Ukuxilongwa okuhlukile kwama-nodules wamaphaphu ahlonzwe nge-computed tomography (CT) kuseyinselele ekusebenzeni komtholampilo.Lapha, sibonisa i-metabolome yomhlaba wonke yamasampula e-serum angu-480, okuhlanganisa izilawuli ezinempilo, izigaxana zamaphaphu amahle, kanye nesiteji I lung adenocarcinoma.I-Adenocarcinomas ibonisa amaphrofayili e-metabolomic ahlukile, kuyilapho ama-nodule angenayo kanye nabantu abanempilo banokufana okuphezulu kumaphrofayili we-metabolomic.Eqenjini lokuthola (n = 306), isethi yama-metabolite angu-27 ikhonjwe ukuze kuhlukaniswe phakathi kwama-nodule ayingozi namabi.I-AUC yemodeli ebandlululayo ekuqinisekiseni kwangaphakathi (n = 104) kanye namaqembu okuqinisekisa kwangaphandle (n = 111) yayingu-0.915 kanye no-0.945, ngokulandelanayo.Ukuhlaziywa kwendlela kwembule ukukhuphuka kwama-metabolites e-glycolytic ahlobene nokuncipha kwe-tryptophan ku-serum yamaphaphu adenocarcinoma uma kuqhathaniswa namaqhuqhuva angenabungozi kanye nokulawula okunempilo, futhi kwaphakamisa ukuthi ukuthatha i-tryptophan kuthuthukisa i-glycolysis kumaseli omdlavuza wamaphaphu.Ucwaningo lwethu luqokomisa ukubaluleka kwe-serum metabolite biomarkers ekuhloleni ingozi yama-nodule yamaphaphu atholwe yi-CT.
Ukuxilongwa kusenesikhathi kubalulekile ukuze kuthuthukiswe amazinga okusinda ezigulini ezinomdlavuza.Imiphumela evela ku-US National Lung Cancer Screening Trial (NLST) kanye ne-European NELSON Study ibonise ukuthi ukuhlolwa nge-dose ephansi ye-computed tomography (LDCT) kunganciphisa kakhulu ukufa komdlavuza wamaphaphu emaqenjini asengozini enkulu1,2,3.Kusukela ekusetshenzisweni okubanzi kwe-LDCT ekuhloleni umdlavuza wamaphaphu, izehlakalo zokutholwa kwe-radiographic ngengozi ye-asymptomatic pulmonary nodules ziye zaqhubeka zanda i-4.Ama-nodule amaphaphu achazwa ngokuthi ama-focal opacities afinyelela ku-3 cm ububanzi obungu-5.Sibhekene nobunzima ekuhloleni amathuba okuba nesifo esiyingozi kanye nokubhekana nenani elikhulu lamaqhuqhuva wamaphaphu atholwe ngengozi ku-LDCT.Imikhawulo ye-CT ingaholela ekuhlolweni kokulandelela njalo kanye nemiphumela emihle engamanga, okuholela ekungeneleleni okungadingekile kanye nokwelashwa ngokweqile6.Ngakho-ke, kunesidingo sokuthuthukisa ama-biomarker anokwethenjelwa futhi awusizo ukuze abone kahle umdlavuza wamaphaphu ezigabeni zokuqala futhi ahlukanise ama-nodule amaningi angenabungozi ekutholweni kokuqala kwe-7.
Ukuhlaziywa okubanzi kwamangqamuzana egazi (i-serum, i-plasma, i-peripheral blood mononuclear cell), okuhlanganisa i-genomics, i-proteomics noma i-DNA methylation8,9,10, kuholele ekukhuleni kwesithakazelo ekutholakaleni kwezimpawu zokuxilonga zomdlavuza wamaphaphu.Phakathi naleso sikhathi, izindlela ze-metabolomics zikala imikhiqizo yokugcina yamaselula ethonywa izenzo ezingapheli kanye nezangaphandle futhi ngenxa yalokho zisetshenziselwa ukubikezela ukuqala kwesifo nomphumela.I-Liquid chromatography-tandem mass spectrometry (LC-MS) iyindlela esetshenziswa kabanzi yocwaningo lwe-metabolomics ngenxa yokuzwela kwayo okuphezulu kanye nobubanzi obukhulu obuguquguqukayo, obungakwazi ukumboza ama-metabolites anezakhiwo ezahlukene ze-physicochemical11,12,13.Nakuba ukuhlaziywa kwe-metabolomic yomhlaba wonke kwe-plasma/serum kusetshenziselwe ukukhomba ama-biomarker ahlotshaniswa nokuxilongwa komdlavuza wamaphaphu14,15,16,17 kanye nempumelelo yokwelapha, i-18 serum metabolite classifiers ukuhlukanisa phakathi kwezigaxana zamaphaphu eziyingozi neziyingozi kusazofundwa kakhulu.-ucwaningo olukhulu.
I-Adenocarcinoma kanye ne-squamous cell carcinoma yizinhlobo ezimbili eziyinhloko zomdlavuza wamaphaphu ongewona omncane (NSCLC).Ukuhlola okuhlukahlukene kwe-CT kubonisa ukuthi i-adenocarcinoma iwuhlobo oluvame kakhulu lwe-histological yomdlavuza wamaphaphu1,19,20,21.Kulolu cwaningo, sisebenzise i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) ukuze senze ukuhlaziywa kwe-metabolomics enanini lamasampula e-serum angu-695, okuhlanganisa ukulawula okunempilo, ama-benign pulmonary nodules, kanye ne-CT-etholwe ≤3 cm.Ukuhlolwa kwe-Stage I lung adenocarcinoma.Sihlonze iphaneli yama-metabolites e-serum ahlukanisa i-adenocarcinoma yamaphaphu kumaqhuqhuva angenabungozi kanye nokulawula okunempilo.Ukuhlaziywa kokunothisa kwendlela kwembula ukuthi i-tryptophan engavamile kanye ne-glucose metabolism yizinguquko ezivamile ku-adenocarcinoma yamaphaphu uma kuqhathaniswa namaqhuqhuva angenabungozi kanye nokulawula okunempilo.Ekugcineni, sisungule futhi saqinisekisa isigaba se-serum metabolic esinemininingwane ephezulu kanye nokuzwela ukuze sihlukanise phakathi kwamaqhubu amaphaphu ayingozi kanye nama-benign atholwe yi-LDCT, engasiza ekuxilongeni okuhlukile kokuqala kanye nokuhlola ubungozi.
Ocwaningweni lwamanje, amasampula e-serum ahambisana nobulili neminyaka aqoqwe ngokuphindaphindiwe kusukela ekulawuleni okunempilo kwe-174, iziguli ezingu-292 ezinama-nodule ama-benign pulmonary, kanye neziguli ezingu-229 ezine-stage I lung adenocarcinoma.Izici zezibalo zezifundo ezingu-695 ziboniswa kuThebula Lokwengeza 1.
Njengoba kuboniswe kuMfanekiso 1a, isamba samasampula e-serum angu-480, okuhlanganisa nokulawula okunempilo kwe-174 (HC), ama-benign nodules (BN) angu-170 (BN), kanye namasampula angu-136 esiteji I lung adenocarcinoma (LA), aqoqwe e-Sun Yat-sen University Cancer Centre.Iqoqo le-Discovery lephrofayili ye-metabolomic engahlosiwe kusetshenziswa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Njengoba kuboniswe Emfanekisweni Owengeziwe woku-1, ama-metabolite ahlukene phakathi kwe-LA ne-HC, i-LA kanye ne-BN ahlonziwe ukuze kusungulwe imodeli yokuhlukanisa futhi kuqhutshekwe nokuhlola ukuhlaziywa kwendlela ehlukile.Amasampula angu-104 aqoqwe yi-Sun Yat-sen University Cancer Centre kanye namasampula angu-111 aqoqwe ezinye izibhedlela ezimbili agunyazwe ngaphakathi nangaphandle, ngokulandelana.
inani labantu bocwaningo kuqoqo lokutholwa elithole ukuhlaziywa kwe-serum metabolomics kusetshenziswa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).b Ukuhlaziywa okunqunyelwe kokubandlulula kwezikwele ezincane (i-PLS-DA) yesamba se-metabolome yamasampula e-serum angu-480 avela eqenjini locwaningo, okuhlanganisa nezilawuli ezinempilo (HC, n = 174), ama-benign nodule (BN, n = 170), kanye ne-stage I lung adenocarcinoma (Los Angeles, n = 136).+ESI, imodi ye-ionization ye-electrospray positive, -ESI, imodi ye-ionization ye-electrospray negative.c–e Ama-Metabolites anenala ehluke kakhulu emaqenjini amabili anikeziwe (ukuhlolwa kwezinga elisayinwe yi-Wilcoxon enemisila emibili, izinga lokutholwa okungamanga elilungisiwe inani lika-p, FDR <0.05) aboniswa ngokubomvu (ukushintsha kokugoqa > 1.2) nokuluhlaza okwesibhakabhaka (ukushintsha kokugoqa <0.83) .) kukhonjiswe emfanekisweni wentaba-mlilo.f Imephu yokushisa yeqoqo le-Hierarchical ebonisa umehluko omkhulu enanini lama-metabolites achazwe phakathi kwe-LA ne-BN.Idatha yomthombo inikezwa ngohlobo lwamafayela edatha yomthombo.
Isamba se-serum metabolome ye-174 HC, 170 BN kanye ne-136 LA eqenjini lokutholwa yahlaziywa kusetshenziswa ukuhlaziywa kwe-UPLC-HRMS.Siqala sibonisa ukuthi amasampuli okulawula ikhwalithi (i-QC) ahlangana ngokuqinile phakathi nendawo yemodeli yokuhlaziywa kwengxenye eyinhloko engagadiwe (PCA), eqinisekisa ukuzinza kokusebenza kocwaningo lwamanje (Umfanekiso Owengeziwe 2).
Njengoba kukhonjisiwe ekuhlaziyweni kokucwasa ngokwengxenye kwezikwele ezincane (PLS-DA) kuMfanekiso 1 b, sithole ukuthi kunomehluko ocacile phakathi kwe-LA ne-BN, i-LA kanye ne-HC ezindleleni ezinhle (+ESI) nezingezinhle (−ESI) ze-electrospray ionization. .eyedwa.Kodwa-ke, awukho umehluko obalulekile otholakele phakathi kwe-BN ne-HC ezimeni ze-+ESI kanye -ESI.
Sithole izici ezihlukene ezingu-382 phakathi kwe-LA ne-HC, izici ezihlukene ezingu-231 phakathi kwe-LA ne-BN, kanye nezici ezingu-95 ezihlukene phakathi kwe-BN ne-HC (ukuhlolwa kwezinga okusayiniwe kwe-Wilcoxon, i-FDR <0.05 nokushintsha okuningi >1.2 noma <0.83) (Umfanekiso .1c-e) )..Amanani aphezulu aphinde achazelwa (Idatha Eyengeziwe 3) kusizindalwazi (mzCloud/HMDB/Chemspider library) ngevelu ye-m/z, isikhathi sokugcina kanye nosesho lwe-spectrum mass mass (imininingwane echazwe esigabeni Sezindlela) 22 .Ekugcineni, i-33 kanye ne-38 yama-metabolites anezichasiselo ezinomehluko omkhulu ngobuningi ahlonziwe ku-LA ngokumelene ne-BN (Umfanekiso we-1f kanye neThebula Lokwengeza 2) kanye ne-LA ngokumelene ne-HC (I-Supplementary Figure 3 kanye ne-Supplementary Table 2), ngokulandelanayo.Ngokuphambene, ama-metabolite ama-3 kuphela anomehluko omkhulu ngobuningi ahlonzwe ku-BN ne-HC (Ithebula Lesengezo 2), ngokuhambisana nokugqagqana phakathi kwe-BN ne-HC ku-PLS-DA.Lawa ma-metabolites ahlukene ahlanganisa izinhlobonhlobo zamakhemikhali ezinto eziphilayo (Umfanekiso Owengeziwe 4).Ihlanganiswe ndawonye, le miphumela ibonisa izinguquko eziphawulekayo ku-serum metabolome ebonisa ukuguqulwa okuyingozi komdlavuza wamaphaphu wesigaba sokuqala uma kuqhathaniswa namaqhuqhuva amaphaphu ayingozi noma izifundo ezinempilo.Phakathi naleso sikhathi, ukufana kwe-serum metabolome ye-BN ne-HC kuphakamisa ukuthi ama-benign pulmonary nodules angase abelane nezici eziningi zezinto eziphilayo nabantu abanempilo.Njengoba kunikezwe ukuthi ukuguqulwa kofuzo kwe-epidermal growth factor receptor (EGFR) kuvamile ku-lung adenocarcinoma subtype 23, siye safuna ukunquma umthelela wokuguqulwa komshayeli ku-serum metabolome.Sibe sesihlaziya iphrofayili ye-metabolomic isiyonke yamacala angama-72 anesimo se-EGFR eqenjini le-adenocarcinoma yamaphaphu.Kuyathakazelisa ukuthi sithole amaphrofayili afanayo phakathi kweziguli eziguquguqukayo ze-EGFR (n = 41) kanye neziguli ze-EGFR zasendle (n = 31) ekuhlaziyweni kwe-PCA (I-Supplementary Figure 5a).Kodwa-ke, sihlonze ama-metabolites angu-7 obuningi bawo obushintshiwe kakhulu ezigulini ezine-EGFR ukuguqulwa uma kuqhathaniswa neziguli ezine-EGFR yohlobo lwasendle (t test, p <0.05 kanye noshintsho olugoqiwe> 1.2 noma <0.83) (Umfanekiso Ongeziwe 5b).Iningi lalawa ma-metabolites (ama-5 kwangu-7) angama-acylcarnitines, adlala indima ebalulekile ezindleleni ze-fatty acid oxidation.
Njengoba kubonisiwe ekuhambeni komsebenzi okuboniswe ku-Figure 2 a, ama-biomarker okuhlukaniswa kwama-nodule atholwe kusetshenziswa ama-opharetha anciphayo nokukhethwa okusekelwe kuma-metabolites angu-33 ahlukene ahlonzwe ku-LA (n = 136) kanye ne-BN (n = 170).Inhlanganisela engcono kakhulu yezinto eziguquguqukayo (i-LASSO) - imodeli yokuhlehliswa kwelogistic kanambambili.Ukuqinisekiswa okuphindwe kashumi kusetshenziswe ukuhlola ukwethembeka kwemodeli.Ukukhetha okuguquguqukayo kanye nokuhlelwa kwepharamitha kulungiswa ngenhlawulo yokwandisa okungenzeka ngepharamitha λ24.Ukuhlaziywa kwe-metabolomics yomhlaba wonke kwenziwa ngokuzimele ekuqinisekiseni kwangaphakathi (n = 104) nokuqinisekiswa kwangaphandle (n = 111) amaqembu ukuze kuhlolwe ukusebenza kwezigaba kwemodeli ebandlululayo.Ngenxa yalokho, ama-metabolites angu-27 ekutholakaleni kwesethi ahlonzwe njengemodeli engcono kakhulu yokubandlulula enenani elikhulu kakhulu le-AUC (Fig. 2b), phakathi kwayo i-9 yayinomsebenzi owandayo kanye nomsebenzi we-18 wehla ku-LA uma kuqhathaniswa ne-BN (Fig. 2c).
Ukugeleza komsebenzi wokwakha isihlukanisi samanodule wamaphaphu, okuhlanganisa ukukhetha iphaneli engcono kakhulu yama-metabolites e-serum kusethi yokutholwa kusetshenziswa imodeli yokuhlehla kokuhamba kanambambili ngokuqinisekisa okuphindwe kashumi kanye nokuhlola ukusebenza okubikezelayo kumasethi okuqinisekisa angaphakathi nangaphandle.b Izibalo zokuqinisekisa okuphambanayo zemodeli yokuhlehla ye-LASSO yokukhethwa kwe-biomarker ye-metabolic.Izinombolo ezinikezwe ngenhla zimelela inani elimaphakathi lama-biomarker akhethiwe kokuthi λ.Ulayini onamachashazi abomvu umele isilinganiso senani le-AUC ku-lambda ehambisanayo.Amabha amaphutha ampunga amelela amanani e-AUC aphansi futhi aphezulu.Ulayini onamachashazi ukhombisa imodeli engcono kakhulu ngama-biomarker angama-27 akhethiwe.I-AUC, indawo engaphansi kwejika le-receiver operating character (ROC).c Songa izinguquko zama-metabolite akhethiwe angama-27 eqenjini le-LA uma kuqhathaniswa neqembu le-BN eqenjini lokutholwa.Ikholomu ebomvu – ukwenza kusebenze.Ikholomu eluhlaza okwesibhakabhaka iwukwehla.I-d–f Receiver operating character (ROC) amajika abonisa amandla emodeli ebandlululayo esuselwe kuzinhlanganisela ze-metabolite ezingama-27 kumasethi okuqinisekisa, angaphakathi, nangaphandle.Idatha yomthombo inikezwa ngohlobo lwamafayela edatha yomthombo.
Imodeli yokubikezela idalwe ngokusekelwe kuma-coefficient wokuhlehla anesisindo walawa ma-metabolites angama-27 (Ithebula Lokwengeza 3).Ukuhlaziywa kwe-ROC okusekelwe kulawa ma-metabolites angu-27 kunikeze indawo engaphansi kwe-curve (AUC) inani le-0.933, ukuzwela kweqembu lokuthola kwakuyi-0.868, futhi ukucaciswa kwakuyi-0.859 (Fig. 2d).Phakathi naleso sikhathi, phakathi kwama-metabolite ahlukene angama-38 anezichasiselo phakathi kwe-LA ne-HC, isethi yama-metabolites ayi-16 athole i-AUC engu-0.902 ngokuzwela okungu-0.801 nokucaciswa okungu-0.856 ekubandlululeni i-LA ku-HC (Umfanekiso Owengeziwe 6a-c).Amanani e-AUC asekelwe emikhawulweni ehlukene yokuguqulwa okugoqiwe kwama-metabolites ahlukene nawo aqhathaniswa.Sithole ukuthi imodeli yokuhlukanisa yenze kangcono kakhulu ekubandlululeni phakathi kwe-LA ne-BN (HC) lapho izinga lokushintsha eligoqiwe lalibekwe ku-1.2 uma kuqhathaniswa nokungu-1.5 noma ku-2.0 (Umfanekiso Owengeziwe 7a,b).Imodeli yokuhlukanisa, esekelwe kumaqembu e-metabolite angu-27, yabuye yaqinisekiswa kumaqoqo angaphakathi nangaphandle.I-AUC yayingu-0.915 (ukuzwela okungu-0.867, ukucaciswa okungu-0.811) kokuqinisekisa kwangaphakathi kanye ne-0.945 (ukuzwela 0.810, ukucaciswa okungu-0.979) kokuqinisekisa kwangaphandle (Fig. 2e, f).Ukuze kuhlolwe ukusebenza kahle kwelabhorethri, amasampula angama-40 avela eqenjini langaphandle ahlaziywa elabhorethri yangaphandle njengoba kuchazwe esigabeni Sezindlela.Ukunemba kwezigaba kuzuze i-AUC engu-0.925 (Umfanekiso Owengeziwe 8).Ngenxa yokuthi i-lung squamous cell carcinoma (LUSC) iwuhlobo oluncane lwesibili oluvame kakhulu lomdlavuza wamaphaphu ongewona omncane (NSCLC) ngemuva kwe-lung adenocarcinoma (LUAD), siphinde sahlola ukusetshenziswa okungaba khona okuqinisekisiwe kwamaphrofayili we-metabolic.I-BN kanye namacala ayi-16 e-LUSC.I-AUC yokubandlulula phakathi kwe-LUSC ne-BN yayingu-0.776 (Umfanekiso Owengeziwe 9), okubonisa ikhono elimpofu uma liqhathaniswa nokubandlulula phakathi kwe-LUAD ne-BN.
Ucwaningo luye lwabonisa ukuthi ubukhulu bama-nodules ezithombeni ze-CT buhlotshaniswa kahle nethuba lokulimala futhi buhlala buyisicaciso esikhulu sokwelashwa kwama-nodule25,26,27.Ukuhlaziywa kwedatha evela eqenjini elikhulu lokuhlola ukuhlolwa kwe-NELSON kubonise ukuthi ingozi yokulimala ezifundweni ezinama-node <5 mm yayifana naleyo kwizifundo ezingenayo ama-node 28.Ngakho-ke, usayizi omncane odinga ukuqapha kwe-CT okuvamile ngu-5 mm, njengoba kunconywe yi-British Thoracic Society (BTS), kanye no-6 mm, njengoba kunconywe i-Fleischner Society 29.Kodwa-ke, ama-nodules amakhulu kune-6 mm futhi angenazo izici ezicacile ezicacile, ezibizwa ngokuthi ama-indeterminate pulmonary nodules (IPN), ahlala eyinselele enkulu ekuhloleni nasekulawuleni emisebenzini yomtholampilo30,31.Ngokulandelayo sihlole ukuthi ingabe usayizi wamanojula uthonye yini amasiginesha e-metabolomic kusetshenziswa amasampula ahlanganisiwe asuka ekutholakaleni nasekuqinisekiseni kwangaphakathi amaqoqo.Ngokugxila kuma-biomarker aqinisekisiwe we-27, siqale saqhathanisa amaphrofayili we-PCA we-HC kanye ne-BN sub-6 mm metabolomes.Sithole ukuthi amaphuzu amaningi wedatha we-HC ne-BN agqine, abonisa ukuthi amazinga e-serum metabolite ayefana kuwo womabili amaqembu (Fig. 3a).Amamephu wesici kuwo wonke amabanga osayizi abahlukene asale elondoloziwe ku-BN ne-LA (Umfanekiso 3b, c), kuyilapho ukuhlukaniswa kubonwe phakathi kwamaqhuqhuva ayingozi kanye nama-benig ebangeni elingu-6–20 mm (Fig. 3d).Leli qembu libe ne-AUC ye-0.927, ukucaciswa kwe-0.868, nokuzwela kwe-0.820 yokubikezela ukonakala kwama-nodules alinganisa u-6 kuya ku-20 mm (Fig. 3e, f).Imiphumela yethu ibonisa ukuthi ohlukanisa ngezigaba angakwazi ukuthwebula izinguquko ze-metabolic ezibangelwa ukuguquka okuyingozi kwangaphambi kwesikhathi, kungakhathaliseki usayizi wamaqhuqhuva.
isikhangiso Ukuqhathaniswa kwamaphrofayili e-PCA phakathi kwamaqembu acacisiwe ngokusekelwe kusigaba se-metabolic sama-metabolites angama-27.CC kanye ne-BN <6 mm.b BN < 6 mm vs BN 6–20 mm.ku-LA 6-20 mm ngokumelene ne-LA 20-30 mm.g BN 6–20 mm kanye 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 Ijika lesici sokusebenza komamukeli (ROC) elibonisa ukusebenza kwemodeli ebandlululayo kumaqhuqhuva angu-6–20 mm.f Amanani angenzeka abalwe ngokusekelwe kumodeli yokuhlehla kwempahla yamaqhuqhuva anesilinganiso esingu-6–20 mm.Ulayini wamachashazi ampunga umele inani elifanele lokusika (0.455).Izinombolo ezingenhla zimelela iphesenti lamacala ahlelelwe i-Los Angeles.Sebenzisa ukuhlolwa koMfundi okunemisila emibili.I-PCA, ukuhlaziywa kwengxenye eyinhloko.Indawo ye-AUC ngaphansi kwejika.Idatha yomthombo inikezwa ngohlobo lwamafayela edatha yomthombo.
Amasampula amane (aneminyaka engu-44-61 ubudala) anobukhulu obufanayo be-nodule yamaphaphu (7-9 mm) aphinde akhethwa ukuze abonise ukusebenza kwemodeli ehlongozwayo yokubikezela ububi (Fig. 4a, b).Ekuhlolweni kokuqala, Ikesi 1 lethulwe njenge-nodule eqinile enezibalo, isici esihlotshaniswa nokulunga, kuyilapho u-Case 2 wethulwa njenge-nodule eqinile kancane enganqunyelwe engenakho izici ezisobala eziyingozi.Imijikelezo emithathu yokulandelela i-CT scans ibonise ukuthi lawa macala ahlala ezinzile esikhathini esiyiminyaka emi-4 futhi ngakho-ke abhekwa njengama-nodule angenangqondo (Fig. 4a).Uma kuqhathaniswa nokuhlolwa komtholampilo kwe-serial CT scans, ukuhlaziywa kwe-metabolite ye-serum enesibhamu esisodwa nemodeli yamanje yokuhlukanisa kuhlonzwe ngokushesha futhi ngokufanelekile lawa maqhuqhuva anobungozi asuselwa kuzithiyo okungenzeka (Ithebula 1).Umfanekiso 4b esimweni sesi-3 ubonisa iqhuqhuva elinezimpawu zokuhoxiswa kwe-pleural, evame ukuhlotshaniswa ne-malignancy32.Icala lesi-4 lethulwe njengeqhuqhuva eliqine ngokwengxenye elinganqunyelwe elingenabo ubufakazi bembangela eyingozi.Zonke lezi zimo zibikezelwe njengeziyingozi ngokwemodeli yokuhlukanisa (Ithebula 1).Ukuhlolwa kwe-lung adenocarcinoma kuboniswe ngokuhlolwa kwe-histopathological ngemva kokuhlinzwa kokukhishwa kwamaphaphu (Fig. 4b).Kusethi yokuqinisekisa yangaphandle, isigaba se-metabolic sibikezele ngokunembile izimo ezimbili zamaqhuqhuva amaphaphu angapheli amakhulu kuno-6 mm (Umfanekiso Owengeziwe 10).
Izithombe ze-CT zefasitela le-axial lamaphaphu amacala amabili ama-benign nodules.Esimeni soku-1, ukuskena kwe-CT ngemva kweminyaka emi-4 kubonise iqhubu eliqinile elilinganisa u-7 mm nokubala engxenyeni engezansi engakwesokudla.Esimeni esingu-2, i-CT scan ngemva kweminyaka engu-5 yembula iqhuqhuva elizinzile, eliqinile kancane elinobubanzi obungu-7 mm engxenyeni engaphezulu kwesokudla.b Izithombe ze-CT zewindi le-axial zamaphaphu kanye nezifundo ze-pathological ezihambisanayo zezimo ezimbili ze-adenocarcinoma yesiteji I ngaphambi kokukhishwa kabusha kwamaphaphu.Ikesi 3 lembule iqhuqhuva elinobubanzi obungu-8 mm engxenyeni engaphezulu kwesokudla ene-pleural retraction.Ikesi 4 lembule i-nodule yengilazi yaphansi eqinile kancane ekala u-9 mm engxenyeni engaphezulu kwesokunxele.Ukungcoliswa kwe-Hematoxylin ne-eosin (H&E) kwezicubu zamaphaphu ezikhishwe kabusha (i-scale bar = 50 μm) ebonisa iphethini yokukhula kwe-acinar ye-lung adenocarcinoma.Imicibisholo ibonisa izigaxana ezitholwe ezithombeni ze-CT.Izithombe ze-H&E ziyizithombe ezimele zezinkambu ezincane (>3) ezihlolwe udokotela wezifo.
Ihlanganiswe ndawonye, imiphumela yethu ibonisa inani elingaba khona lama-biomarker e-serum metabolite ekuxilongweni okuhlukene kwamaqhuqhuva wamaphaphu, okungase kubangele izinselele lapho kuhlolwa ukuhlolwa kwe-CT.
Ngokusekelwe kuphaneli ye-metabolite ehlukile eqinisekisiwe, sifuna ukuhlonza izixhumanisi zebhayoloji zezinguquko ezinkulu ze-metabolic.Ukuhlaziywa kokuthuthukiswa kwendlela ye-KEGG yi-MetaboAnalyst kuhlonze izindlela ezi-6 ezivamile ezishintshwe ngokuphawulekayo phakathi kwamaqembu amabili anikeziwe (i-LA vs. HC ne-LA vs. BN, i-p elungisiwe ≤ 0.001, umphumela> 0.01).Lezi zinguquko zibonakala ngokuphazamiseka kwe-pyruvate metabolism, i-tryptophan metabolism, i-niacin ne-nicotinamide metabolism, i-glycolysis, umjikelezo we-TCA, kanye ne-purine metabolism (Fig. 5a).Sibe sesiqhubeka nokwenza i-metabolomics eqondisiwe ukuze siqinisekise izinguquko ezinkulu sisebenzisa ubuningi obuphelele.Ukunqunywa kwama-metabolites avamile emigwaqweni evame ukushintshwa yi-triple quadrupole mass spectrometry (QQQ) kusetshenziswa izindinganiso ze-metabolite zangempela.Izici zezibalo zesampula yocwaningo lwe-metabolomics zifakiwe Kuthebula Lokwengeza 4. Ngokuhambisana nemiphumela yethu ye-metabolomics yomhlaba wonke, ukuhlaziya kobuningi kuqinisekisile ukuthi i-hypoxanthine ne-xanthine, i-pyruvate, ne-lactate zinyuswe ku-LA uma kuqhathaniswa ne-BN ne-HC (Fig. 5b, c, p <0.05).Kodwa-ke, akukho mehluko obalulekile kulawa ma-metabolites owatholakala phakathi kwe-BN ne-HC.
Ukuhlaziywa kokuthuthukiswa kwendlela ye-KEGG yama-metabolite ahluke kakhulu eqenjini le-LA uma kuqhathaniswa namaqembu e-BN ne-HC.Kusetshenziswe i-Globaltest enemisila emibili, futhi amanani e-p alungiswa kusetshenziswa indlela ye-Holm-Bonferroni (elungisiwe p ≤ 0.001 kanye nosayizi womphumela > 0.01).b–d Iziqephu ze-Violin ezibonisa i-hypoxanthine, i-xanthine, i-lactate, i-pyruvate, namazinga e-tryptophan ku-serum HC, BN, ne-LA kunqunywa i-LC-MS/MS (n = 70 iqembu ngalinye).Imigqa enamachashazi amhlophe namnyama ikhombisa i-median ne-quartile, ngokulandelana.e Itulo leviolin elibonisa i-Log2TPM evamile (imibhalo ngesigidi) isisho se-mRNA se-SLC7A5 ne-QPRT ku-lung adenocarcinoma (n = 513) uma kuqhathaniswa nezicubu ezivamile zamaphaphu (n = 59) kudathasethi ye-LUAD-TCGA.Ibhokisi elimhlophe limelela ububanzi be-interquartile, umugqa omnyama ovundlile phakathi nendawo umele i-median, futhi umugqa omnyama ome mpo osuka ebhokisini umelela u-95% wesikhawu sokuzethemba (CI).f Isiqephu sokuhlanganisa se-Pearson se-SLC7A5 nesisho se-GAPDH ku-lung adenocarcinoma (n = 513) kanye nezicubu zamaphaphu ezivamile (n = 59) kudathasethi ye-TCGA.Indawo empunga imele i-CI engu-95%.r, i-Pearson coefficient yokuhlanganisa.g Amazinga e-tryptophan eselula ajwayelekile kumaseli angu-A549 adluliswa ngokulawula okungaqondile kwe-shRNA (NC) kanye ne-shSLC7A5 (Sh1, Sh2) enqunywe i-LC-MS/MS.Ukuhlaziywa kwezibalo kwamasampuli amahlanu azimele ngokwebhayoloji eqenjini ngalinye kuyethulwa.h Amazinga eselula e-NADt (inani eliphelele le-NAD, okuhlanganisa i-NAD+ ne-NADH) kumaseli angu-A549 (NC) kanye namaseli e-SLC7A5 awisa phansi ama-A549 (Sh1, Sh2).Ukuhlaziywa kwezibalo kwamasampuli amathathu azimele ngokwebhayoloji eqenjini ngalinye kuyethulwa.Umsebenzi we-Glycolytic wamaseli we-A549 ngaphambi nangemva kokugoqa kwe-SLC7A5 kukalwa ngesilinganiso se-extracellular acidification rate (ECAR) (n = 4 amasampula azimele ngokwebhayoloji iqembu ngalinye).2-DG,2-deoxy-D-glucose.Ukuhlolwa kwe-t koMfundi okunemisila emibili kusetshenziswe kokuthi (b–h).Ku-(g–i), amabha wephutha amelela isilinganiso esingu-± SD, ukuhlolwa ngakunye kwenziwa izikhathi ezintathu ngokuzimela futhi imiphumela iyefana.Idatha yomthombo inikezwa ngohlobo lwamafayela edatha yomthombo.
Uma sicabangela umthelela obalulekile we-tryptophan metabolism eguquliwe eqenjini le-LA, siphinde sahlola amazinga e-serum tryptophan emaqenjini e-HC, BN, kanye ne-LA sisebenzisa i-QQQ.Sithole ukuthi i-serum tryptophan yehlisiwe ku-LA uma iqhathaniswa ne-HC noma i-BN (p <0.001, Umfanekiso 5d), okuhambisana nokutholakele kwangaphambilini ukuthi amazinga e-tryptophan ajikelezayo aphansi ezigulini ezinomdlavuza wamaphaphu kunezilawuli ezinempilo ezivela eqenjini lokulawula33,34 ,35.Olunye ucwaningo olusebenzisa i-PET/CT tracer 11C-methyl-L-tryptophan luthole ukuthi isikhathi sokugcina isignali ye-tryptophan kuzicubu zomdlavuza wamaphaphu senyuke kakhulu uma kuqhathaniswa nezilonda ezinobungozi noma izicubu ezijwayelekile36.Sicabanga ukuthi ukwehla kwe-tryptophan ku-LA serum kungase kubonise ukutholwa kwe-tryptophan okusebenzayo ngamaseli omdlavuza wamaphaphu.
Kuyaziwa futhi ukuthi umkhiqizo wokugcina wendlela ye-kynurenine ye-tryptophan catabolism yi-NAD+37,38, okuyi-substrate ebalulekile yokusabela kwe-glyceraldehyde-3-phosphate nge-1,3-bisphosphoglycerate ku-glycolysis39.Ngenkathi izifundo zangaphambilini zigxile endimeni ye-tryptophan catabolism ekulawuleni amasosha omzimba, sifuna ukucacisa ukusebenzisana phakathi kwe-tryptophan dysregulation kanye nezindlela ze-glycolytic ezibonwe ocwaningweni lwamanje.I-Solute transporter umndeni 7 ilungu 5 (SLC7A5) yaziwa njenge-tryptophan transporter43,44,45.I-Quinolinic acid phosphoribosyltransferase (QPRT) iyi-enzyme etholakala ezansi nomfula we-kynurenine eguqula i-quinolinic acid ibe yi-NAMN46.Ukuhlolwa kwedathasethi ye-LUAD TCGA kwembula ukuthi kokubili i-SLC7A5 ne-QPRT zazilawulwa kakhulu kuzicubu zesimila uma kuqhathaniswa nezicubu ezivamile (Fig. 5e).Lokhu kwanda kwabonwa ngezigaba I no-II kanye nezigaba III kanye ne-IV ze-lung adenocarcinoma (Umfanekiso Ongeziwe 11), okubonisa ukuphazamiseka kokuqala kwe-tryptophan metabolism ehambisana ne-tumorigenesis.
Ukwengeza, idathasethi ye-LUAD-TCGA ibonise ukuhlobana okuhle phakathi kwe-SLC7A5 nesisho se-GAPDH mRNA kumasampuli esiguli somdlavuza (r = 0.45, p = 1.55E-26, Umfanekiso 5f).Ngokuphambene, akukho ukuhlobana okubalulekile okutholakele phakathi kwamasignesha ofuzo anjalo kuzicubu zamaphaphu ezivamile (r = 0.25, p = 0.06, Umfanekiso 5f).I-Knockdown ye-SLC7A5 (Figure Supplementary Figure 12) kumaseli e-A549 kunciphisa kakhulu amazinga e-tryptophan yeselula kanye ne-NAD(H) (Umfanekiso 5g, h), okuholela ekusebenzeni kwe-glycolytic okuncishisiwe njengoba kulinganiswa ngesilinganiso se-extracellular acidification rate (ECAR) (Umfanekiso 1).5i).Ngakho-ke, ngokususelwa ekushintsheni kwe-metabolic ekutholweni kwe-serum kanye ne-in vitro, sicabanga ukuthi i-tryptophan metabolism ingase ikhiqize i-NAD+ ngendlela ye-kynurenine futhi ibambe iqhaza elibalulekile ekukhuthazeni i-glycolysis kumdlavuza wamaphaphu.
Ucwaningo luye lwabonisa ukuthi inani elikhulu lama-nodules e-pulmonary angapheli atholwe yi-LDCT angase aholele esidingweni sokuhlolwa okwengeziwe okufana ne-PET-CT, i-lung biopsy, kanye nokwelashwa ngokweqile ngenxa yokuxilongwa okungelona iqiniso kwe-malignancy.31 Njengoba kuboniswe ku-Figure 6, ucwaningo lwethu luhlonze iphaneli lama-metabolites e-serum anenani lokuxilonga elingase lithuthukise ukuhlukaniswa kwengozi kanye nokuphathwa okulandelayo kwama-nodule e-pulmonary atholwe yi-CT.
Izigaxa zamaphaphu zihlolwa kusetshenziswa umthamo ophansi we-computed tomography (LDCT) enezici zokuthwebula eziphakamisa izimbangela eziyingozi noma ezimbi.Umphumela ongaqinisekile wamaqhuqhuva ungaholela ekuvakasheni kokulandelela njalo, ukungenelela okungadingekile, nokwelashwa ngokweqile.Ukufakwa kwezigaba ze-serum metabolic ezinenani lokuxilonga kungase kuthuthukise ukuhlolwa kwengozi kanye nokuphathwa okulandelayo kwamaqhuqhuva wamaphaphu.I-PET positron emission tomography.
Idatha evela ocwaningweni lwase-US NLST kanye nocwaningo lwaseYurophu lwe-NELSON luphakamisa ukuthi ukuhlola amaqembu asengozini enkulu nge-tomography ye-computed tomography (LDCT) enedosi ephansi kungase kunciphise ukufa komdlavuza wamaphaphu1,3.Kodwa-ke, ukuhlolwa kobungozi kanye nokuphathwa komtholampilo okwalandela kwezinombolo ezinkulu zamaqhuqhuva e-pulmonary atholwe yi-LDCT kuseyinselele kakhulu.Umgomo oyinhloko uwukuthuthukisa ukuhlukaniswa okulungile kwamaphrothokholi asuselwe ku-LDCT ngokuhlanganisa ama-biomarker athembekile.
Ama-biomarker athile wamangqamuzana, njengama-metabolite egazi, ahlonzwe ngokuqhathanisa umdlavuza wamaphaphu nezilawuli ezinempilo15,17.Ocwaningweni lwamanje, sigxile ekusetshenzisweni kokuhlaziywa kwe-serum metabolomics ukuhlukanisa phakathi kwamaqhubu amaphaphu ayingozi kanye namabi atholwe ngengozi yi-LDCT.Siqhathanise i-serum metabolome yomhlaba wonke yokulawula okunempilo (HC), ama-benign lung nodules (BN), kanye namasampula esiteji I lung adenocarcinoma (LA) sisebenzisa ukuhlaziywa kwe-UPLC-HRMS.Sithole ukuthi i-HC ne-BN zinamaphrofayili afanayo we-metabolic, kanti i-LA ibonise izinguquko eziphawulekayo uma kuqhathaniswa ne-HC ne-BN.Sihlonze amasethi amabili e-serum metabolites ahlukanisa i-LA ku-HC ne-BN.
Uhlelo lwamanje lokuhlonza olusekelwe ku-LDCT lwamaqhuqhuva ayingozi kanye namabi lusekelwe kakhulu kusayizi, ukuminyana, ukumila kokwakheka komhlaba kanye nezinga lokukhula lamaqhuqhuva ngokuhamba kwesikhathi30.Ucwaningo lwangaphambilini lukhombisile ukuthi ubukhulu bamaqhuqhuva buhlobene kakhulu namathuba okuba nomdlavuza wamaphaphu.Ngisho nasezigulini ezisengozini enkulu, ingozi yokulimala kuma-nodes <6 mm ingu-<1%.Ingozi yokulimala kwamaqhuqhuva anesilinganiso esingu-6 kuye ku-20 mm isuka ku-8% iye ku-64%30.Ngakho-ke, i-Fleischner Society incoma ukunqanyulwa kwedayamitha engu-6 mm ukuze kulandelelwe i-CT evamile.I-29 Kodwa-ke, ukuhlolwa kobungozi kanye nokuphathwa kwamanodule e-pulmonary (IPN) amakhulu kune-6 mm akuzange kwenziwe ngokwanele i-31.Ukuphathwa kwamanje kwesifo senhliziyo esizalwa nakho ngokuvamile kusekelwe ekulindeni okuqaphile ngokuqapha njalo kwe-CT.
Ngokusekelwe ku-metabolome eqinisekisiwe, sibonise ngokokuqala ukugqagqana kwamasiginesha e-metabolomic phakathi kwabantu abanempilo kanye namaqhuqhuva angama-benign <6 mm.Ukufana kwezinto eziphilayo kuhambisana nokutholwe kwangaphambili kwe-CT ukuthi ingozi yokulimala kwama-nodules <6 mm iphansi njengezifundo ezingenayo ama-node.30 Kumele kuqashelwe ukuthi imiphumela yethu iphinde ibonise ukuthi ama-nodules angama-benign <6 mm no-≥6 mm aphakeme. ukufana kumaphrofayili we-metabolomic, okuphakamisa ukuthi incazelo yokusebenza ye-benign etiology iyahambisana kungakhathaliseki ukuthi usayizi wamanodule.Ngakho-ke, amaphaneli wesimanje we-serum metabolite wokuxilonga angase anikeze ukuhlolwa okukodwa njengokuhlolwa kokukhishwa lapho ama-nodules etholwa ekuqaleni ku-CT futhi okungenzeka anciphise ukuqapha kwe-serial.Ngesikhathi esifanayo, iphaneli efanayo yezimpawu ze-biomarker ye-metabolic yahlukanisa amaqhuqhuva ayingozi ≥6 mm ngosayizi kusukela kumaqhuqhuva amahle futhi yanikeza izibikezelo ezinembile zama-IPN anosayizi ofanayo nezici ze-morphological ezingacacile ezithombeni ze-CT.Lesi sigaba se-serum metabolism senza kahle ekubikezeleni ukonakala kwamaqhuqhuva ≥6 mm nge-AUC engu-0.927.Uma sihlanganiswa, imiphumela yethu ibonisa ukuthi amasiginesha e-serum metabolomic ahlukile angase abonise ngokuqondile izinguquko ze-metabolic ezibangelwa isimila futhi abe nenani elingaba khona njengezibikezelo zengozi, ngaphandle kosayizi wamaqhuqhuva.
Ngokuphawulekayo, i-lung adenocarcinoma (LUAD) kanye ne-squamous cell carcinoma (LUSC) yizinhlobo eziyinhloko zomdlavuza wamaphaphu ongewona omncane (NSCLC).Njengoba i-LUSC ihlotshaniswa ngokuqinile nokusetshenziswa kukagwayi47 futhi i-LUAD iwumlando ovame kakhulu wamaqhuqhuva wamaphaphu engozi atholwe ekuhlolweni kwe-CT48, imodeli yethu yokuhlukanisa yakhelwe ngokukhethekile amasampula esiteji I adenocarcinoma.U-Wang nozakwabo baphinde bagxila ku-LUAD futhi bakhombe amasignesha ayisishiyagalolunye e-lipid esebenzisa i-lipidomics ukuhlukanisa umdlavuza wamaphaphu wesigaba sokuqala kubantu abanempilo17.Sihlole imodeli yamanje yokuhlukanisa ezimweni ezingu-16 zesiteji I LUSC kanye namanodule angama-74 futhi sabona ukunemba okuphansi kokubikezela kwe-LUSC (AUC 0.776), okusikisela ukuthi i-LUAD ne-LUSC zingase zibe namasiginesha azo e-metabolomic.Ngempela, i-LUAD kanye ne-LUSC kukhonjiswe ukuthi kwehlukile ku-etiology, imvelaphi yezinto eziphilayo kanye nokuphambuka kofuzo49.Ngakho-ke, ezinye izinhlobo ze-histology kufanele zifakwe kumamodeli wokuqeqesha ukuze kutholwe umdlavuza wamaphaphu ngokusekelwe kubantu ezinhlelweni zokuhlola.
Lapha, sihlonze izindlela eziyisithupha ezivame ukuguqulwa kakhulu ku-lung adenocarcinoma uma kuqhathaniswa nezilawuli ezinempilo namaqhuqhuva amahle.I-Xanthine ne-hypoxanthine ama-metabolites ajwayelekile e-purine metabolic pathway.Ngokuvumelana nemiphumela yethu, i-intermediate ehlotshaniswa ne-purine metabolism yanda kakhulu ku-serum noma izicubu zeziguli ezine-lung adenocarcinoma uma kuqhathaniswa nezilawuli ezinempilo noma iziguli esiteji sokuqala15,50.Amazinga aphezulu e-serum xanthine kanye ne-hypoxanthine angase abonise i-anabolism edingekayo ngamaseli omdlavuza akhula ngokushesha.Ukungasebenzi kahle kwe-glucose metabolism kuwuphawu olwaziwayo lomdlavuza we-metabolism51.Lapha, sibone ukwanda okukhulu kwe-pyruvate ne-lactate eqenjini le-LA uma kuqhathaniswa neqembu le-HC ne-BN, elihambisana nemibiko yangaphambilini yokungahambi kahle kwendlela ye-glycolytic kumaphrofayili we-serum metabolome yeziguli ezingezona ezincane zomdlavuza wamaphaphu (NSCLC) kanye izilawuli ezinempilo.imiphumela iyahambisana52,53.
Okubalulekile, sibone ukuhlobana okuphambene phakathi kwe-pyruvate ne-tryptophan metabolism ku-serum ye-lung adenocarcinomas.Amazinga e-serum tryptophan ancishisiwe eqenjini le-LA uma kuqhathaniswa neqembu le-HC noma le-BN.Kuyathakazelisa ukuthi ucwaningo lwangaphambilini olukhulu olusebenzisa i-cohort oluzoba khona lwathola ukuthi amazinga aphansi e-tryptophan ejikelezayo ahlotshaniswa nengozi eyengeziwe yomdlavuza wamaphaphu 54.I-Tryptophan iyi-amino acid ebalulekile esiyithola ngokuphelele ekudleni.Siphetha ngokuthi ukuncipha kwe-serum tryptophan ku-lung adenocarcinoma kungase kubonise ukuncipha ngokushesha kwale metabolite.Kwaziwa kahle ukuthi umkhiqizo wokugcina we-tryptophan catabolism nge-kynurenine pathway ungumthombo we-de novo NAD+ synthesis.Ngoba i-NAD+ ikhiqizwa ngokuyinhloko ngendlela yokuhlenga, ukubaluleka kwe-NAD+ ku-tryptophan metabolism empilweni nasezifo kusazonqunywa46.Ukuhlaziywa kwethu kwe-database ye-TCGA kubonise ukuthi inkulumo ye-tryptophan transporter solute transporter 7A5 (SLC7A5) yanda kakhulu ku-adenocarcinoma yamaphaphu uma iqhathaniswa nezilawuli ezijwayelekile futhi yayihlotshaniswa kahle nokuvezwa kwe-enzyme ye-glycolytic GAPDH.Ucwaningo lwangaphambilini lugxile kakhulu endimeni ye-tryptophan catabolism ekucindezeleni impendulo ye-antitumor immune40,41,42.Lapha sibonisa ukuthi ukuvinjwa kokuthathwa kwe-tryptophan ngokugoqa i-SLC7A5 kumaseli omdlavuza wamaphaphu kuphumela ekwehleni okulandelayo kwamazinga e-NAD eselula kanye nokuncishiswa okuhambisanayo komsebenzi we-glycolytic.Kafushane, ucwaningo lwethu luhlinzeka ngesisekelo sebhayoloji soshintsho kumetabolism ye-serum ehambisana noshintsho olubi lwe-lung adenocarcinoma.
Ukuguqulwa kwe-EGFR kuyizinguquko ezivame kakhulu zomshayeli ezigulini ezine-NSCLC.Ocwaningweni lwethu, sithole ukuthi iziguli ezine-EGFR mutation (n = 41) zinamaphrofayili e-metabolomic jikelele afana neziguli ezinohlobo lwasendle lwe-EGFR (n = 31), nakuba sithole ukwehla kwamazinga e-serum kwezinye iziguli ze-EGFR eziguquguqukayo ezigulini ze-acylcarnitine.Umsebenzi omisiwe we-acylcarnitines ukuthutha amaqembu e-acyl kusuka ku-cytoplasm kuya ku-matrix ye-mitochondrial, okuholela ekuhlanganiseni kwe-fatty acids ukukhiqiza amandla e-55.Ngokuvumelana nokutholakele kwethu, ucwaningo lwakamuva luphinde lwahlonza amaphrofayili afanayo e-metabolome phakathi kwezimila zohlobo lwasendle lwe-EGFR kanye ne-EGFR ngokuhlaziya i-metabolome yomhlaba wonke yamasampula ezicubu zamaphaphu angu-102 we-adenocarcinoma50.Ngokuthakazelisayo, okuqukethwe kwe-acylcarnitine kuphinde kwatholakala eqenjini le-EGFR le-mutant.Ngakho-ke, noma ngabe izinguquko emazingeni e-acylcarnitine zibonisa izinguquko ze-metabolic ezenziwe yi-EGFR kanye nezindlela ezingaphansi zamangqamuzana zingase zifanele ukutadisha okwengeziwe.
Sengiphetha, ucwaningo lwethu lusungula isigaba se-serum metabolic sokuxilongwa okuhlukile kwamaqhuqhuva wamaphaphu futhi siphakamisa ukuhamba komsebenzi okungathuthukisa ukuhlolwa kobungozi futhi kube lula ukuphathwa komtholampilo okusekelwe ekuhlolweni kwe-CT scan.
Lolu cwaningo lugunyazwe yiKomidi Lezimiso Zokuziphatha leSibhedlela Somdlavuza saseNyuvesi i-Sun Yat-sen, iSibhedlela Esihlanganisiwe Sokuqala sase-Sun Yat-sen University, kanye neKomidi Lezimiso Zokuziphatha lase-Zhengzhou University Cancer Hospital.Emaqenjini okutholakala nawokuqinisekisa angaphakathi, amasera angu-174 avela kubantu abanempilo kanye namasera angama-244 avela kumaqhuqhuva angenabungozi aqoqwe kubantu abahlolwa minyaka yonke eMnyangweni Wokulawula Nokuvimbela Umdlavuza, Isikhungo Somdlavuza saseNyuvesi yaseSun Yat-sen, kanye namaqhuqhuva angama-166.i-serum.I-Stage I lung adenocarcinomas yaqoqwa e-Sun Yat-sen University Cancer Centre.Eqeqebeni lokuqinisekisa langaphandle, kube nezigameko ezingama-48 zamaqhuqhuva amahle, amacala angama-39 e-stage I lung adenocarcinoma evela eSibhedlela Esihlanganisiwe Sokuqala sase-Sun Yat-sen University, kanye namacala angu-24 esiteji I lung adenocarcinoma yaseSibhedlela Somdlavuza saseZhengzhou.Isikhungo somdlavuza seNyuvesi yase-Sun Yat-sen siphinde saqoqa izehlakalo eziyi-16 zomdlavuza wamaphaphu we-squamous cell wesigaba I ukuze kuhlolwe ikhono lokuxilonga lesigaba esimisiwe se-metabolic (izici zesiguli zikhonjiswe kuThebula Lokwengeza lesi-5).Amasampuli avela eqenjini lokutholwa kanye neqembu lokuqinisekisa langaphakathi aqoqwe phakathi kukaJanuwari 2018 noMeyi 2020. Amasampuli eqembu lokuqinisekisa langaphandle aqoqwe phakathi kuka-Agasti 2021 no-Okthoba 2022. Ukuze kuncishiswe ukuchema kobulili, cishe izinombolo ezilinganayo zamacala abesilisa nabesifazane zanikezwa ngalinye. iqembu.Ithimba Lokutholwa Nethimba Lokubuyekeza Kwangaphakathi.Ubulili bombambi qhaza banqunywa ngokusekelwe ekuzibikeni kwakhe.Imvume enolwazi itholwe kubo bonke ababambiqhaza futhi akukho sinxephezelo esinikeziwe.Izihloko ezinezigaxa ezinhle kwakuyilezo ezinesilinganiso esizinzile se-CT scan ku-2 kuya ku-5 iminyaka ngesikhathi sokuhlaziywa, ngaphandle kwecala le-1 elivela kusampula yokuqinisekisa yangaphandle, eyaqoqwa ngaphambi kokuhlinzwa futhi yatholwa yi-histopathology.Ngaphandle kwe-bronchitis engapheli.Amacala e-adenocarcinoma yamaphaphu aqoqwa ngaphambi kokukhishwa kabusha kwamaphaphu futhi aqinisekiswa ngokuxilongwa kwe-pathological.Amasampula egazi lokuzila aqoqwe kumashubhu okuhlukanisa i-serum ngaphandle kwama-anticoagulants.Amasampula egazi ajikijelwa ihora elingu-1 ekamelweni lokushisa bese efakwa phakathi ku-2851 × g imizuzu engu-10 ku-4°C ukuze kuqoqwe i-serum supernatant.Ama-aliquots e-Serum ayenziwe iqhwa ku--80°C kuze kukhishwe i-metabolite.UMnyango Wokuvimbela Umdlavuza kanye Nokuhlolwa Kwezempilo e-Sun Yat-sen University Cancer Centre uqoqe i-serum eningi kubanikeli abanempilo abayi-100, okuhlanganisa nenani elilinganayo labesilisa nabesifazane abaneminyaka engama-40 kuya kwengama-55.Izilinganiso ezilinganayo zesampula ngayinye yabanikeli zahlanganiswa, ichibi eliwumphumela lacashunwa futhi lagcinwa ku -80°C.Ingxube ye-serum isetshenziswe njengento eyinkomba yokulawulwa kwekhwalithi kanye nokumiswa kwedatha.
Iseramu eyireferensi namasampuli okuhlola ancibilika futhi ama-metabolite akhishwa kusetshenziswa indlela yokukhipha ehlanganisiwe (MTBE/methanol/water) 56 .Kafushane, u-50 μl we-serum waxutshwa no-225 μl we-methanol ebandayo eqhweni kanye no-750 μl we-ice-cold methyl tert-butyl ether (MTBE).Hlanganisa ingxube bese uyifukamela eqhweni ihora elingu-1.Amasampula abe esexutshwa ne-vortex exutshwe no-188 μl wamanzi e-MS-grade aqukethe amazinga angaphakathi (13C-lactate, 13C3-pyruvate, 13C-methionine, kanye ne-13C6-isoleucine, athengwe kwa-Cambridge Isotope Laboratories).Ingxube ibe i-centrifuged ku-15,000 × g imizuzu engu-10 ku-4 ° C, futhi isigaba esiphansi sadluliselwa kumashubhu amabili (125 μL ngalinye) ukuze kuhlaziywe i-LC-MS ngezindlela ezinhle nezingezinhle.Ekugcineni, isampuli yahwamuka yaba yoma kusigxivizo se-vacuum esinesivinini esikhulu.
Ama-metabolites omisiwe aphinde ahlanganiswa ku-120 μl we-80% ye-acetonitrile, i-vortexed for 5 min, futhi i-centrifuged ku-15,000 × g imizuzu engu-10 ku-4 ° C.Ama-supernatant adluliselwa ezitsheni zengilazi ye-amber ezinama-microinsert ocwaningo lwe-metabolomics.Ukuhlaziywa kwe-metabolomics okungahlosiwe kuplathifomu ye-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Ama-Metabolites ahlukaniswa kusetshenziswa uhlelo lwe-Dionex Ultimate 3000 UPLC kanye nekholomu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Amanzi).Kumodi ye-ion enhle, izigaba zeselula zazingu-95% (A) no-50% we-acetonitrile (B), ngasinye siqukethe i-10 mmol/L ammonium acetate kanye ne-0.1% ye-formic acid.Kwimodi engalungile, izigaba zeselula A no-B zaziqukethe i-95% kanye ne-50% ye-acetonitrile, ngokulandelana, zombili izigaba zaziqukethe i-10 mmol / L ammonium acetate, i-pH = 9. Uhlelo lwe-gradient lwalumi kanje: 0-0.5 min, 2% B;0.5–12 amaminithi, 2–50% B;12–14 amaminithi, 50–98% B;14–16 amaminithi, 98% B;16–16.1.imiz, 98 –2% B;16.1–20 amaminithi, 2% B. Ikholomu igcinwe ku-40°C kanye nesampula ku-10°C kusampula esizenzakalelayo.Izinga lokugeleza lalingu-0.3 ml/min, umthamo womjovo wawuyi-3 μl.I-Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) enomthombo we-ionization ye-electrospray (ESI) yasetshenziswa ngemodi yokuskena ephelele futhi yahlanganiswa nemodi yokuqapha ye-ddMS2 ukuze kuqoqwe amavolumu amakhulu edatha.Amapharamitha we-MS ahlelwe kanje: i-voltage yesifutho +3.8 kV/- 3.2 kV, izinga lokushisa le-capillary 320°C, igesi evikelayo engu-40 arb, igesi elisizayo elingu-10 arb, izinga lokushisa le-heater elingu-350°C, ibanga lokuskena 70-1050 m/h, isixazululo.70 000. Idatha yatholwa kusetshenziswa i-Xcalibur 4.1 (Thermo Fisher Scientific).
Ukuze kuhlolwe ikhwalithi yedatha, amasampuli okulawula ikhwalithi ehlanganisiwe (QC) enziwe ngokukhipha ama-aliquots angu-10 μL we-supernatant kusampula ngayinye.Imijovo yesampula yokulawula ikhwalithi eyisithupha yahlaziywa ekuqaleni kokulandelana kokuhlaziya ukuze kuhlolwe ukuzinza kohlelo lwe-UPLC-MS.Amasampula okulawula ikhwalithi abe esengeniswa eqoqweni ngezikhathi ezithile.Wonke amaqoqo angu-11 amasampula e-serum kulolu cwaningo ahlaziywa yi-LC-MS.Ama-aliquots engxube ye-serum pool evela kubanikeli abanempilo abayi-100 asetshenziswe njengereferensi kumaqoqo ahlukene ukuze kuqashwe inqubo yokukhipha futhi kulungiswe imiphumela ye-batch-to-batch.Ukuhlaziywa kwe-metabolomics okungahlosiwe kweqembu lokutholwa, iqoqo lokuqinisekisa langaphakathi, neqembu lokuqinisekisa langaphandle kwenziwe e-Metabolomics Center yase-Sun Yat-sen University.Ilabhorethri yangaphandle ye-Guangdong University of Technology Analysis and Testing Center iphinde yahlaziya amasampula angu-40 avela eqenjini langaphandle ukuze ihlole ukusebenza kwemodeli yokuhlukanisa.
Ngemva kokukhipha nokwakhiwa kabusha, inani eliphelele le-serum metabolites likalwa kusetshenziswa i-ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) enomthombo we-electrospray ionization (ESI) kumodi yokuqapha ukusabela okuningi (MRM).Ikholomu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Amanzi) yasetshenziswa ukuhlukanisa ama-metabolites.Isigaba esihambayo sasihlanganisa u-90% (A) no-5% we-acetonitrile (B) ene-10 mmol/L ammonium acetate kanye nesisombululo se-ammonia esingu-0.1%.Uhlelo lwe-gradient belumi kanje: 0–1.5 min, 0% B;1.5–6.5 amaminithi, 0–15% B;6.5–8 amaminithi, 15% B;8–8.5 amaminithi, 15%–0% B;8.5–11.5 amaminithi, 0%B.Ikholomu iye yanakekelwa ku-40 °C kanye nesampula ku-10 °C kusampula esizenzakalelayo.Izinga lokugeleza lalingu-0.3 mL/min kanti umthamo womjovo wawuyi-1 μL.Amapharamitha we-MS ahlelwe kanje: i-capillary voltage ± 3.5 kV, ingcindezi ye-nebulizer engu-35 psi, i-sheath gas flow 12 L/min, izinga lokushisa le-sheath gas 350 ° C, izinga lokushisa legesi elomile lingu-250 ° C, nokugeleza kwegesi yokomisa 14 l/min.Ukuguqulwa kwe-MRM kwe-tryptophan, pyruvate, lactate, hypoxanthine ne-xanthine kwakungu-205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 kanye no-151.0–107.9 ngokulandelana.Idatha yaqoqwa kusetshenziswa i-Mass Hunter B.07.00 (Agilent Technologies).Kumasampuli e-serum, i-tryptophan, i-pyruvate, i-lactate, i-hypoxanthine, ne-xanthine ziye zalinganiswa kusetshenziswa amajika okulinganisa ezixazululo ezijwayelekile zengxube.Kumasampula eseli, okuqukethwe kwe-tryptophan kuye kwajwayezwa ukuze kube izinga langaphakathi nesisindo sephrotheni yeseli.
Ukukhishwa okuphezulu (m/z nesikhathi sokugcinwa (RT)) kwenziwe kusetshenziswa i-Compound Discovery 3.1 ne-TraceFinder 4.0 (i-Thermo Fisher Scientific).Ukuze kuqedwe umehluko ongaba khona phakathi kwamaqoqo, isici ngasinye esiphezulu sesampula sokuhlola sihlukaniswe ukuphakama kwesici sezinto eziyisethenjwa kusukela kunqwaba efanayo ukuze kutholwe inala ehlobene.Ukuchezuka okulinganiselwe okulinganiselwe kwezindinganiso zangaphakathi ngaphambi nangemuva kokumiswa kuboniswa kuThebula Lokwengeza 6. Umehluko phakathi kwamaqembu amabili ubonakale ngezinga lokutholwa okungamanga (FDR<0.05, ukuhlolwa kwezinga okusayinwe yi-Wilcoxon) kanye noshintsho olugoqiwe (>1.2 noma <0.83).Idatha ye-MS eluhlaza yezici ezikhishiwe kanye nedatha ye-MS elungiswe ku-serum iboniswa ku-Supplementary Data 1 kanye ne-Supplementary Data 2, ngokulandelanayo.Isichasiselo esiphezulu senziwe ngokusekelwe emazingeni amane achaziwe okuhlonza, okuhlanganisa ama-metabolite ahlonziwe, ama-puatively annotated compounds, amakilasi ahlanganisiwe anezimpawu ezicacile, kanye nezinhlanganisela ezingaziwa 22.Ngokusekelwe ekusesheni kwesizindalwazi ku-Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), izinhlanganisela zebhayoloji ezinezindinganiso eziqinisekisiwe ze-MS/MS noma izichasiselo eziqondile zokufanisa ku-mzCloud (amaphuzu > 85) noma i-Chemspider ekugcineni zikhethwe njengeziphakathi phakathi kwemetabolome ehlukile.Izichasiselo eziphezulu zesici ngasinye zifakiwe kudatha Eyengeziwe 3. I-MetaboAnalyst 5.0 isetshenziselwe ukuhlaziywa okungaguquguquki kokuchichima kwesamba esijwayelekile se-metabolite.I-MetaboAnalyst 5.0 iphinde yahlola ukuhlaziywa kwendlela ye-KEGG yokucebisa ngokusekelwe kuma-metabolites ahluke kakhulu.Ukuhlaziywa kwengxenye eyinhloko (i-PCA) kanye nokuhlaziywa kokubandlulula kwezikwele ezincane kancane (PLS-DA) kwahlaziywa kusetshenziswa iphakheji yesofthiwe ye-ropls (v.1.26.4) ngokujwayela kwesitaki kanye nokulinganisa okuzenzakalelayo.Imodeli ye-biomarker ye-metabolite elungile yokubikezela ukonakala kwamaqhuqhuva yenziwa kusetshenziswa ukuhlehliswa kwezinto kanambambili ngokuncipha okuphelele nokukhetha opharetha (i-LASSO, iphakheji ye-R v.4.1-3).Ukusebenza kwemodeli ebandlululayo kumasethi okutholwa nokuqinisekisa kuye kwabonakala ngokulinganisa i-AUC ngokusekelwe ekuhlaziyweni kwe-ROC ngokuya ngephakheji ye-pROC (v.1.18.0.).Ukunqunywa kwamathuba amahle kutholwe ngokusekelwe kunkomba enkulu ye-Youden yemodeli (ukuzwela + ukucaciswa - 1).Amasampuli anamanani amancane noma amakhulu kunomkhawulo azobikezelwa njengamaqhuqhuva amahle kanye ne-lung adenocarcinoma, ngokulandelanayo.
Amaseli e-A549 (#CCL-185, Iqoqo Lesiko Lohlobo LwaseMelika) akhuliswe ngendlela ye-F-12K equkethe u-10% we-FBS.Ukulandelana kwe-hairpin emfushane ye-RNA (shRNA) eqondise i-SLC7A5 kanye ne-nontargeting control (NC) kufakwe ku-lentviral vector pLKO.1-puro.Ukulandelana kwe-antisense kwe-shSLC7A5 kungokulandelayo: Sh1 (5′-GGAGAAACCTGATGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGGAACTAT-3′).Ama-antibodies kuya ku-SLC7A5 (#5347) kanye ne-tubulin (#2148) athengwe ku-Cell Signaling Technology.Ama-antibodies ku-SLC7A5 kanye ne-tubulin asetshenziswe ekuhlanjululweni kwe-1:1000 ekuhlaziyweni kwe-blot yaseNtshonalanga.
I-Seahorse XF Glycolytic Stress Test ikala amazinga e-extracellular acidification (ECAR).Esivivinyweni, i-glucose, i-oligomycin A, ne-2-DG inikezwe ngokulandelana ukuze kuhlolwe umthamo we-cell glycolytic njengoba kulinganiswa i-ECAR.
Amaseli angu-A549 adluliselwe ngesilawuli esingakhonjiwe (NC) kanye ne-shSLC7A5 (Sh1, Sh2) afakwe ubusuku bonke ezitsheni ezinobubanzi obungu-10 cm.Ama-metabolites amaseli akhishwa nge-1 ml ye-ice-cold 80% ye-methanol enamanzi.Amaseli esixazululweni se-methanol ayesulwa, aqoqwa eshubhu elisha, futhi afakwa phakathi nendawo ku-15,000 × g imizuzu engu-15 ku-4°C.Qoqa u-800 µl we-supernatant futhi womise usebenzisa isikhonzi se-vacuum esinesivinini esikhulu.Ama-pellets omisiwe e-metabolite abe esehlaziywa amazinga e-tryptophan kusetshenziswa i-LC-MS/MS njengoba kuchazwe ngenhla.Amaleveli e-NAD(H) eselula kumaseli angu-A549 (NC kanye ne-shSLC7A5) akalwe kusetshenziswa ikhithi yombala ye-NAD+/NADH (#K337, BioVision) ngokuya ngemiyalelo yomkhiqizi.Amaleveli amaprotheni akalwa ngesampula ngayinye ukwenza inani lama-metabolite lijwayelekile.
Azikho izindlela zezibalo ezisetshenzisiwe ukuze kutholwe usayizi wesampula kuqala.Ucwaningo lwangaphambilini lwe-metabolomics oluhloselwe ukutholwa kwe-biomarker15,18 luthathwe njengezilinganiso zokunqunywa kosayizi, futhi uma kuqhathaniswa nale mibiko, isampula yethu yayanele.Awekho amasampula akhishiwe eqenjini locwaningo.Amasampula e-Serum anikezwe ngezikhathi ezithile eqenjini lokutholwa (amacala angu-306, i-74.6%) kanye neqembu lokuqinisekisa langaphakathi (amacala angu-104, ama-25.4%) wezifundo ze-metabolomics ezingahlosiwe.Siphinde sakhetha ngokungahleliwe izimo ezingu-70 eqenjini ngalinye kusukela ekutholweni okusethelwe izifundo ezihlosiwe ze-metabolomics.Abaphenyi baphuphuthekisiwe ekunikezelweni kweqembu ngesikhathi sokuqoqwa nokuhlaziywa kwedatha ye-LC-MS.Ukuhlaziywa kwezibalo kwedatha ye-metabolomics nokuhlolwa kweseli kuchazwa ku- Imiphumela, Izinganekwane Zomfanekiso, kanye nezigaba zezindlela.Ukulinganisa kwe-tryptophan yamaselula, i-NADT, nomsebenzi we-glycolytic kwenziwa izikhathi ezintathu ngokuzimela ngemiphumela efanayo.
Ukuze uthole ulwazi olwengeziwe mayelana nomklamo wocwaningo, bheka I-Natural Portfolio Report Abstract ehlobene nalesi sihloko.
Idatha eluhlaza ye-MS yezici ezikhishiwe kanye nedatha ye-MS evamile ye-serum eyireferensi kuboniswa Kudatha Eyengeziwe 1 kanye Nedatha Eyengeziwe 2, ngokulandelanayo.Izichasiselo eziphakeme zezici ezihlukene zethulwa ku-Supplementary Data 3. Idathasethi ye-LUAD TCGA ingalandwa ku-https://portal.gdc.cancer.gov/.Idatha yokufaka yokuhlela igrafu inikezwa kudatha yomthombo.Idatha yomthombo inikezwe lesi sihloko.
I-National Lung Screening Study Group, njll. Ukunciphisa ukufa komdlavuza wamaphaphu ngedosi ephansi ye-computed tomography.I-Northern England.J. Med.365, 395–409 (2011).
I-Kramer, i-BS, i-Berg, i-KD, i-Aberle, i-DR kanye noMprofethi, ukuhlolwa komdlavuza we-PC wamaphaphu kusetshenziswa umthamo ophansi we-helical CT: imiphumela evela ku-National Lung Screening Study (NLST).J. Med.Isikrini 18, 109–111 (2011).
De Koning, HJ, et al.Ukunciphisa ukufa komdlavuza wamaphaphu ngokuhlolwa kwe-CT ye-volumetric esivivinyweni esingahleliwe.I-Northern England.J. Med.382, 503–513 (2020).
Isikhathi sokuthumela: Sep-18-2023