Kev kuaj mob sib txawv ntawm pulmonary nodules txheeb xyuas los ntawm kev suav tomography (CT) tseem yog qhov nyuaj hauv kev kho mob.Ntawm no, peb qhia txog lub ntiaj teb metabolome ntawm 480 cov qauv kuaj ntshav, suav nrog kev tswj hwm kev noj qab haus huv, benign ntsws nodules, thiab theem I ntsws adenocarcinoma.Adenocarcinomas nthuav tawm cov txheej txheem metabolomic tshwj xeeb, thaum benign nodules thiab cov neeg noj qab haus huv muaj qhov sib xws hauv cov kab mob metabolomic.Hauv pab pawg tshawb pom (n = 306), cov txheej txheem ntawm 27 metabolites tau txheeb xyuas qhov sib txawv ntawm benign thiab malignant nodules.Lub AUC ntawm cov qauv kev ntxub ntxaug hauv kev siv hluav taws xob sab hauv (n = 104) thiab validation sab nraud (n = 111) pawg yog 0.915 thiab 0.945, feem.Txoj kev tsom xam pom tias muaj zog glycolytic metabolites cuam tshuam nrog txo tryptophan hauv lub ntsws adenocarcinoma serum piv nrog cov benign nodules thiab tswj kev noj qab haus huv, thiab qhia tias tryptophan uptake txhawb nqa glycolysis hauv ntsws cancer hlwb.Peb txoj kev tshawb fawb qhia txog cov txiaj ntsig ntawm cov tshuaj lom neeg cov tshuaj biomarkers hauv kev ntsuam xyuas qhov kev pheej hmoo ntawm pulmonary nodules kuaj pom los ntawm CT.
Kev kuaj mob ntxov yog qhov tseem ceeb los txhim kho cov kev muaj sia nyob rau cov neeg mob qog noj ntshav.Cov txiaj ntsig tau los ntawm US National Lung Cancer Screening Trial (NLST) thiab European NELSON Txoj Kev Tshawb Fawb tau pom tias kev tshuaj ntsuam xyuas nrog qis-dose suav tomography (LDCT) tuaj yeem txo qis mob qog noj ntshav hauv cov neeg muaj kev pheej hmoo siab 1,2,3.Txij li kev siv LDCT dav dav rau kev kuaj mob qog noj ntshav, qhov tshwm sim ntawm qhov tshwm sim ntawm qhov pom ntawm cov hlab ntsws asymptomatic pulmonary nodules tau nce ntxiv 4.Pulmonary nodules txhais tau tias yog focal opacities mus txog 3 cm nyob rau hauv txoj kab uas hla 5.Peb ntsib teeb meem hauv kev txheeb xyuas qhov yuav tshwm sim ntawm malignancy thiab cuam tshuam nrog ntau tus pulmonary nodules kuaj pom tshwm sim ntawm LDCT.Kev txwv ntawm CT tuaj yeem ua rau muaj kev soj ntsuam ntau zaus thiab cov txiaj ntsig tsis zoo, ua rau muaj kev cuam tshuam tsis tsim nyog thiab kev kho mob ntau dhau 6.Yog li ntawd, yuav tsum tau tsim cov biomarkers txhim khu kev qha thiab muaj txiaj ntsig zoo txhawm rau txheeb xyuas qhov mob qog noj ntshav hauv lub ntsws thaum ntxov thiab sib txawv feem ntau cov nodules ntawm qhov pib tshawb pom 7.
Kev tsom xam molecular ntawm cov ntshav (cov ntshav, ntshav, ntshav peripheral cov ntshav mononuclear hlwb), suav nrog genomics, proteomics lossis DNA methylation8,9,10, tau ua rau muaj kev txaus siab rau kev tshawb pom ntawm biomarkers kuaj mob ntsws cancer.Lub caij no, cov txheej txheem metabolomics ntsuas ntsuas cov khoom kawg ntawm tes uas cuam tshuam los ntawm endogenous thiab exogenous ua thiab yog li siv los kwv yees kab mob pib thiab tshwm sim.Liquid chromatography-tandem mass spectrometry (LC-MS) yog ib txoj kev siv dav rau cov kev tshawb fawb metabolomics vim nws qhov rhiab heev thiab loj dynamic ntau yam, uas tuaj yeem npog cov metabolites nrog cov khoom sib txawv physicochemical 11,12,13.Txawm hais tias lub ntiaj teb metabolomic tsom xam ntawm plasma / serum tau siv los txheeb xyuas cov cim biomarkers cuam tshuam nrog kev kuaj mob ntsws cancer14,15,16,17 thiab kev kho mob, 18 cov tshuaj metabolite hauv cov ntshav kom paub qhov txawv ntawm benign thiab malignant ntsws nodules tseem yuav tsum tau kawm ntau.- kev tshawb fawb loj.
Adenocarcinoma thiab squamous cell carcinoma yog ob lub ntsiab subtypes ntawm non-small cell lung cancer (NSCLC).Ntau yam kev tshuaj ntsuam xyuas CT qhia tias adenocarcinoma yog hom mob ntsws cancer ntau tshaj plaws ntawm 1,19,20,21.Hauv txoj kev tshawb no, peb tau siv ultra-performance kua chromatography-high-resolution mass spectrometry (UPLC-HRMS) los ua metabolomics tsom xam ntawm tag nrho ntawm 695 cov qauv ntshav, suav nrog kev tswj kev noj qab haus huv, benign pulmonary nodules, thiab CT-nrhiav ≤3 cm.Kev tshuaj ntsuam rau theem I ntsws adenocarcinoma.Peb tau txheeb xyuas lub vaj huam sib luag ntawm cov tshuaj lom neeg hauv cov ntshav uas paub qhov txawv ntawm lub ntsws adenocarcinoma los ntawm benign nodules thiab tswj kev noj qab haus huv.Txoj kev txhim kho kev txheeb xyuas tau qhia tias qhov txawv txav ntawm tryptophan thiab qabzib metabolism yog ib qho kev hloov pauv hauv lub ntsws adenocarcinoma piv nrog cov benign nodules thiab tswj kev noj qab haus huv.Thaum kawg, peb tau tsim thiab siv tau cov tshuaj ntsuam xyuas cov ntshav hauv cov ntshav uas muaj qhov tshwj xeeb thiab rhiab heev kom paub qhov txawv ntawm malignant thiab benign pulmonary nodules kuaj pom los ntawm LDCT, uas yuav pab tau rau kev kuaj mob ntxov thiab kev ntsuas kev pheej hmoo.
Hauv kev tshawb fawb tam sim no, cov qauv kuaj ntshav ntawm poj niam txiv neej thiab hnub nyoog sib txawv tau sau rov qab los ntawm 174 kev tswj hwm kev noj qab haus huv, 292 cov neeg mob uas muaj cov kab mob ntsws tsis zoo, thiab 229 tus neeg mob uas muaj theem I ntsws adenocarcinoma.Cov yam ntxwv ntawm cov pej xeem ntawm 695 cov ntsiab lus muaj nyob rau hauv Cov Lus Qhia Ntxiv 1.
Raws li pom nyob rau hauv daim duab 1a, tag nrho ntawm 480 cov qauv kuaj ntshav, suav nrog 174 kev tswj kev noj qab haus huv (HC), 170 benign nodules (BN), thiab 136 theem kuv lub ntsws adenocarcinoma (LA) cov qauv, tau sau ntawm Sun Yat-sen University Cancer Center.Discovery cohort rau untargeted metabolomic profileing siv ultra-performance kua chromatography-high-resolution mass spectrometry (UPLC-HRMS).Raws li pom hauv Daim Duab Ntxiv 1, qhov sib txawv ntawm cov metabolites ntawm LA thiab HC, LA thiab BN tau raug txheeb xyuas los tsim cov qauv kev faib tawm thiab tshawb nrhiav qhov sib txawv ntawm txoj hauv kev.104 cov qauv sau los ntawm Sun Yat-sen University Cancer Center thiab 111 cov qauv sau los ntawm ob lub tsev kho mob tau raug rau kev lees paub sab hauv thiab sab nraud, raws li.
Ib txoj kev tshawb fawb cov pej xeem nyob rau hauv lub discovery cohort uas underwent lub ntiaj teb no serum metabolomics tsom xam siv ultra-performance kua chromatography-high-resolution mass spectrometry (UPLC-HRMS).b Ib feem tsawg squares cais cais (PLS-DA) ntawm tag nrho cov metabolome ntawm 480 cov qauv kuaj ntshav los ntawm kev tshawb fawb cohort, suav nrog kev tswj kev noj qab haus huv (HC, n = 174), benign nodules (BN, n = 170), thiab theem I ntsws adenocarcinoma (Los Angeles, n = 136).+ ESI, zoo electrospray ionization hom, -ESI, tsis zoo electrospray ionization hom.c–e Metabolites nrog ntau qhov sib txawv hauv ob pawg (ob-tailed Wilcoxon kos npe rau qeb xeem, qhov kev tshawb pom tsis tseeb hloov p tus nqi, FDR <0.05) yog pom hauv liab (fold hloov> 1.2) thiab xiav (fold hloov <0.83) .) pom nyob rau ntawm lub roob hluav taws.f Hierarchical clustering tshav kub daim ntawv qhia qhia qhov sib txawv tseem ceeb nyob rau hauv tus naj npawb ntawm annotated metabolites ntawm LA thiab BN.Cov ntaub ntawv qhov chaw yog muab nyob rau hauv daim ntawv ntawm cov ntaub ntawv cov ntaub ntawv.
Tag nrho cov serum metabolome ntawm 174 HC, 170 BN thiab 136 LA hauv pawg tshawb nrhiav tau txheeb xyuas siv UPLC-HRMS tsom xam.Peb ua ntej qhia tias kev tswj xyuas qhov zoo (QC) cov qauv ntsuas nruj nruj ntawm qhov chaw ntawm qhov kev soj ntsuam tus thawj tswj hwm tsis saib xyuas (PCA) qauv, lees paub qhov ruaj khov ntawm qhov kev kawm tam sim no qhov kev ua tau zoo (Cov duab ntxiv 2).
Raws li pom nyob rau hauv ib feem tsawg squares-discriminant tsom xam (PLS-DA) nyob rau hauv daim duab 1 b, peb pom tau hais tias muaj qhov sib txawv tseeb ntawm LA thiab BN, LA thiab HC nyob rau hauv zoo (+ESI) thiab tsis zoo (-ESI) electrospray ionization hom. .cais.Txawm li cas los xij, tsis muaj qhov sib txawv tseem ceeb ntawm BN thiab HC hauv + ESI thiab -ESI cov xwm txheej.
Peb pom 382 qhov sib txawv ntawm LA thiab HC, 231 qhov sib txawv ntawm LA thiab BN, thiab 95 qhov sib txawv ntawm BN thiab HC (Wilcoxon kos npe rau qeb xeem, FDR <0.05 thiab ntau qhov kev hloov pauv> 1.2 lossis <0.83) (Daim duab .1c-e )..Peaks tau piav qhia ntxiv (Cov Ntaub Ntawv Ntxiv 3) tawm tsam cov ntaub ntawv (mzCloud/HMDB/Chemspider lub tsev qiv ntawv) los ntawm m/z tus nqi, lub sijhawm khaws cia thiab fragmentation huab hwm coj spectrum tshawb nrhiav (cov ntsiab lus piav qhia hauv Tshooj Lus) 22 .Thaum kawg, 33 thiab 38 annotated metabolites nrog qhov sib txawv tseem ceeb hauv kev nplua nuj tau raug txheeb xyuas rau LA piv rau BN (Daim duab 1f thiab Cov Lus Ntxiv 2) thiab LA piv rau HC (Cov Duab Ntxiv 3 thiab Cov Lus Ntxiv 2), feem.Hauv qhov sib piv, tsuas yog 3 cov metabolites uas muaj qhov sib txawv tseem ceeb hauv kev nplua nuj tau pom nyob rau hauv BN thiab HC (Cov Lus Qhia Ntxiv 2), raws li kev sib tshooj ntawm BN thiab HC hauv PLS-DA.Cov metabolites sib txawv no suav nrog ntau yam biochemicals (Cov duab ntxiv 4).Muab ua ke, cov txiaj ntsig no qhia tau tias muaj kev hloov pauv tseem ceeb hauv cov ntshav ntshav metabolome uas cuam tshuam txog kev hloov pauv ntawm lub ntsws cancer thaum ntxov piv nrog cov mob ntsws benign lossis cov neeg noj qab haus huv.Lub caij no, qhov zoo sib xws ntawm cov ntshav ntshav metabolome ntawm BN thiab HC qhia tias benign pulmonary nodules yuav qhia tau ntau yam kab mob lom neeg nrog rau cov neeg noj qab haus huv.Muab hais tias epidermal growth factor receptor (EGFR) gene mutations yog muaj nyob rau hauv lub ntsws adenocarcinoma subtype 23, peb nrhiav los txiav txim qhov cuam tshuam ntawm kev hloov ntawm tus neeg tsav tsheb mus rau hauv cov ntshav metabolome.Tom qab ntawd peb txheeb xyuas tag nrho cov metabolomic profile ntawm 72 tus neeg mob nrog EGFR xwm txheej hauv pawg ntsws adenocarcinoma.Qhov zoo siab, peb pom cov ntaub ntawv sib piv ntawm EGFR mutant cov neeg mob (n = 41) thiab EGFR cov neeg mob qus (n = 31) hauv PCA tsom xam (Cov duab ntxiv 5a).Txawm li cas los xij, peb tau txheeb xyuas 7 cov metabolites uas nws qhov kev nplua nuj tau hloov pauv ntau hauv cov neeg mob uas muaj EGFR hloov pauv piv rau cov neeg mob uas muaj hom tsiaj qus EGFR (t test, p <0.05 thiab fold hloov> 1.2 lossis <0.83) (Cov duab ntxiv 5b).Feem ntau ntawm cov metabolites (5 tawm ntawm 7) yog acylcarnitines, uas ua lub luag haujlwm tseem ceeb hauv txoj kev fatty acid oxidation.
Raws li tau piav qhia hauv cov haujlwm ua haujlwm tau pom hauv daim duab 2 a, biomarkers rau kev faib cov nodule tau txais kev siv cov neeg ua haujlwm qis qis thiab xaiv raws li 33 qhov sib txawv metabolites pom hauv LA (n = 136) thiab BN (n = 170).Kev sib xyaw ua ke zoo tshaj plaws ntawm qhov sib txawv (LASSO) - binary logistic regression qauv.Kaum-fold cross-validation tau siv los ntsuas qhov kev ntseeg tau ntawm tus qauv.Kev xaiv tsis sib xws thiab qhov ntsuas tsis tu ncua yog hloov kho los ntawm qhov muaj peev xwm ua kom lub txim loj tshaj plaws nrog rau qhov ntsuas λ24.Ntiaj teb no metabolomics tsom xam tau ua ntxiv ntawm nws tus kheej nyob rau hauv lub internal validation (n = 104) thiab sab nraud validation (n = 111) pab pawg los ntsuam xyuas cov kev faib ua feem ntawm cov qauv kev ntxub ntxaug.Raws li qhov tshwm sim, 27 metabolites hauv cov txheej txheem tshawb pom tau pom tias yog tus qauv kev ntxub ntxaug zoo tshaj plaws nrog qhov loj tshaj plaws AUC tus nqi (Fig. 2b), ntawm cov uas 9 tau nce kev ua si thiab 18 txo kev ua haujlwm hauv LA piv rau BN (Fig. 2c).
Kev ua haujlwm rau kev tsim cov kab mob pulmonary nodule classifier, suav nrog xaiv cov vaj huam sib luag zoo tshaj plaws ntawm cov tshuaj ntshav hauv cov ntshav hauv qhov kev tshawb pom uas siv cov qauv binary logistic regression los ntawm kaum-fold cross-validation thiab ntsuam xyuas kev ua tau zoo hauv cov txheej txheem sab hauv thiab sab nraud.b Hla-validation txheeb cais ntawm LASSO regression qauv rau metabolic biomarker xaiv.Cov lej muab saum toj no sawv cev rau qhov nruab nrab ntawm cov biomarkers xaiv ntawm qhov muab λ.Cov kab liab dotted sawv cev rau qhov nruab nrab AUC tus nqi ntawm cov lambda sib raug.Grey yuam kev bar sawv cev qhov tsawg kawg nkaus thiab siab tshaj AUC qhov tseem ceeb.Cov kab dotted qhia txog tus qauv zoo tshaj plaws nrog 27 tus neeg xaiv biomarkers.AUC, cheeb tsam nyob rau hauv lub receiver operating yam ntxwv (ROC) nkhaus.c Fold pauv ntawm 27 cov metabolites xaiv hauv pawg LA piv nrog BN pawg hauv pawg tshawb pom.Kab liab - ua kom.Cov kab xiav yog qhov poob.d-f Tus neeg txais kev ua haujlwm yam ntxwv (ROC) nkhaus qhia lub zog ntawm tus qauv kev ntxub ntxaug raws li 27 metabolite sib xyaw ua ke hauv kev tshawb pom, sab hauv, thiab sab nraud validation teev.Cov ntaub ntawv qhov chaw yog muab nyob rau hauv daim ntawv ntawm cov ntaub ntawv cov ntaub ntawv.
Tus qauv kwv yees tau tsim raws li qhov hnyav regression coefficients ntawm cov 27 metabolites (Cov Lus Ntxiv 3).ROC tsom xam raws li cov 27 metabolites yielded thaj tsam nyob rau hauv lub nkhaus (AUC) tus nqi ntawm 0.933, discovery pab pawg neeg rhiab heev yog 0.868, thiab tshwj xeeb yog 0.859 (Fig. 2d).Lub caij no, ntawm 38 qhov sib txawv ntawm cov metabolites ntawm LA thiab HC, cov txheej txheem ntawm 16 metabolites ua tiav AUC ntawm 0.902 nrog rhiab heev ntawm 0.801 thiab qhov tshwj xeeb ntawm 0.856 hauv kev ntxub ntxaug LA los ntawm HC (Cov Duab Ntxiv 6a-c).AUC qhov tseem ceeb raws li qhov sib txawv ntawm qhov hloov pauv hloov pauv rau qhov sib txawv metabolites kuj tau muab piv.Peb pom tias tus qauv kev faib tawm ua tau zoo tshaj plaws hauv kev ntxub ntxaug ntawm LA thiab BN (HC) thaum qhov kev hloov pauv qib tau teem rau 1.2 piv rau 1.5 lossis 2.0 (Cov duab ntxiv 7a,b).Cov qauv kev faib tawm, raws li 27 pawg metabolite, tau raug lees paub ntxiv hauv pawg sab hauv thiab sab nraud.AUC yog 0.915 (rhiab heev 0.867, qhov tshwj xeeb 0.811) rau kev siv sab hauv thiab 0.945 (rhiab heev 0.810, tshwj xeeb 0.979) rau kev siv sab nraud (Fig. 2e, f).Txhawm rau ntsuas kev ua haujlwm ntawm kev ua haujlwm, 40 cov qauv los ntawm pawg neeg sab nraud tau txheeb xyuas hauv chav kuaj sab nraud raws li tau piav qhia hauv Tshooj Txheej Txheem.Kev faib tawm qhov tseeb tau ua tiav AUC ntawm 0.925 (Cov duab ntxiv 8).Vim hais tias lub ntsws squamous cell carcinoma (LUSC) yog qhov thib ob feem ntau subtype ntawm non-small cell lung cancer (NSCLC) tom qab mob ntsws adenocarcinoma (LUAD), peb kuj tau kuaj qhov muaj peev xwm siv tau ntawm cov kab mob metabolic.BN thiab 16 kis ntawm LUSC.AUC ntawm kev ntxub ntxaug ntawm LUSC thiab BN yog 0.776 (Cov duab ntxiv 9), qhia tias muaj peev xwm tsis zoo piv rau kev ntxub ntxaug ntawm LUAD thiab BN.
Cov kev tshawb fawb tau pom tias qhov loj ntawm nodules ntawm CT cov duab zoo sib xws nrog qhov yuav ua rau mob qog noj ntshav thiab tseem yog qhov tseem ceeb ntawm kev kho mob nodule25,26,27.Kev soj ntsuam ntawm cov ntaub ntawv los ntawm pawg loj ntawm NELSON kev tshuaj ntsuam xyuas tau pom tias qhov kev pheej hmoo ntawm malignancy nyob rau hauv cov ncauj lus nrog nodes <5 mm yog txawm zoo ib yam li cov uas tsis muaj nodes 28.Yog li, qhov tsawg kawg nkaus uas yuav tsum tau saib xyuas CT li niaj zaus yog 5 hli, raws li kev pom zoo los ntawm British Thoracic Society (BTS), thiab 6 hli, raws li kev pom zoo los ntawm Fleischner Society 29.Txawm li cas los xij, cov nodules loj dua 6 hli thiab tsis muaj cov yam ntxwv zoo, hu ua indeterminate pulmonary nodules (IPN), tseem yog ib qho kev sib tw loj hauv kev ntsuam xyuas thiab kev tswj xyuas hauv kev kho mob 30,31.Peb tom ntej no tau tshuaj xyuas seb qhov loj me me cuam tshuam rau cov kab mob metabolomic uas siv cov qauv sib xyaw los ntawm kev tshawb pom thiab cov ntawv pov thawj sab hauv.Kev tsom mus rau 27 cov cim biomarkers, peb thawj zaug piv cov PCA profiles ntawm HC thiab BN sub-6 hli metabolomes.Peb pom tias feem ntau ntawm cov ntaub ntawv cov ntsiab lus rau HC thiab BN sib tshooj, ua kom pom tias cov ntshav qab zib metabolite zoo sib xws hauv ob pawg (Fig. 3a).Cov ntawv qhia tshwj xeeb thoob plaws ntau qhov sib txawv tseem khaws cia hauv BN thiab LA (Daim duab 3b, c), qhov sib cais tau pom ntawm cov kab mob tsis zoo thiab cov benign nodules nyob rau hauv 6-20 mm (Fig. 3d).Cov pawg no muaj AUC ntawm 0.927, qhov tshwj xeeb ntawm 0.868, thiab rhiab heev ntawm 0.820 rau kev kwv yees qhov tsis zoo ntawm cov nodules ntsuas 6 txog 20 hli (Fig. 3e, f).Peb cov txiaj ntsig tau pom tias cov khoom lag luam tuaj yeem ntes cov kev hloov pauv hauv metabolic tshwm sim los ntawm kev hloov pauv sai sai, tsis hais qhov loj me.
ad Kev sib piv ntawm PCA profiles ntawm cov pab pawg raws li cov txheej txheem metabolic ntawm 27 metabolites.CC thiab BN <6 mm.b BN < 6 hli vs BN 6-20 mm.hauv LA 6-20 mm vs LA 20–30 mm.g BN 6-20 mm thiab LA 6-20 mm.GC, n = 174;BN < 6 hli, n = 153;BN 6-20 hli, n = 91;LA 6–20 mm, n = 89;LA 20–30 mm, n = 77. e Receiver kev khiav hauj lwm yam ntxwv (ROC) nkhaus qhia cov qauv kev ua tau zoo rau cov nodules 6–20 mm.f Cov txiaj ntsig yuav raug suav nrog raws li tus qauv logistic regression rau nodules ntsuas 6-20 mm.Cov kab grey dotted sawv cev rau qhov zoo tshaj plaws txiav tawm tus nqi (0.455).Cov lej saum toj no sawv cev rau feem pua ntawm cov xwm txheej tau npaj rau Los Angeles.Siv ob-tailed Student's t test.PCA, kev txheeb xyuas qhov tseem ceeb.AUC cheeb tsam hauv qab qhov nkhaus.Cov ntaub ntawv qhov chaw yog muab nyob rau hauv daim ntawv ntawm cov ntaub ntawv cov ntaub ntawv.
Plaub qhov qauv (hnub nyoog 44–61 xyoo) uas muaj qhov loj ntawm pulmonary nodule zoo sib xws (7–9 mm) tau raug xaiv ntxiv los piav qhia txog kev ua tau zoo ntawm cov qauv kev kwv yees kev mob qog noj ntshav (Fig. 4a, b).Ntawm qhov kev tshuaj ntsuam thawj zaug, Case 1 tau nthuav tawm raws li cov khoom nodule nrog calcification, ib qho kev cuam tshuam nrog benignity, whereas Case 2 nthuav tawm raws li ib qho kev txiav txim siab ib nrab ntawm cov nodule uas tsis muaj qhov pom tseeb benign nta.Peb qhov kev soj ntsuam CT soj ntsuam tau pom tias cov xwm txheej no tseem nyob ruaj khov nyob rau lub sijhawm 4 xyoo thiab yog li ntawd suav tias yog benign nodules (Fig. 4a).Piv nrog rau kev soj ntsuam kev soj ntsuam ntawm serial CT scans, ib leeg-shot serum metabolite tsom xam nrog cov qauv classifier tam sim no sai thiab raug txheeb xyuas cov nodules benign raws li cov kev txwv tsis pub muaj (Table 1).Daim duab 4b nyob rau hauv rooj plaub 3 qhia tau hais tias lub nodule nrog cov tsos mob ntawm pleural retraction, uas feem ntau yog txuam nrog malignancy32.Case 4 nthuav tawm raws li kev txiav txim siab ib nrab ntawm cov pob txha uas tsis muaj pov thawj ntawm qhov ua rau tsis zoo.Tag nrho cov xwm txheej no tau kwv yees ua phem raws li tus qauv cais (Table 1).Kev soj ntsuam ntawm lub ntsws adenocarcinoma tau pom los ntawm kev kuaj histopathological tom qab kev phais mob ntsws (daim duab 4b).Rau cov txheej txheem validation sab nraud, tus metabolic classifier tau raug kwv yees ob kis ntawm indeterminate lub ntsws nodules loj dua 6 hli (Cov duab ntxiv 10).
CT duab ntawm lub qhov rais axial ntawm lub ntsws ntawm ob kis ntawm benign nodules.Nyob rau hauv rooj plaub 1, CT scan tom qab 4 xyoos pom tau tias muaj qhov ruaj khov nodule ntsuas 7 mm nrog calcification nyob rau hauv txoj cai qis lobe.Nyob rau hauv rooj plaub 2, CT scan tom qab 5 xyoos tau qhia tias muaj qhov ruaj khov, ib nrab ntawm cov khoom nodule nrog txoj kab uas hla ntawm 7 hli nyob rau sab xis sab sauv.b Axial qhov rais CT cov duab ntawm lub ntsws thiab cov kev tshawb fawb txog kev sib raug zoo ntawm ob kis ntawm theem I adenocarcinoma ua ntej lub ntsws resection.Case 3 tau nthuav tawm ib lub nodule nrog txoj kab uas hla ntawm 8 mm nyob rau hauv txoj cai sab sauv lobe nrog pleural retraction.Case 4 tau nthuav tawm ib feem hauv av-iav nodule ntsuas 9 mm nyob rau sab laug sab sauv.Hematoxylin thiab eosin (H&E) staining ntawm resected ntsws ntaub so ntswg (scale bar = 50 μm) qhia tus acinar loj hlob qauv ntawm ntsws adenocarcinoma.Cov xub qhia cov nodules pom ntawm CT duab.H&E cov duab yog cov duab sawv cev ntawm ntau yam (> 3) microscopic teb tshuaj xyuas los ntawm tus kws kho mob.
Muab ua ke, peb cov txiaj ntsig tau qhia txog qhov muaj txiaj ntsig ntawm cov tshuaj lom neeg cov tshuaj biomarkers hauv kev kuaj mob sib txawv ntawm cov qog pulmonary nodules, uas yuav ua rau muaj teeb meem thaum ntsuas CT kev tshuaj ntsuam.
Raws li kev txheeb xyuas qhov sib txawv ntawm cov metabolite vaj huam sib luag, peb nrhiav kev txheeb xyuas cov kev sib raug zoo ntawm cov metabolic loj.KEGG pathway enrichment analysis los ntawm MetaboAnalyst tau txheeb xyuas 6 txoj hauv kev hloov pauv loj ntawm ob pawg (LA vs. HC thiab LA vs. BN, kho p ≤ 0.001, cuam tshuam > 0.01).Cov kev hloov pauv no tau tshwm sim los ntawm kev cuam tshuam hauv pyruvate metabolism, tryptophan metabolism, niacin thiab nicotinamide metabolism, glycolysis, TCA cycle, thiab purine metabolism (Fig. 5a).Tom qab ntawd peb tau ua tiav cov phiaj xwm metabolomics txhawm rau txheeb xyuas cov kev hloov pauv loj uas siv qhov ntsuas qhov tseeb.Kev txiav txim siab ntawm cov metabolites hauv cov kev hloov pauv feem ntau los ntawm triple quadrupole mass spectrometry (QQQ) siv cov qauv metabolite tseeb.Cov yam ntxwv ntawm cov kab mob metabolomics txoj kev tshawb fawb lub hom phiaj yog suav nrog hauv Cov Lus Qhia Ntxiv 4. Raws li peb cov txiaj ntsig thoob ntiaj teb metabolomics, kev soj ntsuam ntau tau lees paub tias hypoxanthine thiab xanthine, pyruvate, thiab lactate tau nce hauv LA piv rau BN thiab HC (Fig. 5b, c, p <0.05).Txawm li cas los xij, tsis muaj qhov sib txawv tseem ceeb hauv cov metabolites no tau pom ntawm BN thiab HC.
KEGG txoj kev txhim kho kev txheeb xyuas ntawm cov metabolites sib txawv hauv LA pawg piv rau BN thiab HC pawg.Ib tug ob-tailed Globaltest tau siv, thiab p qhov tseem ceeb tau hloov kho siv txoj kev Holm-Bonferroni (hloov p ≤ 0.001 thiab cov nyhuv loj> 0.01).b–d Violin plots qhia hypoxanthine, xanthine, lactate, pyruvate, thiab tryptophan theem hauv ntshav HC, BN, thiab LA txiav txim los ntawm LC-MS/MS (n = 70 ib pab pawg).Dawb thiab dub dotted kab qhia qhov nruab nrab thiab quartile, feem.e Violin zaj duab xis uas qhia txog qhov ua tau zoo Log2TPM (transcripts per lab) mRNA qhia ntawm SLC7A5 thiab QPRT hauv ntsws adenocarcinoma (n = 513) piv rau lub ntsws li qub (n = 59) hauv LUAD-TCGA dataset.Lub thawv dawb sawv cev rau qhov sib txawv ntawm qhov sib txawv, kab rov tav dub hauv nruab nrab sawv cev rau qhov nruab nrab, thiab cov kab dub ntsug txuas ntxiv los ntawm lub thawv sawv cev rau 95% kev ntseeg siab lub sijhawm (CI).f Pearson correlation plot ntawm SLC7A5 thiab GAPDH qhia hauv ntsws adenocarcinoma (n = 513) thiab lub ntsws li qub (n = 59) hauv TCGA dataset.Cov cheeb tsam grey sawv cev rau 95% CI.r, Pearson correlation coefficient.g Normalized cellular tryptophan theem hauv A549 hlwb transfected nrog nonspecific shRNA tswj (NC) thiab shSLC7A5 (Sh1, Sh2) txiav txim los ntawm LC-MS/MS.Kev txheeb xyuas ntawm tsib tus qauv kev ywj pheej lom neeg hauv txhua pab pawg tau nthuav tawm.h Cellular theem ntawm NADt (tag nrho NAD, suav nrog NAD + thiab NADH) hauv A549 hlwb (NC) thiab SLC7A5 knockdown A549 hlwb (Sh1, Sh2).Kev txheeb xyuas ntawm peb cov qauv kev ywj pheej lom neeg hauv txhua pab pawg tau nthuav tawm.i Glycolytic kev ua ntawm A549 hlwb ua ntej thiab tom qab SLC7A5 knockdown yog ntsuas los ntawm extracellular acidification tus nqi (ECAR) (n = 4 biologically ywj siab kuaj ib pawg).2-DG, 2-deoxy-D-glucose.Ob-tailed Student's t test tau siv hauv (b–h).Hauv (g–i), cov cim yuam kev sawv cev rau qhov txhais tau tias ± SD, txhua qhov kev sim tau ua peb zaug ntawm nws tus kheej thiab cov txiaj ntsig tau zoo sib xws.Cov ntaub ntawv qhov chaw yog muab nyob rau hauv daim ntawv ntawm cov ntaub ntawv cov ntaub ntawv.
Xav txog qhov cuam tshuam tseem ceeb ntawm kev hloov pauv tryptophan metabolism hauv LA pab pawg, peb kuj tau soj ntsuam cov ntshav tryptophan hauv HC, BN, thiab LA pawg siv QQQ.Peb pom tias cov ntshav tryptophan tau txo qis hauv LA piv nrog HC lossis BN (p < 0.001, Daim duab 5d), uas zoo ib yam nrog cov kev tshawb pom yav dhau los uas cov qib tryptophan muaj qis dua hauv cov neeg mob ntsws cancer dua li kev noj qab haus huv los ntawm pawg tswj hwm 33,34 ,35 ib.Lwm txoj kev tshawb fawb siv PET/CT tracer 11C-methyl-L-tryptophan pom tias lub sij hawm tryptophan tuav lub sij hawm nyob rau hauv lub ntsws cancer cov ntaub so ntswg tau nce loj dua piv rau benign lesions los yog ib txwm cov ntaub so ntswg36.Peb xav tias qhov txo qis hauv tryptophan hauv LA ntshav tuaj yeem cuam tshuam txog kev ua haujlwm ntawm tryptophan los ntawm lub ntsws cancer hlwb.
Nws kuj tseem paub tias cov khoom kawg ntawm kynurenine txoj hauv kev ntawm tryptophan catabolism yog NAD + 37,38, uas yog ib qho tseem ceeb substrate rau cov tshuaj tiv thaiv ntawm glyceraldehyde-3-phosphate nrog 1,3-bisphosphoglycerate hauv glycolysis39.Thaum cov kev tshawb fawb yav dhau los tau tsom mus rau lub luag haujlwm ntawm tryptophan catabolism hauv kev tiv thaiv kev tiv thaiv kab mob, peb tau nrhiav kev qhia txog kev sib cuam tshuam ntawm tryptophan dysregulation thiab glycolytic txoj hauv kev pom hauv txoj kev tshawb fawb tam sim no.Solute transporter family 7 member 5 (SLC7A5) paub tias yog tryptophan transporter43,44,45.Quinolinic acid phosphoribosyltransferase (QPRT) yog ib qho enzyme nyob hauv qab ntawm txoj kev kynurenine uas hloov quinolinic acid rau NAMN46.Kev tshuaj xyuas ntawm LUAD TCGA cov ntaub ntawv qhia tau hais tias ob qho tib si SLC7A5 thiab QPRT tau nce qib hauv cov ntaub so ntswg piv rau cov ntaub so ntswg ib txwm (Fig. 5e).Qhov kev nce no tau pom nyob rau hauv theem I thiab II nrog rau theem III thiab IV ntawm lub ntsws adenocarcinoma (Cov duab ntxiv 11), qhia tias muaj kev cuam tshuam ntxov hauv tryptophan metabolism cuam tshuam nrog tumorigenesis.
Tsis tas li ntawd, LUAD-TCGA dataset tau pom qhov sib raug zoo ntawm SLC7A5 thiab GAPDH mRNA qhia hauv cov neeg mob qog noj ntshav (r = 0.45, p = 1.55E-26, Daim duab 5f).Nyob rau hauv sib piv, tsis muaj kev sib raug zoo tseem ceeb ntawm xws li gene kos npe nyob rau hauv lub ntsws cov ntaub so ntswg (r = 0.25, p = 0.06, daim duab 5f).Knockdown ntawm SLC7A5 (Ntxiv daim duab 12) hauv A549 hlwb txo qis cellular tryptophan thiab NAD(H) ntau ntau (Daim duab 5g, h), ua rau muaj kev cuam tshuam glycolytic raws li ntsuas los ntawm extracellular acidification tus nqi (ECAR) (Daim duab 1).5 ib).Yog li, raws li kev hloov pauv hauv cov ntshav hauv cov ntshav thiab kuaj hauv vitro, peb xav tias tryptophan metabolism tuaj yeem tsim NAD + los ntawm txoj kev kynurenine thiab ua lub luag haujlwm tseem ceeb hauv kev txhawb nqa glycolysis hauv ntsws cancer.
Cov kev tshawb fawb tau pom tias muaj ntau qhov tsis paub meej pulmonary nodules kuaj pom los ntawm LDCT tuaj yeem ua rau xav tau kev kuaj ntxiv xws li PET-CT, kev kuaj mob ntsws, thiab kev kho mob ntau dhau vim qhov kuaj pom tsis zoo ntawm malignancy.31 Raws li pom hauv daim duab 6, peb txoj kev tshawb fawb tau txheeb xyuas ib lub vaj huam sib luag ntawm cov tshuaj lom neeg metabolites uas muaj peev xwm kuaj xyuas tus nqi uas tuaj yeem txhim kho kev pheej hmoo stratification thiab kev tswj xyuas tom qab ntawm pulmonary nodules kuaj pom los ntawm CT.
Pulmonary nodules raug soj ntsuam siv cov koob tshuaj qis qis (LDCT) nrog cov duab kos duab qhia txog qhov ua rau tsis zoo lossis tsis zoo.Qhov tshwm sim tsis meej ntawm cov nodules tuaj yeem ua rau muaj kev rov qab mus xyuas ntau zaus, kev cuam tshuam tsis tsim nyog, thiab kev kho mob ntau dhau.Kev suav nrog cov tshuaj hauv cov ntshav hauv cov ntshav nrog cov txiaj ntsig kev kuaj mob tuaj yeem txhim kho kev soj ntsuam kev pheej hmoo thiab kev tswj xyuas tom qab ntawm pulmonary nodules.PET positron emission tomography.
Cov ntaub ntawv los ntawm US NLST txoj kev tshawb fawb thiab European NELSON txoj kev tshawb fawb qhia tias kev tshuaj xyuas cov pab pawg muaj kev pheej hmoo siab nrog cov tshuaj qis qis qis (LDCT) tuaj yeem txo qis mob qog noj ntshav 1,3.Txawm li cas los xij, kev soj ntsuam kev pheej hmoo thiab kev tswj xyuas tom ntej ntawm cov neeg coob coob ntawm cov qog pulmonary nodules kuaj pom los ntawm LDCT tseem yog qhov nyuaj tshaj plaws.Lub hom phiaj tseem ceeb yog txhawm rau txhim kho qhov kev faib tawm kom raug ntawm LDCT-raws li kev cai uas twb muaj lawm los ntawm kev koom nrog cov biomarkers txhim khu kev qha.
Qee cov tshuaj molecular biomarkers, xws li cov ntshav metabolites, tau raug txheeb xyuas los ntawm kev sib piv cov qog nqaij hlav cancer nrog kev noj qab haus huv 15,17.Hauv txoj kev tshawb fawb tam sim no, peb tau tsom mus rau kev siv cov tshuaj ntsuam xyuas cov ntshav hauv cov ntshav kom paub qhov txawv ntawm benign thiab malignant pulmonary nodules tshwm sim los ntawm LDCT.Peb muab piv rau lub ntiaj teb cov ntshav ntshav metabolome ntawm kev noj qab haus huv tswj (HC), benign ntsws nodules (BN), thiab theem I ntsws adenocarcinoma (LA) cov qauv siv UPLC-HRMS tsom xam.Peb pom tias HC thiab BN muaj cov kab mob metabolic zoo sib xws, thaum LA tau pom cov kev hloov pauv tseem ceeb piv rau HC thiab BN.Peb tau txheeb xyuas ob pawg ntawm cov piam thaj hauv cov ntshav uas sib txawv LA ntawm HC thiab BN.
Tam sim no LDCT-raws li kev txheeb xyuas cov txheej txheem rau benign thiab malignant nodules feem ntau yog nyob ntawm qhov loj, ntom, morphology thiab kev loj hlob ntawm cov nodules dhau sijhawm 30.Cov kev tshawb fawb yav dhau los tau pom tias qhov loj ntawm nodules muaj feem cuam tshuam nrog qhov yuav ua rau mob qog noj ntshav.Txawm nyob rau hauv cov neeg mob uas muaj kev pheej hmoo siab, qhov kev pheej hmoo ntawm malignancy nyob rau hauv cov nodes <6 mm yog <1%.Qhov kev pheej hmoo ntawm malignancy rau nodules ntsuas 6 mus rau 20 mm yog li ntawm 8% mus rau 64% 30.Yog li ntawd, Fleischner Society pom zoo kom txiav txoj kab uas hla ntawm 6 hli rau CT raws li niaj hnub.29 Txawm li cas los xij, kev soj ntsuam kev pheej hmoo thiab kev tswj xyuas cov kab mob pulmonary nodules (IPN) loj dua 6 hli tsis tau ua tiav 31.Kev tswj hwm tus kab mob hauv lub plawv tam sim no feem ntau yog ua raws li kev saib xyuas nrog kev saib xyuas CT nquag.
Raws li cov metabolome validated, peb tau pom thawj zaug qhov sib tshooj ntawm metabolomic kos npe ntawm cov neeg noj qab haus huv thiab benign nodules <6 hli.Cov kev sib raug zoo ntawm cov kab mob lom tau zoo ib yam nrog cov kev tshawb pom yav dhau los ntawm CT uas qhov kev pheej hmoo ntawm malignancy rau nodules <6 mm yog tsawg li rau cov kev kawm uas tsis muaj nodes.30 Nws yuav tsum tau muab sau tseg tias peb cov qhabnias kuj qhia tau hais tias benign nodules <6 mm thiab ≥6 mm muaj siab. zoo sib xws hauv metabolomic profiles, qhia tias kev ua haujlwm txhais ntawm benign etiology zoo ib yam tsis hais qhov loj me.Yog li, niaj hnub tshuaj ntsuam xyuas cov tshuaj metabolite panels tuaj yeem muab ib qho kev ntsuas ib zaug raws li txoj cai-tawm kev sim thaum nodules tau pib kuaj pom ntawm CT thiab muaj peev xwm txo qis kev soj ntsuam xyuas.Nyob rau tib lub sijhawm, tib lub vaj huam sib luag ntawm metabolic biomarkers txawv qhov malignant nodules ≥6 mm loj los ntawm cov nodules benign thiab muab kev kwv yees raug tseeb rau IPNs ntawm qhov loj me thiab tsis meej morphological nta ntawm CT duab.Qhov no cov tshuaj metabolism hauv cov ntshav tau ua tau zoo hauv kev kwv yees qhov tsis zoo ntawm nodules ≥6 hli nrog AUC ntawm 0.927.Ua ke, peb cov txiaj ntsig tau qhia tias cov cim tshwj xeeb hauv cov kab mob metabolic tshwj xeeb tuaj yeem cuam tshuam txog kev hloov pauv ntawm cov qog nqaij hlav thaum ntxov thiab muaj peev xwm muaj txiaj ntsig raws li kev pheej hmoo, tsis muaj qhov loj me.
Qhov tseem ceeb, lub ntsws adenocarcinoma (LUAD) thiab squamous cell carcinoma (LUSC) yog hom tseem ceeb ntawm cov qog nqaij hlav tsis me me (NSCLC).Muab hais tias LUSC muaj kev cuam tshuam nrog kev haus luam yeeb47 thiab LUAD yog qhov feem ntau histology ntawm qhov teeb meem ntawm lub ntsws nodules kuaj pom ntawm CT screening48, peb cov qauv classifier tau tsim tshwj xeeb rau theem I adenocarcinoma kuaj.Wang thiab cov npoj yaig kuj tau tsom mus rau LUAD thiab tau txheeb xyuas cuaj lub npe lipid siv lipidomics kom paub qhov txawv ntawm qhov mob ntsws cancer ntawm cov neeg noj qab haus huv17.Peb tau sim cov qauv kev faib tawm tam sim no ntawm 16 tus neeg mob ntawm theem I LUSC thiab 74 benign nodules thiab pom cov LUSC qhov tseeb qhov tseeb (AUC 0.776), qhia tias LUAD thiab LUSC tej zaum yuav muaj lawv tus kheej metabolomic kos npe.Tseeb tiag, LUAD thiab LUSC tau pom tias sib txawv hauv etiology, keeb kwm ntawm keeb kwm thiab caj ces aberrations49.Yog li ntawd, lwm hom histology yuav tsum tau muab tso rau hauv cov qauv kev cob qhia rau cov pej xeem-raws li kev kuaj mob ntsws cancer hauv kev tshuaj ntsuam xyuas.
Ntawm no, peb tau txheeb xyuas txog rau txoj hauv kev hloov pauv ntau tshaj plaws hauv lub ntsws adenocarcinoma piv nrog kev tswj hwm kev noj qab haus huv thiab benign nodules.Xanthine thiab hypoxanthine yog cov metabolites ntawm purine metabolic txoj kev.Raws li peb cov txiaj ntsig, cov neeg nruab nrab cuam tshuam nrog purine metabolism tau nce ntau hauv cov ntshav los yog cov ntaub so ntswg ntawm cov neeg mob ntsws adenocarcinoma piv nrog kev noj qab haus huv los yog cov neeg mob ntawm preinvasive theem 15,50.Kev nce qib hauv cov ntshav xanthine thiab hypoxanthine tuaj yeem cuam tshuam qhov anabolism xav tau los ntawm cov qog nqaij hlav qog nqaij hlav sai sai.Dysregulation ntawm qabzib metabolism yog ib tug zoo-paub lub cim ntawm cancer metabolism51.Ntawm no, peb tau pom muaj qhov nce ntxiv hauv pyruvate thiab lactate hauv LA pawg piv nrog HC thiab BN pawg, uas zoo ib yam nrog cov ntawv tshaj tawm dhau los ntawm glycolytic txoj kev txawv txav hauv cov ntshav metabolome profiles ntawm cov neeg mob uas tsis yog mob ntsws cancer (NSCLC) thiab tswj kev noj qab haus huv.cov txiaj ntsig tau zoo ib yam 52,53.
Qhov tseem ceeb, peb tau pom qhov sib cuam tshuam ntawm pyruvate thiab tryptophan metabolism hauv cov ntshav ntawm lub ntsws adenocarcinomas.Cov qib ntshav tryptophan tau txo qis hauv pawg LA piv nrog HC lossis BN pawg.Interestingly, ib tug yav tas los loj-scale kev tshawb fawb siv ib tug yav tom ntej cohort pom tau hais tias tsawg theem ntawm circulating tryptophan yog txuam nrog ib tug muaj zog kev pheej hmoo ntawm mob ntsws cancer 54.Tryptophan yog ib qho tseem ceeb amino acid uas peb tau txais tag nrho los ntawm cov zaub mov.Peb xaus lus tias cov ntshav tryptophan depletion hauv ntsws adenocarcinoma tuaj yeem cuam tshuam sai ntawm cov metabolite no.Nws paub zoo tias cov khoom kawg ntawm tryptophan catabolism ntawm txoj kev kynurenine yog lub hauv paus ntawm de novo NAD + synthesis.Vim tias NAD + yog tsim los ntawm txoj kev khaws cia, qhov tseem ceeb ntawm NAD + hauv tryptophan metabolism hauv kev noj qab haus huv thiab kab mob tseem yuav txiav txim siab46.Peb qhov kev soj ntsuam ntawm TCGA cov ntaub ntawv tau pom tias qhov kev qhia ntawm tryptophan transporter solute transporter 7A5 (SLC7A5) tau nce ntau hauv lub ntsws adenocarcinoma piv nrog cov kev tswj ib txwm muaj thiab muaj kev cuam tshuam zoo nrog kev qhia ntawm glycolytic enzyme GAPDH.Cov kev tshawb fawb yav dhau los tau tsom mus rau lub luag haujlwm ntawm tryptophan catabolism hauv kev tawm tsam cov tshuaj tiv thaiv kab mob tiv thaiv kab mob 40,41,42.Ntawm no peb qhia tau tias inhibition ntawm tryptophan uptake los ntawm knockdown ntawm SLC7A5 nyob rau hauv lub ntsws cancer hlwb ua rau ib tug tom qab txo nyob rau hauv cellular NAD theem thiab ib tug concomitant attenuation ntawm glycolytic kev ua si.Hauv cov ntsiab lus, peb txoj kev tshawb fawb muab lub hauv paus lom neeg rau kev hloov pauv hauv cov ntshav cov metabolism cuam tshuam nrog kev hloov pauv ntawm lub ntsws adenocarcinoma.
EGFR kev hloov pauv yog qhov kev hloov pauv ntawm tus tsav tsheb ntau tshaj plaws hauv cov neeg mob NSCLC.Hauv peb txoj kev tshawb fawb, peb pom tias cov neeg mob uas muaj EGFR hloov pauv (n = 41) muaj tag nrho cov txheej txheem metabolomic zoo ib yam li cov neeg mob uas muaj hom tsiaj qus EGFR (n = 31), txawm hais tias peb pom cov ntshav qis qis ntawm qee cov neeg mob EGFR mutant hauv cov neeg mob acylcarnitine.Lub luag haujlwm tsim ntawm acylcarnitines yog thauj acyl pawg los ntawm cytoplasm mus rau hauv mitochondrial matrix, ua rau oxidation ntawm fatty acids los tsim lub zog 55.Raws li peb qhov kev tshawb pom, ib txoj kev tshawb fawb tsis ntev los no kuj tau txheeb xyuas cov metabolome zoo sib xws ntawm EGFR mutant thiab EGFR cov qog nqaij hlav qus los ntawm kev txheeb xyuas lub ntiaj teb metabolome ntawm 102 lub ntsws adenocarcinoma cov ntaub so ntswg kuaj 50.Interestingly, cov ntsiab lus acylcarnitine kuj pom nyob rau hauv EGFR mutant pawg.Yog li, txawm hais tias kev hloov pauv hauv cov qib acylcarnitine cuam tshuam txog EGFR-vim cov kev hloov pauv hauv metabolic thiab cov hauv paus ntawm cov txheej txheem molecular yuav ua tau zoo rau kev kawm ntxiv.
Hauv kev xaus, peb txoj kev tshawb fawb tau tsim kom muaj cov tshuaj lom neeg hauv cov ntshav rau kev kuaj mob sib txawv ntawm pulmonary nodules thiab tawm tswv yim txog kev ua haujlwm uas tuaj yeem ua kom zoo tshaj qhov kev ntsuas kev pheej hmoo thiab pab tswj kev kho mob raws li kev tshuaj ntsuam CT scan.
Txoj kev tshawb no tau pom zoo los ntawm Pawg Saib Xyuas Kev Ncaj Ncees ntawm Sun Yat-sen University Cancer Tsev Kho Mob, Thawj Tsev Kho Mob ntawm Sun Yat-sen University, thiab Pawg Saib Xyuas Kev Ncaj Ncees ntawm Zhengzhou University Cancer Hospital.Hauv kev tshawb pom thiab cov pab pawg muaj kev lees paub, 174 sera los ntawm cov tib neeg noj qab haus huv thiab 244 sera los ntawm benign nodules tau sau los ntawm cov tib neeg uas tau mus kuaj mob txhua xyoo ntawm Lub Tsev Haujlwm Saib Xyuas Kev Tiv Thaiv thiab Tiv Thaiv Kab Mob, Sun Yat-sen University Cancer Center, thiab 166 benign nodules.ntshav qab zib.Theem I ntsws adenocarcinomas tau sau los ntawm Sun Yat-sen University Cancer Center.Nyob rau sab nraud validation cohort, muaj 48 tus neeg mob ntawm benign nodules, 39 mob ntawm theem kuv lub ntsws adenocarcinoma los ntawm First Affiliated Tsev Kho Mob ntawm Sun Yat-sen University, thiab 24 mob ntawm theem I ntsws adenocarcinoma los ntawm Zhengzhou Cancer Tsev Kho Mob.Sun Yat-sen University Cancer Center kuj tau sau 16 tus neeg mob ntawm theem I squamous cell ntsws cancer los kuaj lub peev xwm kuaj mob ntawm tus tsim metabolic classifier (tus yam ntxwv ntawm tus neeg mob tau pom nyob rau hauv Daim Ntawv Ntxiv 5).Cov piv txwv los ntawm pawg neeg tshawb nrhiav thiab pawg neeg pov thawj sab hauv tau raug sau thaum Lub Ib Hlis 2018 txog Lub Tsib Hlis 2020. Cov qauv rau pawg neeg saib xyuas sab nraud tau sau thaum Lub Yim Hli 2021 txog Lub Kaum Hli 2022. Txhawm rau txo qis kev tsis ncaj ncees ntawm poj niam txiv neej, kwv yees li tus naj npawb ntawm cov txiv neej thiab poj niam sib npaug. pawg.Discovery Team thiab Internal Review Team.Cov neeg koom nrog poj niam txiv neej tau txiav txim siab raws li daim ntawv qhia tus kheej.Cov ntaub ntawv pom zoo tau txais los ntawm txhua tus neeg koom nrog thiab tsis muaj nyiaj them poob haujlwm.Cov ncauj lus uas muaj benign nodules yog cov uas muaj cov qhab nia CT ruaj khov ntawm 2 mus rau 5 xyoo thaum lub sijhawm tshuaj xyuas, tshwj tsis yog rau 1 rooj plaub los ntawm cov qauv siv sab nraud, uas tau sau ua ntej thiab kuaj xyuas los ntawm histopathology.Nrog rau kev zam ntawm mob bronchitis.Cov mob ntsws adenocarcinoma tau sau ua ntej lub ntsws resection thiab paub tseeb los ntawm kev kuaj mob pathological.Cov ntshav yoo mov tau sau rau hauv cov hlab ntshav sib cais yam tsis muaj cov tshuaj tiv thaiv coagulants.Cov ntshav kuaj ntshav tau txhaws rau 1 teev nyob rau hauv chav tsev kub thiab tom qab ntawd centrifuged ntawm 2851 × g rau 10 feeb ntawm 4 ° C txhawm rau sau cov ntshav qab zib.Serum aliquots tau khov ntawm -80 ° C kom txog thaum metabolite extraction.Lub Tsev Haujlwm Saib Xyuas Kev Tiv Thaiv Kab Mob thiab Kev Tshuaj Kho Mob ntawm Sun Yat-sen University Cancer Center tau sau cov kua dej los ntawm 100 tus neeg noj qab haus huv, suav nrog cov txiv neej thiab poj niam muaj hnub nyoog 40 txog 55 xyoo sib npaug.Qhov sib npaug ntawm txhua tus qauv pub dawb tau muab sib xyaw, lub pas dej ua ke tau muab faib thiab khaws cia ntawm -80 ° C.Cov kua dej sib tov tau siv los ua cov ntaub ntawv siv rau kev tswj kom zoo thiab cov ntaub ntawv txheem.
Cov ntaub ntawv pov thawj thiab cov qauv kuaj tau thawed thiab cov metabolites tau muab rho tawm los ntawm kev sib xyaw ua ke (MTBE / methanol / dej) 56 .Luv luv, 50 μl ntawm cov ntshav yog tov nrog 225 μl ntawm cov dej khov-txias methanol thiab 750 μl ntawm cov dej khov-txias methyl tert-butyl ether (MTBE).Do cov sib tov thiab incubate on dej khov rau 1 teev.Cov qauv no tau muab sib xyaw thiab vortex sib xyaw nrog 188 μl ntawm MS-qib dej uas muaj cov qauv sab hauv (13C-lactate, 13C3-pyruvate, 13C-methionine, thiab 13C6-isoleucine, yuav los ntawm Cambridge Isotope Laboratories).Qhov sib tov yog ces centrifuged ntawm 15,000 × g rau 10 min ntawm 4 ° C, thiab theem qis tau hloov mus rau hauv ob lub raj (125 μL txhua) rau LC-MS tsom xam hauv hom zoo thiab tsis zoo.Thaum kawg, tus qauv tau evaporated rau dryness nyob rau hauv ib tug high-ceev lub tshuab nqus tsev concentrator.
Cov metabolites qhuav tau rov tsim dua hauv 120 μl ntawm 80% acetonitrile, vortexed rau 5 min, thiab centrifuged ntawm 15,000 × g rau 10 min ntawm 4 ° C.Supernatants tau pauv mus rau hauv amber iav vials nrog microinserts rau kev tshawb fawb metabolomics.Untargeted metabolomics tsom xam ntawm ib qho kev ua tau zoo ntawm cov kua chromatography-high-resolution mass spectrometry (UPLC-HRMS) platform.Metabolites tau muab cais siv Dionex Ultimate 3000 UPLC system thiab ACQUITY BEH Amide kem (2.1 × 100 mm, 1.7 μm, Dej).Hauv hom ion zoo, cov theem mobile yog 95% (A) thiab 50% acetonitrile (B), txhua tus muaj 10 mmol / L ammonium acetate thiab 0.1% formic acid.Hauv hom tsis zoo, cov theem ntawm tes A thiab B muaj 95% thiab 50% acetonitrile, raws li, ob theem muaj 10 mmol/L ammonium acetate, pH = 9. Qhov kev pab cuam gradient yog raws li nram no: 0–0.5 min, 2% B;0.5-12 feeb, 2-50% B;12–14 feeb, 50–98% B;14–16 feeb, 98% B;16-16.1 Nws.min, 98-2% B;16.1–20 min, 2% B. Cov kem tau khaws cia ntawm 40 ° C thiab cov qauv ntawm 10 ° C hauv autosampler.Tus nqi ntws yog 0.3 ml / min, lub ntim ntim yog 3 μl.Q-Exactive Orbitrap huab hwm coj spectrometer (Thermo Fisher Scientific) nrog lub electrospray ionization (ESI) qhov chaw tau ua nyob rau hauv tag nrho scan hom thiab ua ke nrog ddMS2 xyuas hom los sau cov ntaub ntawv loj.MS tsis tau teev tseg raws li hauv qab no: tshuaj tsuag voltage +3.8 kV /- 3.2 kV, capillary kub 320 ° C, shielding gas 40 arb, auxiliary gas 10 arb, sojntsuam rhaub kub 350 ° C, scan ntau 70-1050 m / h, daws teeb meem.70 000. Cov ntaub ntawv tau txais los ntawm Xcalibur 4.1 (Thermo Fisher Scientific).
Txhawm rau soj ntsuam cov ntaub ntawv zoo, cov qauv tswj kev tswj hwm zoo (QC) tau tsim los ntawm kev tshem tawm 10 μL aliquots ntawm supernatant los ntawm txhua tus qauv.Rau qhov zoo tswj cov qauv txhaj tshuaj tau txheeb xyuas thaum pib ntawm kev tshuaj ntsuam xyuas raws li kev ntsuas kev ruaj ntseg ntawm UPLC-MS system.Cov qauv tswj kev ua tau zoo yog tom qab ntawd muab tso rau hauv lub batch.Tag nrho 11 pawg ntawm cov qauv ntshav hauv qhov kev tshawb fawb no tau txheeb xyuas los ntawm LC-MS.Aliquots ntawm lub pas dej da dej sib tov los ntawm 100 tus neeg noj qab haus huv tau siv los ua cov khoom siv hauv cov khoom siv los saib xyuas cov txheej txheem rho tawm thiab kho rau cov teebmeem batch-rau-batch.Untargeted metabolomics tsom xam ntawm kev tshawb pom cohort, internal validation cohort, thiab sab nraud validation cohort tau ua nyob rau hauv lub Metabolomics Center ntawm Sun Yat-sen University.Lub chaw soj nstuam sab nraud ntawm Guangdong University of Technology Analysis thiab Test Center kuj tau tshuaj xyuas 40 cov qauv los ntawm pawg sab nraud los ntsuas qhov ua tau zoo ntawm cov qauv cais.
Tom qab kev rho tawm thiab rov tsim dua tshiab, kev ntsuas qhov tseeb ntawm cov tshuaj lom neeg hauv cov ntshav tau raug ntsuas los ntawm kev siv ultra-siab ua kua chromatography-tandem huab hwm coj spectrometry (Agilent 6495 triple quadrupole) nrog ib qho electrospray ionization (ESI) qhov chaw hauv ntau hom tshuaj tiv thaiv (MRM).Ib qho ACQUITY BEH Amide kem (2.1 × 100 mm, 1.7 μm, Dej) tau siv los cais cov metabolites.Cov theem mobile muaj 90% (A) thiab 5% acetonitrile (B) nrog 10 mmol / L ammonium acetate thiab 0.1% ammonia daws.Qhov kev pab cuam gradient tau raws li hauv qab no: 0–1.5 min, 0% B;1.5–6.5 feeb, 0–15% B;6.5–8 feeb, 15% B;8–8.5 feeb, 15%–0% B;8.5–11.5 feeb, 0%B.Kem tau khaws cia ntawm 40 ° C thiab cov qauv ntawm 10 ° C hauv autosampler.Tus nqi ntws yog 0.3 mL / min thiab txhaj tshuaj yog 1 μL.MS tsis tau teem caij raws li hauv qab no: capillary voltage ± 3.5 kV, nebulizer siab 35 psi, sheath gas flow 12 L/min, sheath gas temperature 350°C, drying gas temperature 250°C, and drying gas flow 14 l/min.MRM hloov pauv ntawm tryptophan, pyruvate, lactate, hypoxanthine thiab xanthine yog 205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 thiab 151.0–107.9 sib.Cov ntaub ntawv tau sau los ntawm Mass Hunter B.07.00 (Agilent Technologies).Rau cov qauv kuaj ntshav, tryptophan, pyruvate, lactate, hypoxanthine, thiab xanthine tau suav nrog siv calibration curves ntawm cov qauv sib tov tov.Rau cov qauv ntawm tes, cov ntsiab lus tryptophan tau normalized rau cov qauv sab hauv thiab cell protein ntau.
Peak extraction (m / z thiab tuav lub sijhawm (RT)) tau ua los ntawm Compound Discovery 3.1 thiab TraceFinder 4.0 (Thermo Fisher Scientific).Txhawm rau tshem tawm qhov sib txawv ntawm cov batch, txhua tus yam ntxwv ncov ntawm cov qauv kuaj tau muab faib los ntawm cov yam ntxwv ncov ntawm cov khoom siv los ntawm tib batch kom tau txais cov txheeb ze ntau.Cov txheej txheem sib txawv ntawm cov qauv sab hauv ua ntej thiab tom qab kev tsim qauv tau pom nyob rau hauv Daim Ntawv Ntxiv 6. Qhov sib txawv ntawm ob pawg tau pom los ntawm qhov kev tshawb pom tsis tseeb (FDR<0.05, Wilcoxon kos npe xeem qib) thiab hloov pauv (> 1.2 lossis <0.83).Raw MS cov ntaub ntawv ntawm cov yam ntxwv uas tau muab rho tawm thiab siv cov ntaub ntawv kho MS cov ntaub ntawv tau qhia hauv Cov Ntaub Ntawv Ntxiv 1 thiab Cov Ntaub Ntawv Ntxiv 2, raws li.Peak annotation tau ua raws li plaub theem ntawm kev txheeb xyuas, suav nrog kev txheeb xyuas cov metabolites, putatively annotated compounds, putatively characterized compound classes, and unknown compounds 22 .Raws li cov ntaub ntawv tshawb fawb hauv Compound Discovery 3.1 (mzCloud, HMDB, Chempider), cov tshuaj lom neeg lom neeg nrog MS / MS sib piv cov qauv siv tau los yog cov ntsiab lus sib tw hauv mzCloud (cov qhab nia> 85) lossis Chempider thaum kawg raug xaiv los ua cov nruab nrab ntawm cov metabolome sib txawv.Peak annotations rau txhua qhov tshwj xeeb muaj nyob rau hauv Cov Ntaub Ntawv Ntxiv 3. MetaboAnalyst 5.0 tau siv rau kev tshuaj xyuas qhov tsis sib xws ntawm cov lej-normalized metabolite abundance.MetaboAnalyst 5.0 kuj tau soj ntsuam KEGG txoj kev txhim kho kev txheeb xyuas raws li cov metabolites sib txawv.Tus Thawj Saib Xyuas Kev Tshawb Fawb (PCA) thiab ib feem tsawg kawg ntawm kev sib cais sib cais (PLS-DA) tau txheeb xyuas siv ropls software pob (v.1.26.4) nrog pawg normalization thiab autoscaling.Qhov zoo tshaj plaws metabolite biomarker qauv rau kev kwv yees nodule malignancy tau tsim siv binary logistic regression nrog tsawg kawg nkaus shrinkage thiab xaiv tus neeg teb xov tooj (LASSO, R pob v.4.1-3).Qhov kev ua tau zoo ntawm cov qauv kev ntxub ntxaug hauv kev tshawb nrhiav thiab kev siv tau cov teeb meem tau tshwm sim los ntawm kev kwv yees AUC raws li kev txheeb xyuas ROC raws li cov pob pPROC (v.1.18.0.).Qhov kev txiav txim siab zoo tshaj plaws tau txais los ntawm qhov siab tshaj plaws Youden Performance index ntawm tus qauv (rhiab heev + qhov tshwj xeeb - 1).Cov qauv uas muaj nuj nqis tsawg dua lossis ntau dua qhov pib yuav raug kwv yees li benign nodules thiab ntsws adenocarcinoma, raws li.
A549 hlwb (#CCL-185, American Hom Culture Collection) tau loj hlob hauv F-12K nruab nrab uas muaj 10% FBS.luv hairpin RNA (shRNA) sequences targeting SLC7A5 and a nontargeting control (NC) tau muab tso rau hauv lentiviral vector pLKO.1-puro.Lub antisense sequences ntawm shSLC7A5 yog raws li nram no: Sh1 (5′-GGAGAAAACCTGATGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGAACTAT-3′).Cov tshuaj tiv thaiv rau SLC7A5 (#5347) thiab tubulin (#2148) tau yuav los ntawm Cell Signaling Technology.Cov tshuaj tiv thaiv kab mob rau SLC7A5 thiab tubulin tau siv los ntawm dilution ntawm 1: 1000 rau Western blot tsom xam.
Seahorse XF Glycolytic Stress Test ntsuas cov qib ntawm cov cellular acidification (ECAR).Hauv kev ntsuam xyuas, cov piam thaj, oligomycin A, thiab 2-DG tau muab tshuaj ua ntu zus los kuaj cov cellular glycolytic muaj peev xwm raws li ntsuas los ntawm ECAR.
A549 hlwb transfected nrog non-targeting tswj (NC) thiab shSLC7A5 (Sh1, Sh2) tau plated hmo ntuj nyob rau hauv 10 cm txoj kab uas hla tais diav.Cell metabolites tau muab rho tawm nrog 1 ml ntawm dej khov-txias 80% aqueous methanol.Cov hlwb hauv cov tshuaj methanol raug muab tshem tawm, sau rau hauv lub raj tshiab, thiab centrifuged ntawm 15,000 × g rau 15 min ntawm 4 ° C.Sau 800 µl ntawm supernatant thiab qhuav siv lub tshuab nqus tsev siab ceev.Cov pellets qhuav metabolite tau raug tshuaj xyuas rau qib tryptophan siv LC-MS / MS raws li tau piav qhia saum toj no.Cellular NAD (H) qib hauv A549 hlwb (NC thiab shSLC7A5) tau ntsuas los ntawm cov khoom siv NAD + / NADH colorimetric (#K337, BioVision) raws li cov chaw tsim khoom cov lus qhia.Cov qib protein tau ntsuas rau txhua tus qauv kom normalize cov metabolites.
Tsis muaj cov txheej txheem txheeb cais tau siv los txiav txim siab ua ntej tus qauv loj.Cov kev tshawb fawb metabolomics yav dhau los tsom rau biomarker discovery15,18 tau raug suav tias yog cov qauv ntsuas rau kev txiav txim siab loj, thiab piv rau cov ntaub ntawv no, peb cov qauv tsim nyog.Tsis muaj cov qauv raug cais tawm ntawm pawg neeg tshawb fawb.Cov qauv kuaj ntshav tau muab tso rau hauv pawg tshawb pom (306 tus neeg mob, 74.6%) thiab pab pawg kuaj xyuas sab hauv (104 tus neeg mob, 25.4%) rau cov kev tshawb fawb metabolomics tsis tau pom zoo.Peb kuj tau xaiv 70 qhov xwm txheej los ntawm txhua pab pawg los ntawm qhov kev tshawb pom tau teeb tsa rau cov kev tshawb fawb txog metabolomics.Cov neeg tshawb xyuas tau ua qhov muag tsis pom kev rau pab pawg ua haujlwm thaum lub sijhawm LC-MS cov ntaub ntawv sau thiab tshuaj xyuas.Kev txheeb xyuas ntawm cov ntaub ntawv metabolomics thiab cov kev sim ntawm tes tau piav qhia hauv cov txiaj ntsig, Daim duab Legends, thiab Cov Txheej Txheem.Kev ntsuas ntawm cellular tryptophan, NADT, thiab glycolytic kev ua haujlwm tau ua peb zaug ntawm nws tus kheej nrog cov txiaj ntsig zoo ib yam.
Yog xav paub ntxiv txog kev tsim qauv, saib Natural Portfolio Report Abstract txuam nrog rau kab lus no.
Cov ntaub ntawv nyoos MS ntawm cov yam ntxwv uas tau muab rho tawm thiab cov ntaub ntawv MS normalized ntawm cov ntaub ntawv siv ntshav tau qhia hauv Cov Ntaub Ntawv Ntxiv 1 thiab Cov Ntaub Ntawv Ntxiv 2, raws li.Peak annotations for differential features are nthuav tawm hauv Cov Ntaub Ntawv Ntxiv 3. Cov ntaub ntawv LUAD TCGA tuaj yeem rub tawm los ntawm https://portal.gdc.cancer.gov/.Cov ntaub ntawv input rau plotting lub graph yog muab nyob rau hauv cov ntaub ntawv qhov chaw.Cov ntaub ntawv tau muab rau cov kab lus no.
National Lung Screening Study Group, thiab lwm yam. Txo cov qog nqaij hlav cancer hauv lub ntsws nrog kev siv tshuaj qis qis.Northern England.J. Med.365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR thiab Yaj Saub, PC Kev kuaj mob ntsws cancer siv cov tshuaj qis helical CT: tau los ntawm National Lung Screening Study (NLST).J. Med.Screen 18, 109–111 (2011).
De Koning, HJ, et al.Txo kev mob qog noj ntshav hauv lub ntsws nrog kev kuaj ntsuas CT hauv kev sim randomized.Northern England.J. Med.382, 503–513 (2020).
Post lub sij hawm: Sep-18-2023