p o eins o e all exhibi ed signi ican en ichmen in pa hways ela ed o in ec ion, in lamma ion, and complemen and coagula ion pa hways. sEVs uniquely displayed signi ican en ichmen in p o easome and ibosome ac o s while no en ichmen in glycolysis and glycogenesis ac o s was obse ed in con as o he o he BAL ac ions (Figu e 1). P o easome and ibosome ac o s a e known as abundan ac o s in cance due o inc eased p o ein u no e . Al hough lEV ac ions esul ed in he lowes amoun o iden i ica ions, i p esen ed unique unc ional en ichmen ela ed o, e.g., chemokine and synapse signaling in cance . In u n, he wo esicles con aining ac ions displayed he maximum sepa a ion in e ms o sample clus e ing. None o he ac ions ully encapsula ed all en iched unc ional ca ego ies. P o eomics analysis o sEV ac ions showed he highes numbe o iden i ied p o eins and he highes di e si y o unc ional en iched KEGG pa hways. In his iew, we ha e analyzed he p o eome o sEVs o bioma ke s disco e y in a case- con ol s udy o 12 con ols and 12 lung cance cases. Figu e 1. P o eome o e iew in b onchoal eola la age (BAL) ac ions. Venn diag ams compa ing cance (YES) e sus con ol (NO) in: (a) sEVs, (b) BAL, (c) lEVs, and (d) DB; (e) Hea -map o KEGG Figu e 1. P o eome o e iew in b onchoal eola la age (BAL) ac ions. Venn diag ams compa ing cance (YES) e sus con ol (NO) in: ( a ) sEVs, ( b ) BAL, ( c ) lEVs, and ( d ) DB; ( e ) Hea -map o KEGG pa hway en ichmen analysis. Func ional en ichmen analysis was pe o med using R. p- alue o each KEGG pa hway was <0.05. The shades o he colo s e lec ed he –log10 (p- alue) o he en ichmen analysis o p o eins iden i ied in di e en subcellula BAL ac ions o NO (whi e) and YES (black) cance s a us samples. La ge ex acellula esicles (lEVs), BAL esicle-deple ed (DB), acellula BAL (BAL), small ex acellula esicles (sEVs). Cance s 2020,12, 3450 5 o 18 2.3. Baseline Cha ac e is ic o Clinical Samples o he En ichmen o sEVs To explo e he po en ial o sEVs p o eome abili y o classi y clinical samples, we selec ed 12 con ols (non-lung cance cases) and 12 lung cance samples in a case-con ol design (Table 1) om ou p e ious analyzed coho o 91 pa ien s suspec ed o lung cance [ 13 ]. The lung cance s a us e lec s he diagnosis a e wo yea s o ollow-up. App oxima ely 42% o he cance pa ien s died wi hin he wo yea ollow-up pe iod. The selec ion o samples o sEV cha ac e iza ion was op imized o p e en s a is ical associa ion o age, gende , smoking s a us, and expe imen al ba ch e ec s wi h lung cance s a us. As expec ed, he lung cance s a us displayed an associa ion o wo-yea su i al and cance s aging. sEVs om he 24 clinical samples we e isola ed ollowing he p o ocol ou lined in (Figu e S1). Table 1. Baseline cha ac e is ic o he clinical samples. NO (N=12) YES (N=12) To al (N=24) pValue Age 0.355 <55 4 (33.3%) 1 (8.3%) 5 (12.5%) >55 1 (8.3%) 1 (8.3%) 2 (8.3%) NA 7 (58.3%) 10 (83.3%) 17 (70.8%) Smoking His o y 0.370 Cu en smoke 4 (33.3%) 3 (25.0%) 7 (29.2%) Fo me smoke 2 (16.7%) 6 (50.0%) 8 (33.3%) Nonsmoke 2 (16.7%) 1 (8.3%) 3 (12.5%) Unknown 4 (33.3%) 2 (16.7%) 6 (25.0%) Gende 0.100 NA 0 (0.0%) 1 (8.3%) 1 (4.2%) F 8 (66.7%) 3 (25.0%) 11 (45.8%) M 4 (33.3%) 8 (66.7%) 12 (50.0%) Ba ch 0.390 1 2 (16.7%) 5 (41.7%) 7 (29.2%) 2 9 (75.0%) 6 (50.0%) 15 (62.5%) 3 1 (8.3%) 1 (8.3%) 2 (8.3%) S age <0.001 2 0 (0.0%) 1 (8.3%) 1 (4.2%) 3 0 (0.0%) 1 (8.3%) 1 (4.2%) 4 0 (0.0%) 5 (41.7%) 5 (20.8%) NA 1 (8.3%) 5 (41.7%) 6 (25.0%) No 11 (91.7%) 0 (0.0%) 11 (45.8%) S a us 0.012 Ali e 12 (100.0%) 7 (58.3%) 19 (79.2%) Dead 0 (0.0%) 5 (41.7%) 5 (20.8%) 2.4. Nanopa icle T acking Analysis o Isola ed EVs The en iched sEVs we e cha ac e ized by nanopa icle acking analysis (Figu e 2and Figu e S3). Figu e 2displays ep esen a i e pa icle size dis ibu ion o one con ol (a) and one lung cance sample (b). All he no malized pa icle dis ibu ions a e plo ed in Figu e S3 and colo -coded wi h clinical s a us. The dis ibu ions display high simila i y ac oss samples. Based on a - es on he no malized coun s o each pa icle size bin o 0.5 nm, no s a is ical di e ences be ween he dis ibu ions o con ol e sus cance we e obse ed (Figu e S3). All dis ibu ions displayed a maximum peak a ound 100 nm, which ep esen s he expec ed size mode o sEVs. The p ecise posi ion o he maximum peak di e ed sligh ly be ween samples. The di e en samples displayed sligh ly di e en peak ops a la ge pa icle sizes likely ep esen ing di e en ypes o non- esicle pa icles such as p o ein complexes, agg ega es, and e en ually i us pa icles. A compa ison o o e all EV p o ein con en Cance s 2020,12, 3450 6 o 18 and o e all o al pa icle coun s om con ol e sus cance samples showed no signi ican di e ences (Figu e 2c,d). Cance s 2020, 12, x FOR PEER REVIEW 6 o 20 clinical s a us. The dis ibu ions display high simila i y ac oss samples. Based on a - es on he no malized coun s o each pa icle size bin o 0.5 nm, no s a is ical di e ences be ween he dis ibu ions o con ol e sus cance we e obse ed (Figu e S3). All dis ibu ions displayed a maximum peak a ound 100 nm, which ep esen s he expec ed size mode o sEVs. The p ecise posi ion o he maximum peak di e ed sligh ly be ween samples. The di e en samples displayed sligh ly di e en peak ops a la ge pa icle sizes likely ep esen ing di e en ypes o non- esicle pa icles such as p o ein complexes, agg ega es, and e en ually i us pa icles. A compa ison o o e all EV p o ein con en and o e all o al pa icle coun s om con ol e sus cance samples showed no signi ican di e ences (Figu e 2c,d). Figu e 2. Nanopa icle acking analysis o sEV samples: (a) pa icle coun s e sus pa icle size in nm o a con ol sample; (b) and a lung cance sample; (c) p o ein concen a ion in sEVs e sus cance s a us; (d) pa icle concen a ion e sus cance s a us. 2.5. T ansmission Elec on Mic oscopy and O e all P o eome o EVs In acco dance wi h nanopa icle acking analysis, TEM analysis o sEVs e ealed esicles o simila sizes a ound 100 nm (Figu e 3a). Wes e n blo analysis o he exosome ma ke CD63 compa ing BAL o sEVs ac ion showed a o y old en ichmen (Figu e S4). All iden i ied p o eins om con ols and lung cance in sEV we e compa ed o p o eins deposi ed in he exosome da abase ExoCa a, showing an o e lap >70% o he wo di e en cance s a us samples (Figu e 3b). Howe e , such compa isons exclude p o ein quan i a ion in o ma ion and consequen ly a e no sui ed o Figu e 2. Nanopa icle acking analysis o sEV samples: ( a ) pa icle coun s e sus pa icle size in nm o a con ol sample; ( b ) and a lung cance sample; ( c ) p o ein concen a ion in sEVs e sus cance s a us; (d) pa icle concen a ion e sus cance s a us. 2.5. T ansmission Elec on Mic oscopy and O e all P o eome o EVs In acco dance wi h nanopa icle acking analysis, TEM analysis o sEVs e ealed esicles o simila sizes a ound 100 nm (Figu e 3a). Wes e n blo analysis o he exosome ma ke CD63 compa ing BAL o sEVs ac ion showed a o y old en ichmen (Figu e S4). All iden i ied p o eins om con ols and lung cance in sEV we e compa ed o p o eins deposi ed in he exosome da abase ExoCa a, showing an o e lap >70% o he wo di e en cance s a us samples (Figu e 3b). Howe e , such compa isons exclude p o ein quan i a ion in o ma ion and consequen ly a e no sui ed o assessing he exosome con en in he sEV ac ions. We, he e o e, de eloped a bioin o ma ics me hodology o quan i a i ely es ima e exosome con en in sEV ac ions (Figu e 3c). The hea map displays a e age iBAQ alues anked om 0 o 1 o equen ly epo ed exosome ma ke s in he li e a u e and he 10 mos equen ly epo ed p o eins in ExoCa a. The quan i a i e alues om cell line sEVs we e ob ained by e-analyzing p e iously published MS da a and se e as e e ence samples. The e e ence da a o sEVs om cell lines we e ob ained by wo di e en EV en ichmen me hodologies: ( 1) PEG -based p ecipi a ion [ 19 ] and (2) di e en ial ul a-cen i uga ion [ 20 ]. The wo me hods esul ed in he iden i ica ion and quan i ica ion o simila exosome ma ke s. We compa ed he quan i a i e p o eomics da a on sEVs Cance s 2020,12, 3450 7 o 18 om human clinical samples o he e e ence da a on sEVs om cell lines. This cons i u es a p uden MS-based assessmen o exosome con en . The clinical BAL sEVs isola ed in his s udy exhibi ed simila exp ession le els o exosome ma ke s when compa ed o he p e ious da a om sEVs isola ed om cell lines (Figu e 3c) [ 19 , 20 ]. Addi ionally, he le el o non EV p o eins is conside ably lowe in BAL sEVs compa ed o cell lines sEVs en iched using he wo me hods while PEG isola ed EVs om cell lines displayed he highes le el o non EV p o ein ma ke s. Cance s 2020, 12, x FOR PEER REVIEW 7 o 20 assessing he exosome con en in he sEV ac ions. We, he e o e, de eloped a bioin o ma ics me hodology o quan i a i ely es ima e exosome con en in sEV ac ions (Figu e 3c). The hea map displays a e age iBAQ alues anked om 0 o 1 o equen ly epo ed exosome ma ke s in he li e a u e and he 10 mos equen ly epo ed p o eins in ExoCa a. The quan i a i e alues om cell line sEVs we e ob ained by e-analyzing p e iously published MS da a and se e as e e ence samples. The e e ence da a o sEVs om cell lines we e ob ained by wo di e en EV en ichmen me hodologies: (1) PEG-based p ecipi a ion [19] and (2) di e en ial ul a-cen i uga ion [20]. The wo me hods esul ed in he iden i ica ion and quan i ica ion o simila exosome ma ke s. We compa ed he quan i a i e p o eomics da a on sEVs om human clinical samples o he e e ence da a on sEVs om cell lines. This cons i u es a p uden MS-based assessmen o exosome con en . The clinical BAL sEVs isola ed in his s udy exhibi ed simila exp ession le els o exosome ma ke s when compa ed o he p e ious da a om sEVs isola ed om cell lines (Figu e 3c) [19,20]. Addi ionally, he le el o non EV p o eins is conside ably lowe in BAL sEVs compa ed o cell lines sEVs en iched using he wo me hods while PEG isola ed EVs om cell lines displayed he highes le el o non EV p o ein ma ke s. Figu e 3. Ex acellula ehicle (EV) quali y assessmen : (a) ep esen a i e ansmission elec on mic oscopy images o en iched sEVs; (b) Venn diag am indica ing o e all p o ein iden i ica ion o e lap wi h ExoCa a da abase; (c) MS-based quan i a i e compa ison be ween en iched BAL sEVs Figu e 3. Ex acellula ehicle (EV) quali y assessmen : ( a ) ep esen a i e ansmission elec on mic oscopy images o en iched sEVs; ( b ) Venn diag am indica ing o e all p o ein iden i ica ion o e lap wi h ExoCa a da abase; ( c ) MS-based quan i a i e compa ison be ween en iched BAL sEVs and cell line isola ed EVs o equen ly epo ed exosome p o ein ma ke s and 10 mos abundan exosome ma ke om ExoCa a. Red labeled p o eins indica e non EV p o eins. 2.6. Dys egula ed P o eins in BAL Exosomes P incipal componen analysis o all he quan i a i e alues sepa a ed NO e sus YES cance samples based on h ee di e en p incipal componen s (Figu e S5). The s a is ical analysis o iBAQ exp ession alues by he R package limma applying a co ec ion o gende and smoking esul ed in 166 genes signi ican ly egula ed (p- alue <0.05) and one p o ein a e co ec ion o mul iple es ing (adjus ed p- alue <0.05) be ween cance posi i e and nega i e s a us (Table S1 and Figu e 4a,b). Cance s 2020,12, 3450 8 o 18 P e iously, we epo ed 133 signi ican egula ed p o eins a e co ec ion o mul iple es ing [ 13 ], in which 91 acellula BAL samples we e analyzed. We specula e ha his di e ence p ima ily esul s om he di e ence in he numbe o cases s udied (24 e sus 91) a he han e lec ing BAL sEVs po en ial as a bioma ke sou ce. In o he wo ds, due o cance he e ogenei y, a highe numbe o samples a e equi ed o de ine p o eins signi ican ly dys egula ed a e co ec ion o mul iple es ing. To u he explo e p o ein he e ogenei y o sEVs samples, uniquely iden i ied p o eins in each sample we e ex ac ed. Figu e 4c depic s he numbe o unique p o eins, inding 607 unique p o eins (Table S2) in o al o sEVs om lung cance e sus 176 o con ol. Coun ing he numbe o uniquely iden i ied p o eins pe sample e ealed ha s age IV cance and mo ali y displayed a signi ican endency o con ain mo e unique p o eins iden i ied compa ed o o he cases (p- alue =0.006, Figu e 4c). The p- alue was calcula ed based on Wilcoxon ank-sum es wi hou emo ing any ou lie s. This end o inc eased p o ein complexi y in cance was con i med in lung cance issue (Figu e S6b) bu no o acellula BAL p o eome (Figu e S6a). Unique cance sEVs p o eins displayed KEGG unc ional egula ion in pa hways associa ed wi h cance (Figu e 4d). 1 Figu e 4. Dys egula ed lung cance p o eins in BAL EVs: ( a ) Volcano plo summa izing he main dys egula ed p o eins whe e –log10 p alues we e co ec ed o gende and smoking; ( b ) op egula ed p o ein a e co ec ion o mul iple es ing; ( c ) numbe o unique p o eins pe sample ( ed ba s indica e lung cance and black ba s ep esen con ols); ( d ) KEGG unc ional en ichmen o cance unique p o eins. The numbe s in he ba s ep esen he numbe o cance unique p o eins in he unc ional ca ego y. The dashed line indica es he p- alue h eshold (<0.05). Cance s 2020,12, 3450 9 o 18 2.7. Comple e Func ional Regula ion Comple e unc ional egula ion analysis es s o signi ican egula ion o en i ies (genes, p o eins) wi hin a unc ional g oup oge he wi h signi ican en ichmen in e ms o he iden i ica ion o a gi en unc ional anno a ed g oup [ 21 ]. The me hodology combines unc ional egula ion and en ichmen analysis in o a single isual summa y. Figu e 5compa es he esul s om he comple e unc ional egula ion analysis o acellula BAL, sEVs, lung umo issue, and mouse xeno ansplan lung umo issue. Fo each unc ional g oup, he numbe o p o eins iden i ied in each sample ype is displayed as well as he o al numbe o p o eins in he espec i e ca ego y. A s a is ical es is pe o med by assessing he signi icance o he en ichmen and he signi icance o he o e all egula ion o he ca ego y. Comple e unc ional egula ion analysis e ealed highe simila i y be ween sEVs and umo issue when compa ed o acellula BAL (Figu e 5). The la ge unc ional g oups up- egula ed in bo h issue and sEVs include NADP binding and ERK pa hway. ECM ecep o in e ac ion cons i u es he la ges down- egula ed unc ional ca ego y common o issue and sEVs (Figu e 5). Se e al unc ional ca ego ies e ealed e e se egula ion when compa ing sEVs wi h umo issue, o example, iglyce ide me abolic p ocesses. DNMT3B complex showed up- egula ion in lung cance o sEVs and umo issue. Abe an DNA me hyla ion caused by he DNMT3B complex in lung cance is well es ablished and p oposed as a possible he apeu ic a ge . Cance s 2020, 12, x FOR PEER REVIEW 10 o 20 umo issue, o example, iglyce ide me abolic p ocesses. DNMT3B complex showed up- egula ion in lung cance o sEVs and umo issue. Abe an DNA me hyla ion caused by he DNMT3B complex in lung cance is well es ablished and p oposed as a possible he apeu ic a ge . Figu e 5. Comple e unc ional egula ion and en ichmen analysis o p o eins iden i ied in acellula BAL (BAL), small EVs, lung umo issue (TissueTN), and xeno ansplan lung umo issue (TissueXN). The colo code in he hea map e lec s he egula ion le el (black un egula ed, g een signi ican up- egula ed in umo , and ed signi ican ly down- egula ed in umo ). The ex in he cells indica es he numbe o p o eins iden i ied in each unc ional ca ego y, he numbe o p o eins anno a ed in he gi en unc ional ca ego y and he signi icance le el o egula ion, and signi icance le el o en ichmen (* p <0.05, ** p < 0.01, and *** p < 0.001). 2.8. Quan i a i e E alua ion o Immune Cell Lineage Ma ke s BAL con ains cells om a ious sou ces like umo cells, epi helial cells, immune cells, and ib oblas s [22,23]. Based on he R package MCPcoun e [24], we es ima ed he a e age abundance sco es o ma ke s o eigh di e en immune and wo s omal cell popula ions (Figu e 6). MCPcoun e iden i ied six p o ein ma ke s in BAL, 15 in sEVs, and 17 in issue samples. In BAL sEVs, B lineage ma ke s esul ed in he highes a e age abundance sco es. BAL sEVs om lung cance end o display highe abundance sco es o monocy ic lineage and ib oblas s han con ols (Figu e 6). This end was no con i med o acellula BAL p o eome (Figu e S7a). Ma ke s o eigh immune and wo s omal cell popula ions could sepa a e samples in o no mal, umo , and xeno ansplan umo issues (Figu e S7b). Figu e 5. Comple e unc ional egula ion and en ichmen analysis o p o eins iden i ied in acellula BAL (BAL), small EVs, lung umo issue (TissueTN), and xeno ansplan lung umo issue (TissueXN). The colo code in he hea map e lec s he egula ion le el (black un egula ed, g een signi ican up- egula ed in umo , and ed signi ican ly down- egula ed in umo ). The ex in he cells indica es he numbe o p o eins iden i ied in each unc ional ca ego y, he numbe o p o eins anno a ed in he gi en unc ional ca ego y and he signi icance le el o egula ion, and signi icance le el o en ichmen (* p<0.05, ** p<0.01, and *** p<0.001). Cance s 2020,12, 3450 16 o 18 Re e ences 1. Fidle , M.M.; B ay, F.; Soe joma a am, I. The global cance bu den and human de elopmen : A e iew. Scand. J. Public Heal h 2018,46, 27–36. [C ossRe ] 2. Ba a, J.A.; Powell, C.A.; Wisni esky, J.P. Global Epidemiology o Lung Cance . Ann. Glob. Heal h 2019 ,85, 8. [C ossRe ] [PubMed] 3. Ko, E.C.; Raben, D.; Fo men i, S.C. The In eg a ion o Radio he apy wi h Immuno he apy o he T ea men o Non-Small Cell Lung Cance . Clin. Cance Res. O . J. Am. Assoc. Cance Res. 2018 ,24, 5792–5806. [C ossRe ] [PubMed] 4. Ma hiesen, R. MS-Based Bioma ke Disco e y in B onchoal eola La age Fluid o Lung Cance . P o eom. Clin. Appl. 2020,14, e1900077. [C ossRe ] 5. Lee, H.; G oo , M.; Pinilla-Ve a, M.; F edenbu gh, L.E.; Jin, Y. Iden i ica ion o miRNA- ich esicles in b onchoal eola la age luid: Insigh s in o he unc ion and he e ogenei y o ex acellula esicles. J. Con ol. Release O . J. Con ol. Release Soc. 2019,294, 43–52. [C ossRe ] 6. Venosa, A.; Ka zen, J.; Tome , Y.; Kopp, M.; Jamil, S.; Russo, S.J.; Muluge a, S.; Bee s, M.F. Epi helial Exp ession o an In e s i ial Lung Disease-Associa ed Mu a ion in Su ac an P o ein-C Modula es Rec ui men and Ac i a ion o Key Myeloid Cell Popula ions in Mice. J. Immunol. 2019 ,202, 2760–2771. [C ossRe ] [PubMed] 7. Lu, R.; Zhao, X.; Li, J.; Niu, P.; Yang, B.; Wu, H.; Wang, W.; Song, H.; Huang, B.; Zhu, N.; e al. Genomic cha ac e isa ion and epidemiology o 2019 no el co ona i us: Implica ions o i us o igins and ecep o binding. Lance 2020,395, 565–574. [C ossRe ] 8. Zhang, C.; Yu, W.; Wang, L.; Zhao, M.; Guo, Q.; L , S.; Hu, X.; Lou, J. DNA Me hyla ion Analysis o he SHOX2 and RASSF1A Panel in B onchoal eola La age Fluid o Lung Cance Diagnosis. J. Cance 2017 ,8, 3585–3591. [C ossRe ] 9. Walmsley, S.; C uickshank-Quinn, C.; Quinn, K.; Zhang, X.; Pe ache, I.; Bowle , R.P.; Reisdo ph, R.; Reisdo ph, N. A p o o ypic small molecule da abase o b onchoal eola la age-based me abolomics. Sci. Da a 2018,5, 180060. [C ossRe ] 10. Gomes, S.; Ca adas, B.; Fe ei a, J.C.; Ma ques, P.I.; Mon ei o, C.; Sucena, M.; Sousa, C.; Vaz Rod igues, L.; Teixei a, G.; Pin o, P.; e al. P o iling o lung mic obio a discloses di e ences in adenoca cinoma and squamous cell ca cinoma. Sci. Rep. 2019,9, 12838. [C ossRe ] 11. Oume aci, T.; Schmid , B.; Wol , T.; Zapa ka, M.; Pich, A.; B o s, B.; Eils, R.; Fleischhacke , M.; Schlegelbe ge , B.; on Neuho , N. B onchoal eola la age luid o lung cance pa ien s: Mapping he uncha ed wa e s using p o eomics echnology. Lung Cance 2011,72, 136–138. [C ossRe ] [PubMed] 12. Pas o , M.D.; Nogal, A.; Molina-Pinelo, S.; Melendez, R.; Salinas, A.; Gonzalez De la Pena, M.; Ma in-Juan, J.; Co al, J.; Ga cia-Ca bone o, R.; Ca ne o, A.; e al. Iden i ica ion o p o eomic signa u es associa ed wi h lung cance and COPD. J. P o eom. 2013,89, 227–237. [C ossRe ] [PubMed] 13. Ca alho, A.S.; Cuco, C.M.; La a eda, C.; Miguel, F.; Ven u a, M.; Almeida, S.; Pin o, P.; de Ab eu, T.T.; Rod igues, L.V.; Seixas, S.; e al. B onchoal eola La age P o eomics in Pa ien s wi h Suspec ed Lung Cance . Sci. Rep. 2017,7, 42190. [C ossRe ] [PubMed] 14. Alma oodi, S.A.; McDonald, C.F.; Collins, A.L.; Da by, I.A.; Pounio is, D.S. Quan i a i e p o eomics o b onchoal eola la age luid in lung adenoca cinoma. Cance Genom. P o eom. 2015,12, 39–48. 15. O ea, I.; Rod iguez-A iza, A.; Chicano-Gal ez, E.; A enas Vacas, M.S.; Ju ado Gamez, B. Disco e y o po en ial p o ein bioma ke s o lung adenoca cinoma in b onchoal eola la age luid by SWATH MS da a-independen acquisi ion and a ge ed da a ex ac ion. J. P o eom. 2016,138, 106–114. [C ossRe ] 16. Sim, S.Y.; Choi, Y.R.; Lee, J.H.; Lim, J.M.; Lee, S.E.; Kim, K.P.; Kim, J.Y.; Lee, S.H.; Kim, M.S. In-Dep h P o eomic Analysis o Human B onchoal eola La age Fluid owa d he Bioma ke Disco e y o Lung Cance s. P o eom. Clin. Appl. 2019,13, e1900028. [C ossRe ] 17. Ca alho, A.S.; Bae a, H.; Sil a, B.C.; Mo aes, M.C.S.; Bodo, C.; Beck, H.C.; Rod iguez, M.S.; Sa aswa , M.; Pandey, A.; Ma hiesen, R. Ex a-cellula esicles ca y p o eome o cance hallma ks. F on . Biosci. 2020 ,25, 398–436. 18. Li, L.; Wei, Y.; To, C.; Zhu, C.Q.; Tong, J.; Pham, N.A.; Taylo , P.; Igna chenko, V.; Igna chenko, A.; Zhang, W.; e al. In eg a ed omic analysis o lung cance e eals me abolism p o eome signa u es wi h p ognos ic impac . Na . Commun. 2014,5, 5469. [C ossRe ] Cance s 2020,12, 3450 17 o 18 19. Hu wi z, S.N.; Ride , M.A.; Bundy, J.L.; Liu, X.; Singh, R.K.; Meckes, D.G., J . P o eomic p o iling o NCI-60 ex acellula esicles unco e s common p o ein ca go and cance ype-speci ic bioma ke s. Onco a ge 2016 , 7, 86999–87015. [C ossRe ] 20. Hoshino, A.; Kim, H.S.; Bojma , L.; Gyan, K.E.; Cio i, M.; He nandez, J.; Zambi inis, C.P.; Rod igues, G.; Molina, H.; Heissel, S.; e al. Ex acellula Vesicle and Pa icle Bioma ke s De ine Mul iple Human Cance s. Cell 2020,182, 1044–1061. [C ossRe ] 21. Ca alho, A.S.; Molina, H.; Ma hiesen, R. New insigh s in o unc ional egula ion in MS-based d ug p o iling. Sci. Rep. 2016,6, 18826. [C ossRe ] [PubMed] 22. Pollock, K.; Alba es, L.; Wend , C.; Hubel, A. Isola ion o ib oblas s and epi helial cells in b onchoal eola la age (BAL). Exp. Lung Res. 2013,39, 146–154. [C ossRe ] 23. Liao, M.; Liu, Y.; Yuan, J.; Wen, Y.; Xu, G.; Zhao, J.; Cheng, L.; Li, J.; Wang, X.; Wang, F.; e al. Single-cell landscape o b onchoal eola immune cells in pa ien s wi h COVID-19. Na . Med. 2020 ,26, 842–844. [C ossRe ] [PubMed] 24. Bech , E.; Gi aldo, N.A.; Lac oix, L.; Bu a d, B.; Ela ouci, N.; Pe i p ez, F.; Sel es, J.; Lau en -Puig, P.; Sau es-F idman, C.; F idman, W.H.; e al. Es ima ing he popula ion abundance o issue-in il a ing immune and s omal cell popula ions using gene exp ession. Genome Biol. 2016,17, 218. [C ossRe ] [PubMed] 25. Webbe , J.; Clay on, A. How pu e a e you esicles? J. Ex acell. Vesicles 2013,2. [C ossRe ] [PubMed] 26. Pu cell, A.W.; Rama a hinam, S.H.; Te ne e, N. Mass spec ome y-based iden i ica ion o MHC-bound pep ides o immunopep idomics. Na . P o oc. 2019,14, 1687–1707. [C ossRe ] 27. Bassani-S e nbe g, M.; Ba nea, E.; Bee , I.; A i i, I.; Ka z, T.; Admon, A. Soluble plasma HLA pep idome as a po en ial sou ce o cance bioma ke s. P oc. Na l. Acad. Sci. USA 2010,107, 18769–18776. [C ossRe ] 28. C je ko ic, A.; Jang, S.C.; Konecna, B.; Hoog, J.L.; Sihlbom, C.; Lasse , C.; Lo all, J. De ailed Analysis o P o ein Topology o Ex acellula Vesicles-E idence o Uncon en ional Memb ane P o ein O ien a ion. Sci. Rep. 2016,6, 36338. [C ossRe ] 29. Robe s, P.J.; De , C.J. Ta ge ing he Ra -MEK-ERK mi ogen-ac i a ed p o ein kinase cascade o he ea men o cance . Oncogene 2007,26, 3291–3310. [C ossRe ] 30. Chiu, L.Y.; Hsin, I.L.; Yang, T.Y.; Sung, W.W.; Chi, J.Y.; Chang, J.T.; Ko, J.L.; Sheu, G.T. The ERK-ZEB1 pa hway media es epi helial-mesenchymal ansi ion in peme exed esis an lung cance cells wi h supp ession by inca alkaloids. Oncogene 2017,36, 242–253. [C ossRe ] 31. Yang, Y.F.; Chang, Y.C.; Jan, Y.H.; Yang, C.J.; Huang, M.S.; Hsiao, M. Squalene syn hase p omo es he in asion o lung cance cells ia he os eopon in/ERK pa hway. Oncogenesis 2020,9, 78. [C ossRe ] [PubMed] 32. Jones, G.G.; Del Rio, I.B.; Sa i, S.; Seke im, A.; Young, L.C.; Ha ig, N.; A eso Zubiau , I.; El-Bah awy, M.A.; Hynds, R.E.; Lei, W.; e al. SHOC2 phospha ase-dependen RAF dime iza ion media es esis ance o MEK inhibi ion in RAS-mu an cance s. Na . Commun. 2019,10, 2532. [C ossRe ] 33. Maacha, S.; Bha , A.A.; Jimenez, L.; Raza, A.; Ha is, M.; Uddin, S.; G i el, J.C. Ex acellula esicles-media ed in e cellula communica ion: Roles in he umo mic oen i onmen and an i-cance d ug esis ance. Mol. Cance 2019,18, 55. [C ossRe ] [PubMed] 34. Aung, T.; Chapuy, B.; Vogel, D.; Wenzel, D.; Oppe mann, M.; Lahmann, M.; Weinhage, T.; Menck, K.; Hup eld, T.; Koch, R.; e al. Exosomal e asion o humo al immuno he apy in agg essi e B-cell lymphoma modula ed by ATP-binding casse e anspo e A3. P oc. Na l. Acad. Sci. USA 2011,108, 15336–15341. [C ossRe ] [PubMed] 35. Os le , K.R.; Da is, E.M.; Payne, S.L.; Gosalia, B.B.; Exposi o-Cespedes, J.; Le Beau, M.M.; Godley, L.A. Cance cells exp ess abe an DNMT3B ansc ip s encoding unca ed p o eins. Oncogene 2007 ,26, 5553–5563. [C ossRe ] [PubMed] 36. Cheng, Y.; He, C.; Wang, M.; Ma, X.; Mo, F.; Yang, S.; Han, J.; Wei, X. Ta ge ing epigene ic egula o s o cance he apy: Mechanisms and ad ances in clinical ials. Signal T ansduc . Ta ge . The . 2019,4, 62. [C ossRe ] 37. Sandhu, R.; Ri enba k, A.G.; Coleman, W.B. Enhancemen o chemo he apeu ic e icacy in hype me hyla o b eas cance cells h ough a ge ed and pha macologic inhibi ion o DNMT3b. B eas Cance Res. T ea . 2012 , 131, 385–399. [C ossRe ] 38. Wu, C.; Guo, E.; Ming, J.; Sun, W.; Nie, X.; Sun, L.; Peng, S.; Luo, M.; Liu, D.; Zhang, L.; e al. Radia ion-Induced DNMT3B P omo es Radio esis ance in Nasopha yngeal Ca cinoma h ough Me hyla ion o p53 and p21. Mol. The . Oncoly ics 2020,17, 306–319. [C ossRe ] 39. Yao, P.; Sun, H.; Xu, C.; Chen, T.; Zou, B.; Jiang, P.; Du, W. E idence o a di ec c oss- alk be ween malic enzyme and he pen ose phospha e pa hway ia s uc u al in e ac ions. J. Biol. Chem. 2017 ,292, 17113–17120. [C ossRe ] Cance s 2020,12, 3450 18 o 18 40. Desg osellie , J.S.; Che esh, D.A. In eg ins in cance : Biological implica ions and he apeu ic oppo uni ies. Na . Re . Cance 2010,10, 9–22. [C ossRe ] 41. Hamidi, H.; Pie ila, M.; I aska, J. The complexi y o in eg ins in cance and new scopes o he apeu ic a ge ing. B . J. Cance 2016,115, 1017–1023. [C ossRe ] [PubMed] 42. Domagala-Kulawik, J.; Guzman, J.; Cos abel, U. Immune cells in b onchoal eola la age in pe iphe al lung cance —Analysis o 140 cases. Respi . In . Re . Tho ac. Dis. 2003,70, 43–48. [C ossRe ] [PubMed] 43. B uno, T.C.; Ebne , P.J.; Moo e, B.L.; Squalls, O.G.; Waugh, K.A.; E uslano , E.B.; Singhal, S.; Mi chell, J.D.; F anklin, W.A.; Me ick, D.T.; e al. An igen-P esen ing In a umo al B Cells A ec CD4(+) TIL Pheno ypes in Non-Small Cell Lung Cance Pa ien s. Cance Immunol. Res. 2017,5, 898–907. [C ossRe ] [PubMed] 44. Ca mi, Y.; Spi ze , M.H.; Linde, I.L.; Bu , B.M.; P es wood, T.R.; Pe lman, N.; Da idson, M.G.; Kenkel, J.A.; Segal, E.; Pusapa i, G.V.; e al. Allogeneic IgG combined wi h dend i ic cell s imuli induce an i umou T-cell immuni y. Na u e 2015,521, 99–104. [C ossRe ] [PubMed] 45. MacMahon, H.; Aus in, J.H.; Gamsu, G.; He old, C.J.; Je , J.R.; Naidich, D.P.; Pa z, E.F., J .; Swensen, S.J. Guidelines o managemen o small pulmona y nodules de ec ed on CT scans: A s a emen om he Fleischne Socie y. Radiology 2005,237, 395–400. [C ossRe ] [PubMed] 46. Wisniewski, J.R.; Zougman, A.; Naga aj, N.; Mann, M. Uni e sal sample p epa a ion me hod o p o eome analysis. Na . Me hods 2009,6, 359–362. [C ossRe ] 47. Ca alho, A.S.; Ribei o, H.; Voabil, P.; Penque, D.; Jensen, O.N.; Molina, H.; Ma hiesen, R. Global mass spec ome y and ansc ip omics a ay based d ug p o iling p o ides no el insigh in o glucosamine induced endoplasmic e iculum s ess. Mol. Cell. P o eom. MCP 2014,13, 3294–3307. [C ossRe ] 48. Cox, J.; Mann, M. MaxQuan enables high pep ide iden i ica ion a es, indi idualized p.p.b.- ange mass accu acies and p o eome-wide p o ein quan i ica ion. Na . Bio echnol. 2008,26, 1367–1372. [C ossRe ] 49. Ma hiesen, R.; P ie o, G.; Amo im, A.; Alo ia, K.; Fullaondo, A.; Ca alho, A.S.; A izmendi, J.M. SIR: De e minis ic p o ein in e ence om pep ides assigned o MS da a. J. P o eom. 2012 ,75, 4176–4183. [C ossRe ] 50. Hackenbe g, M.; Ma hiesen, R. Anno a ion-Modules: A ool o inding signi ican combina ions o mul isou ce anno a ions o gene lis s. Bioin o ma ics 2008,24, 1386–1393. [C ossRe ] 51. Gu, Z.; Eils, R.; Schlesne , M. Complex hea maps e eal pa e ns and co ela ions in mul idimensional genomic da a. Bioin o ma ics 2016,32, 2847–2849. [C ossRe ] [PubMed] 52. Kanehisa, M.; Fu umichi, M.; Tanabe, M.; Sa o, Y.; Mo ishima, K. KEGG: New pe spec i es on genomes, pa hways, diseases and d ugs. Nucleic Acids Res. 2017,45, D353–D361. [C ossRe ] [PubMed] 53. Gene On ology Conso ium. The Gene On ology Resou ce: 20 yea s and s ill GOing s ong. Nucleic Acids Res . 2019,47, D330–D338. [C ossRe ] [PubMed] 54. Goel, R.; Ha sha, H.C.; Pandey, A.; P asad, T.S. Human P o ein Re e ence Da abase and Human P o einpedia as esou ces o phosphop o eome analysis. Mol. Biosys . 2012,8, 453–463. [C ossRe ] [PubMed] 55. Ruepp, A.; B aune , B.; Dunge -Kal enbach, I.; F ishman, G.; Mon one, C.; S ansky, M.; Waegele, B.; Schmid , T.; Doudieu, O.N.; S ump len, V.; e al. CORUM: The comp ehensi e esou ce o mammalian p o ein complexes. Nucleic Acids Res. 2008,36, D646–D650. [C ossRe ] 56. Libe zon, A.; Bi ge , C.; Tho aldsdo i , H.; Ghandi, M.; Mesi o , J.P.; Tamayo, P. The Molecula Signa u es Da abase (MSigDB) hallma k gene se collec ion. Cell Sys . 2015,1, 417–425. [C ossRe ] 57. Pe ez-Ri e ol, Y.; Cso das, A.; Bai, J.; Be nal-Llina es, M.; Hewapa hi ana, S.; Kundu, D.J.; Inugan i, A.; G iss, J.; Maye , G.; Eisenache , M.; e al. The PRIDE da abase and ela ed ools and esou ces in 2019: Imp o ing suppo o quan i ica ion da a. Nucleic Acids Res. 2018,47, D442–D450. [C ossRe ] Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. © 2020 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).