Ukuxilongwa okwahlukileyo kwee-pulmonary nodules ezichongiweyo yi-computed tomography (CT) kusengumngeni kwindlela yokusebenza kweklinikhi. Apha, sichaza i-metabolome yehlabathi yeesampuli ze-serum ezingama-480, kubandakanya ulawulo olusempilweni, ii-benign lung nodules, kunye ne-stage I lung adenocarcinoma. Ii-Adenocarcinomas zibonisa iiprofayili ze-metabolomic ezizodwa, ngelixa ii-benign nodules kunye nabantu abasempilweni banokufana okuphezulu kwiiprofayili ze-metabolomic. Kwiqela lokufumanisa (n = 306), kwachongwa iseti yee-metabolites ezingama-27 ukuze kwahlulwe phakathi kwee-benign kunye nee-malignant nodules. I-AUC yemodeli yokwahlula kwi-internal validation (n = 104) kunye ne-external validation (n = 111) amaqela yayingama-0.915 kunye nama-0.945, ngokulandelelana. Uhlalutyo lwendlela lubonise ukwanda kwee-glycolytic metabolites ezinxulumene ne-tryptophan enciphileyo kwi-lung adenocarcinoma serum xa kuthelekiswa nee-benign nodules kunye nolawulo olusempilweni, kwaye yacebisa ukuba uptake ye-tryptophan ikhuthaza i-glycolysis kwiiseli zomhlaza wemiphunga. Uphononongo lwethu lugxininisa ixabiso lee-biomarkers ze-serum metabolite ekuvavanyeni umngcipheko wee-nodules ze-pulmonary ezifunyenwe yi-CT.
Ukuxilongwa kwangoko kubalulekile ukuphucula amazinga okusinda kwizigulane ezinomhlaza. Iziphumo ezivela kwi-US National Lung Cancer Screening Trial (NLST) kunye ne-European NELSON Study zibonise ukuba ukuhlolwa nge-computed tomography (LDCT) yedosi ephantsi kunokunciphisa kakhulu ukufa komhlaza wemiphunga kumaqela asemngciphekweni ophezulu1,2,3. Ukusukela oko kwasetyenziswa kakhulu i-LDCT ekuhlolweni komhlaza wemiphunga, ukuvela kweziphumo ze-radiographic ezingalindelekanga zee-pulmonary nodules ezingenazimpawu kuqhubeke kwanda 4. Ii-pulmonary nodules zichazwa njenge-focal opacities ukuya kuthi ga kwi-3 cm ububanzi 5. Sijongene nobunzima ekuhloleni amathuba okuba ne-malignancy kunye nokujongana nenani elikhulu lee-pulmonary nodules ezifunyenwe ngengozi kwi-LDCT. Ukunqongophala kwe-CT kunokukhokelela kwiimviwo zokulandelela rhoqo kunye neziphumo ezingezizo, ezikhokelela ekungeneleleni okungeyomfuneko kunye nonyango olugqithisileyo6. Ke ngoko, kukho imfuneko yokuphuhlisa ii-biomarkers ezithembekileyo neziluncedo zokuchonga ngokuchanekileyo umhlaza wemiphunga kumanqanaba okuqala kunye nokwahlula uninzi lwee-benign nodules ekufumaneni kokuqala 7.
Uhlalutyo olupheleleyo lwemolekyuli yegazi (i-serum, i-plasma, iiseli ze-mononuclear zegazi ezingaphandle), kubandakanya i-genomics, i-proteomics okanye i-DNA methylation8,9,10, kukhokelele ekukhuleni komdla ekufumaneni ii-biomarkers zokuxilonga umhlaza wemiphunga. Okwangoku, iindlela ze-metabolomics zilinganisa iimveliso zokuphela kweseli eziphenjelelwa zizenzo ze-endogenous kunye nezangaphandle kwaye ke ngoko zisetyenziselwa ukuqikelela ukuqala kwesifo kunye nesiphumo. I-Liquid chromatography-tandem mass spectrometry (LC-MS) yindlela esetyenziswa kakhulu kwizifundo ze-metabolomics ngenxa yobuntununtunu bayo obuphezulu kunye noluhlu olukhulu lwe-dynamic, olunokugubungela ii-metabolites ezineempawu ezahlukeneyo ze-physicochemical11,12,13. Nangona uhlalutyo lwe-metabolomic lwehlabathi lwe-plasma/serum lusetyenzisiwe ukuchonga ii-biomarkers ezinxulumene nokuxilongwa komhlaza wemiphunga14,15,16,17 kunye nokusebenza kakuhle konyango, ii-serum metabolite classifiers ezili-18 zokwahlula phakathi kwee-nodules zemiphunga ezinobungozi nezinobungozi zisafuna ukufundwa kakhulu. -uphando olukhulu.
I-Adenocarcinoma kunye ne-squamous cell carcinoma zezona ntlobo zimbini ziphambili zomhlaza wemiphunga ongengoweseli encinci (i-NSCLC). Uvavanyo olwahlukeneyo lwe-CT screening lubonisa ukuba i-adenocarcinoma lolona hlobo luqhelekileyo lomhlaza wemiphunga1,19,20,21. Kolu phononongo, sisebenzise i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) ukwenza uhlalutyo lwe-metabolomics kwiisampuli ze-serum ezingama-695, kubandakanya ulawulo olusempilweni, amaqhuqhuva e-pulmonary angenabungozi, kunye ne-CT-detected ≤3 cm. Uvavanyo lwe-lung adenocarcinoma yeSigaba soku-1. Sichonge iphaneli yee-serum metabolites ezahlula i-lung adenocarcinoma kumaqhuqhuva angenabungozi kunye nolawulo olusempilweni. Uhlalutyo lwendlela yokucebisa lutyhile ukuba i-tryptophan engaqhelekanga kunye ne-glucose metabolism ziinguqu eziqhelekileyo kwi-lung adenocarcinoma xa kuthelekiswa namaqhuqhuva angenabungozi kunye nolawulo olusempilweni. Okokugqibela, simisele saza saqinisekisa i-serum metabolic classifier eneenkcukacha eziphezulu kunye nobuntununtunu bokwahlula phakathi kwamaqhuqhuva e-pulmonary anobungozi nangenabungozi afunyenwe yi-LDCT, anokunceda ekuxilongweni okwahlukileyo kwangoko kunye novavanyo lomngcipheko.
Kolu phononongo lwangoku, iisampulu ze-serum ezihambelanayo ngokwesini nangobudala ziqokelelwe ngasemva kwiilawuli ezili-174 ezisempilweni, izigulane ezingama-292 ezinee-nodules ze-pulmonary ezingenobungozi, kunye nezigulane ezingama-229 ezine-lung adenocarcinoma yesigaba sokuqala. Iimpawu zabantu abangama-695 ziboniswe kwiTheyibhile eyoNgezelelweyo 1.
Njengoko kubonisiwe kuMfanekiso 1a, iisampulu ze-serum ezingama-480 zizonke, kuquka iisampulu ezili-174 ze-healthy control (HC), iisampulu ezili-170 ze-benign nodules (BN), kunye nesampulu ezili-136 ze-stage I lung adenocarcinoma (LA), ziqokelelwe kwiZiko loMhlaza leYunivesithi yaseSun Yat-sen. Iqela lokufumanisa i-metabolomic profiling engajoliswanga kusetyenziswa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Njengoko kubonisiwe kuMfanekiso oNcedisayo 1, ii-metabolites ezahlukeneyo phakathi kwe-LA kunye ne-HC, i-LA kunye ne-BN zichongiwe ukuze kusekwe imodeli yokwahlulahlula kunye nokuphonononga ngakumbi uhlalutyo lwendlela eyahlukileyo. Iisampulu ezili-104 eziqokelelwe yiZiko loMhlaza leYunivesithi yaseSun Yat-sen kunye nesampulu ezili-111 eziqokelelwe zizibhedlele ezibini zivavanywe ngaphakathi nangaphandle, ngokwahlukeneyo.
a Iqela labantu abafundayo kwi-discovery cohort abaye bavavanywa nge-serum metabolomics yehlabathi jikelele besebenzisa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). b Uhlalutyo lwe-Partial least squares discriminant (PLS-DA) ye-total metabolome yeesampuli ze-serum ezingama-480 ezivela kwi-study cohort, kubandakanya ulawulo olusempilweni (HC, n = 174), amaqhuqhuva angenabungozi (BN, n = 170), kunye ne-stage I lung adenocarcinoma (Los Angeles, n = 136). +ESI, i-positive electrospray ionization mode, -ESI, i-negative electrospray ionization mode. c–e IiMetabolites ezine-extra significantly varigities kumaqela amabini anikiweyo (uvavanyo lwe-Wilcoxon olusayinwe nge-two-tailed rank test, false discovery rate adjusted p value, FDR <0.05) ziboniswe ngombala obomvu (fold change > 1.2) kunye nohlaza okwesibhakabhaka (fold change < 0.83). ) ziboniswe kwi-volcano graphic. Imephu yobushushu beqela le-hierarchical ebonisa umahluko omkhulu kwinani lee-metabolites ezichazwe phakathi kwe-LA kunye ne-BN. Idatha yomthombo inikezelwa ngohlobo lweefayile zedatha yomthombo.
I-metabolome iyonke ye-serum ye-174 HC, i-170 BN kunye ne-136 LA kwiqela lokufumanisa ihlalutywe kusetyenziswa uhlalutyo lwe-UPLC-HRMS. Okokuqala sibonisa ukuba iisampulu zolawulo lomgangatho (QC) ziqokelelana ngokuqinileyo embindini wemodeli yohlalutyo lwecandelo eliphambili elingalawulwayo (i-PCA), okuqinisekisa uzinzo lokusebenza kophando lwangoku (Umfanekiso ongezelelweyo 2).
Njengoko kubonisiwe kuhlalutyo oluncinci lwe-partial least squares-discriminant (PLS-DA) kuMfanekiso 1 b, sifumanise ukuba kukho umahluko ocacileyo phakathi kwe-LA kunye ne-BN, i-LA kunye ne-HC kwiindlela ze-ionization ze-electrospray ezi-positive (+ESI) kunye ne-negative (−ESI). Nangona kunjalo, akukho mahluko abalulekileyo afunyenweyo phakathi kwe-BN kunye ne-HC kwiimeko ze-+ESI kunye ne--ESI.
Sifumene iimpawu zomahluko ezingama-382 phakathi kwe-LA kunye ne-HC, iimpawu zomahluko ezingama-231 phakathi kwe-LA kunye ne-BN, kunye neempawu zomahluko ezingama-95 phakathi kwe-BN kunye ne-HC (uvavanyo lwezinga olusayinwe yiWilcoxon, i-FDR <0.05 kunye notshintsho oluninzi >1.2 okanye <0.83) (Umfanekiso .1c-e). . Iincochoyi zichazwe ngakumbi (Idatha eyongezelelweyo 3) ngokuchasene nesiseko sedatha (ilayibrari ye-mzCloud/HMDB/Chemspider) ngexabiso le-m/z, ixesha lokugcina kunye nokukhangela i-mass spectrum yokwahlulwahlulwa (iinkcukacha ezichazwe kwicandelo leeNdlela) 22. Okokugqibela, ii-metabolites ezingama-33 kunye nama-38 ezichazwe ngeenkcukacha ezinomahluko omkhulu ngobuninzi zichongiwe kwi-LA ngokuchasene ne-BN (Umfanekiso 1f kunye neTheyibhile eyongezelelweyo 2) kunye ne-LA ngokuchasene ne-HC (Umfanekiso eyongezelelweyo 3 kunye neTheyibhile eyongezelelweyo 2), ngokwahlukeneyo. Ngokwahlukileyo koko, zi-metabolites ezi-3 kuphela ezinomahluko omkhulu ngobuninzi ezichongiweyo kwi-BN nakwi-HC (Itheyibhile eyoNgezelelweyo 2), ehambelana nokudibana phakathi kwe-BN kunye ne-HC kwi-PLS-DA. Ezi metabolites zahlukileyo zigubungela uluhlu olubanzi lwee-biochemicals (Umfanekiso oNgezelelweyo 4). Xa zizonke, ezi ziphumo zibonisa utshintsho olukhulu kwi-serum metabolome ebonisa utshintsho olubi lomhlaza wemiphunga kwinqanaba lokuqala xa kuthelekiswa nee-benign lung nodules okanye abantu abasempilweni. Okwangoku, ukufana kwe-serum metabolome ye-BN kunye ne-HC kubonisa ukuba ii-benign pulmonary nodules zinokwabelana ngeempawu ezininzi zebhayoloji nabantu abasempilweni. Ngenxa yokuba utshintsho lwe-epidermal growth factor receptor (EGFR) gene luqhelekile kwi-lung adenocarcinoma subtype 23, sifuna ukufumanisa impembelelo yotshintsho lomqhubi kwi-serum metabolome. Emva koko sihlalutye iprofayili iyonke ye-metabolomic yamatyala angama-72 anesimo se-EGFR kwiqela le-lung adenocarcinoma. Okunomdla kukuba, sifumene iiprofayili ezifanayo phakathi kwezigulane ezine-EGFR mutant (n = 41) kunye nezigulane ze-EGFR wild-type (n = 31) kuhlalutyo lwe-PCA (Umfanekiso oNcedisayo 5a). Nangona kunjalo, sichonge ii-metabolites ezi-7 ezibuninzi bazo butshintshe kakhulu kwizigulane ezine-EGFR mutation xa kuthelekiswa nezigulane ezine-EGFR wild-type (t test, p < 0.05 kunye ne-fold change > 1.2 okanye < 0.83) (Umfanekiso oNcedisayo 5b). Uninzi lwezi metabolites (5 kwi-7) yi-acylcarnitines, edlala indima ebalulekileyo kwiindlela ze-oxidation ye-fatty acid.
Njengoko kubonisiwe kumsebenzi oboniswe kuMfanekiso 2 a, ii-biomarkers zokuhlelwa kwamaqhuqhuva zifunyenwe kusetyenziswa ii-operators ze-shrinkage ezingaphantsi kwe-absolute kunye nokukhetha okusekwe kwi-33 differential metabolites ezichongiweyo kwi-LA (n = 136) kunye ne-BN (n = 170). Eyona ndibaniselwano ilungileyo yeenguqu (i-LASSO) - imodeli ye-binary logistic regression. Ukuqinisekiswa kwe-cross-validation okuphindwe kalishumi kusetyenzisiwe ukuvavanya ukuthembeka kwemodeli. Ukukhethwa kwe-variable kunye nokulungiswa kweparameter kulungiswa ngesohlwayo sokwandisa amathuba ngeparameter λ24. Uhlalutyo lwe-metabolomics lwehlabathi lwenziwe ngokuzimeleyo kumaqela okuqinisekiswa kwangaphakathi (n = 104) kunye nokuqinisekiswa kwangaphandle (n = 111) ukuvavanya ukusebenza kokuhlelwa kwemodeli ye-discriminant. Ngenxa yoko, ii-metabolites ezingama-27 kwiseti yokufumanisa zichongiwe njengemodeli ye-discriminant engcono kakhulu enexabiso elikhulu le-AUC eliphakathi (Umzobo 2b), phakathi kwazo ezi-9 zazinomsebenzi owandisiweyo kwaye ezili-18 umsebenzi wehlile kwi-LA xa kuthelekiswa ne-BN (Umzobo 2c).
Ukuhamba komsebenzi wokwakha i-pulmonary nodule classifier, kubandakanya ukukhetha iphaneli engcono kakhulu ye-serum metabolites kwiseti yokufumanisa kusetyenziswa imodeli yokuguqula i-binary logistic ngokusebenzisa ukuqinisekiswa okuphindwe kalishumi kunye nokuvavanya ukusebenza kokuqikelela kwiiseti zokuqinisekisa zangaphakathi nangaphandle. b Izibalo zokuqinisekisa ezinqamlezileyo zemodeli yokuguqula i-LASSO yokukhetha i-biomarker ye-metabolic. Amanani anikiweyo apha ngasentla amele inani eliqhelekileyo lee-biomarkers ezikhethiweyo kwi-λ enikiweyo. Umgca onamachaphaza abomvu umele ixabiso eliphakathi le-AUC kwi-lambda ehambelanayo. Iibha zempazamo ezingwevu zimele amaxabiso amancinci kunye naphezulu e-AUC. Umgca onamachaphaza ubonisa imodeli engcono kakhulu ene-27 biomarkers ezikhethiweyo. I-AUC, indawo engaphantsi kwe-receiver operating characteristic (ROC) curve. c Utshintsho olugobileyo lwee-metabolites ezikhethiweyo ezingama-27 kwiqela le-LA xa kuthelekiswa neqela le-BN kwiqela lokufumanisa. Ikholamu ebomvu - ukusebenza. Ikholamu eluhlaza okwesibhakabhaka kukwehla. d–f Ii-curves ze-Receiver operating characteristic (ROC) ezibonisa amandla emodeli yokwahlula ngokusekelwe kwi-27 metabolite combinations kwi-discovery, internal, kunye ne-external validation sets. Idatha yomthombo inikezelwa ngendlela yeefayile zedatha yomthombo.
Imodeli yokuqikelela yenziwe ngokusekelwe kwii-coefficients ze-regression ezilinganisiweyo zezi metabolites ezingama-27 (Itheyibhile eyoNgezelelweyo 3). Uhlalutyo lwe-ROC olusekelwe kwezi metabolites ezingama-27 luvelise indawo ephantsi kwexabiso le-curve (AUC) le-0.933, uvakalelo lweqela lokufumanisa yayiyi-0.868, kwaye ukucaciswa yayiyi-0.859 (Umzobo 2d). Okwangoku, phakathi kwee-metabolites ezingama-38 ezichaziweyo phakathi kwe-LA kunye ne-HC, iseti yee-metabolites ezili-16 ifumene i-AUC ye-0.902 enovakalelo lwe-0.801 kunye nokucaciswa kwe-0.856 ekuhlukaniseni i-LA kwi-HC (Umfanekiso oyoNgezelelweyo 6a-c). Amaxabiso e-AUC asekelwe kwimida eyahlukeneyo yotshintsho lwe-fold kwii-metabolites ezahlukeneyo nawo athelekiswa. Sifumanise ukuba imodeli yohlulo isebenze ngcono ekuhlukaniseni phakathi kwe-LA kunye ne-BN (HC) xa inqanaba lotshintsho lwe-fold limiselwe kwi-1.2 ngokuchasene ne-1.5 okanye i-2.0 (Umfanekiso oyoNgezelelweyo 7a,b). Imodeli yokwahlulahlula, esekelwe kumaqela angama-27 e-metabolite, iqinisekiswe ngakumbi kwii-cohorts zangaphakathi nezangaphandle. I-AUC yayiyi-0.915 (uvakalelo 0.867, ukucaciswa 0.811) yokuqinisekiswa kwangaphakathi kunye ne-0.945 (uvakalelo 0.810, ukucaciswa 0.979) yokuqinisekiswa kwangaphandle (Umzobo 2e, f). Ukuvavanya ukusebenza kakuhle kwe-interlaboratory, iisampuli ezingama-40 ezivela kwi-cohort yangaphandle zahlalutywa kwilabhoratri yangaphandle njengoko kuchaziwe kwicandelo leeNdlela. Ukuchaneka kokwahlulahlula kufezekise i-AUC ye-0.925 (Umfanekiso oNcedisayo 8). Ngenxa yokuba i-lung squamous cell carcinoma (LUSC) lolona hlobo lwesibini oluqhelekileyo lomhlaza wemiphunga we-non-small cell lung cancer (NSCLC) emva kwe-lung adenocarcinoma (LUAD), sikwavavanyile ukusetyenziswa okuqinisekisiweyo kweeprofayili ze-metabolic. I-BN kunye namatyala ali-16 e-LUSC. I-AUC yocalucalulo phakathi kwe-LUSC kunye ne-BN yayiyi-0.776 (Umfanekiso ongezelelweyo 9), nto leyo ebonisa ubuchule obuphantsi xa kuthelekiswa nocalucalulo phakathi kwe-LUAD kunye ne-BN.
Izifundo zibonise ukuba ubungakanani bee-nodules kwimifanekiso ye-CT bunxulumene kakuhle namathuba okuba ne-malignancy kwaye buhlala buyinto ephambili ebangela unyango lwee-nodules25,26,27. Uhlalutyo lwedatha evela kwiqela elikhulu le-NELSON screening study lubonise ukuba umngcipheko we-malignancy kubantu abane-nodes <5 mm wawufana nowe-subsexuals ezingenazo ii-nodes28. Ke ngoko, ubungakanani obuncinci obufuna ukubekwa esweni rhoqo kwe-CT yi-5 mm, njengoko kucetyisiwe yiBritish Thoracic Society (BTS), kunye ne-6 mm, njengoko kucetyisiwe yiFleischner Society29. Nangona kunjalo, ii-nodules ezinkulu kune-6 mm kwaye ezingenazo iimpawu ezicacileyo ezingabonakaliyo, ezibizwa ngokuba zii-indeterminate pulmonary nodules (IPN), zihlala zingumngeni omkhulu kuvavanyo kunye nolawulo kwi-clinical practice30,31. Okulandelayo sihlolisise ukuba ubungakanani bee-nodules buchaphazele na i-metabolomic signatures sisebenzisa iisampulu ezidityanisiweyo ezivela kwii-cohorts zokufumanisa kunye nokuqinisekiswa kwangaphakathi. Sigxile kwi-biomarkers ezingama-27 eziqinisekisiweyo, siqale sathelekisa iiprofayili ze-PCA ze-HC kunye ne-BN sub-6 mm metabolomes. Sifumanise ukuba uninzi lwamanqaku edatha ye-HC kunye ne-BN ahambelana, nto leyo ebonisa ukuba amanqanaba e-serum metabolite ayefana kuwo omabini amaqela (Umzobo 3a). Iimaphu zeempawu kuzo zonke iintlobo ezahlukeneyo zobukhulu zahlala zigciniwe kwi-BN nakwi-LA (Umzobo 3b, c), ngelixa ukwahlukana kwabonwa phakathi kwamaqhuqhuva anobungozi kunye nalawo angenabungozi kuluhlu lwe-6-20 mm (Umzobo 3d). Eli qela lalinomlinganiselo we-AUC we-0.927, ubunyani be-0.868, kunye nobuthathaka be-0.820 bokuqikelela umhlaza wamaqhuqhuva obungakanani be-6 ukuya kwi-20 mm (Umzobo 3e, f). Iziphumo zethu zibonisa ukuba i-classifier inokubamba utshintsho lwe-metabolic olubangelwa kukutshintsha kwe-malignant kwangoko, nokuba ubungakanani bamaqhuqhuva bunjani.
Intengiso Uthelekiso lweeprofayili ze-PCA phakathi kwamaqela athile asekelwe kwi-metabolic classifier ye-27 metabolites. CC kunye ne-BN < 6 mm. b BN < 6 mm vs BN 6–20 mm. kwi-LA 6–20 mm xa kuthelekiswa ne-LA 20–30 mm. g BN 6–20 mm kunye ne-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 I-Receiver operating characteristic (ROC) curve ebonisa ukusebenza kwemodeli yokwahlula kwii-nodules 6–20 mm. f Amaxabiso okunokwenzeka abalwe ngokusekelwe kwimodeli yokubuyela umva kwe-logistic kwii-nodules ezilinganisa i-6–20 mm. Umgca ompunga onamachaphaza umele ixabiso elifanelekileyo lokunqunyulwa (0.455). Amanani angentla amele ipesenti yamatyala aqikelelweyo eLos Angeles. Sebenzisa uvavanyo lwe-t lomfundi olunemisila emibini. I-PCA, uhlalutyo lwecandelo eliphambili. Indawo ye-AUC phantsi kwegophe. Idatha yomthombo inikezelwa ngohlobo lweefayile zedatha yomthombo.
Iisampuli ezine (ezineminyaka engama-44-61 ubudala) ezinobukhulu obufanayo bee-pulmonary nodules (7-9 mm) zikhethwe ngakumbi ukubonisa ukusebenza kwemodeli yokuqikelela umhlaza ecetywayo (Umzobo 4a, b). Ekuhlolweni kokuqala, iTyala 1 labonakala njenge-solid nodule ene-calcification, uphawu oluhambelana nokungabi nabungozi, ngelixa iTyala 2 labonakala njenge-indeterminate partial solid nodule engenazo iimpawu ezicacileyo ezinobungozi. Ii-CT scans ezintathu ezilandelayo zibonise ukuba la matyala ahlala ezinzile kwixesha leminyaka emi-4 kwaye ngenxa yoko athathwa njengee-benign nodules (Umzobo 4a). Xa kuthelekiswa novavanyo lweklinikhi lwee-serial CT scans, uhlalutyo lwe-single-shot serum metabolite kunye nemodeli yangoku yokwahlulahlula ngokukhawuleza nangokuchanekileyo zichonge ezi-benign nodules ngokusekelwe kwimida enokwenzeka (Itheyibhile 1). Umfanekiso 4b kwimeko yesi-3 ubonisa i-nodule eneempawu zokurhoxa kwe-pleural, edla ngokunxulunyaniswa ne-malignancy32. Ityala 4 labonakala njenge-indeterminate partial solid nodule engenabungqina besizathu esinobungozi. Zonke ezi meko zaxelwa kwangaphambili njenge-malignant ngokwemodeli yokwahlulahlula (Itheyibhile 1). Uvavanyo lwe-lung adenocarcinoma luboniswe ngovavanyo lwe-histopathological emva kotyando lokususwa kwemiphunga (Umzobo 4b). Kwiseti yokuqinisekisa yangaphandle, i-metabolism classifier ixele ngokuchanekileyo iimeko ezimbini ze-indeterminate lung nodules ezinkulu kune-6 mm (Umfanekiso ongezelelweyo 10).
Imifanekiso ye-CT yefestile ye-axial yemiphunga yamatyala amabini ee-nodules ezingenobungozi. Kwimeko yoku-1, i-CT scan emva kweminyaka emi-4 ibonise i-nodule eqinileyo ezinzileyo enomlinganiselo we-7 mm ene-calcification kwi-lobe esezantsi yasekunene. Kwimeko yesi-2, i-CT scan emva kweminyaka emi-5 ibonise i-nodule eqinileyo, enxalenye iqinileyo enobubanzi be-7 mm kwi-lobe ephezulu yasekunene. b I-Axial window Imifanekiso ye-CT yemiphunga kunye nezifundo ze-pathological ezifanayo zamatyala amabini e-stage I adenocarcinoma ngaphambi kokususwa kwemiphunga. Kwimeko yesi-3 ibonise i-nodule enobubanzi be-8 mm kwi-lobe ephezulu yasekunene ene-pleural retraction. Kwimeko yesi-4 ibonise i-nodule yeglasi yomhlaba eqinileyo enomlinganiselo we-9 mm kwi-lobe ephezulu yasekhohlo. Ukufakwa kwe-Hematoxylin kunye ne-eosin (H&E) kwizicubu zemiphunga ezisusiweyo (scale bar = 50 μm) ebonisa ipateni yokukhula kwe-acinar ye-lung adenocarcinoma. Iintolo zibonisa ii-nodules ezifunyenwe kwimifanekiso ye-CT. Imifanekiso ye-H&E yimifanekiso emele amasimi amaninzi (>3) e-microscopic ahlolwe yi-pathologist.
Xa zizonke, iziphumo zethu zibonisa ixabiso elinokubakho lee-biomarkers ze-serum metabolite kwi-diagnostic differential ye-pulmonary nodules, ezinokubangela imingeni xa kuvavanywa ukuhlolwa kwe-CT.
Ngokusekelwe kwiphaneli ye-metabolite eyahlukileyo eqinisekisiweyo, sifuna ukuchonga ii-correlates zebhayoloji zotshintsho olukhulu lwe-metabolic. Uhlalutyo lwe-KEGG pathway enrichment lwe-MetaboAnalyst luchonge iindlela ezi-6 eziqhelekileyo ezitshintshileyo kakhulu phakathi kwamaqela amabini anikiweyo (i-LA vs. HC kunye ne-LA vs. BN, ilungisiwe i-p ≤ 0.001, isiphumo > 0.01). Olu tshintsho lubonakaliswe kukuphazamiseka kwi-metabolism ye-pyruvate, i-tryptophan metabolism, i-niacin kunye ne-nicotinamide metabolism, i-glycolysis, umjikelo we-TCA, kunye ne-purine metabolism (Umzobo 5a). Emva koko senze i-metabolomics ejoliswe kuyo ukuqinisekisa utshintsho olukhulu sisebenzisa i-absolute quantification. Ukuchonga ii-metabolites eziqhelekileyo kwiindlela eziguquliweyo eziqhelekileyo yi-triple quadrupole mass spectrometry (QQQ) sisebenzisa imigangatho ye-metabolite yokwenyani. Iimpawu zedemografi zesampulu yophando lwe-metabolomics zifakiwe kwiTheyibhile eyoNgezelelweyo 4. Ngokuhambelana neziphumo zethu zehlabathi ze-metabolomics, uhlalutyo lobungakanani luqinisekisile ukuba i-hypoxanthine kunye ne-xanthine, i-pyruvate, kunye ne-lactate zonyukile kwi-LA xa kuthelekiswa ne-BN kunye ne-HC (Umzobo 5b, c, p <0.05). Nangona kunjalo, akukho mahluko abalulekileyo kwezi metabolites afunyenweyo phakathi kwe-BN kunye ne-HC.
Uhlalutyo lwe-KEGG pathway enrichment lwe-metabolites ezahlukeneyo kakhulu kwiqela le-LA xa kuthelekiswa namaqela e-BN kunye ne-HC. Kusetyenziswe uvavanyo lweGlobal olunemisila emibini, kwaye amaxabiso e-p ahlengahlengiswa kusetyenziswa indlela yeHolm-Bonferroni (ilungisiwe i-p ≤ 0.001 kunye nobukhulu besiphumo > 0.01). b–d Iiploti zeViolin ezibonisa amanqanaba e-hypoxanthine, xanthine, lactate, pyruvate, kunye ne-tryptophan kwi-serum HC, BN, kunye ne-LA emiselwe yi-LC-MS/MS (n = 70 kwiqela ngalinye). Imigca emhlophe nemnyama enamachaphaza ibonisa i-median kunye ne-quartile, ngokulandelelana. e Iploti yeViolin ebonisa ukubonakaliswa kwe-mRNA ye-Log2TPM eqhelekileyo (iitranscripts ngesigidi) ye-SLC7A5 kunye ne-QPRT kwi-lung adenocarcinoma (n = 513) xa kuthelekiswa nezicubu zemiphunga eziqhelekileyo (n = 59) kwi-dataset ye-LUAD-TCGA. Ibhokisi emhlophe imele uluhlu lwe-interquartile, umgca omnyama othe tye embindini umele i-median, kwaye umgca omnyama othe nkqo ovela ebhokisini umele i-95% confidence interval (CI). f I-Pearson correlation plot ye-SLC7A5 kunye ne-GAPDH expression kwi-lung adenocarcinoma (n = 513) kunye ne-normal lung tissue (n = 59) kwi-dataset ye-TCGA. Indawo engwevu imele i-95% CI. r, i-Pearson coefficient coefficient. g Amanqanaba e-tryptophan eseli aqhelekileyo kwiiseli ze-A549 adluliselwe ngolawulo lwe-shRNA olungenazo ii-specific (NC) kunye ne-shSLC7A5 (Sh1, Sh2) emiselwe yi-LC-MS/MS. Uhlalutyo lwezibalo lweesampuli ezintlanu ezizimeleyo ngokwebhayoloji kwiqela ngalinye luveziwe. h Amanqanaba eeseli ze-NADt (i-NAD iyonke, kuquka i-NAD+ kunye ne-NADH) kwiiseli ze-A549 (NC) kunye neeseli ze-SLC7A5 knockdown A549 (Sh1, Sh2). Uhlalutyo lwezibalo lweesampuli ezintathu ezizimeleyo ngokwebhayoloji kwiqela ngalinye luveziwe. Umsebenzi we-Glycolytic weeseli ze-A549 ngaphambi nasemva kokuwa kwe-SLC7A5 ulinganiswe ngesantya se-extracellular acidification (ECAR) (n = iisampuli ezi-4 ezizimeleyo ngokwebhayoloji kwiqela ngalinye). I-2-DG,2-deoxy-D-glucose. Uvavanyo lwe-t lomfundi olunemisila emibini lusetyenziswe kwi-(b–h). Kwi-(g–i), iibha zempazamo zimele i-± SD ephakathi, uvavanyo ngalunye lwenziwe kathathu ngokuzimeleyo kwaye iziphumo bezifana. Idatha yomthombo inikezelwa ngohlobo lweefayile zedatha yomthombo.
Ukuqwalasela impembelelo ebalulekileyo yotshintsho kwimetabolism ye-tryptophan kwiqela le-LA, sikwavavanya amanqanaba e-serum tryptophan kumaqela e-HC, BN, kunye ne-LA sisebenzisa i-QQQ. Sifumanise ukuba i-serum tryptophan yehlisiwe kwi-LA xa ithelekiswa ne-HC okanye i-BN (p < 0.001, Umfanekiso 5d), ohambelana neziphumo zangaphambili zokuba amanqanaba e-tryptophan ajikelezayo aphantsi kwizigulane ezinomhlaza wemiphunga kunakwii-control ezisempilweni ezivela kwiqela lolawulo33,34,35. Olunye uphando olusebenzisa i-PET/CT tracer 11C-methyl-L-tryptophan lufumanise ukuba ixesha lokugcina i-tryptophan signal kwizicubu zomhlaza wemiphunga landa kakhulu xa kuthelekiswa nezilonda ezingalunganga okanye izicubu eziqhelekileyo36. Sicinga ukuba ukwehla kwe-tryptophan kwi-serum ye-LA kunokubonisa ukufunxwa kwe-tryptophan esebenzayo ziiseli zomhlaza wemiphunga.
Kwakhona kwaziwa ukuba imveliso yokugqibela yendlela ye-kynurenine ye-tryptophan catabolism yi-NAD+37,38, eyisiseko esibalulekileyo sempendulo ye-glyceraldehyde-3-phosphate kunye ne-1,3-bisphosphoglycerate kwi-glycolysis39. Ngelixa izifundo zangaphambili zigxile kwindima ye-tryptophan catabolism kulawulo lomzimba, sifuna ukucacisa ukusebenzisana phakathi kwe-tryptophan dysregulation kunye neendlela ze-glycolytic ezibonwe kolu phononongo lwangoku. I-Solute transporter family 7 member 5 (SLC7A5) yaziwa ngokuba yi-tryptophan transporter43,44,45. I-Quinolinic acid phosphoribosyltransferase (QPRT) yi-enzyme efumaneka ngezantsi kwendlela ye-kynurenine eguqula i-quinolinic acid ibe yi-NAMN46. Ukuhlolwa kwedatha ye-LUAD TCGA kubonise ukuba zombini i-SLC7A5 kunye ne-QPRT zinyuswe kakhulu kwizicubu ze-tumor xa kuthelekiswa nezicubu eziqhelekileyo (Umzobo 5e). Olu nyuko lubonwe kwizigaba I kunye ne-II kunye nezigaba III kunye ne-IV ze-lung adenocarcinoma (Umfanekiso Ongezelelweyo 11), obonisa ukuphazamiseka kwangethuba kwi-metabolism ye-tryptophan enxulumene ne-tumorigenesis.
Ukongeza, iseti yedatha ye-LUAD-TCGA ibonise unxibelelwano oluhle phakathi kwe-SLC7A5 kunye nokubonakaliswa kwe-GAPDH mRNA kwiisampuli zezigulane ezinomhlaza (r = 0.45, p = 1.55E-26, Umfanekiso 5f). Ngokwahlukileyo koko, akukho lwalamano lubalulekileyo olufunyenweyo phakathi kwezi signatures zejini kwizicubu zemiphunga eziqhelekileyo (r = 0.25, p = 0.06, Umfanekiso 5f). Ukuthotywa kwe-SLC7A5 (Umfanekiso ongezelelweyo 12) kwiiseli ze-A549 kunciphisa kakhulu amanqanaba e-tryptophan yeselula kunye ne-NAD(H) (Umfanekiso 5g,h), okubangela umsebenzi we-glycolytic onciphileyo njengoko ulinganiswa yi-extracellular acidification rate (ECAR) (Umfanekiso 1). 5i). Ke ngoko, ngokusekelwe kutshintsho lwe-metabolic kwi-serum kunye nokufunyanwa kwe-in vitro, sicinga ukuba i-tryptophan metabolism inokuvelisa i-NAD+ ngendlela ye-kynurenine kwaye idlale indima ebalulekileyo ekukhuthazeni i-glycolysis kumhlaza wemiphunga.
Izifundo zibonise ukuba inani elikhulu lee-nodules ze-pulmonary ezingaziwayo ezifunyenwe yi-LDCT zinokukhokelela kwisidingo sovavanyo olongezelelweyo olufana ne-PET-CT, i-lung biopsy, kunye nonyango olugqithisileyo ngenxa yokuxilongwa kwe-malignancy engalunganga.31 Njengoko kubonisiwe kuMfanekiso 6, uphando lwethu luchonge iphaneli yee-metabolites ze-serum ezinexabiso elinokubakho lokuxilonga elinokuphucula ukwahlulwahlulwa komngcipheko kunye nolawulo olulandelayo lwee-nodules ze-pulmonary ezifunyenwe yi-CT.
Amaqhuqhuva eempumlo avavanywa kusetyenziswa i-computed tomography (LDCT) eneempawu zomfanekiso ezibonisa izizathu ezingezizo okanye ezimbi. Isiphumo esingaqinisekanga samaqhuqhuva sinokukhokelela ekutyelelweni rhoqo, ukungenelela okungeyomfuneko, kunye nonyango olugqithisileyo. Ukufakwa kwe-serum metabolic classifiers ezinexabiso lokuxilonga kunokuphucula uvavanyo lomngcipheko kunye nolawulo olulandelayo lwamaqhuqhuva eempumlo. I-PET positron emission tomography.
Idatha evela kuphando lwe-US NLST kunye nophando lwe-European NELSON lubonisa ukuba ukuhlolwa kwamaqela asengozini ephezulu nge-computed tomography (LDCT) yedosi ephantsi kunokunciphisa ukufa kwabantu abanomhlaza wemiphunga1,3. Nangona kunjalo, uvavanyo lomngcipheko kunye nolawulo lweklinikhi olulandelayo lwamanani amakhulu ee-pulmonary nodules ezifunyenwe yi-LDCT zihlala zingumngeni omkhulu. Injongo ephambili kukuphucula ukuhlelwa ngokuchanekileyo kweenkqubo ezikhoyo ezisekelwe kwi-LDCT ngokubandakanya ii-biomarkers ezinokuthenjwa.
Ezinye ii-biomarkers zemolekyuli, ezifana nee-metabolites zegazi, zichongiwe ngokuthelekisa umhlaza wemiphunga kunye nee-controls eziphilileyo15,17. Kolu phononongo lwangoku, sigxile ekusetyenzisweni kohlalutyo lwe-serum metabolomics ukwahlula phakathi kwee-nodules ze-pulmonary ezingalunganga nezingalunganga ezifunyenwe ngengozi yi-LDCT. Sithelekise iisampulu ze-serum metabolome yehlabathi yolawulo olusempilweni (HC), ii-nodules ze-lung ezingalunganga (BN), kunye neesampulu ze-lung adenocarcinoma (LA) zesigaba sokuqala sisebenzisa uhlalutyo lwe-UPLC-HRMS. Sifumanise ukuba i-HC kunye ne-BN zineprofayili ezifanayo ze-metabolic, ngelixa i-LA ibonise utshintsho olukhulu xa kuthelekiswa ne-HC kunye ne-BN. Sichonge iiseti ezimbini zee-serum metabolites ezahlula i-LA kwi-HC kunye ne-BN.
Inkqubo yokuchonga amaqhuqhuva angengobungozi nanobungozi ngokusekwe kwi-LDCT isekelwe kakhulu kubukhulu, uxinano, imo kunye nesantya sokukhula kwamaqhuqhuva ngokuhamba kwexesha. Izifundo zangaphambili zibonise ukuba ubukhulu bamaqhuqhuva bunxulumene kakhulu nethuba lomhlaza wemiphunga. Kwanakwizigulane ezisengozini enkulu, umngcipheko womhlaza kumaqhuqhuva angaphantsi kwe-6 mm yi-<1%. Umngcipheko womhlaza kumaqhuqhuva aphakathi kwe-6 ukuya kwi-20 mm ususela kwi-8% ukuya kwi-64%30. Ke ngoko, iFleischner Society icebisa ububanzi be-cutoff obuyi-6 mm ukuze kulandelwe i-CT rhoqo. 29 Nangona kunjalo, uvavanyo lomngcipheko kunye nolawulo lwamaqhuqhuva angenasiphelo (IPN) amakhulu kune-6 mm alwenziwanga ngokwaneleyo 31. Ulawulo lwangoku lwesifo sentliziyo oluhlala luhleli lusekelwe ekulindeni ngononophelo kunye nokujonga rhoqo i-CT.
Ngokusekelwe kwi-metabolome eqinisekisiweyo, sibonise okokuqala ukudibana kweempawu ze-metabolomic phakathi kwabantu abaphilileyo kunye nama-nodules angenabungozi <6 mm. Ukufana kwezinto eziphilayo kuhambelana neziphumo zangaphambili ze-CT zokuba umngcipheko womhlaza wamaqhuqhuva <6 mm uphantsi njengakwizifundo ezingenamaqhuqhuva.30 Kufuneka kuqatshelwe ukuba iziphumo zethu zibonisa nokuba amaqhuqhuva angenabungozi <6 mm kunye ≥6 mm afana kakhulu kwiiprofayili ze-metabolomic, nto leyo ebonisa ukuba inkcazo yokusebenza ye-etiology engalunganga iyahambelana nokuba ubukhulu bamaqhuqhuva bunjani. Ke ngoko, iiphaneli ze-serum metabolite zokuxilonga zanamhlanje zinokubonelela ngovavanyo olunye njengovavanyo olumiselweyo xa amaqhuqhuva efunyanwa okokuqala kwi-CT kwaye anokunciphisa ukujonga ngokulandelelana. Kwangaxeshanye, iphaneli efanayo yee-biomarkers ze-metabolic yahlula amaqhuqhuva anobungozi ≥6 mm ngobukhulu kumaqhuqhuva angenabungozi kwaye yabonelela ngoqikelelo oluchanekileyo lwee-IPN ezinobukhulu obufanayo kunye neempawu ze-morphological ezingacacanga kwimifanekiso ye-CT. Olu hlobo lwe-serum metabolism lusebenze kakuhle ekuqikeleleni umhlaza wamaqhuqhuva ≥6 mm nge-AUC ye-0.927. Xa zizonke, iziphumo zethu zibonisa ukuba iimpawu ze-serum metabolomic ezizodwa zinokubonakalisa ngokukodwa utshintsho lwe-metabolic olubangelwa yi-tumor kwaye zibe nexabiso elinokubakho njengezibikezeli zomngcipheko, ngaphandle kobukhulu be-nodule.
Okuphawulekayo kukuba, i-lung adenocarcinoma (LUAD) kunye ne-squamous cell carcinoma (LUSC) zezona ntlobo ziphambili zomhlaza wemiphunga ongengoweseli encinci (NSCLC). Ngenxa yokuba i-LUSC inxulunyaniswa kakhulu nokusetyenziswa kwecuba47 kwaye i-LUAD yeyona histology ixhaphakileyo yee-lung nodules ezingalindelekanga ezifunyenwe kwi-CT screening48, imodeli yethu yokwahlulahlula yakhelwe ngokukodwa iisampuli ze-adenocarcinoma yesigaba soku-1. UWang kunye noogxa bakhe bagxile kwi-LUAD kwaye bachonga iimpawu ezilithoba ze-lipid besebenzisa i-lipidomics ukwahlula umhlaza wemiphunga wesigaba sokuqala kubantu abaphilileyo17. Sivavanye imodeli yokwahlulahlula yangoku kwiimeko ezili-16 zesigaba soku-1 se-LUSC kunye nee-nodules ezingama-74 ezingalunganga kwaye sabona ukuchaneka kokuqikelela kwe-LUSC okuphantsi (AUC 0.776), nto leyo ebonisa ukuba i-LUAD kunye ne-LUSC zinokuba neempawu zazo ze-metabolomic. Enyanisweni, i-LUAD kunye ne-LUSC zibonakalisiwe ukuba zahlukile kwi-etiology, imvelaphi yebhayoloji kunye nokuphazamiseka kwemfuza49. Ngoko ke, ezinye iintlobo ze-histology kufuneka zifakwe kwiimodeli zoqeqesho zokufumanisa umhlaza wemiphunga ngokusekelwe kubemi kwiinkqubo zokuhlola.
Apha, sichonge iindlela ezintandathu ezihlala zitshintsha kwi-lung adenocarcinoma xa kuthelekiswa nolawulo olusempilweni kunye namaqhuqhuva angenabungozi. I-Xanthine kunye ne-hypoxanthine zii-metabolites eziqhelekileyo zendlela ye-purine metabolic. Ngokuhambelana neziphumo zethu, ii-intermediates ezinxulumene ne-purine metabolism zandiswe kakhulu kwi-serum okanye kwizicubu zezigulane ezine-lung adenocarcinoma xa kuthelekiswa nolawulo olusempilweni okanye izigulane ezikwinqanaba lokuqala lokuhlasela15,50. Amanqanaba aphezulu e-serum xanthine kunye ne-hypoxanthine anokubonisa i-anabolism efunekayo kwiiseli zomhlaza ezikhula ngokukhawuleza. Ukungasebenzi kakuhle kwe-glucose metabolism luphawu olwaziwayo lwe-cancer metabolism51. Apha, sibonile ukwanda okukhulu kwe-pyruvate kunye ne-lactate kwiqela le-LA xa kuthelekiswa neqela le-HC kunye ne-BN, elihambelana neengxelo zangaphambili zokungahambi kakuhle kwendlela ye-glycolytic kwiprofayili ye-serum metabolome yezigulane zomhlaza wemiphunga ezingezizo ezincinci (i-NSCLC) kunye nolawulo olusempilweni. iziphumo ziyafana52,53.
Okubalulekileyo, sibonile ulwalamano oluchaseneyo phakathi kwe-pyruvate kunye ne-tryptophan metabolism kwi-serum ye-lung adenocarcinomas. Amanqanaba e-serum tryptophan ancitshisiwe kwiqela le-LA xa kuthelekiswa neqela le-HC okanye le-BN. Okunomdla kukuba, uphando olukhulu lwangaphambili olusebenzisa iqela eliza kujonga lufumanise ukuba amanqanaba aphantsi e-tryptophan ejikelezayo adibene nomngcipheko okhulayo womhlaza wemiphunga 54. I-Tryptophan yi-amino acid ebalulekileyo esiyifumana ngokupheleleyo ekutyeni. Sigqiba kwelokuba ukuncipha kwe-serum tryptophan kwi-lung adenocarcinoma kunokubonakalisa ukuncipha ngokukhawuleza kwale metabolite. Kuyaziwa ukuba imveliso yokugqibela ye-tryptophan catabolism ngendlela ye-kynurenine ngumthombo wokwenziwa kwe-de novo NAD+. Ngenxa yokuba i-NAD+ iveliswa ngokuyintloko ngendlela yokusindisa, ukubaluleka kwe-NAD+ kwi-tryptophan metabolism kwimpilo nakwizifo kusafuneka kumiselwe46. Uhlalutyo lwethu lwedathabheyisi ye-TCGA lubonise ukuba ukubonakaliswa kwe-tryptophan transporter solute transporter 7A5 (SLC7A5) kwanda kakhulu kwi-lung adenocarcinoma xa kuthelekiswa nolawulo oluqhelekileyo kwaye kwahambelana kakuhle nokubonakaliswa kwe-glycolytic enzyme GAPDH. Izifundo zangaphambili zigxile kakhulu kwindima ye-tryptophan catabolism ekucinezeleni impendulo yomzimba yokulwa nomhlaza40,41,42. Apha sibonisa ukuba ukuthintela ukufunxwa kwe-tryptophan ngokunciphisa i-SLC7A5 kwiiseli zomhlaza wemiphunga kubangela ukwehla okulandelayo kwamanqanaba e-NAD eseli kunye nokuncipha komsebenzi we-glycolytic ngaxeshanye. Ngamafutshane, uphando lwethu lubonelela ngesiseko sebhayoloji sotshintsho kwi-serum metabolism enxulumene notshintsho olubi lwe-lung adenocarcinoma.
Utshintsho lwe-EGFR lolona tshintsho luqhelekileyo kwizigulane ezine-NSCLC. Kuphononongo lwethu, sifumanise ukuba izigulane ezine-EGFR mutation (n = 41) zine-metabolomic profiles iyonke efana nezigulane ezine-wild-type EGFR (n = 31), nangona sifumene amanqanaba e-serum aphantsi kwezinye izigulane ezine-EGFR mutant kwizigulane ezine-acylcarnitine. Umsebenzi omiselweyo we-acylcarnitines kukuthutha amaqela e-acyl ukusuka kwi-cytoplasm ukuya kwi-mitochondrial matrix, okukhokelela kwi-oxidation ye-fatty acids ukuvelisa amandla 55. Ngokuhambelana neziphumo zethu, uphando lwakutshanje lukwachonge ii-metabolome profiles ezifanayo phakathi kwe-EGFR mutant kunye ne-EGFR wild-type tumors ngokuhlalutya i-metabolome yehlabathi yeesampuli zezicubu ze-lung adenocarcinoma eziyi-1025. Okunomdla kukuba, umxholo we-acylcarnitine nawo ufunyenwe kwiqela le-EGFR mutant. Ke ngoko, nokuba utshintsho kumanqanaba e-acylcarnitine lubonisa utshintsho lwe-metabolic olubangelwa yi-EGFR kunye neendlela ze-molecular ezingaphantsi lunokufaneleka ukuba lufundwe ngakumbi.
Ukuqukumbela, uphando lwethu luseka indlela yokwahlulahlula i-serum metabolic classifier yokuchonga umahluko wee-pulmonary nodules kwaye lucebisa indlela yokusebenza enokwenza ngcono uvavanyo lomngcipheko kwaye iququzelele ulawulo lweklinikhi olusekelwe kuvavanyo lwe-CT scan.
Olu phononongo luvunyiwe yiKomiti yeMithetho yoBuchule beSibhedlele soMhlaza seYunivesithi yaseSun Yat-sen, iSibhedlele sokuQala esiManyeneyo seYunivesithi yaseSun Yat-sen, kunye neKomiti yeMithetho yoBuchule beSibhedlele soMhlaza seYunivesithi yaseZhengzhou. Kumaqela okufunyanwa nokuqinisekiswa kwangaphakathi, ama-sera ali-174 kubantu abaphilileyo kunye nama-sera angama-244 avela kuma-nodule angenabungozi aqokelelwe kubantu abavavanywa rhoqo ngonyaka kwiSebe loLawulo loMhlaza kunye nokuThintela, iZiko loMhlaza leYunivesithi yaseSun Yat-sen, kunye nama-nodule angenabungozi ali-166. I-serum. Ama-lung adenocarcinoma eSigaba soku-1 aqokelelwe kwiZiko loMhlaza leYunivesithi yaseSun Yat-sen. Kwiqela lokuqinisekiswa kwangaphandle, bekukho amatyala angama-48 ama-nodule angenabungozi, amatyala angama-39 e-lung adenocarcinoma yeSigaba soku-1 evela kwiSibhedlele sokuQala esiManyeneyo seYunivesithi yaseSun Yat-sen, kunye namatyala angama-24 e-lung adenocarcinoma yeSigaba soku-1 evela kwiSibhedlele soMhlaza saseZhengzhou. IZiko loMhlaza leYunivesithi iSun Yat-sen likwaqokelele amatyala ali-16 omhlaza wemiphunga we-squamous cell wesigaba sokuqala ukuze kuvavanywe amandla okuxilonga we-metabolic classifier esekwe (iimpawu zesigulana ziboniswe kwiTheyibhile eyoNgezelelweyo 5). Iisampulu ezivela kwi-discovery cohort kunye ne-internal validation cohort zaqokelelwa phakathi kukaJanuwari 2018 noMeyi 2020. Iisampulu ze-external validation cohort zaqokelelwa phakathi kuka-Agasti 2021 no-Okthobha 2022. Ukunciphisa ukukhetha ngokwesini, malunga nenani elilinganayo lamatyala amadoda namabhinqa anikwa kwi-cohort nganye. Iqela loFumaniso kunye neQela loHlolo lwangaphakathi. Isini sabathathi-nxaxheba samiselwa ngokusekelwe kwingxelo yabo. Imvume enolwazi yafunyanwa kubo bonke abathathi-nxaxheba kwaye akukho mbuyekezo inikezelweyo. Abantu abane-benign nodules ngabo babene-CT scan score ezinzileyo kwiminyaka emi-2 ukuya kwemi-5 ngexesha lohlalutyo, ngaphandle kwetyala eli-1 kwisampulu yoqinisekiso lwangaphandle, eyaqokelelwa ngaphambi kotyando kwaye yafunyaniswa yi-histopathology. Ngaphandle kwe-bronchitis engapheliyo. Amatyala e-lung adenocarcinoma aqokelelwa ngaphambi kokususwa kwemiphunga kwaye aqinisekiswa ngokuxilongwa kwe-pathological. Iisampuli zegazi zokuzila ukutya zaqokelelwa kwiityhubhu zokwahlukanisa i-serum ngaphandle kwezixhobo zokuthintela ukugabha kwegazi. Iisampuli zegazi zavalwa iyure e-1 kubushushu begumbi zaza zafakwa kwi-centrifuge kwi-2851 × g imizuzu eli-10 kwi-4°C ukuze kuqokelelwe i-serum supernatant. Ii-serum aliquots zaqandiswa kwi--80°C de kwakhutshwa i-metabolite. ISebe loThintelo loMhlaza kunye noVavanyo lwezonyango leSun Yat-sen University Cancer Center laqokelela ichibi le-serum kubanikeli abali-100 abasempilweni, kuquka inani elilinganayo lamadoda nabafazi abaneminyaka engama-40 ukuya kwengama-55 ubudala. Iivolumu ezilinganayo zesampuli nganye yabanikeli zaxutywa, ichibi eliphumayo laxutywa laza lagcinwa kwi--80°C. Umxube we-serum wasetyenziswa njengesixhobo sokubhekisa kulawulo lomgangatho kunye nomgangatho wedatha.
Iisampulu ze-serum kunye neesampuli zovavanyo ezisetyenzisiweyo zanyibilikiswa kwaye ii-metabolites zakhutshwa kusetyenziswa indlela yokukhupha edibeneyo (MTBE/methanol/water) 56. Ngamafutshane, i-50 μl yeserum yaxutywa ne-225 μl ye-methanol ebandayo kunye ne-750 μl ye-methyl tert-butyl ether ebandayo (MTBE). Xuba umxube kwaye uwufake kwi-ice iyure e-1. Emva koko iisampulu zaxutywa kwaye i-vortex yaxutywa ne-188 μl yamanzi e-MS-grade aqulethe imigangatho yangaphakathi (13C-lactate, 13C3-pyruvate, 13C-methionine, kunye ne-13C6-isoleucine, ethengwe kwiCambridge Isotope Laboratories). Umxube emva koko waxutywa kwi-centrifuge kwi-15,000 × g imizuzu eli-10 kwi-4 °C, kwaye isigaba esisezantsi sadluliselwa kwiityhubhu ezimbini (125 μL nganye) ukuze kuhlalutywe i-LC-MS ngeendlela ezilungileyo nezingalunganga. Ekugqibeleni, isampuli yapheliswa yaba ngumphunga yaze yoma kwi-vacuum concentrator enesantya esiphezulu.
Iimetabolites ezomileyo zaphinda zahlanganiswa kwi-120 μl ye-80% ye-acetonitrile, zaxutywa imizuzu emi-5, zaza zaxutywa kwi-centrifuge kwi-15,000 × g imizuzu eli-10 kwi-4°C. Ii-Supernatants zadluliselwa kwiibhotile zeglasi ze-amber ezinee-microinserts kwizifundo ze-metabolomics. Uhlalutyo lwe-metabolomics olungakhange lujoliswe kwiqonga le-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). IiMetabolites zahlulwe kusetyenziswa inkqubo ye-Dionex Ultimate 3000 UPLC kunye nekholamu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Waters). Kwimo ye-ion e-positive, ii-mobile phases zaziyi-95% (A) kunye ne-50% ye-acetonitrile (B), nganye iqulethe i-10 mmol/L ammonium acetate kunye ne-0.1% formic acid. Kwimo engalunganga, ii-mobile phases A kunye ne-B ziqulathe i-acetonitrile engama-95% kunye ne-50%, ngokulandelelana, zombini ezi zigaba ziqulathe i-10 mmol/L ammonium acetate, pH = 9. Inkqubo ye-gradient yayimi ngolu hlobo lulandelayo: 0–0.5 min, 2% B; 0.5–12 min, 2–50% B; 12–14 min, 50–98% B; 14–16 min, 98% B; 16–16.1. min, 98–2% B; 16.1–20 min, 2% B. Ikholamu igcinwe kwi-40°C kwaye isampuli ikwi-10°C kwi-autosampler. Isantya sokuhamba yayiyi-0.3 ml/min, umthamo we-injection yayiyi-3 μl. I-Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) ene-electrospray ionization (ESI) source isetyenzisiwe kwi-full scan mode kwaye idityaniswe ne-ddMS2 monitoring mode ukuqokelela idatha eninzi. Iiparameter ze-MS zimiselwe ngolu hlobo lulandelayo: i-spray voltage +3.8 kV/- 3.2 kV, i-capillary temperature 320°C, i-shielding gas 40 arb, i-assistant gas 10 arb, i-probe heater temperature 350°C, i-scanning range 70–1050 m/h, i-resolution. 70 000. Idatha ifunyenwe kusetyenziswa i-Xcalibur 4.1 (Thermo Fisher Scientific).
Ukuvavanya umgangatho wedatha, iisampulu zolawulo lomgangatho ezidityanisiweyo (QC) zenziwe ngokususa ii-aliquots ezili-10 μL ze-supernatant kwisampulu nganye. Ii-injections ezintandathu zesampulu zolawulo lomgangatho zahlalutywa ekuqaleni kolandelelwano lohlalutyo ukuvavanya uzinzo lwenkqubo ye-UPLC-MS. Iisampulu zolawulo lomgangatho emva koko zifakwa rhoqo kwi-batch. Zonke iibhetshi ezili-11 zeesampulu ze-serum kolu phononongo zahlalutywa yi-LC-MS. Ii-aliquots zomxube we-serum pool ezivela kubanikeli abali-100 abaphilileyo zisetyenziswe njengezinto zokubhekisa kwiibhetshi ezahlukeneyo ukujonga inkqubo yokukhupha kunye nokulungelelanisa iziphumo zebhetshi ukuya kwibhetshi. Uhlalutyo lwe-metabolomics olungajoliswanga lwe-discovery cohort, i-internal validation cohort, kunye ne-external validation cohort lwenziwe kwiMetabolomics Center yeSun Yat-sen University. Ilebhu yangaphandle yeGuangdong University of Technology Analysis and Testing Center ikwahlalutye iisampulu ezingama-40 ezivela kwi-external cohort ukuvavanya ukusebenza kwemodeli yokwahlulahlula.
Emva kokukhupha nokuhlaziya, umlinganiselo opheleleyo wee-serum metabolites ulinganiswe kusetyenziswa i-ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) ene-electrospray ionization (ESI) source kwimo yokujonga i-reaction ezininzi (MRM). Ikholamu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Waters) yasetyenziswa ukwahlula ii-metabolites. Isigaba esihambayo sasiquka i-90% (A) kunye ne-5% ye-acetonitrile (B) ene-10 mmol/L ammonium acetate kunye nesisombululo se-0.1% ye-ammonia. Inkqubo ye-gradient yayilandelayo: 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. Ikholamu igcinwe kwi-40 °C kwaye isampuli igcinwe kwi-10 °C kwi-autosampler. Izinga lokuhamba yayiyi-0.3 mL/min kwaye umthamo we-injection yayiyi-1 μL. Iiparameter ze-MS zibekwe ngolu hlobo lulandelayo: i-capillary voltage ±3.5 kV, uxinzelelo lwe-nebulizer 35 psi, ukuhamba kwegesi yesheath 12 L/min, ubushushu begesi yesheath 350°C, ubushushu begesi yokomisa 250°C, kunye nokuhamba kwegesi yokomisa 14 l/min. Ukuguqulwa kwe-MRM kwe-tryptophan, i-pyruvate, i-lactate, i-hypoxanthine kunye ne-xanthine bekuyi-205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 kunye ne-151.0–107. 9 ngokwahlukeneyo. Idatha iqokelelwe kusetyenziswa iMass Hunter B.07.00 (Agilent Technologies). Kwiisampuli ze-serum, i-tryptophan, i-pyruvate, i-lactate, i-hypoxanthine, kunye ne-xanthine zalinganiswa kusetyenziswa ii-calibration curves zezisombululo zomxube oqhelekileyo. Kwiisampuli zeseli, umxholo we-tryptophan walungiswa waba ngumgangatho wangaphakathi kunye nobunzima beproteni yeseli.
Ukukhupha i-peak extraction (m/z kunye nexesha lokugcina (RT)) kwenziwe kusetyenziswa i-Compound Discovery 3.1 kunye ne-TraceFinder 4.0 (Thermo Fisher Scientific). Ukususa umahluko onokubakho phakathi kwee-batches, i-characteristic peak nganye yesampulu yovavanyo yahlulwe yi-characteristic peak yezinto ezisetyenzisiweyo ezivela kwi-batch efanayo ukuze kufunyanwe ubuninzi obuhambelanayo. Ukuphambuka okuqhelekileyo kwemigangatho yangaphakathi ngaphambi nasemva kokumiswa kuboniswe kwiTheyibhile eyoNgezelelweyo 6. Umahluko phakathi kwamaqela amabini ubonakaliswe yi-false discovery rate (FDR<0.05, Wilcoxon signed rank test) kunye ne-fold change (>1.2 okanye <0.83). Idatha ye-MS eluhlaza yeempawu ezikhutshiweyo kunye nedatha ye-MS elungisiweyo nge-serum iboniswe kwi-Supplementary Data 1 kunye ne-Supplementary Data 2, ngokulandelanayo. I-peak annotation yenziwe ngokusekelwe kumanqanaba amane achaziweyo okuchonga, kubandakanya i-metabolites echongiweyo, ii-putatively annotated compounds, ii-putatively characterised compound classes, kunye nee-compounds ezingaziwayo 22. Ngokusekelwe kuphendlo lwedathabheyisi kwiCompound Discovery 3.1 (mzCloud, HMDB, Chemspider), iikhompawundi zebhayoloji ezinemigangatho eqinisekisiweyo yokufanisa i-MS/MS okanye iinkcazo ezihambelanayo ngqo kwi-mzCloud (amanqaku > 85) okanye iChemspider ekugqibeleni zikhethwe njengee-intermediate phakathi kwe-differential metabolome. Iinkcazo ze-peak zesici ngasinye zifakiwe kwi-Supplementary Data 3. I-MetaboAnalyst 5.0 isetyenziselwe uhlalutyo lwe-univariate lwe-sum-normalized metabolite abundance. I-MetaboAnalyst 5.0 ikwavavanye uhlalutyo lwe-KEGG pathway enrichment olusekelwe kwi-metabolites ezahlukeneyo kakhulu. Uhlalutyo lwe-Principal component (PCA) kunye ne-partial least squares discriminant analysis (PLS-DA) zihlalutywe kusetyenziswa i-ropls software package (v.1.26.4) kunye ne-stack normalization kunye ne-autoscaling. I-optimal metabolar model yokuqikelela i-nodule monitancy yenziwe kusetyenziswa i-binary logistic regression kunye ne-least absolute shrinkage kunye ne-selection operator (LASSO, R package v.4.1-3). Ukusebenza kwemodeli yokwahlulahlula kwiiseti zokufumanisa nokuqinisekisa kwaphawulwa ngokuqikelela i-AUC ngokusekelwe kuhlalutyo lwe-ROC ngokwephakheji ye-pROC (v.1.18.0.). Ukunciphisa amathuba afanelekileyo kufunyenwe ngokusekelwe kwi-Youden index ephezulu yemodeli (uvakalelo + ukucaciswa - 1). Iisampulu ezinamaxabiso angaphantsi okanye amakhulu kunomda ziya kuqikelelwa njengee-nodules ezingenobungozi kunye ne-lung adenocarcinoma, ngokwahlukeneyo.
Iiseli ze-A549 (#CCL-185, iAmerican Type Culture Collection) zikhuliswe kwi-F-12K medium equlethe i-10% FBS. Iisequences ze-RNA (shRNA) ezimfutshane ezijolise kwi-SLC7A5 kunye ne-nontargeting control (NC) zifakwe kwi-lentiviral vector pLKO.1-puro. Iisequences ze-antisense ze-shSLC7A5 zezi zilandelayo: Sh1 (5′-GGAGAAAACCTGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGGAACTAT-3′). Ii-antibodies kwi-SLC7A5 (#5347) kunye ne-tubulin (#2148) zithengwe kwi-Cell Signaling Technology. Ii-antibodies kwi-SLC7A5 kunye ne-tubulin zisetyenziswe kwi-dilution ye-1:1000 kuhlalutyo lwe-Western blot.
Uvavanyo lwe-Seahorse XF Glycolytic Stress lulinganisa amanqanaba e-extracellular acidification (ECAR). Kuvavanyo, i-glucose, i-oligomycin A, kunye ne-2-DG zinikwe ngokulandelelana ukuvavanya amandla e-cell glycolytic njengoko kulinganiswa yi-ECAR.
Iiseli ze-A549 ezifakwe i-non-targeting control (NC) kunye ne-shSLC7A5 (Sh1, Sh2) zafakwa ubusuku bonke kwizitya ezinobubanzi obuyi-10 cm. Ii-metabolites zeeseli zakhutshwa nge-1 ml ye-methanol yamanzi ebandayo ebandayo eyi-80%. Iiseli kwisisombululo se-methanol zakhutshelwa ngaphandle, zaqokelelwa kwityhubhu entsha, zaza zafakwa kwi-centrifuge kwi-15,000 × g imizuzu eli-15 kwi-4°C. Qokelela i-800 µl ye-supernatant kwaye zome kusetyenziswa i-vacuum concentrator enesantya esiphezulu. Ii-pellets ze-metabolite ezomileyo zahlalutywa emva koko ukuze kujongwe amanqanaba e-tryptophan kusetyenziswa i-LC-MS/MS njengoko kuchaziwe apha ngasentla. Amanqanaba e-NAD(H) eeseli ze-A549 (NC kunye ne-shSLC7A5) alinganiswe kusetyenziswa i-quantitative NAD+/NADH colorimetric kit (#K337, BioVision) ngokwemiyalelo yomenzi. Amanqanaba eeprotheyini alinganiswe kwisampulu nganye ukuze kulungiswe ubungakanani bee-metabolites.
Akukho ndlela zezibalo ezisetyenzisiweyo ukumisela ubungakanani besampulu kwangaphambili. Izifundo zangaphambili ze-metabolomics ezijolise ekufumaneni i-biomarker15,18 zithathwe njengemilinganiselo yokumisela ubungakanani, kwaye xa kuthelekiswa nezi ngxelo, isampulu yethu yayanele. Akukho sampuli zikhutshiweyo kwi-cohort yophando. Iisampuli ze-serum zabelwa ngokungacwangciswanga kwiqela lokufumanisa (iimeko ezingama-306, 74.6%) kunye neqela lokuqinisekisa langaphakathi (iimeko ezili-104, 25.4%) kwizifundo ze-metabolomics ezingajoliswanga. Sikwakhethe ngokungacwangciswanga iimeko ezingama-70 kwiqela ngalinye kwiseti yokufumanisa kwizifundo ze-metabolomics ezijolisiweyo. Abaphandi baphoswe kukwabelwa kweqela ngexesha lokuqokelelwa kunye nohlalutyo lwedatha ye-LC-MS. Uhlalutyo lwezibalo lwedatha ye-metabolomics kunye novavanyo lweseli luchazwe kwicandelo leZiphumo, iMifanekiso, kunye neendlela. Ukulinganiswa kwe-tryptophan yeselula, i-NADT, kunye nomsebenzi we-glycolytic kwenziwe kathathu ngokuzimeleyo kunye neziphumo ezifanayo.
Ukuze ufumane ulwazi oluthe kratya malunga noyilo lophando, jonga i-Natural Portfolio Report Abstract enxulumene neli nqaku.
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Ixesha lokuthumela: Septemba-18-2023