Gutandukanya itandukaniro rya nodules zifata ibimenyetso byerekanwe na tomografiya yabazwe (CT) bikomeje kuba ingorabahizi mubikorwa byubuvuzi.Hano, turanga metabolome yisi yose ya serumu 480, harimo kugenzura ubuzima bwiza, nodules nziza yibihaha, hamwe nicyiciro cya I ibihaha adenocarcinoma.Adenocarcinoma yerekana imyirondoro idasanzwe ya metabolomic, mu gihe nodules nziza n'abantu bafite ubuzima bwiza bafite aho bahurira na profili ya metabolomic.Mu itsinda ryubuvumbuzi (n = 306), hamenyekanye metabolite 27 yo gutandukanya nodules nziza kandi mbi.AUC y'icyitegererezo cy'ivangura mu kwemeza imbere (n = 104) no kwemeza hanze (n = 111) byari 0.915 na 0.945.Isesengura ry’inzira ryagaragaje ko metabolite yiyongera ya glycoltique ifitanye isano no kugabanuka kwa tryptophan muri serumu y'ibihaha adenocarcinoma ugereranije na nodules nziza ndetse no kugenzura ubuzima bwiza, ikanagaragaza ko gufata tryptophan bitera glycolysis mu ngirabuzimafatizo za kanseri y'ibihaha.Ubushakashatsi bwacu bugaragaza agaciro ka biomarkers ya serum metabolite mugusuzuma ibyago byo guhumeka neza byamenyekanye na CT.
Kwipimisha hakiri kare ni ngombwa kugirango ubuzima bwa kanseri burokoke.Ibyavuye mu bushakashatsi bwakozwe na Leta Zunze Ubumwe za Amerika (NLST) hamwe n’ubushakashatsi bw’ibihugu by’i Burayi NELSON bwerekanye ko kwisuzumisha hamwe na tomografiya nkeya (LDCT) bishobora kugabanya cyane impfu za kanseri y'ibihaha mu matsinda afite ibyago byinshi 1,2,3.Kuva ikoreshwa rya LDCT mu gusuzuma kanseri y'ibihaha, umubare w'ibyagaragaye kuri radiografiya iterwa na nodules ya asimptomatic pulmonary nodules wakomeje kwiyongera 4.Imitsi ihumeka isobanurwa nkibintu byibanda kuri cm 3 z'umurambararo 5.Duhura ningorane zo gusuzuma amahirwe yo kurwara nabi no guhangana numubare munini wimpyiko zagaragaye kuri LDCT.Imipaka ya CT irashobora gutuma umuntu akurikirana ibizamini kenshi hamwe nibisubizo byiza-byiza, biganisha ku gutabara bitari ngombwa no gukabya6.Niyo mpamvu, hakenewe guteza imbere biomarkers yizewe kandi yingirakamaro kugirango tumenye neza kanseri yibihaha mugihe cyambere no gutandukanya nodules nyinshi nziza mugihe cyo gutahura 7.
Isesengura ryuzuye rya molekuline yamaraso (serumu, plasma, peripheri yamaraso mononuclear selile), harimo genomics, proteomics cyangwa ADN methylation ya ADN 8,9,10, byatumye abantu bashishikazwa no kuvumbura ibimenyetso byerekana ibimenyetso bya kanseri yibihaha.Hagati aho, uburyo bwa metabolomics bupima ibicuruzwa biva mu ngirabuzimafatizo biterwa n’ibikorwa bya endogenous na exogenous bityo bigakoreshwa mu guhanura indwara n’ingaruka zabyo.Amazi ya chromatografiya-tandem mass spectrometrie (LC-MS) nuburyo bukoreshwa cyane mubushakashatsi bwa metabolomics bitewe nubukangurambaga bwabwo bwinshi hamwe nimbaraga nini cyane, bushobora gutwikira metabolite ifite imiterere itandukanye ya fiziki ya chimique11,12,13.Nubwo isesengura rya metabolomic ku isi ryakozwe na plasma / serumu ryakoreshejwe kugirango hamenyekane biomarkers ifitanye isano no gusuzuma kanseri y'ibihaha14,15,16,17 hamwe no kuvura neza, ibyiciro 18 bya serumu metabolite itandukanya ibihaha byiza kandi bibi bikomeje kwigwa cyane.-ubushakashatsi bukomeye.
Adenocarcinoma na kanseri y'uturemangingo ni ubwoko bubiri bw'ingenzi bwa kanseri y'ibihaha itari ntoya (NSCLC).Ibizamini bitandukanye byo gusuzuma CT byerekana ko adenocarcinoma aribwo bwoko bwa kanseri yibihaha ya kanseri y'ibihaha1,19,20,21.Muri ubu bushakashatsi, twifashishije ultra-imikorere yamazi ya chromatografiya-y-imiterere-nini ya mass spectrometrie (UPLC-HRMS) kugirango dukore isesengura rya metabolomics kuri serumu zose hamwe 695, harimo kugenzura ubuzima bwiza, nodules nziza, na CT yamenyekanye cm3.Kugaragaza Icyiciro cya I ibihaha adenocarcinoma.Twabonye akanama ka metabolite ya serumu itandukanya ibihaha adenocarcinoma na nodules nziza kandi igenzura neza.Isesengura ryitunganyirize rya Pathway ryerekanye ko tryptophan idasanzwe na glucose metabolism ari ibintu bisanzwe bihinduka muri adenocarcinoma y'ibihaha ugereranije na nodules nziza no kugenzura neza.Hanyuma, twashizeho kandi twemeza serumu metabolike itondekanya ibintu byihariye kandi byunvikana kugirango tumenye itandukaniro riri hagati ya LDCT mbi, ishobora gufasha mugupima hakiri kare no gusuzuma ingaruka.
Mu bushakashatsi burimo gukorwa, ingero za serumu zihuye n’imyaka hamwe n’imyaka byakusanyirijwe mu buryo butandukanye bivuye ku 174 igenzura neza, abarwayi 292 barwaye imitsi y’imitsi, n’abarwayi 229 bafite icyiciro cya mbere cy’ibihaha adenocarcinoma.Ibiranga demokarasi biranga amasomo 695 bigaragara mu mbonerahamwe ya 1.
Nkuko bigaragara ku gishushanyo cya 1a, icyitegererezo cya serumu 480, harimo 174 yo kugenzura ubuzima bwiza (HC), 170 nodules nodules (BN), hamwe n’icyiciro cya 136 I I ibihaha adenocarcinoma (LA), byakusanyirijwe mu kigo cya kanseri cya kaminuza ya Sun Yat-sen.Ivumburwa rya cohort ya metabolomic idateganijwe ukoresheje umwirondoro wa ultra-imikorere y'amazi ya chromatografiya-ikemurwa cyane na mass spectrometrie (UPLC-HRMS).Nkuko bigaragara mu gishushanyo cya 1, metabolite itandukanye hagati ya LA na HC, LA na BN byagaragaye kugirango ishyireho icyitegererezo kandi ikore ubushakashatsi bwisesengura ryinzira zitandukanye.Ingero 104 zegeranijwe n’ikigo cya kanseri cya kaminuza ya Sun Yat-sen hamwe n’ingero 111 zegeranijwe n’ibindi bitaro bibiri zapimwe imbere n’imbere.
ubushakashatsi bwabaturage mubyiciro byavumbuwe byakorewe isesengura rya serumu metabolomics kwisi yose ukoresheje ultra-imikorere yamazi ya chromatografiya-ikemurwa cyane na mass spectrometrie (UPLC-HRMS).b Igice gito byibuze isesengura rishingiye ku ivangura (PLS-DA) rya metabolome yose hamwe ya 480 ya serumu yavuye mubushakashatsi bwakozwe, harimo kugenzura ubuzima bwiza (HC, n = 174), nodules nziza (BN, n = 170), nicyiciro cya I ibihaha adenocarcinoma (Los Angeles, n = 136).+ ESI, uburyo bwiza bwa electrospray ionisiyoneri, -ESI, uburyo bubi bwa electrospray ionisation.C. .) yerekanwe ku gishushanyo cy'ibirunga.f Ikarita yubushyuhe ikurikirana yerekana itandukaniro rigaragara mumibare ya metabolite yatangajwe hagati ya LA na BN.Inkomoko yamakuru yatanzwe muburyo bwamadosiye yamakuru.
Serumu metabolome yose hamwe ya 174 HC, 170 BN na 136 LA mumatsinda yubuvumbuzi yasesenguwe hakoreshejwe isesengura rya UPLC-HRMS.Turabanza kwerekana ko kugenzura ubuziranenge (QC) ingero zifatika hagati yikigereranyo cyibanze kitagenzuwe (PCA), cyemeza ko imikorere yubushakashatsi iriho (Ishusho yinyongera 2).
Nkuko bigaragara mu isesengura ryibice byibuze-ivangura (PLS-DA) ku gishushanyo cya 1 b, twasanze hari itandukaniro rigaragara hagati ya LA na BN, LA na HC muburyo bwiza (+ ESI) nibibi (−ESI) uburyo bwa electrospray ionisation .mu bwigunge.Ariko, nta tandukaniro rikomeye ryabonetse hagati ya BN na HC mubihe + ESI na -ESI.
Twabonye ibintu 382 bitandukanye bitandukanye hagati ya LA na HC, 231 itandukaniro hagati ya LA na BN, nibintu 95 bitandukanye hagati ya BN na HC (Wilcoxon yasinywe ikizamini cyamanota, FDR <0.05 nimpinduka nyinshi> 1.2 cyangwa <0.83) (Ishusho .1c-e )..Impinga zongeye gusobanurwa (Amakuru yinyongera 3) arwanya data base (mzCloud / HMDB / Isomero rya Chemspider) nagaciro ka m / z, igihe cyo kugumya no gucamo ibice rusange (ibisobanuro birambuye mubice byuburyo) 22.Hanyuma, 33 na 38 zanditseho metabolite zifite itandukaniro rinini mubwinshi byagaragaye kuri LA na BN (Igicapo 1f na Imbonerahamwe Yinyongera 2) na LA na HC (Ishusho yinyongera 3 nimbonerahamwe 2).Ibinyuranye, metabolite 3 gusa ifite itandukaniro rinini mubwinshi yagaragaye muri BN na HC (Imbonerahamwe yinyongera 2), ijyanye no guhuza BN na HC muri PLS-DA.Izi metabolite zitandukanye zitandukanya ibintu byinshi bya biohimiki (Ishusho yinyongera 4).Ufatiye hamwe, ibisubizo byerekana impinduka zikomeye muri serumu metabolome yerekana ihinduka ribi rya kanseri y'ibihaha hakiri kare ugereranije nibihaha byiza cyangwa ingingo nziza.Hagati aho, isano ya serumu metabolome ya BN na HC yerekana ko nodules nziza yimpyiko ishobora gusangira ibintu byinshi biranga abantu bafite ubuzima bwiza.Bitewe nuko ihinduka ryimiterere ya epidermal (EGFR) ihinduka ryimiterere ya gene ikunze kugaragara mubihaha adenocarcinoma subtype ya 23, twashatse kumenya ingaruka ziterwa na mutation ya shoferi kuri serumu metabolome.Twahise dusesengura imiterere rusange ya metabolomic yimanza 72 hamwe na EGFR mumatsinda ya adenocarcinoma yibihaha.Igishimishije, twabonye imyirondoro igereranijwe hagati yabarwayi ba mutant ba EGFR (n = 41) nabarwayi bo mu bwoko bwa EGFR (n = 31) mubisesengura PCA (Ishusho yinyongera 5a).Ariko, twabonye metabolite 7 zifite ubwinshi bwahinduwe cyane kubarwayi bafite ihinduka rya EGFR ugereranije n’abarwayi bafite ubwoko bwa EGFR bwo mu gasozi (t test, p <0.05 no guhinduka inshuro> 1.2 cyangwa <0.83) (Ishusho yinyongera 5b).Ubwinshi muri metabolite (5 kuri 7) ni acylcarnitine, igira uruhare runini munzira ya aside irike.
Nkuko bigaragarira mubikorwa byerekanwe ku gishushanyo cya 2 a, biomarkers yo gushyira mu byiciro nodule yabonetse hakoreshejwe byibuze kugabanya kugabanuka no guhitamo gushingiye kuri metabolite 33 itandukanye yagaragaye muri LA (n = 136) na BN (n = 170).Ibyiza bihuza impinduka (LASSO) - binary logistic regression moderi.Inshuro icumi zambukiranya-mikoreshereze yakoreshejwe mu kugerageza kwizerwa kwicyitegererezo.Guhitamo guhinduka hamwe nibisanzwe byahinduwe nibihano bishoboka cyane hamwe nibisobanuro λ24.Isesengura rya metabolomics ku isi ryakozwe kandi ryigenga mu kwemeza imbere (n = 104) no kwemeza hanze (n = 111) kugira ngo hamenyekane imikorere y’imiterere y’ivangura.Kubera iyo mpamvu, metabolite 27 zo mu bushakashatsi zavumbuwe zagaragaye nk’icyitegererezo cyiza kivangura gifite agaciro kanini cyane AUC (Ishusho 2b), muri bo 9 bariyongereye ibikorwa naho 18 bagabanuka muri LA ugereranije na BN (Ishusho 2c).
Workflow yo kubaka impyisi nodule itondekanya, harimo guhitamo icyiza cyiza cya serumu metabolite muguvumbura ukoresheje uburyo bwa binary logistic regression moderi ukoresheje inshuro icumi zambukiranya-kwemeza no gusuzuma imikorere iteganijwe mumbere no hanze yemewe.b Kwambukiranya-mibare yerekana uburyo bwa LASSO bwisubiramo bwo guhitamo biomarker ya metabolike.Imibare yatanzwe hejuru yerekana impuzandengo ya biomarkers yatoranijwe kuri given.Umurongo utudomo utukura ugereranya impuzandengo ya AUC kuri lambda ihuye.Ikosa ryibara ryerekana ibara ntarengwa na AUC agaciro.Umurongo utudomo werekana icyitegererezo cyiza hamwe na 27 byatoranijwe biomarkers.AUC, agace munsi yabakiriye ikora ibiranga (ROC) umurongo.c Guhinduka gukabije kwa 27 metabolite yatoranijwe mumatsinda ya LA ugereranije nitsinda rya BN mumatsinda yo kuvumbura.Inkingi itukura - gukora.Inkingi yubururu nigabanuka.d - f Kwakira ibikorwa biranga (ROC) umurongo werekana imbaraga za moderi ivangura ishingiye kuri 27 metabolite ikomatanya mubuvumbuzi, imbere, hamwe no kwemeza hanze.Inkomoko yamakuru yatanzwe muburyo bwamadosiye yamakuru.
Icyitegererezo cyo guhanura cyakozwe hashingiwe ku bipimo biremereye byo gusubira inyuma kwi metabolite 27 (Imbonerahamwe 3).Isesengura ROC rishingiye kuri metabolite 27 ryatanze agace kari munsi yu murongo (AUC) agaciro ka 0.933, itsinda ryivumbuye ryiyumvamo ni 0.868, naho umwihariko ni 0.859 (Ishusho 2d).Hagati aho, muri 38 zasobanuwe zitandukanye za metabolite hagati ya LA na HC, itsinda rya metabolite 16 ryageze kuri AUC ya 0.902 ifite sensibilité ya 0.801 hamwe na 0.856 mu kuvangura LA na HC (Ishusho yinyongera 6a-c).Indangagaciro za AUC zishingiye ku bubiko butandukanye bwo guhindura imipaka itandukanye ya metabolite nayo yagereranijwe.Twabonye ko uburyo bwo gutondekanya ibyiciro bwitwaye neza mugutandukanya LA na BN (HC) mugihe urwego rwo guhindura inshuro rwashyizwe kuri 1.2 na 1.5 cyangwa 2.0 (Ishusho yinyongera 7a, b).Icyitegererezo cyo gutondekanya, gishingiye ku matsinda 27 ya metabolite, cyarushijeho kwemezwa imbere mu gihugu no hanze.AUC yari 0.915 (sensibilité 0.867, umwihariko 0.811) kugirango yemeze imbere na 0.945 (sensitivite 0.810, umwihariko 0.979) kugirango yemeze hanze (Ishusho 2e, f).Kugirango hamenyekane imikorere myiza, ingero 40 ziva muri cohort zasesenguwe muri laboratoire yo hanze nkuko byasobanuwe mu gice cya Methods.Ibyiciro byukuri byageze kuri AUC ya 0.925 (Ishusho yinyongera 8).Kuberako ibihaha squamous selile carcinoma (LUSC) nubwoko bwa kabiri bukunze kwibasirwa na kanseri y'ibihaha itari mito mito (NSCLC) nyuma y'ibihaha adenocarcinoma (LUAD), twagerageje kandi kwemeza akamaro ko gukoresha imiterere ya metabolike.BN n'imanza 16 za LUSC.AUC ivangura hagati ya LUSC na BN yari 0.776 (Ishusho yinyongera 9), yerekana ubushobozi buke ugereranije nivangura hagati ya LUAD na BN.
Ubushakashatsi bwerekanye ko ubunini bwa nodules ku mashusho ya CT bufitanye isano neza n’uko hashobora kubaho indwara mbi kandi bikomeza kuba intandaro yo kuvura node 25.26,27.Isesengura ryamakuru yaturutse mu itsinda rinini ry’ubushakashatsi bwa NELSON bwerekanye ko ibyago byo kurwara nabi mu masomo afite imitwe <5 mm ndetse byari bisa nkibyo mu masomo adafite 28.Kubwibyo, ingano ntarengwa isaba gukurikirana CT buri gihe ni mm 5, nkuko byasabwe na Sosiyete y'Abongereza Thoracic (BTS), na mm 6, nkuko byasabwe na Sosiyete Fleischner 29.Nyamara, nodules irenze mm 6 kandi idafite ibimenyetso byiza, bita indeterminate pulmonary nodules (IPN), iracyari ikibazo gikomeye mugusuzuma no gucunga mubikorwa byubuvuzi30,31.Ubukurikira twasuzumye niba ingano ya nodule yagize uruhare mu gusinya metabolomic dukoresheje ingero zahujwe zivumbuwe hamwe n’imbere yo kwemeza imbere.Twibanze kuri 27 biomarkers yemewe, twabanje kugereranya imyirondoro ya PCA ya HC na BN sub-6 mm metabolomes.Twabonye ko ingingo nyinshi zamakuru kuri HC na BN zuzuzanya, byerekana ko urugero rwa serumu metabolite rwasaga mumatsinda yombi (Ishusho 3a).Ikarita iranga ubunini butandukanye yagumye ibitswe muri BN na LA (Igishusho 3b, c), mugihe itandukaniro ryagaragaye hagati ya nodules mbi kandi nziza mu ntera ya mm 6-20 (Ishusho 3d).Iri tsinda ryari rifite AUC ya 0.927, umwihariko wa 0.868, hamwe na sensibilité ya 0.820 yo guhanura ububi bwa nodules bupima mm 6 kugeza kuri 20 (Ishusho 3e, f).Ibisubizo byacu byerekana ko urutonde rushobora gufata impinduka ziterwa no guhinduka hakiri kare, tutitaye ku bunini bwa node.
ad Kugereranya imyirondoro ya PCA hagati yitsinda ryihariye rishingiye kuri metabolic classifier ya 27 metabolite.CC na BN <6 mm.b BN <6 mm vs BN 6–20 mm.muri LA 6-20 mm na LA 20-30 mm.g BN 6-20 mm na LA 6–20 mm.GC, n = 174;BN <6 mm, n = 153;BN 6-20 mm, n = 91;LA 6-20 mm, n = 89;LA 20-30 mm, n = 77. e Kwakira ibikorwa biranga (ROC) umurongo werekana imikorere yicyitegererezo ivangura kuri nodules 6-20 mm.f Indangagaciro zishoboka zabazwe hashingiwe ku buryo bwa logistique yo gusubira inyuma kuri nodules ipima mm 6-20.Umurongo utudomo wumurongo ugereranya agaciro keza (0.455).Imibare iri hejuru yerekana ijanisha ryimanza ziteganijwe i Los Angeles.Koresha ikizamini cyumurizo wumunyeshuri.PCA, isesengura ryibanze.Agace ka AUC munsi yumurongo.Inkomoko yamakuru yatanzwe muburyo bwamadosiye yamakuru.
Ingero enye (zifite imyaka 44-61) zifite ubunini busa na node (7-9 mm) zatoranijwe kugirango zerekane imikorere yuburyo buteganijwe bwo guhanura indwara (Ishusho 4a, b).Mugusuzuma kwambere, Urubanza 1 rwerekanwe nkumutwe ukomeye hamwe no kubara, ikintu kijyanye no kugirira neza, mugihe urubanza 2 rwerekanwe nkumutwe utazwi neza kandi udafite ibimenyetso bigaragara.Inzira eshatu zo gukurikirana CT scan zerekanye ko izo manza zagumye zihamye mugihe cyimyaka 4 bityo zifatwa nkizuru ryiza (Ishusho 4a).Ugereranije nisuzuma ryamavuriro ya CT scan, isesengura rimwe rya serumu metabolite hamwe na moderi iriho ubu byihuse kandi byerekana neza izo node nziza zishingiye ku mbogamizi zishoboka (Imbonerahamwe 1).Igishushanyo 4b mugihe cya 3 cyerekana nodule ifite ibimenyetso byo gusubira inyuma, bikunze kuba bifitanye isano na malignance32.Urubanza rwa 4 rwatanzwe nkumutwe utazwi neza udafite ibimenyetso byerekana impamvu nziza.Izi manza zose zahanuwe nkibibi ukurikije icyitegererezo (Imbonerahamwe 1).Isuzuma rya adenocarcinoma y'ibihaha ryerekanwe no gusuzuma histopathologique nyuma yo kubagwa ibihaha (Ishusho 4b).Kubijyanye no kwemeza hanze, ibyiciro bya metabolike byahanuye neza inshuro ebyiri zifata ibihaha bitamenyekanye kurenza mm 6 (Ishusho yinyongera 10).
CT amashusho yidirishya rya axial yibihaha byimanza ebyiri za node nziza.Mugihe cya 1, CT scan nyuma yimyaka 4 yerekanaga nodule ihamye ipima mm 7 hamwe na calcisation muburyo bwiburyo bwo hepfo.Mugihe cya 2, CT scan nyuma yimyaka 5 yerekanye nodule ihamye, igice gikomeye hamwe na diameter ya mm 7 mumurongo wo hejuru wiburyo.b Idirishya rya Axial CT amashusho yibihaha hamwe nubushakashatsi bujyanye nibibazo bibiri byicyiciro cya I adenocarcinoma mbere yo kwanga ibihaha.Urubanza rwa 3 rwerekanye node ifite umurambararo wa mm 8 mu gice cyo hejuru cyiburyo cyo kwisubiza inyuma.Urubanza rwa 4 rwerekanye igice gikomeye cyubutaka-ikirahure nodule ipima mm 9 mumurongo wo hejuru wibumoso.Hematoxyline na eosine (H&E) irangi ryumubiri wibihaha (umunzani bar = 50 μ m) byerekana uburyo bwo gukura kwa acinari yibihaha adenocarcinoma.Imyambi yerekana nodules yagaragaye kumashusho ya CT.H&E amashusho ni amashusho yerekana imirima myinshi (> 3) microscopique imirima yasuzumwe na patologue.
Dufatiye hamwe, ibisubizo byacu byerekana agaciro gashoboka ka serumu metabolite biomarkers mugupima itandukaniro rya nodules zifata ibihaha, bishobora guteza ibibazo mugihe cyo gusuzuma CT.
Dushingiye ku cyemezo cyemewe cyo gutandukanya metabolite, twashatse kumenya isano iri hagati y’ibinyabuzima ihinduka ry’imihindagurikire.Isesengura ryinzira ya KEGG ryakozwe na MetaboAisesengura ryerekanye inzira 6 zisanzwe zahinduwe kuburyo bugaragara hagati yaya matsinda yombi (LA na HC na LA na BN, yahinduwe p ≤ 0.001, ingaruka> 0.01).Izi mpinduka zaranzwe n’imivurungano ya pyruvate metabolism, tryptophan metabolism, niacin na nicotinamide metabolism, glycolysis, cycle TCA, na metabolism ya purine (Ishusho 5a).Twongeye gukora metabolomics igamije kugenzura impinduka zikomeye dukoresheje umubare wuzuye.Kumenya metabolite isanzwe muburyo busanzwe bwahinduwe munzira eshatu quadrupole mass spectrometry (QQQ) ukoresheje ibipimo byukuri bya metabolite.Imiterere y’imiterere y’icyitegererezo cy’ubushakashatsi bwa metabolomics yashyizwe mu mbonerahamwe y’inyongera 4. Bihuye n’ibisubizo byatanzwe na metabolomics ku isi, isesengura ry’imibare ryemeje ko hypoxanthine na xanthine, pyruvate, na lactate byiyongereye muri LA ugereranije na BN na HC (Ishusho 5b, c, p <0.05).Ariko, nta tandukaniro rikomeye muri izo metabolite ryabonetse hagati ya BN na HC.
Isesengura ryinzira ya KEGG isesengura metabolite itandukanye cyane mumatsinda ya LA ugereranije nitsinda rya BN na HC.Imirongo ibiri umurizo Globaltest yarakoreshejwe, kandi p agaciro karahinduwe hakoreshejwe uburyo bwa Holm-Bonferroni (byahinduwe p ≤ 0.001 nubunini bwingaruka> 0.01).b - d Ibibanza bya Violin byerekana hypoxanthine, xanthine, lactate, pyruvate, na tryptophan murwego rwa serumu HC, BN, na LA byagenwe na LC-MS / MS (n = 70 kuri buri tsinda).Imirongo yera n'umukara yerekana utudomo twerekana median na quartile.e Umugambi wa Violin werekana Log2TPM isanzwe (transcript kuri miriyoni) mRNA imvugo ya SLC7A5 na QPRT mumihaha adenocarcinoma (n = 513) ugereranije nibice bisanzwe byibihaha (n = 59) muri dataset ya LUAD-TCGA.Agasanduku cyera kagereranya intera itandukanye, umurongo wirabura utambitse hagati ugereranya hagati, naho umurongo wumukara uhagaze uva mu gasanduku ugereranya 95% intera intera (CI).f Pearson ihuza umugambi wa SLC7A5 na GAPDH mumagambo adenocarcinoma yibihaha (n = 513) hamwe nibice bisanzwe byibihaha (n = 59) muri dataset ya TCGA.Agace kijimye kerekana 95% CI.r, Coefficient ya Pearson.g Urwego rusanzwe rwa selile tripitofani muri selile A549 yandujwe hamwe na shRNA idafite akamaro (NC) na shSLC7A5 (Sh1, Sh2) byagenwe na LC-MS / MS.Isesengura mibare ryibintu bitanu byigenga byibinyabuzima muri buri tsinda birerekanwa.h Urwego rwa selire ya NADt (NAD yose, harimo NAD + na NADH) muri selile A549 (NC) na SLC7A5 gukubita selile A549 (Sh1, Sh2).Isesengura mibare ryibintu bitatu byigenga byibinyabuzima muri buri tsinda birerekanwa.i Igikorwa cya Glycolytike ya selile A549 mbere na nyuma ya SLC7A5 gukomanga byapimwe nigipimo cya aside irike idasanzwe (ECAR) (n = 4 icyitegererezo cyigenga cyibinyabuzima kuri buri tsinda).2-DG, 2-deoxy-D-glucose.Ikizamini cyimirizo ibiri yumunyeshuri yakoreshejwe muri (b - h).Muri (g - i), utubari twibeshya twerekana uburyo ± SD, buri igeragezwa ryakozwe inshuro eshatu wigenga kandi ibisubizo byari bisa.Inkomoko yamakuru yatanzwe muburyo bwamadosiye yamakuru.
Twihweje ingaruka zikomeye ziterwa na traptophan metabolisme yahinduwe mumatsinda ya LA, twasuzumye kandi serumu tryptophan murwego rwa HC, BN, na LA dukoresheje QQQ.Twasanze serumu tryptophan yagabanutse muri LA ugereranije na HC cyangwa BN (p <0.001, Igicapo 5d), ibyo bikaba bihuye nubushakashatsi bwabanje bwerekana ko kuzenguruka kwa tripitofani biri hasi ku barwayi ba kanseri y'ibihaha kuruta kubigenzura neza bivuye mu itsinda rishinzwe kugenzura 33,34 , 35.Ubundi bushakashatsi bwakoresheje PET / CT tracer 11C-methyl-L-tryptophan bwerekanye ko igihe cyo kugumana ibimenyetso bya tripitofani mu ngingo za kanseri y'ibihaha cyiyongereye cyane ugereranije n'ibikomere byiza cyangwa tissue zisanzwe36.Turakekeranya ko kugabanuka kwa tryptophan muri serumu ya LA bishobora kwerekana uburyo bwo gufata tryptophan ikora na kanseri yibihaha.
Birazwi kandi ko ibicuruzwa byanyuma byinzira ya kynurenine ya tryptophan catabolism ari NAD + 37,38, ikaba ari insimburangingo ikomeye kugirango reaction ya glyceraldehyde-3-fosifate hamwe na 1,3-bisphosphoglycerate muri glycolysis39.Mugihe ubushakashatsi bwibanze bwibanze ku ruhare rwa catabolisme ya tryptophan mu kugenzura ubudahangarwa bw'umubiri, twashatse gusobanura imikoranire hagati ya dysptulation tryptophan n'inzira ya glycolitike yagaragaye muri ubu bushakashatsi.Umuryango utwara abagenzi 7 abanyamuryango 5 (SLC7A5) uzwiho kuba utwara tripitofani 43,44,45.Acide Quinolinic fosiforiibosyltransferase (QPRT) ni enzyme iherereye hepfo yumuhanda wa kynurenine uhindura aside ya cinoline na NAMN46.Ubugenzuzi bwakozwe na LUAD TCGA dataset bwerekanye ko SLC7A5 na QPRT byombi byagenzuwe cyane mubice byibyimba ugereranije nuduce dusanzwe (Ishusho 5e).Ubu bwiyongere bwagaragaye mu cyiciro cya I na II kimwe no mu cyiciro cya III n'icya IV bya adenocarcinoma y'ibihaha (Ishusho y'inyongera 11), byerekana imvururu hakiri kare muri metabolism ya tryptophan ifitanye isano na tumorigenez.
Byongeye kandi, dataset ya LUAD-TCGA yerekanye isano iri hagati ya SLC7A5 na GAPDH mRNA imvugo yabarwayi ba kanseri (r = 0.45, p = 1.55E-26, Ishusho 5f).Ibinyuranye, nta sano rikomeye ryabonetse hagati yimikono ya gene mubice bisanzwe by ibihaha (r = 0,25, p = 0.06, Ishusho 5f).Gukomanga kwa SLC7A5 (Ishusho yinyongera 12) muri selile A549 byagabanije cyane urwego rwa tripitofani ya selile na NAD (H) (Ishusho 5g, h), bituma ibikorwa bya glycolitike byiyongera nkuko byapimwe nigipimo cya aside irike idasanzwe (ECAR) (Ishusho 1).5i).Rero, dushingiye kumihindagurikire ya metabolike muri serumu no muri vitro detection, turakeka ko metabolism ya tryptophan ishobora kubyara NAD + binyuze munzira ya kynurenine kandi ikagira uruhare runini muguteza glycolise muri kanseri yibihaha.
Ubushakashatsi bwerekanye ko umubare munini wa nodules zidasobanutse neza zagaragaye na LDCT zishobora gutuma hakenerwa ubundi bushakashatsi nka PET-CT, ibihaha biopsy, hamwe no gukabya bitewe no gusuzuma ibinyoma-bibi byerekana indwara mbi.31 Nkuko bigaragara ku gishushanyo cya 6, ubushakashatsi bwacu bwerekanye itsinda rya serumu metabolite ifite agaciro gasuzumwa gashobora kunoza ibyiciro ndetse no gucunga neza imitsi yimitsi yagaragaye na CT.
Indwara y'ibihaha isuzumwa hifashishijwe ibipimo bike bya tomografiya (LDCT) hamwe n'ibiranga amashusho byerekana impamvu nziza cyangwa mbi.Ingaruka zitazwi za nodules zirashobora gutuma umuntu akurikiranwa kenshi, gutabarwa bitari ngombwa, no gukabya.Kwinjizamo serumu metabolike itondekanya bifite agaciro ko kwisuzumisha birashobora kunoza isuzuma ryibyago hamwe nubuyobozi bukurikira bwimitsi ihaha.PET positron yoherejwe na tomografiya.
Imibare yavuye mu bushakashatsi bwakozwe na Amerika NLST hamwe n’ubushakashatsi bw’ibihugu by’i Burayi NELSON yerekana ko gusuzuma amatsinda afite ibyago byinshi hamwe na tomografiya nkeya (LDCT) bishobora kugabanya impfu za kanseri y'ibihaha1,3.Nyamara, gusuzuma ibyago hamwe nubuyobozi bukurikiraho bwo kuvura umubare munini wimpyiko zimpanuka zagaragaye na LDCT ziracyari ingorabahizi.Intego nyamukuru nuguhindura ibyiciro byukuri bya LDCT ishingiye kuri protocole ushyiramo biomarkers yizewe.
Bimwe mubimenyetso bya biomarkers, nka metabolite yamaraso, byagaragaye mugereranya kanseri yibihaha hamwe nubuzima bwiza15,17.Mu bushakashatsi burimo, twibanze ku gushyira mu bikorwa isesengura rya metabolomics ya serumu kugirango tumenye itandukaniro ryiza na malignant pulmonary nodules byagaragaye na LDCT.Twagereranije serumu metabolome kwisi yose igenzura neza (HC), nodules nziza yibihaha (BN), nicyiciro cya I ibihaha adenocarcinoma (LA) dukoresheje isesengura rya UPLC-HRMS.Twasanze HC na BN bafite imyirondoro ya metabolike isa, mugihe LA yerekanye impinduka zikomeye ugereranije na HC na BN.Twabonye ibice bibiri bya serumu metabolite itandukanya LA na HC na BN.
Gahunda ya LDCT ishingiye kuri gahunda ya nodules nziza kandi mbi cyane ishingiye ku bunini, ubucucike, morphologie no kwiyongera kwa node mu gihe30.Ubushakashatsi bwibanze bwerekanye ko ingano ya nodules ifitanye isano rya bugufi na kanseri y'ibihaha.Ndetse no mu barwayi bafite ibyago byinshi, ibyago byo kurwara nabi muri node <6 mm ni <1%.Ibyago byo kurwara nabi kuri nodules bipima mm 6 kugeza kuri 20 biri hagati ya 8% na 64% 30.Kubwibyo, Umuryango wa Fleischner urasaba kugabanya diameter ya mm 6 kugirango CT ikurikirane.29 Nyamara, gusuzuma ibyago no gucunga neza imitsi itamenyekana (IPN) irenze mm 6 ntabwo byakozwe bihagije 31.Ubu buryo bwo kuvura indwara z'umutima zavutse mubisanzwe bishingiye kubitegereza neza hamwe no gukurikirana CT kenshi.
Dushingiye kuri metabolome yemewe, twerekanye kunshuro yambere guhuza umukono wa metabolomic hagati yabantu bafite ubuzima bwiza na nodules nziza <6 mm.Guhuza ibinyabuzima bihuye nubushakashatsi bwa CT bwabanje kwerekana ko ibyago byo kurwara nabi kuri node <6 mm biri hasi nkibisanzwe bitagira aho bihurira.30 Twabibutsa ko ibisubizo byacu byerekana kandi ko nodules nziza <6 mm na mm6 mm zifite hejuru guhuza imyirondoro ya metabolomic, byerekana ko ibisobanuro byimikorere ya benig etiologiya bihoraho hatitawe ku bunini bwa nodule.Niyo mpamvu, isuzuma rya kijyambere rya serumu metabolite rishobora gutanga igisubizo kimwe nkikizamini kidasuzumwa mugihe nodules yabanje kugaragara kuri CT kandi birashobora kugabanya gukurikirana ikurikiranwa.Muri icyo gihe, akanama kamwe ka biomarkers ya metabolike yatandukanije nodules mbi ≥6 mm z'ubunini na nodules nziza kandi itanga ubuhanuzi nyabwo kuri IPN zifite ubunini busa nibiranga morfologiya bidasobanutse kumashusho ya CT.Iyi serumu metabolism classifier yakoze neza muguhishurira ububi bwa nodules mm6 mm hamwe na AUC ya 0.927.Dufatiye hamwe, ibisubizo byacu byerekana ko imikono idasanzwe ya serumu metabolomic ishobora kwerekana cyane cyane impinduka ziterwa na metabolike hakiri kare kandi bifite agaciro nkibishobora guhanura ingaruka, bititaye ku bunini bwa node.
Ikigaragara ni uko adenocarcinoma y'ibihaha (LUAD) na kanseri y'udukoko twa kanseri (LUSC) ari ubwoko nyamukuru bwa kanseri y'ibihaha itari ntoya (NSCLC).Urebye ko LUSC ifitanye isano cyane no gukoresha itabi47 kandi LUAD nizo zikunze kugaragara cyane mumateka yibihaha yibihaha byagaragaye kuri CT kwerekanwa48, icyitegererezo cyacu cyubatswe kubwicyiciro cya I adenocarcinoma.Wang na bagenzi be bibanze kandi kuri LUAD kandi berekana imikono icyenda ya lipide ikoresheje lipidomique kugirango itandukanye kanseri yibihaha hakiri kare nabantu bafite ubuzima bwiza17.Twagerageje kwerekana ibyiciro byerekana ibyiciro 16 byicyiciro cya I LUSC na node nziza 74 kandi twarebye neza LUSC yo guhanura neza (AUC 0.776), byerekana ko LUAD na LUSC bashobora kuba bafite umukono wa metabolomic.Mubyukuri, LUAD na LUSC byagaragaye ko bitandukanye muri etiologiya, inkomoko y'ibinyabuzima na aberrations 49.Kubwibyo, ubundi bwoko bwamateka bugomba gushyirwa muburyo bwo guhugura abaturage bashingiye kuri kanseri y'ibihaha muri gahunda yo gusuzuma.
Hano, twabonye inzira esheshatu zahinduwe cyane mubihaha adenocarcinoma ugereranije no kugenzura neza hamwe na nodules nziza.Xanthine na hypoxanthine ni metabolite isanzwe yinzira ya metabolike.Dukurikije ibisubizo byacu, abahuza bifitanye isano na metabolisme ya purine bariyongereye cyane muri serumu cyangwa ingirangingo z'abarwayi bafite ibihaha adenocarcinoma ugereranije no kugenzura neza cyangwa abarwayi ku cyiciro kibanziriza 15,50.Urwego rwo hejuru rwa serumu xanthine na hypoxanthine rushobora kwerekana anabolism isabwa na selile ya kanseri ikwirakwira vuba.Kugabanya metabolisme ya glucose nikintu kizwi cyane kiranga kanseri ya metabolisme 51.Hano, twabonye ubwiyongere bukabije bwa pyruvate na lactate mu itsinda rya LA ugereranije nitsinda rya HC na BN, ibyo bikaba bihuye na raporo zabanjirije iyi zerekana inzira ya glycolytike idasanzwe muri serumu metabolome yerekana abarwayi ba kanseri y'ibihaha itari ntoya (NSCLC) na kugenzura neza.ibisubizo birahuye 52,53.
Icy'ingenzi, twabonye isano iri hagati ya pyruvate na tryptophan metabolism muri serumu ya adenocarcinoma y'ibihaha.Urwego rwa tryptophan rwaragabanutse mu itsinda rya LA ugereranije nitsinda rya HC cyangwa BN.Igishimishije, ubushakashatsi bunini bwakoresheje ubushakashatsi bwerekanye ko urwego ruto rwa tripitofani ruzenguruka rwatewe no kwiyongera kwa kanseri y'ibihaha 54.Tryptophan ni aside amine yingenzi tubona rwose mubiryo.Twanzuye ko kugabanuka kwa serumu tryptophan mu bihaha adenocarcinoma bishobora kwerekana kugabanuka vuba kwa metabolite.Birazwi neza ko ibicuruzwa byanyuma bya tryptophan catabolism binyuze munzira ya kynurenine nisoko ya de novo NAD + synthesis.Kuberako NAD + ikorwa cyane cyane binyuze munzira yo gukiza, akamaro ka NAD + muri metabolism ya tryptophan mubuzima nuburwayi biracyamenyekana46.Isesengura ry’ububiko bwa TCGA ryerekanye ko imvugo y’abatwara tryptophan itwara abantu 7A5 (SLC7A5) yariyongereye cyane mu bihaha adenocarcinoma ugereranije n’ubugenzuzi busanzwe kandi byari bifitanye isano neza n’imvugo ya glycoltique enzyme GAPDH.Ubushakashatsi bwibanze bwibanze ahanini ku ruhare rwa tryptophan catabolism mu guhashya ubudahangarwa bw'umubiri 40,41,42.Hano turerekana ko kubuza gufata tryptophan ukomanga kuri SLC7A5 mu ngirangingo za kanseri y'ibihaha bituma igabanuka rya selile ya selile NAD hamwe no guhuza ibikorwa bya glycolitike.Muri make, ubushakashatsi bwacu butanga ishingiro ryibinyabuzima byimpinduka za serumu metabolism zijyanye no guhindura nabi ibihaha adenocarcinoma.
Ihinduka rya EGFR ni ihinduka rikomeye ry’abashoferi ku barwayi bafite NSCLC.Mu bushakashatsi bwacu, twasanze abarwayi bafite mutation ya EGFR (n = 41) bafite imiterere rusange ya metabolomic isa n’abarwayi bafite ubwoko bwa EGFR bwo mu gasozi (n = 31), nubwo twasanze igabanuka rya serumu ya bamwe mu barwayi ba mutant ba EGFR ku barwayi ba acylcarnitine.Igikorwa cyashyizweho na acylcarnitine ni ugutwara amatsinda ya acyl avuye muri cytoplazme muri matrise ya mitochondrial, biganisha kuri okiside ya aside irike kugirango itange ingufu 55.Dukurikije ibyo twabonye, ubushakashatsi buherutse kandi bwagaragaje imiterere isa na metabolome hagati ya mutant ya EGFR na EGFR yibibyimba byo mu gasozi hifashishijwe isesengura rya metabolome ku isi ya 102 y'ibihaha adenocarcinoma.Igishimishije, ibirimo acylcarnitine nabyo byabonetse mumatsinda ya mutant ya EGFR.Kubwibyo, niba impinduka murwego rwa acylcarnitine zigaragaza impinduka za EGFR ziterwa na metabolike hamwe ninzira ya molekile yinzira irashobora gukomeza ubushakashatsi.
Mu gusoza, ubushakashatsi bwacu bushiraho serumu metabolic classifier kugirango isuzume itandukaniro rya nodules yimpyiko kandi itanga igitekerezo cyakazi gishobora kunoza isuzuma ryibyago no koroshya imiyoborere yubuvuzi bushingiye kuri CT scan.
Ubu bushakashatsi bwemejwe na komite ishinzwe imyitwarire y’ibitaro bya Kaminuza bya Sun Yat-sen, ibitaro bya mbere bishamikiye kuri kaminuza ya Sun Yat-sen, na komite ishinzwe imyitwarire y’ibitaro bya kanseri bya kaminuza ya Zhengzhou.Mu matsinda yo kuvumbura no kwemeza imbere, sera 174 zabantu bafite ubuzima bwiza na 244 sera ziva muri nodules zegeranijwe ku bantu bakora ibizamini by’ubuvuzi buri mwaka mu ishami rishinzwe kurwanya no gukumira kanseri, ikigo cya kanseri cya kaminuza ya Sun Yat-sen, na node 166.serumu.Icyiciro cya I ibihaha adenocarcinoma yakusanyirijwe mu kigo cya kanseri ya kaminuza ya Sun Yat-sen.Muri cohort yo kwemeza hanze, habaye ibibazo 48 bya nodules nziza, 39 byicyiciro cya I ibihaha adenocarcinoma yo mu bitaro bya mbere bishamikiye kuri kaminuza ya Sun Yat-sen, naho 24 byo mu cyiciro cya I ibihaha I adenocarcinoma yo mu bitaro bya Kanseri ya Zhengzhou.Ikigo cya Kanseri ya Sun Yat-sen cyakusanyije kandi ibibazo 16 byo mu cyiciro cya mbere cya kanseri y'ibihaha ya kanseri y'ibihaha kugira ngo isuzume ubushobozi bwo gusuzuma imiterere ya metabolike yashyizweho (ibiranga abarwayi bigaragara mu mbonerahamwe ya 5).Ingero zivuye mu itsinda ryavumbuwe hamwe n’itsinda ry’imbere mu gihugu zegeranijwe hagati ya Mutarama 2018 na Gicurasi 2020. Ingero z’itsinda ryemewe ryo hanze zegeranijwe hagati ya Kanama 2021 na Ukwakira 2022. Kugira ngo hagabanuke ihohoterwa rishingiye ku gitsina, umubare w’imanza zingana n’abagabo n’abagore wahawe buri wese cohort.Ikipe yo Kuvumbura hamwe nitsinda ryimbere.Uburinganire bwabitabiriye bwagenwe hashingiwe kuri raporo yonyine.Uruhushya rubimenyeshejwe rwabonetse kubitabiriye amahugurwa bose kandi nta ndishyi zatanzwe.Ibintu bifite nodules nziza ni abafite amanota meza ya CT scan kumyaka 2 kugeza kuri 5 mugihe cyo gusesengura, usibye ikibazo 1 cyatanzwe nicyitegererezo cyo kwemeza hanze, cyakusanyirijwe mbere kandi kigasuzumwa na histopathology.Usibye bronchite idakira.Indwara y'ibihaha adenocarcinoma yakusanyirijwe mbere yo kwanga ibihaha kandi byemejwe no gusuzuma indwara.Amaraso yisonzesha yakusanyirijwe mumiyoboro yo gutandukanya serumu nta anticoagulants.Ingero zamaraso zashyizwe kumasaha 1 mubushyuhe bwicyumba hanyuma zishyirwa kuri 2851 × g muminota 10 kuri 4 ° C kugirango zegerane serumu ndengakamere.Serumu alikoti yahagaritswe kuri -80 ° C kugeza ikuwemo metabolite.Ishami rishinzwe gukumira kanseri no gusuzuma ubuvuzi ikigo cya kanseri cya kaminuza ya Yat Yat sen yakusanyije pisine ya serumu ku baterankunga 100 bafite ubuzima bwiza, harimo umubare w’abagabo n’abagore bangana kuva ku myaka 40 kugeza 55.Ingano ingana ya buri cyitegererezo cyabaterankunga yaravanze, pisine yavuyemo yarayibitswe kandi ibikwa kuri -80 ° C.Serumu ivanze yakoreshejwe nkibikoresho byo kugenzura ubuziranenge no guhuza amakuru.
Serumu yerekana hamwe nicyitegererezo cyakuweho kandi metabolite yakuwe hakoreshejwe uburyo bwo kuvoma hamwe (MTBE / methanol / amazi) 56.Muri make, 50 μl za serumu zavanze na 225 μl za methanol ikonje cyane na 750 μl ya methyl tert-butyl ether (MTBE).Koresha imvange hanyuma ushire kurubura kumasaha 1.Ingero zavanze hanyuma vortex ivangwa na 188 μl y'amazi yo mu rwego rwa MS arimo ibipimo by'imbere (13C-lactate, 13C3-pyruvate, 13C-methionine, na 13C6-isoleucine, yaguzwe muri Laboratoire ya Cambridge Isotope).Uruvange rwahise rushyirwa kuri 15,000 × g muminota 10 kuri 4 ° C, hanyuma icyiciro cyo hasi cyimurirwa mumiyoboro ibiri (125 μL buri) kugirango LC-MS isesengurwe muburyo bwiza kandi bubi.Hanyuma, icyitegererezo cyahinduwe kugirango cyumuke mumashanyarazi yihuta.
Metabolite yumye yongeye gushyirwaho muri 120 μl ya 80% ya acetonitrile, ikazunguruka iminota 5, hanyuma igashyirwa kuri 15,000 × g mu minota 10 kuri 4 ° C.Supernatants yimuriwe mumashanyarazi ya amber hamwe na microinserts yo kwiga metabolomics.Isesengura rya metabolomics idateganijwe kuri ultra-imikorere yamazi ya chromatografiya-ihanitse cyane ya mass spectrometrie (UPLC-HRMS).Metabolite yatandukanijwe ikoresheje Dionex Ultimate 3000 UPLC na ACQUITY BEH Amide inkingi (2,1 × 100 mm, 1,7 mm, Amazi).Muburyo bwiza bwa ion, ibyiciro bigendanwa byari 95% (A) na 50% acetonitrile (B), buri kimwe kirimo acetate ya mmol / L 10 na acide 0.1%.Muburyo bubi, icyiciro kigendanwa A na B cyarimo 95% na 50% acetonitrile, kimwe, ibyiciro byombi birimo acetate ya mmol / L 10 ya ammonium, pH = 9. Gahunda ya gradient yari iteye itya: 0–0.5 min, 2% B;0.5-12 min, 2-50% B;Iminota 12–14, 50-98% B;Iminota 14-16, 98% B;16–16.1.min, 98–2% B;16.1–20 min, 2% B. Inkingi yagumishijwe kuri 40 ° C hamwe nicyitegererezo kuri 10 ° C muri autosampler.Igipimo cyo gutembera cyari 0.3 ml / min, ingano yo gutera inshinge 3 μl.Q-Ikora Orbitrap mass spectrometer (Thermo Fisher Scientific) hamwe na electrospray ionisation (ESI) yakoreshwaga muburyo bwuzuye bwo gusikana kandi ihujwe nuburyo bwo gukurikirana ddMS2 kugirango ikusanyirize hamwe amakuru menshi.Ibipimo bya MS byashyizweho kuburyo bukurikira: spray voltage +3.8 kV / - 3,2 kV, ubushyuhe bwa capillary 320 ° C, gukingira gaze 40 arb, gaze yingoboka 10 arb, ubushyuhe bwa probe ubushyuhe 350 ° C, gusikana 70-1050 m / h, imyanzuro.70 000. Amakuru yabonetse hakoreshejwe Xcalibur 4.1 (Thermo Fisher Scientific).
Kugirango hamenyekane ubuziranenge bwamakuru, icyitegererezo cyiza (QC) cyakozwe mugukuraho 10 μL aliquots ya supernatant kuri buri cyitegererezo.Inshinge esheshatu zo kugenzura ubuziranenge bwasesenguwe mu ntangiriro yurukurikirane rwisesengura kugirango hamenyekane ituze rya sisitemu ya UPLC-MS.Ingero zo kugenzura ubuziranenge noneho zinjizwa mugihe runaka.Ibyiciro 11 byose bya serumu muri ubu bushakashatsi byasesenguwe na LC-MS.Aliquots ya serumu ivanze nabaterankunga 100 bazima yakoreshejwe nkibikoresho byerekanwe mubice bitandukanye kugirango ikurikirane uburyo bwo kuyikuramo no guhindura ingaruka zicyiciro.Isesengura rya metabolomics ridafite intego yo kuvumbura ibice, kwemeza imbere, hamwe no kwemeza hanze byakorewe mu kigo cya Metabolomics Centre ya Sun Yat-sen University.Laboratoire yo hanze ya Guangdong University of Technology Analysis and Testing Centre nayo yasesenguye ingero 40 ziva mu itsinda ryo hanze kugirango isuzume imikorere yicyitegererezo.
Nyuma yo gukuramo no kongera kwiyubaka, ibipimo byuzuye bya serumu metabolite byapimwe hifashishijwe ultra-high performance fluid chromatography-tandem mass spectrometrie (Agilent 6495 triple quadrupole) hamwe na electrospray ionisation (ESI) muburyo bwinshi bwo gukurikirana (MRM).AKARERE KA BEH Hagati yinkingi (2,1 × 100 mm, 1,7 mm, Amazi) yakoreshejwe mugutandukanya metabolite.Icyiciro kigendanwa cyari kigizwe na 90% (A) na 5% acetonitrile (B) hamwe na acetate ya mmol / L 10 na ammonium 0.1%.Gahunda ya gradient yari iteye itya: 0-1.5 min, 0% B;1.5-6.5 min, 0-15% B;6.5-8 min, 15% B;8-8,5 min, 15% –0% B;8.5–11.5 min, 0% B.Inkingi yagumishijwe kuri 40 ° C hamwe nicyitegererezo kuri 10 ° C muri autosampler.Igipimo cyo gutembera cyari 0.3 mL / min naho inshinge zatewe ni 1 μL.Ibipimo bya MS byashyizweho kuburyo bukurikira: capillary voltage ± 3,5 kV, umuvuduko wa nebulizer 35 psi, gazi ya sheath 12 L / min, ubushyuhe bwa gaze ya sheath 350 ° C, kumisha ubushyuhe bwa gaze 250 ° C, no kumisha gaze 14 l / min.Ihinduka rya MRM rya tryptophan, pyruvate, lactate, hypoxanthine na xanthine byari 205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 na 151.0-107.9.Amakuru yakusanyijwe hakoreshejwe Mass Hunter B.07.00 (Agilent Technologies).Kuburugero rwa serumu, tryptophan, pyruvate, lactate, hypoxanthine, na xanthine byapimwe hakoreshejwe kalibrasi yumurongo wibisubizo bisanzwe bivangwa.Kuburugero rwakagari, ibintu bya tryptophan byari bisanzwe mubisanzwe imbere hamwe na proteine selile.
Gukuramo impinga (m / z nigihe cyo kugumana (RT)) byakozwe hakoreshejwe Compound Discovery 3.1 na TraceFinder 4.0 (Thermo Fisher Scientific).Kugira ngo ukureho itandukaniro rishobora kuba hagati yitsinda, buri mpinga iranga ikigereranyo cyikigereranyo yagabanijwe nimpinga iranga ibikoresho byerekanwe kuva murwego rumwe kugirango ubone ubwinshi ugereranije.Kugereranya gutandukana kurwego rwimbere mbere na nyuma yubuziranenge byerekanwe mumbonerahamwe yinyongera 6. Itandukaniro riri hagati yaya matsinda yombi ryaranzwe nigipimo cyo kuvumbura ibinyoma (FDR <0.05, Wilcoxon yasinywe ikizamini cyurwego) hamwe no guhindura inshuro (> 1.2 cyangwa <0.83).Amakuru ya MS yibintu byakuweho hamwe na serumu yakosowe na MS yerekanwa muri Data wongeyeho 1 hamwe namakuru yinyongera 2.Peak annotation yakozwe hashingiwe ku nzego enye zasobanuwe zo kumenyekanisha, harimo metabolite yamenyekanye, ibivugwa mu buryo bwanditse, ibyiciro biranga ibyiciro, hamwe n’ibintu bitazwi 22.Ukurikije ubushakashatsi bwakozwe mububiko bwavumbuwe 3.1 (mzCloud, HMDB, Chemspider), ibinyabuzima hamwe na MS / MS bihuye nibipimo byemewe cyangwa ibisobanuro bihuye neza muri mzCloud (amanota> 85) cyangwa Chemspider amaherezo byatoranijwe nkumuhuza hagati ya metabolome itandukanye.Impanuro yibisobanuro kuri buri kintu cyashyizwe mumibare yinyongera 3. MetaboAnalyst 5.0 yakoreshejwe mugusesengura rimwe ryumubare-usanzwe wa metabolite ubwinshi.MetaboAsesengura 5.0 yanasuzumye isesengura ryitunganyirizwa rya KEGG rishingiye kuri metabolite zitandukanye.Isesengura ryibanze ryibanze (PCA) hamwe nisesengura ryibice byibuze kwaduka (PLS-DA) byasesenguwe hifashishijwe porogaramu ya ropls (v.1.26.4) hamwe nibisanzwe hamwe na autoscaling.Uburyo bwiza bwa metabolite biomarker yo guhanura ububi bwa nodule bwakozwe hakoreshejwe binary logistic regression hamwe byibuze kugabanuka no guhitamo (LASSO, R pack v.4.1-3).Imikorere yicyitegererezo kivangura mugushakisha no kwemeza yaranzwe no kugereranya AUC ishingiye ku isesengura rya ROC ukurikije pROC (v.1.18.0.).Uburyo bwiza bwo kugabanuka bwabonetse hashingiwe ku gipimo ntarengwa cya Youden cyerekana icyitegererezo (sensitivite + yihariye - 1).Ingero zifite agaciro gake cyangwa karenze urubibi zizahanurwa nka nodules nziza na adenocarcinoma y'ibihaha.
Ingirabuzimafatizo A549 (# CCL-185, Icyegeranyo cy’umuco w’Abanyamerika) cyakuze mu buryo bwa F-12K burimo 10% FBS.Imisatsi migufi RNA (shRNA) ikurikiranye yibasiye SLC7A5 hamwe nigenzura ridahwitse (NC) byinjijwe mumashanyarazi ya lentiviral pLKO.1-puro.Urutonde rwa antisense ya shSLC7A5 nuburyo bukurikira: Sh1 (5′-GGAGAAACCTGATGAACAGTT-3 ′), Sh2 (5′-GCCGTGGACTTCGGGAACTAT-3 ′).Antibodies kuri SLC7A5 (# 5347) na tubuline (# 2148) zaguzwe muri tekinoroji yerekana ibimenyetso.Antibodies kuri SLC7A5 na tubuline byakoreshejwe muguhindura 1: 1000 kugirango isesengura ryiburengerazuba.
Ikizamini cya Seahorse XF Glycolytic Stress gipima aside irike idasanzwe (ECAR).Mu isuzuma, glucose, oligomycine A, na 2-DG byatanzwe bikurikiranye kugirango bipime ubushobozi bwa selile glycolitike nkuko byapimwe na ECAR.
Utugingo ngengabuzima A549 twandujwe no kugenzura tutagamije (NC) na shSLC7A5 (Sh1, Sh2) twaraye dushyizwe hamwe mu masahani ya cm 10.Metabolite selile yakuwe hamwe na ml 1 yubukonje bwa metani 80%.Ingirabuzimafatizo zo mu gisubizo cya methanol zarakuweho, zegeranijwe mu muyoboro mushya, hanyuma zishyirwa kuri 15.000 × g mu minota 15 kuri 4 ° C.Kusanya 800 µl yindengakamere kandi yumutse ukoresheje umuvuduko mwinshi wa vacuum.Pellette yumye yumye noneho yasesenguwe kurwego rwa tripitofani ukoresheje LC-MS / MS nkuko byasobanuwe haruguru.Urwego rwa selile NAD (H) muri selile A549 (NC na shSLC7A5) rwapimwe hifashishijwe ibikoresho bya NAD + / NADH ibara ryinshi (# K337, BioVision) ukurikije amabwiriza yabakozwe.Urwego rwa poroteyine rwapimwe kuri buri cyitegererezo kugirango rusanzwe rusanzwe rwa metabolite.
Nta buryo bwibarurishamibare bwakoreshejwe kugirango hamenyekane mbere ingano yubunini.Ubushakashatsi bwa metabolomics bwambere bugamije kuvumbura biomarker15,18 byafashwe nkibipimo ngenderwaho mu kugena ingano, kandi ugereranije nizi raporo, icyitegererezo cyacu cyari gihagije.Nta byitegererezo byakuwe mu itsinda ry’inyigisho.Ingero za serumu zahawe itsinda ryivumbuwe (imanza 306, 74,6%) hamwe nitsinda ryimbere ryimbere (imanza 104, 25.4%) kubushakashatsi bwa metabolomics butateganijwe.Twahisemo kandi gutoranya imanza 70 muri buri tsinda uhereye kubuvumbuzi bwashyizweho kubushakashatsi bwa metabolomics.Abashakashatsi bahumye amaso imirimo yo mu matsinda mugihe cyo gukusanya no gusesengura amakuru ya LC-MS.Isesengura mibare yamakuru ya metabolomics hamwe nubushakashatsi bwakagari byasobanuwe mubisubizo bijyanye, imigani y'ibishushanyo, hamwe nuburyo bukoreshwa.Umubare wibikorwa bya selile tryptophan, NADT, na glycolitike byakozwe inshuro eshatu wigenga hamwe nibisubizo bimwe.
Kubindi bisobanuro bijyanye nigishushanyo mbonera cyo kwiga, reba Raporo Kamere ya Portfolio Raporo ijyanye niyi ngingo.
Amakuru yibanze ya MS yibintu byakuweho hamwe nibisanzwe MS yamakuru ya serumu yerekanwe byerekanwe mumibare yinyongera 1 hamwe namakuru yinyongera 2.Impanuro yibisobanuro kubintu bitandukanye itangwa mumibare yinyongera 3. Dataset ya LUAD TCGA irashobora gukurwa kuri https://portal.gdc.cancer.gov/.Iyinjiza ryamakuru yo gutegura igishushanyo gitangwa mumasoko yamakuru.Inkomoko yamakuru yatanzwe kuriyi ngingo.
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Igihe cyo kohereza: Nzeri-18-2023