Ukuxilongwa okwahlukileyo kwee-nodule ze-pulmonary ezichongiweyo nge-computed tomography (CT) ihlala ingumngeni ekusebenzeni kwekliniki.Apha, sibonakalisa i-metabolome yehlabathi jikelele yeesampulu ze-serum ze-480, kubandakanywa ulawulo olunempilo, amaqhuqhuva e-benign lung, kunye nesigaba I lung adenocarcinoma.I-Adenocarcinomas ibonisa iiprofayili ezizodwa ze-metabolomic, ngelixa ii-nodules ezinobungozi kunye nabantu abasempilweni banokufana okuphezulu kwiiprofayili ze-metabolomic.Kwiqela lokufumanisa (n = 306), isethi ye-metabolites ye-27 ichongiwe ukuhlula phakathi kwee-nodules ezinobungozi kunye nezibi.I-AUC yemodeli yocalucalulo ekuqinisekiseni kwangaphakathi (n = 104) kunye nokuqinisekiswa kwangaphandle (n = 111) amaqela ngu-0.915 kunye ne-0.945, ngokulandelanayo.Uhlalutyo lwendlela lubonakalise ukonyuka kwe-glycolytic metabolites ehambelana nokuncipha kwe-tryptophan kwiserum ye-adenocarcinoma ye-lung xa kuthelekiswa namaqhuqhuva angenabungozi kunye nolawulo olusempilweni, kwaye yacebisa ukuba ukufunyanwa kwe-tryptophan kukhuthaza i-glycolysis kwiiseli zomhlaza wemiphunga.Uphononongo lwethu lubonisa ixabiso le-serum metabolite biomarkers ekuhloleni umngcipheko we-pulmonary nodules efunyenwe yi-CT.
Ukuxilongwa kwangethuba kubalulekile ekuphuculeni amazinga okuphila kwizigulane ezinomhlaza.Iziphumo ezivela kwi-US National Lung Cancer Screening Trial (NLST) kunye ne-European NELSON Study ibonise ukuba ukuhlolwa nge-dose ephantsi ye-computed tomography (LDCT) kunokunciphisa kakhulu ukufa komhlaza wemiphunga kumaqela anobungozi obukhulu1,2,3.Ukusukela ekusetyenzisweni okubanzi kwe-LDCT yokuhlola umhlaza wemiphunga, izehlo zokufunyaniswa kwe-radiographic ngengozi ye-asymptomatic pulmonary nodules ziye zaqhubeka zanda i-4.Amaqhuqhuva e-pulmonary achazwa njenge-opacities ekugxilwe kuyo ukuya kwi-3 cm ububanzi be-5.Sijongene nobunzima ekuhloleni ukuba nokwenzeka kokwenzakala kunye nokujongana nenani elikhulu lamaqhuqhuva emiphunga afunyaniswe ngengozi kwi-LDCT.Ukulinganiselwa kwe-CT kunokukhokelela kwiimviwo ezilandelelweyo rhoqo kunye neziphumo ezingezizo zobuxoki, ezikhokelela kungenelelo olungeyomfuneko kunye nonyango olugqithisileyo6.Ngoko ke, kukho imfuneko yokuphuhlisa i-biomarkers ethembekileyo kunye neluncedo ukuchonga ngokuchanekileyo umhlaza wemiphunga kwizigaba zokuqala kunye nokwahlula ama-nodules amaninzi ekufumaneni kokuqala kwe-7.
Uhlalutyo olubanzi lweemolekyuli zegazi (i-serum, i-plasma, i-peripheral blood mononuclear cells), kuquka i-genomics, i-proteomics okanye i-DNA methylation8,9,10, ikhokelele ekukhuleni komdla ekufumaneni i-biomarkers yokuxilongwa komhlaza wemiphunga.Okwangoku, iindlela ze-metabolomics zilinganisa iimveliso zokuphela kweeseli eziphenjelelwa zizenzo ezingapheliyo kunye nezangaphandle kwaye ke zisetyenziselwa ukuqikelela ukuqala kwesifo kunye nesiphumo.I-Liquid chromatography-tandem mass spectrometry (LC-MS) yindlela esetyenziswa ngokubanzi kwizifundo ze-metabolomics ngenxa yovakalelo oluphezulu kunye noluhlu olukhulu oluguquguqukayo, olunokugubungela i-metabolites eneempawu ezahlukeneyo ze-physicochemical11,12,13.Nangona uhlalutyo lwe-metabolomic yehlabathi jikelele lwe-plasma / serum lusetyenziselwe ukuchonga iimpawu ze-biomarkers ezinxulumene nokuxilongwa komhlaza wemiphunga14,15,16,17 kunye nempumelelo yonyango, i-18 serum metabolite classifiers ukwahlula phakathi kwe-benign kunye ne-nodules ye-lung enobungozi kusafuneka kufundwe kakhulu.-uphando olukhulu.
I-Adenocarcinoma kunye ne-squamous cell carcinoma zezona ntlobo ziphambili zomhlaza wemiphunga weseli engeyiyo encinci (NSCLC).Iimvavanyo ezahlukeneyo ze-CT screening zibonisa ukuba i-adenocarcinoma lolona hlobo luqhelekileyo lwe-histological yomhlaza wemiphunga1,19,20,21.Kolu phononongo, sisebenzise i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) ukwenza uhlalutyo lwe-metabolomics kwiisampulu ze-serum ze-695 zizonke, kubandakanywa nokulawula okunempilo, i-benign pulmonary nodules, kunye ne-CT-ifunyenwe ≤3 cm.Uvavanyo lweSigaba I lung adenocarcinoma.Sichonge iphaneli ye-metabolites ye-serum eyahlula i-lung adenocarcinoma kwii-nodules ezinobungozi kunye nokulawula okunempilo.Uhlalutyo lwendlela yokutyebisa lubonise ukuba i-tryptophan engaqhelekanga kunye ne-glucose metabolism lutshintsho oluqhelekileyo kwi-lung adenocarcinoma xa kuthelekiswa namaqhuqhuva anobungozi kunye nolawulo olusempilweni.Okokugqibela, siye saseka kwaye saqinisekisa udidi lwe-serum metabolic olucacileyo oluphezulu kunye novakalelo lokwahlula phakathi kwamaqhuqhuva abi kunye ne-benign pulmonary afunyenwe yi-LDCT, enokunceda ekuxilongeni kwangaphambili okwahlukileyo kunye novavanyo lomngcipheko.
Kuphononongo lwangoku, iisampulu zeserum ezihambelana nesondo kunye nobudala ziye zaqokelelwa ngokuphindaphindiweyo ukusuka kulawulo olusempilweni lwe-174, izigulane ezingama-292 ezinamaqhuqhuva e-benign pulmonary, kunye nezigulane ezingama-229 ezinenqanaba I lung adenocarcinoma.Iimpawu zedemografi yezifundo ezingama-695 ziboniswa kwiTheyibhile eyoNgezelelweyo 1.
Njengoko kubonisiwe kwi-Figure 1a, i-sampulu ye-serum ye-480 iyonke, kubandakanywa i-174 yolawulo olunempilo (HC), i-170 ye-benign nodules (BN), kunye ne-136 isigaba se-I lung adenocarcinoma (LA) iisampulu, zaqokelelwa kwi-Sun Yat-sen University Cancer Centre.Iqela lokufumanisa iprofayili yemetabolomic engajoliswanga kusetyenziswa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Njengoko kubonisiwe kwi-Supplementary Figure 1, i-metabolites eyahlukileyo phakathi kwe-LA kunye ne-HC, i-LA kunye ne-BN ichongiwe ukuseka imodeli yokuhlela kunye nokuphonononga uhlalutyo lwendlela eyahlukileyo.Iisampulu ze-104 eziqokelelwe yi-Sun Yat-sen University Cancer Centre kunye neesampuli ze-111 eziqokelelwe zezinye izibhedlele ezimbini ziphantsi kokuqinisekiswa kwangaphakathi nangaphandle, ngokulandelanayo.
Inani labemi bophononongo kwiqela lokufumanisa eliye lenziwa uhlalutyo lwe-serum metabolomics yehlabathi kusetyenziswa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).b Ngokuyinxenye ubuncinane izikwere ucalucalulo (PLS-DA) ye-metabolome iyonke 480 iisampulu serum ukusuka kwiqela lophononongo, kuquka ulawulo olusempilweni (HC, n = 174), amaqhuqhuva benign (BN, n = 170), kunye nenqanaba I lung adenocarcinoma (Los Angeles, n = 136).+ESI, imowudi ye-ionization ye-electrospray efanelekileyo, -ESI, imowudi ye-ionization ye-electrospray engalunganga.c–e I-Metabolites enobuninzi obahluke kakhulu kumaqela amabini anikiweyo (i-Wilcoxon enemisila emibini yovavanyo lwenqanaba elisayiniweyo, izinga lokufunyanwa kobuxoki elihlengahlengisiweyo ixabiso le-p, i-FDR <0.05) iboniswe ngobomvu (utshintsho oluphindwe kabini> 1.2) kunye noluhlaza okwesibhakabhaka (ukutshintsha kwe-fold <0.83) .) eboniswe kumzobo wentaba-mlilo.f Imephu yobushushu ehlanganisiweyo yeHierarchical ebonisa umahluko omkhulu kwinani leemetabolites ezichaziweyo phakathi kwe-LA kunye ne-BN.Idatha yomthombo inikwe ngohlobo lweefayile zomthombo wedatha.
I-metabolome ye-serum epheleleyo ye-174 HC, i-170 BN kunye ne-136 LA kwiqela lokufumanisa yahlalutywa ngokusebenzisa uhlalutyo lwe-UPLC-HRMS.Okokuqala sibonisa ukuba iisampuli zokulawula umgangatho (i-QC) zidibanisa ngokuqinileyo kwiziko lohlalutyo lwecandelo eliyintloko (PCA) elingagadwanga, eliqinisekisa ukuzinza kokusebenza kophando lwangoku (umzobo owongezelelweyo 2).
Njengoko kubonisiwe kuhlalutyo oluncinci lwezikwere-zocalucalulo (PLS-DA) kuMfanekiso 1 b, sifumanise ukuba kukho umahluko ocacileyo phakathi kwe-LA kunye ne-BN, i-LA kunye ne-HC kwi-positive (+ESI) kunye ne-negative (−ESI) ye-electrospray ionization modes. .kwadwa.Nangona kunjalo, akukho ntlukwano ebalulekileyo efunyenweyo phakathi kwe-BN kunye ne-HC kwi- +ESI kunye ne -ESI iimeko.
Sifumene iimpawu ze-382 ezihlukeneyo phakathi kwe-LA kunye ne-HC, iimpawu ze-231 ezihlukeneyo phakathi kwe-LA kunye ne-BN, kunye ne-95 yeempawu ezahlukileyo phakathi kwe-BN kunye ne-HC (i-Wilcoxon isayinwe uvavanyo lwenqanaba, i-FDR <0.05 kunye noshintsho oluninzi> 1.2 okanye <0.83) (Umfanekiso .1c-e) )..Iincopho zachazwa ngakumbi (iDatha eyoNgezelelweyo yesi-3) ngokuchasene nesiseko sedatha (mzCloud/HMDB/Chemspider library) ngexabiso le-m/z, ixesha lokugcinwa kunye nokuqhekeka kokukhangela kobuninzi be-spectrum (iinkcukacha ezichazwe kwicandelo leNdlela) 22.Ekugqibeleni, i-33 kunye ne-38 i-metabolites echazwe kunye nentlukwano enkulu yobuninzi ichongiwe kwi-LA ngokubhekiselele kwi-BN (Umfanekiso we-1f kunye neThebhile eyoNgezelelweyo ye-2) kunye ne-LA ngokubhekiselele kwi-HC (i-Supplementary Figure 3 kunye ne-Supplementary Table 2), ngokulandelanayo.Ngokwahlukileyo, kuphela i-metabolites ye-3 eneeyantlukwano ezinkulu kubuninzi zichongiwe kwi-BN kunye ne-HC (iThebhile eyoNgezelelweyo ye-2), ehambelana nokugqithiswa phakathi kwe-BN kunye ne-HC kwi-PLS-DA.Ezi metabolites ezihlukeneyo zigubungela uluhlu olubanzi lweekhemikhali ze-biochemicals (umzobo owongezelelweyo 4).Zithathiwe kunye, ezi ziphumo zibonisa utshintsho olubalulekileyo kwi-serum metabolome ebonisa utshintsho olubi lomhlaza wemiphunga wenqanaba lokuqala xa kuthelekiswa namaqhuqhuva emiphunga okanye izifundo ezisempilweni.Ngeli xesha, ukufana kwe-serum metabolome ye-BN kunye ne-HC ibonisa ukuba i-benign pulmonary nodules inokwabelana ngeempawu ezininzi zebhayoloji nabantu abasempilweni.Ngenxa yokuba i-epidermal growth factor receptor (EGFR) i-gene mutations ixhaphake kwi-lung adenocarcinoma subtype 23, siye safuna ukufumanisa impembelelo yokuguqulwa komqhubi kwi-serum metabolome.Emva koko sihlalutye iphrofayili ye-metabolomic jikelele yeemeko ze-72 kunye nesimo se-EGFR kwiqela le-lung adenocarcinoma.Okubangel 'umdla kukuba, sifumene iiprofayili ezifaniswayo phakathi kwezigulane eziguquguqukayo ze-EGFR (n = 41) kunye nezigulane ze-EGFR zasendle (n = 31) kuhlalutyo lwe-PCA (i-Supplementary Figure 5a).Nangona kunjalo, sichonge i-metabolites ye-7 ubuninzi bayo baguqulwa kakhulu kwizigulane ezinokuguqulwa kwe-EGFR xa kuthelekiswa nezigulane ezinohlobo lwasendle lwe-EGFR (uvavanyo lwe-t, p <0.05 kunye nokuguqulwa kwe-fold> 1.2 okanye <0.83) (i-Supplementary Figure 5b).Uninzi lwala metabolites (5 ngaphandle kwe-7) yi-acylcarnitines, edlala indima ebalulekileyo kwiindlela ze-acid oxidation.
Njengoko kubonisiwe ekuhambeni komsebenzi kuboniswe kuMzobo 2 a, i-biomarkers yokuhlelwa kwamaqhuqhuva afunyenwe kusetyenziswa abaqhubi abancinci be-shrinkage kunye nokukhethwa ngokusekelwe kwi-33 ye-metabolites eyahlukileyo echongiweyo kwi-LA (n = 136) kunye ne-BN (n = 170).Eyona ndibaniselwano yezinto eziguquguqukayo (i-LASSO) - imodeli ye-binary logistic regression model.Ukuqinisekiswa okuphindwe kabini kusetyenziswe ukuvavanya ukuthembeka kwemodeli.Ukhetho oluguquguqukayo kunye nohlengahlengiso lweparameter luhlengahlengiswa ngesohlwayo sokwandisa okunokwenzeka ngeparameter λ24.Uhlalutyo lwe-metabolomics lwehlabathi lwenziwa ngokuzimeleyo ekuqinisekiseni kwangaphakathi (n = 104) kunye nokuqinisekiswa kwangaphandle (n = 111) amaqela ukuvavanya ukusebenza kokuhlelwa komzekelo wokucalula.Ngenxa yoko, i-metabolites ye-27 ekufumaneni isethi yachongwa njengeyona ndlela ibalaseleyo yokucalula kunye nexabiso elikhulu le-AUC (umzobo 2b), phakathi kwayo i-9 yandisa umsebenzi kunye ne-18 yehla umsebenzi kwi-LA xa kuthelekiswa ne-BN (umzobo 2c).
Ukuhamba komsebenzi wokwakha i-pulmonary nodule classifier, kubandakanywa nokukhetha eyona ipaneli ye-serum metabolites kwiseti yokufumanisa kusetyenziswa imodeli yokubuyela umva kokubini ngokusebenzisa ukuqinisekiswa okuphindwe kalishumi kunye nokuvavanya ukusebenza kwangaphambili kwiiseti zokuqinisekisa zangaphakathi nangaphandle.b Iinkcukacha-manani ezinqamlezileyo zemodeli yokubuyisela i-LASSO yokukhethwa kwe-biomarker ye-metabolic.Amanani anikiweyo ngasentla amele umndilili yenani leempawu zebhayoloji ezikhethiweyo kwindawo enikiweyo λ.Umgca onamachaphaza obomvu umele umndilili wexabiso le-AUC kwi-lambda ehambelanayo.Iimpazamo zeBar ezingwevu zibonisa ubuncinci kunye nowona maxabiso aphezulu e-AUC.Umgca onamachaphaza ubonisa eyona modeli ilungileyo kunye ne-27 ekhethiweyo ye-biomarkers.I-AUC, indawo ephantsi kophawu lokusebenza lwe-receiver (ROC) curve.c Songa utshintsho lwe-27 ekhethiweyo ye-metabolites kwiqela le-LA xa kuthelekiswa neqela le-BN kwiqela lokufumanisa.Ikholamu ebomvu - ukuvuselela.Uluhlu oluluhlaza luyancipha.I-d-f I-receiver yokusebenza ye-receiver (ROC) iijika ezibonisa amandla emodeli ecalulayo esekelwe kwi-27 ye-metabolite indibaniselwano ekufumaneni, iiseti zangaphakathi, kunye nangaphandle zokuqinisekisa.Idatha yomthombo inikwe ngohlobo lweefayile zomthombo wedatha.
Imodeli yokuxela kwangaphambili yenziwe ngokusekelwe kwi-coefficients yokubuyisela ubunzima be-metabolites ye-27 (iThebhile eyoNgezelelweyo 3).Uhlalutyo lwe-ROC olusekelwe kula ma-metabolites e-27 luvelise indawo ephantsi kwe-curve (AUC) ixabiso le-0.933, ukuqonda kweqela lokufumanisa kwakuyi-0.868, kwaye i-0.859 ethile (umzobo 2d).Okwangoku, phakathi kwe-38 i-metabolites ephawulekayo ephawulekayo phakathi kwe-LA kunye ne-HC, isethi ye-metabolites ye-16 iphumelele i-AUC ye-0.902 ngovakalelo lwe-0.801 kunye neenkcukacha ze-0.856 ekucaluleni i-LA kwi-HC (i-Supplementary Figure 6a-c).Amaxabiso e-AUC asekwe kwimigangatho eyahlukeneyo yokutshintsha kweemetabolites ezahlukeneyo nazo zathelekiswa.Sifumene ukuba imodeli yokuhlelwa yenza kakuhle ekucaluleni phakathi kwe-LA kunye ne-BN (HC) xa inqanaba lokutshintsha kwe-fold lisethelwe kwi-1.2 ngokubhekiselele kwi-1.5 okanye i-2.0 (i-Supplementary Figure 7a,b).Imodeli yokwahlula, esekelwe kumaqela e-metabolite e-27, yaqinisekiswa ngakumbi kumaqela angaphakathi nangaphandle.I-AUC yayingu-0.915 (ubuntununtunu be-0.867, i-0.811 ethile) yokuqinisekiswa kwangaphakathi kunye ne-0.945 (ubuntununtunu be-0.810, i-specific 0.979) yokuqinisekiswa kwangaphandle (umzobo 2e, f).Ukuvavanya ukusebenza kakuhle kwe-interlaboratory, iisampuli ze-40 ezivela kwiqela langaphandle zihlalutywe kwibhubhoratri yangaphandle njengoko kuchazwe kwicandelo leNdlela.Ukuchaneka kokuhlelwa kuphumelele i-AUC ye-0.925 (umzobo owongezelelweyo 8).Ngenxa yokuba i-lung squamous cell carcinoma (LUSC) yolona hlobo lwesibini luqhelekileyo lomhlaza wemiphunga ongeyonxalenye encinci yeseli (NSCLC) emva kwe-lung adenocarcinoma (LUAD), siye savavanya usetyenziso oluqinisekisiweyo lweprofayili ye-metabolic.I-BN kunye ne-16 iimeko ze-LUSC.I-AUC yocalucalulo phakathi kwe-LUSC kunye ne-BN yayingu-0.776 (umzobo owongezelelweyo we-9), ebonisa isakhono esihlwempuzekileyo xa kuthelekiswa nocalucalulo phakathi kwe-LUAD kunye ne-BN.
Uphononongo lubonise ukuba ubukhulu bamaqhuqhuva kwimifanekiso ye-CT buhambelana ngokufanelekileyo kunye nokwenzeka kobugwenxa kwaye buhlala bungummiselo omkhulu wonyango lwe-nodule25,26,27.Uhlalutyo lwedatha evela kwiqela elikhulu lokuhlola ukuhlolwa kwe-NELSON lubonise ukuba umngcipheko wokuxhatshazwa kwizifundo ezinama-nodes <5 mm wawufana nokuba kwizifundo ezingenazo ii-nodes ze-28.Ngoko ke, ubungakanani obuncinci obufuna ukubeka iliso rhoqo kwi-CT yi-5 mm, njengoko kucetyiswa yi-British Thoracic Society (BTS), kunye ne-6 mm, njengoko kunconywa yi-Fleischner Society 29.Nangona kunjalo, ama-nodules amakhulu kune-6 mm kwaye ngaphandle kweempawu ezicacileyo ezicacileyo, ezibizwa ngokuba yi-indeterminate pulmonary nodules (IPN), zihlala zingumngeni omkhulu ekuvavanyeni nasekulawuleni kwikliniki30,31.Siye savavanya ngokulandelayo ukuba ubungakanani bamaqhuqhuva buphembelele utyikityo lwe-metabolomic kusetyenziswa iisampulu ezidityanisiweyo ukusuka ekufumaneni nasekuqinisekiseni kwangaphakathi amaqela.Ukugxininisa kwii-biomarkers eziqinisekisiweyo ze-27, saqala ngokuthelekisa iiprofayili ze-PCA ze-HC kunye ne-BN sub-6 mm metabolomes.Sifumene ukuba uninzi lwamanqaku edatha ye-HC kunye ne-BN idibene, ebonisa ukuba amanqanaba e-metabolite ye-serum ayefana kumaqela omabini (umzobo 3a).Imephu yeempawu kwiindidi ezahlukeneyo zobukhulu zahlala zigcinwe kwi-BN kunye ne-LA (Umfanekiso 3b, c), kanti ukuhlukana kwabonwa phakathi kwama-nodules anobungozi kunye ne-benign kwi-6-20 mm ububanzi (Umfanekiso 3d).Eli qela line-AUC ye-0.927, i-specific ye-0.868, kunye novelwano lwe-0.820 yokuxela kwangaphambili ububi bee-nodules ezilinganisa i-6 ukuya kwi-20 mm (Umfanekiso 3e, f).Iziphumo zethu zibonisa ukuba umntu ohlelayo unokubamba utshintsho lwemetabolism olubangelwa lutshintsho olubi lwangaphambili, nokuba lungakanani na ubungakanani bamaqhuqhuva.
ad Ukuthelekiswa kweeprofayili ze-PCA phakathi kwamaqela achaziweyo ngokusekelwe kuluhlu lwe-metabolic ye-27 metabolites.I-CC kunye ne-BN <6 mm.b BN < 6 mm vs BN 6-20 mm.kwi-LA 6-20 mm ngokuchasene ne-LA 20-30 mm.g BN 6-20 mm kunye LA 6-20 mm.GC, n = 174;BN < 6 mm, n = 153;BN 6-20 mm, n = 91;LA 6-20 mm, n = 89;I-LA 20–30 mm, n = 77. e I-receiver yokusebenza kwe-receiver (ROC) ijika ebonisa imodeli yokusebenza ecalucalulo kwii-nodules 6-20 mm.f Amaxabiso anokubakho abalwe ngokusekwe kwimodeli yokubuyisela umva kwiiqhuqhuva ezinomlinganiselo we-6-20 mm.Umgca onamachaphaza angwevu umele elona xabiso lifanelekileyo lokunqunyulwa (0.455).Amanani angasentla amele ipesenti yamatyala aqikelelweyo eLos Angeles.Sebenzisa uvavanyo lwe-t olunemisila emibini.I-PCA, uhlalutyo lwecandelo eliphambili.Indawo ye-AUC phantsi kwegophe.Idatha yomthombo inikwe ngohlobo lweefayile zomthombo wedatha.
Iisampulu ezine (ezineminyaka eyi-44-61 iminyaka) kunye nobukhulu obufanayo be-nodule ye-pulmonary (7-9 mm) zaphinda zakhethwa ukubonisa ukusebenza kwemodeli ecetywayo yokubikezela ububi (Umfanekiso 4a, b).Ekuhlolweni kokuqala, i-Case 1 ibonakaliswe njengeqhuqhuva eliqinileyo kunye nokubala, uphawu oluhambelana nokulunga, ngelixa i-Case 2 iboniswe njengeqhuqhuva eliqinileyo elingenammiselo kwaye akukho mpawu zicacileyo.Imijikelezo emithathu yokulandelela i-CT scans ibonise ukuba la matyala ahlala ezinzile kwixesha le-4 kwaye ngoko ke athathwa njengama-nodules angenayo (umzobo 4a).Xa kuthelekiswa novavanyo lwezonyango lwe-serial CT scans, uhlalutyo lwe-serum ye-metabolite ene-shot-shot kunye nemodeli yangoku yokuhlelwa ngokukhawuleza nangokuchanekileyo ichongiwe la maqhuqhuva angenabungozi asekelwe kwizithintelo ezinokwenzeka (Itheyibhile 1).Umzobo we-4b kwimeko ye-3 ubonisa iqhuqhuva elineempawu zokurhoxa kwe-pleural, edla ngokunxulunyaniswa ne-malignancy32.Ityala lesi-4 libonakaliswe njengeqhuqhuva eliqinileyo ngokuyinxenye engenabungqina besizathu esilungileyo.Zonke ezi meko zaqikelelwa njengeziyingozi ngokwemodeli yohlelo (Itheyibhile 1).Uvavanyo lwe-lung adenocarcinoma lubonakaliswe ngokuhlolwa kwe-histopathological emva kokuhlinzwa kwe-lung rection (Umfanekiso 4b).Kwiseti yokuqinisekisa yangaphandle, i-classifier ye-metabolic ichaze ngokuchanekileyo iimeko ezimbini ze-lung nodules ezingapheliyo ezinkulu kune-6 mm (umzobo owongezelelweyo 10).
Imifanekiso ye-CT yefestile ye-axial yemiphunga yeemeko ezimbini ze-benign nodules.Kwimeko ye-1, i-CT scan emva kweminyaka emi-4 ibonise i-nodule eqinile ezinzileyo yokulinganisa i-7 mm kunye ne-calcification kwi-lobe esezantsi ekunene.Kwimeko yesi-2, i-CT scan emva kweminyaka emi-5 iveze iqhuqhuva elizinzile, eliqina kancinci kunye nobubanzi be-7 mm kwilobe ephezulu ngasekunene.b Imifanekiso ye-CT ye-Axial yefestile yemiphunga kunye nezifundo ze-pathological ezihambelanayo kwiimeko ezimbini zenqanaba I-adenocarcinoma phambi kokutshatyalaliswa kwemiphunga.Ityala lesi-3 liveze iqhuqhuva elinobubanzi obuyi-8 mm kwilobe engaphezulu yasekunene ene-pleural retraction.Ityala lesi-4 libonakalise iqhuqhuva leglasi eqinileyo elingana ne-9 mm kwilobe ephezulu ngasekhohlo.I-Hematoxylin kunye ne-eosin (H&E) ukungcoliswa kwezicubu zemiphunga ezihlaziyiweyo (i-scale bar = 50 μm) ebonisa ipateni yokukhula kwe-acinar ye-lung adenocarcinoma.Iintolo zibonisa amaqhuqhuva afunyenwe kwimifanekiso yeCT.Imifanekiso ye-H & E yimifanekiso emele yemimandla emininzi (> 3) ye-microscopic ehlolwe yi-pathologist.
Zithathiwe kunye, iziphumo zethu zibonisa ixabiso elinokubakho le-serum metabolite biomarkers kwi-diagnostic eyahlukileyo ye-pulmonary nodules, enokuthi ibangele imingeni xa ivavanya ukuhlolwa kwe-CT.
Ngokusekwe kwiphaneli yemetabolite eqinisekisiweyo eqinisekisiweyo, siye safuna ukuchonga ulungelelwaniso lwebhayoloji yotshintsho olukhulu lwemetaboli.Uhlalutyo lwendlela ye-KEGG yokutyebisa i-MetaboAnalyst ichongiwe i-6 eqhelekileyo iguqulwe ngokuphawulekayo kwiindlela phakathi kwamaqela amabini anikezelweyo (i-LA vs. HC kunye ne-LA vs. BN, i-p ≤ 0.001, isiphumo> i-0.01 ilungisiwe).Olu tshintsho lubonakaliswe ngokuphazamiseka kwi-pyruvate metabolism, i-tryptophan metabolism, i-niacin kunye ne-nicotinamide metabolism, i-glycolysis, umjikelezo we-TCA, kunye ne-purine metabolism (Umfanekiso 5a).Siye saphinda senza i-metabolomics ekujoliswe kuyo ukuze siqinisekise utshintsho olukhulu sisebenzisa ubungakanani obupheleleyo.Ukumiselwa kweemetabolites eziqhelekileyo kwiindlela eziqhelekileyo ezitshintshwe nge-triple quadrupole mass spectrometry (QQQ) usebenzisa imigangatho ye-metabolite eyiyo.Iimpawu ze-demographic zesampula ekujoliswe kuyo kwi-metabolomics zifakwe kwi-Supplementary Table 4. Ngokuhambelana neziphumo zethu ze-metabolomics zehlabathi, uhlalutyo lobuninzi luqinisekisile ukuba i-hypoxanthine kunye ne-xanthine, i-pyruvate, kunye ne-lactate zonyuswa kwi-LA xa kuthelekiswa ne-BN kunye ne-HC (Umfanekiso 5b, c, p <0.05).Nangona kunjalo, akukho mahluko abalulekileyo kule metabolites afunyenwe phakathi kwe-BN kunye ne-HC.
Uhlalutyo lwendlela ye-KEGG yokuphucula i-metabolites ehluke kakhulu kwiqela le-LA xa kuthelekiswa namaqela e-BN kunye ne-HC.I-Globaltest enemisila emibini isetyenzisiwe, kwaye amaxabiso e-p ahlengahlengiswa kusetyenziswa indlela yeHolm-Bonferroni (uhlengahlengiso p ≤ 0.001 kunye nobukhulu besiphumo> 0.01).b-d Iiplani zeViolin ezibonisa i-hypoxanthine, i-xanthine, i-lactate, i-pyruvate, kunye namanqanaba e-tryptophan kwi-serum HC, BN, kunye ne-LA egqitywe yi-LC-MS / MS (n = 70 kwiqela ngalinye).Imigca echokoziweyo emhlophe namnyama ibonisa i-median kunye ne-quartile, ngokulandelanayo.e Iploti yeViolin ebonisa i-Log2TPM eqhelekileyo (imibhalo ngesigidi) inkcazo ye-mRNA ye-SLC7A5 kunye ne-QPRT kwi-lung adenocarcinoma (n = 513) xa kuthelekiswa nezicubu zemiphunga eziqhelekileyo (n = 59) kwi-LUAD-TCGA dataset.Ibhokisi emhlophe imele uluhlu lwe-interquartile, umgca omnyama othe tye embindini umele i-median, kwaye umgca omnyama othe nkqo osuka ebhokisini umele i-95% yexesha lokuzithemba (CI).f I-Pearson correlation plot ye-SLC7A5 kunye nokubonakaliswa kwe-GAPDH kwi-lung adenocarcinoma (n = 513) kunye nezicubu zemiphunga eziqhelekileyo (n = 59) kwi-dataset ye-TCGA.Indawo engwevu imele i-95% CI.r, Pearson coefficient yokulungelelanisa.g Amanqanaba e-tryptophan eselula aqhelekileyo kwiiseli ze-A549 ezidluliselwe ngolawulo olungabonakaliyo lwe-shRNA (NC) kunye ne-shSLC7A5 (Sh1, Sh2) egqitywe yi-LC-MS/MS.Uhlalutyo lwamanani lweesampulu ezintlanu ezizimeleyo ngokwebhayoloji kwiqela ngalinye zibonisiwe.h Amanqanaba eeSewula ze-NADt (i-NAD iyonke, kuquka i-NAD + kunye ne-NADH) kwiiseli ze-A549 (NC) kunye ne-SLC7A5 yokugoqa iiseli ze-A549 (Sh1, Sh2).Uhlalutyo lwamanani lweesampulu ezintathu ezizimeleyo ngokwebhayoloji kwiqela ngalinye zibonisiwe.i Glycolytic umsebenzi weeseli A549 ngaphambi nasemva SLC7A5 knockdown kulinganiswa extracellular acidification rate (ECAR) (n = 4 iisampulu ezizimeleyo biologically kwiqela ngalinye).I-2-DG, i-2-deoxy-D-glucose.Uvavanyo loMfundi olunemisila emibini lusetyenziswe ku-(b–h).Kwi-(g-i), imivalo yempazamo imele intsingiselo ± SD, umfuniselo ngamnye wenziwa kathathu ngokuzimeleyo kwaye iziphumo ziyafana.Idatha yomthombo inikwe ngohlobo lweefayile zomthombo wedatha.
Ukuqwalasela impembelelo ebalulekileyo yokuguqulwa kwe-tryptophan metabolism kwiqela le-LA, siye savavanya amanqanaba e-serum tryptophan kumaqela e-HC, BN, kunye ne-LA esebenzisa i-QQQ.Sifumene ukuba i-serum tryptophan yancitshiswa kwi-LA xa kuthelekiswa ne-HC okanye i-BN (p <0.001, Umfanekiso we-5d), ehambelana neziphumo zangaphambili ukuba amanqanaba e-tryptophan ajikelezayo aphantsi kwizigulane ezinomdlavuza wemiphunga kunokulawulwa okunempilo kwiqela lolawulo33,34 ,35.Olunye uphando olusebenzisa i-PET / CT tracer 11C-methyl-L-tryptophan yafumanisa ukuba ixesha lokugcinwa kwesignali ye-tryptophan kwizicubu zomhlaza wemiphunga yanda kakhulu xa kuthelekiswa nezilonda ezinobungozi okanye izicubu eziqhelekileyo36.Siqikelela ukuba ukuhla kwe-tryptophan kwi-serum ye-LA kunokubonakalisa ukufunyanwa kwe-tryptophan esebenzayo ziiseli zomhlaza wemiphunga.
Kwakhona kwaziwa ukuba imveliso yokuphela kwendlela ye-kynurenine ye-tryptophan catabolism yi-NAD + 37,38, eyona substrate ebalulekileyo yokusabela kwe-glyceraldehyde-3-phosphate kunye ne-1,3-bisphosphoglycerate kwi-glycolysis39.Ngelixa uphononongo lwangaphambili lujolise kwindima ye-tryptophan catabolism kulawulo lwe-immune, siye safuna ukucacisa unxibelelwano phakathi kwe-tryptophan dysregulation kunye neendlela ze-glycolytic ezibonwe kuphononongo lwangoku.Usapho lweSolute transporter 7 ilungu 5 (SLC7A5) yaziwa ngokuba yi-tryptophan transporter43,44,45.I-Quinolinic acid i-phosphoribosyltransferase (QPRT) yi-enzyme ehlala ezantsi kwendlela ye-kynurenine eguqula i-quinolinic acid kwi-NAMN46.Ukuhlolwa kwe-LUAD TCGA dataset ibonise ukuba zombini i-SLC7A5 kunye ne-QPRT zilawulwa kakhulu kwizicubu ze-tumor xa kuthelekiswa nezicubu eziqhelekileyo (umzobo 5e).Olu lwando lubonwe kwizigaba ze-I kunye ne-II kunye nezigaba ze-III kunye ne-IV ze-lung adenocarcinoma (i-Supplementary Figure 11), ebonisa ukuphazamiseka kwangaphambili kwi-tryptophan metabolism ehambelana ne-tumorigenesis.
Ukongezelela, i-LUAD-TCGA dataset ibonise ulungelelwaniso oluhle phakathi kwe-SLC7A5 kunye ne-GAPDH mRNA ibonakaliso kwiisampuli zesigulane somhlaza (r = 0.45, p = 1.55E-26, Umfanekiso 5f).Ngokwahlukileyo, akukho kulungelelaniswa okubalulekileyo kufunyenwe phakathi kwezisayino zemfuza kwizicubu zemiphunga eziqhelekileyo (r = 0.25, p = 0.06, Umfanekiso 5f).I-Knockdown ye-SLC7A5 (i-Supplementary Figure 12) kwiiseli ze-A549 zanciphisa kakhulu i-tryptophan yeselula kunye ne-NAD (H) amanqanaba (Umfanekiso we-5g, h), okubangelwa umsebenzi we-glycolytic owenziweyo njengoko ulinganiswe yi-extracellular acidification rate (ECAR) (Umfanekiso 1).5i).Ke, ngokusekwe kutshintsho lwemetabolism kwiserum kunye nokufunyanwa kwe-vitro, siqikelela ukuba i-tryptophan metabolism inokuvelisa i-NAD+ ngendlela ye-kynurenine kwaye idlale indima ebalulekileyo ekukhuthazeni i-glycolysis kumhlaza wemiphunga.
Uphononongo lubonise ukuba inani elikhulu leengqungquthela ze-pulmonary ezingabonakaliyo ezifunyenwe yi-LDCT zingakhokelela kwisidingo sovavanyo olongezelelweyo olufana ne-PET-CT, i-lung biopsy, kunye nokugqithisa ngenxa yokuxilongwa kobuxoki bobubi.31 Njengoko kuboniswe kuMfanekiso 6, uphando lwethu luchonge iphaneli ye-metabolites ye-serum enexabiso elinokuthi liphucule i-stratification yengozi kunye nokulawulwa okulandelayo kwee-nodule ze-pulmonary ezifunyenwe yi-CT.
Amaqhuqhuva e-pulmonary avavanywa kusetyenziswa idosi ephantsi ye-computed tomography (LDCT) eneempawu zokucinga ezibonisa izizathu ezinobungozi okanye ezimbi.Isiphumo esingaqinisekanga samaqhuqhuva singakhokelela kutyelelo lokulandelelana rhoqo, ungenelelo olungeyomfuneko, kunye nokunyangwa ngokugqithisileyo.Ukubandakanywa kwe-serum metabolic classifiers ngexabiso lokuxilonga kunokuphucula uvavanyo lomngcipheko kunye nolawulo olulandelayo lweenodule ze-pulmonary.I-PET positron emission tomography.
Idatha evela kuphononongo lwe-NLST yase-US kunye nophononongo lwaseYurophu lwe-NELSON lucebisa ukuba ukuhlolwa kwamaqela anobungozi obuphezulu kunye nedosi ephantsi ye-computed tomography (LDCT) kunokunciphisa ukufa komhlaza wemiphunga1,3.Nangona kunjalo, uvavanyo lomngcipheko kunye nolawulo lweklinikhi olwalandelayo lwamanani amakhulu amaqhuqhuva emiphungeni afunyenwe yi-LDCT ahlala engowona mngeni.Eyona njongo iphambili kukwandisa ulwahlulo oluchanekileyo lwemigaqo ekhoyo esekwe kwi-LDCT ngokubandakanya ii-biomarkers ezithembekileyo.
Ezinye iimpawu ze-molecular biomarkers, ezifana ne-metabolites yegazi, ziye zachongwa ngokuthelekisa umhlaza wemiphunga kunye nokulawula okunempilo15,17.Kuphononongo lwangoku, sigxininise ekusetyenzisweni kohlalutyo lwe-serum metabolomics ukwahlula phakathi kwe-benign kunye ne-malignant nodules ye-pulmonary efunyenwe ngengozi yi-LDCT.Siqhathanise i-metabolome ye-serum yehlabathi yolawulo olunempilo (HC), i-benign lung nodules (BN), kunye ne-stage I lung adenocarcinoma (LA) iisampulu usebenzisa uhlalutyo lwe-UPLC-HRMS.Sifumene ukuba i-HC kunye ne-BN zineeprofayili ze-metabolic ezifanayo, ngelixa i-LA ibonise utshintsho oluphawulekayo xa kuthelekiswa ne-HC kunye ne-BN.Sichonge iiseti ezimbini ze-serum metabolites ezahlula i-LA kwi-HC kunye ne-BN.
Inkqubo yokuchonga yangoku esekwe kwi-LDCT yamaqhuqhuva anobungozi kunye namaqhuqhuva anobungozi isekelwe ikakhulu kubungakanani, ubuninzi, i-morphology kunye nezinga lokukhula kwamaqhuqhuva ngexesha30.Uphononongo lwangaphambili lubonise ukuba ubukhulu bamaqhuqhuva bunxulumene ngokusondeleyo nokubakho komhlaza wemiphunga.Kwanakwizigulane ezinobungozi obuphezulu, umngcipheko we-malignancy kwi-nodes <6 mm ngu-<1%.Umngcipheko wokulimala kwamaqhuqhuva anomlinganiselo we-6 ukuya kwi-20 mm ukusuka kwi-8% ukuya kwi-64% ye-30.Ngoko ke, i-Fleischner Society incoma i-cutoff diameter ye-6 mm yokulandelela i-CT yesiqhelo.I-29 Nangona kunjalo, ukuhlolwa komngcipheko kunye nokulawulwa kwee-nodules ze-pulmonary (IPN) ezinkulu kune-6 mm ayenziwanga ngokwaneleyo i-31.Ulawulo lwangoku lwesifo senhliziyo esizalwa ngokuqhelekileyo lusekelwe ekulindeni okulindileyo kunye nokubeka iliso rhoqo kwi-CT.
Ngokusekelwe kwi-metabolome eqinisekisiweyo, sibonise okokuqala ukugqithiswa kweesignesha ze-metabolomic phakathi kwabantu abaphilileyo kunye ne-benign nodules <6 mm.Ukufana kwezinto eziphilayo kuhambelana neziphumo ze-CT zangaphambili ukuba umngcipheko wokuxhatshazwa kwama-nodules <6 mm uphantsi njengezifundo ezingenazo ii-nodes.30 Kufuneka kuqatshelwe ukuba iziphumo zethu zibonisa ukuba i-nodules enobungozi <6 mm kunye ne-≥6 mm iphezulu. ukufana kwiiprofayili ze-metabolomic, ebonisa ukuba inkcazo yokusebenza ye-benign etiology iyahambelana kungakhathaliseki ubungakanani be-nodule.Ngaloo ndlela, iipaneli ze-serum ze-metabolite zanamhlanje zinokubonelela ngovavanyo olulodwa njengovavanyo olulawulayo xa ama-nodules afunyanwa okokuqala kwi-CT kwaye anokunciphisa ukubeka iliso kwe-serial.Kwangaxeshanye, ipaneli efanayo yee-biomarkers ze-metabolic yahlula amaqhuqhuva anobungozi ≥6 mm ngobukhulu ukusuka kumaqhuqhuva angenabungozi kwaye yanikezela ngoqikelelo oluchanekileyo lwee-IPN zobukhulu obufanayo kunye neempawu ezingaqondakaliyo ze-morphological kwimifanekiso ye-CT.Lo mdidi we-metabolism we-serum uqhube kakuhle ekuqikeleleni ububi bamaqhuqhuva ≥6 mm nge-AUC ye-0.927.Zithathiwe kunye, iziphumo zethu zibonisa ukuba utyikityo olulodwa lwe-serum ye-metabolomic lunokubonisa ngokuthe ngqo utshintsho lwe-metabolism olubangelwa lithumba kwaye lube nexabiso elinokubakho njengokuqikelela umngcipheko, ngokuzimeleyo kubungakanani bamaqhuqhuva.
Ngokucacileyo, i-lung adenocarcinoma (LUAD) kunye ne-squamous cell carcinoma (LUSC) zezona ntlobo zomhlaza wemiphunga weseli engeyincinci (NSCLC).Ngenxa yokuba i-LUSC inyanyaniswa ngamandla nokusetyenziswa kwecuba47 kunye ne-LUAD yeyona histology ixhaphakileyo yamaqhuqhuva emiphunga afunyaniswe kuvavanyo lwe-CT48, imodeli yethu yokuhlelwa yayakhelwe ngokukodwa iisampulu zenqanaba I adenocarcinoma.UWang kunye noogxa nabo bagxininise kwi-LUAD kwaye bachonge iisayinwe ezilithoba ze-lipid zisebenzisa i-lipidomics ukwahlula umhlaza wemiphunga wenqanaba lokuqala kubantu abasempilweni17.Sivavanye imodeli yohlelo lwangoku kumatyala ali-16 enqanaba I LUSC kunye namaqhuqhuva angama-74 kwaye sabona ukuchaneka kwengqikelelo ephantsi ye-LUSC (AUC 0.776), ecebisa ukuba i-LUAD kunye ne-LUSC zinokuba nezazo iisayino ze-metabolomic.Ewe, i-LUAD kunye ne-LUSC zibonakaliswe ukuba zahlukile kwi-etiology, imvelaphi yebhayoloji kunye nokuphambuka kwemfuza49.Ke ngoko, ezinye iindidi ze-histology kufuneka zibandakanywe kwiimodeli zoqeqesho zokufumana umhlaza wemiphunga esekwe ngabantu kwiinkqubo zovavanyo.
Apha, sichonge iindlela ezintandathu ezihlala zitshintshwa rhoqo kwi-lung adenocarcinoma xa kuthelekiswa nolawulo olusempilweni kunye namaqhuqhuva angenabungozi.I-Xanthine kunye ne-hypoxanthine ziimetabolites eziqhelekileyo ze-purine metabolic pathway.Ngokuhambelana neziphumo zethu, i-intermediates ehambelana ne-purine metabolism yanda kakhulu kwi-serum okanye izicubu zezigulane ezine-lung adenocarcinoma xa kuthelekiswa nokulawula okunempilo okanye izigulane kwisigaba sokuqala15,50.Ukuphakama kwe-serum xanthine kunye namanqanaba e-hypoxanthine anokubonisa i-anabolism efunekayo ngokwandisa ngokukhawuleza iiseli zomhlaza.I-dysregulation ye-glucose metabolism luphawu olwaziwayo lomhlaza wemetabolism51.Apha, siye sabona ukwanda okukhulu kwe-pyruvate kunye ne-lactate kwiqela le-LA xa kuthelekiswa ne-HC kunye neqela le-BN, elihambelanayo neengxelo zangaphambili ze-glycolytic pathway abnormalities kwiiprofayili ze-serum ze-metabolome ze-non-small cell lung cancer (NSCLC) izigulane kunye ulawulo olunempilo.Iziphumo ziyangqinelana52,53.
Okubalulekileyo, siye sabona ulungelelwaniso oluphambeneyo phakathi kwe-pyruvate kunye ne-tryptophan metabolism kwi-serum ye-lung adenocarcinomas.Amanqanaba e-tryptophan eSerum aye ancitshiswa kwiqela le-LA xa kuthelekiswa ne-HC okanye iqela le-BN.Kuyathakazelisa ukuba uphando olunzulu lwangaphambili olusebenzisa i-cohort olulindelekileyo lufumene ukuba amanqanaba aphantsi e-tryptophan ejikelezayo adibaniswa nomngcipheko wokwanda komhlaza wamaphaphu 54.I-Tryptophan yi-amino acid ebalulekileyo esiyifumana ngokupheleleyo ekutyeni.Siphetha ukuba i-serum tryptophan depletion kwi-lung adenocarcinoma ingabonakalisa ukutshatyalaliswa ngokukhawuleza kwale metabolite.Kuyaziwa ukuba isiphelo semveliso ye-tryptophan catabolism ngendlela ye-kynurenine ngumthombo we-de novo NAD + synthesis.Kuba i-NAD+ iveliswa ikakhulu ngendlela yokuhlangula, ukubaluleka kwe-NAD+ kwi-tryptophan metabolism kwimpilo nakwisifo kusafuneka kumiselwe46.Uhlalutyo lwethu lwe-database ye-TCGA lubonise ukuba ukubonakaliswa kwe-tryptophan transporter solute transporter 7A5 (SLC7A5) kwandiswe kakhulu kwi-lung adenocarcinoma xa kuthelekiswa nolawulo oluqhelekileyo kwaye yayihambelana kakuhle nokubonakaliswa kwe-enzyme ye-glycolytic GAPDH.Izifundo zangaphambili zijolise ikakhulu kwindima ye-tryptophan catabolism ekucinezeleni impendulo ye-immune ye-antitumor40,41,42.Apha sibonisa ukuba ukuvinjwa kokuthathwa kwe-tryptophan ngokunkqonkqoza kwe-SLC7A5 kwiiseli zomhlaza wemiphunga kukhokelela ekunciphiseni okulandelayo kumanqanaba e-NAD yeselula kunye nokuthomalaliswa okuhambelanayo komsebenzi we-glycolytic.Isishwankathelo, uphononongo lwethu lubonelela ngesiseko sebhayoloji sotshintsho kwi-serum metabolism ehambelana notshintsho olubi lwe-lung adenocarcinoma.
Ukuguqulwa kwe-EGFR yeyona nto ixhaphakileyo kwiinguqu zomqhubi kwizigulane ezine-NSCLC.Kuphononongo lwethu, sifumene ukuba izigulane ezinokuguqulwa kwe-EGFR (n = 41) zineeprofayili ze-metabolomic ngokubanzi ezifana nezigulane ezinohlobo lwasendle lwe-EGFR (n = 31), nangona sifumene amanqanaba e-serum aphantsi kwezinye izigulane ze-EGFR eziguquguqukayo kwizigulane ze-acylcarnitine.Umsebenzi omiselweyo we-acylcarnitines kukuthutha amaqela e-acyl ukusuka kwi-cytoplasm ukuya kwi-matrix ye-mitochondrial, ekhokelela kwi-oxidation ye-fatty acids ukuvelisa amandla e-55.Ngokuhambelana neziphumo zethu, uphononongo lwakutsha nje luchonge iiprofayili zemetabolome ezifanayo phakathi kwe-EGFR eguqukileyo kunye ne-EGFR yasendle yohlobo lwamathumba ngokuhlalutya imetabolome yehlabathi ye-102 lung adenocarcinoma tissue samples50.Kuyathakazelisa ukuba umxholo we-acylcarnitine ufunyenwe kwiqela le-EGFR eliguqukileyo.Ngoko ke, ingaba utshintsho kumanqanaba e-acylcarnitine lubonisa utshintsho lwe-metabolic olubangelwa yi-EGFR kunye neendlela eziphantsi kwee-molecular pathways zinokufaneleka ukufundwa okuqhubekayo.
Ukuqukumbela, uphononongo lwethu luseka i-serum metabolic classifier yokuxilongwa ngokwahlukileyo kwe-pulmonary nodules kwaye iphakamisa ukuhamba komsebenzi okunokuthi kuphuculwe uvavanyo lomngcipheko kunye nokwenza lula ulawulo lweklinikhi olusekwe kuvavanyo lwe-CT scan.
Olu pho nonongo luvunyiwe yiKomidi yezokuziphatha yeSibhedlele seCancer yeYunivesithi yaseSun Yat-sen, iSibhedlele sokuQala esiBambiseneyo seYunivesithi yaseSun Yat-sen, kunye neKomidi yezokuziphatha yeSibhedlele seCancer yeYunivesithi yaseZhengzhou.Kumaqela okufumanisa kunye nokuqinisekiswa kwangaphakathi, i-sera ye-174 evela kubantu abaphilileyo kunye neesera ezingama-244 ezivela kwii-nodules ezinobungozi zaqokelelwa kubantu abaphantsi kovavanyo lwezonyango zonyaka kwiSebe loLawulo lweCancer kunye noThintelo, iZiko leCancer yeYunivesithi yaseSun Yat-sen, kunye ne-166 ye-benign nodules.serum.Inqanaba I lung adenocarcinomas zaqokelelwa kwiZiko loMhlaza leYunivesithi yaseSun Yat-sen.Kwiqela lokuqinisekisa langaphandle, kwakukho iimeko ze-48 ze-nodules ezinobungozi, iimeko ze-39 zenqanaba I lung adenocarcinoma ukusuka kwiSibhedlele esiBambiseneyo sokuQala seYunivesithi yase-Sun Yat-sen, kunye namatyala angama-24 enqanaba I lung adenocarcinoma evela kwisibhedlele somhlaza saseZhengzhou.IZiko loMhlaza leYunivesithi yaseSun Yat-sen nalo liqokelele iimeko ezili-16 zesigaba I squamous cell somhlaza wemiphunga ukuvavanya isakhono sokuxilonga sohlelo olusekiweyo lwe-metabolic classifier (iimpawu zesigulana zibonisiwe kwiTheyibhile eyoNgezelelweyo yesi-5).Iisampulu ezivela kwiqela lokufumanisa kunye neqela loqinisekiso lwangaphakathi ziqokelelwe phakathi kukaJanuwari 2018 kunye noMeyi 2020. Iisampulu zeqela loqinisekiso lwangaphandle zaqokelelwa phakathi kuka-Agasti 2021 kunye no-Okthobha 2022. Ukunciphisa ucalucalulo lwesini, malunga namanani alinganayo amatyala abesilisa nabasetyhini anikezelwe kumntu ngamnye. iqela.Iqela loFundo kunye neQela loPhononongo lwaNgaphakathi.Isini somthathi-nxaxheba samiselwa ngokusekelwe kwingxelo yakhe.Imvume enolwazi ifunyenwe kubo bonke abathathi-nxaxheba kwaye akukho mbuyekezo inikezelweyo.Izifundo ezine-nodules ezinobunzima zazinamanqaku azinzileyo e-CT scan kwi-2 kwiminyaka eyi-5 ngexesha lokuhlalutya, ngaphandle kwe-1 imeko evela kwisampuli yokuqinisekisa yangaphandle, eyaqokelelwa kwangaphambili kwaye yafunyaniswa yi-histopathology.Ngaphandle kwe-bronchitis engapheliyo.Iimeko ze-Lung adenocarcinoma zaqokelelwa ngaphambi kokutshatyalaliswa kwemiphunga kwaye ziqinisekiswe ngokuxilongwa kwe-pathological.Iisampulu zegazi lokuzila ngokukhawuleza zaqokelelwa kwiityhubhu zokuhlukana kwe-serum ngaphandle kwe-anticoagulants.Iisampulu zegazi zahluthwa ngeyure ye-1 kwindawo yokushisa kwaye emva koko i-centrifuged kwi-2851 × g imizuzu eyi-10 kwi-4 ° C ukuqokelela i-serum supernatant.I-aliquots yeSerum yayikhenkcezwe kwi -80 ° C de kutsalwe i-metabolite.ISebe loThintelo lweCancer kunye noHlolo lwezoNyango lwe-Sun Yat-sen University Cancer Centre liqokelele i-pool ye-serum evela kubaxhasi be-100 abaphilileyo, kubandakanywa inani elilinganayo lamadoda nabasetyhini abaneminyaka eyi-40 ukuya kwi-55 iminyaka.Umthamo olinganayo wesampulu yomnikeli ngamnye waxutywa, ichibi elifunyenweyo lacatshulwa kwaye lagcinwa kwi -80 ° C.Umxube we-serum usetyenziswe njengezinto ezibhekiselele kulawulo lomgangatho kunye nokulinganisa idatha.
I-serum yereferensi kunye neesampuli zokuvavanya zancibilika kwaye i-metabolites yachithwa kusetyenziswa indlela yokudibanisa edibeneyo (MTBE / methanol / amanzi) i-56.Ngokufutshane, i-50 μl yeserum yaxutywa ne-225 μl yemethanol ebandayo yomkhenkce kunye ne-750 μl ye-methyl tert-butyl ether yomkhenkce ebandayo (MTBE).Gxuma umxube kwaye udibanise kwiqhwa ngeyure eli-1.Iisampuli zaye zaxutywa kunye ne-vortex exutywe ne-188 μl yamanzi e-MS-grade aqulethe imigangatho yangaphakathi (13C-lactate, 13C3-pyruvate, 13C-methionine, kunye ne-13C6-isoleucine, ethengwe kwi-Cambridge Isotope Laboratories).Umxube wawufakwe kwi-centrifuged kwi-15,000 × g ye-10 min kwi-4 ° C, kwaye isigaba esisezantsi sadluliselwa kwiibhubhu ezimbini (i-125 μL nganye) kuhlalutyo lwe-LC-MS kwiindlela ezilungileyo nezimbi.Ekugqibeleni, isampulu yakhutshwa ngumphunga ukuze yome kwindawo yokucoca enesantya esiphezulu.
I-metabolites eyomileyo ihlaziywe kwi-120 μl ye-80% ye-acetonitrile, i-vortexed for 5 min, kwaye i-centrifuged kwi-15,000 × g ye-10 min kwi-4 ° C.Ii-supernatants zakhutshelwa kwiingqayi zeglasi e-amber ezinemincindi encinci kwizifundo zemetabolomics.Uhlalutyo olungajoliswanga lwe-metabolomics kwi-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) iqonga.I-Metabolites yahlulwa kusetyenziswa inkqubo ye-Dionex Ultimate 3000 UPLC kunye nekholamu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Amanzi).Kwimodi ye-ion enhle, izigaba ezihambahambayo ziyi-95% (A) kunye ne-50% ye-acetonitrile (B), nganye iqulethe i-10 mmol / L i-ammonium acetate kunye ne-0.1% ye-asidi ye-formic.Kwimo engafanelekanga, izigaba ze-mobile A kunye ne-B ziqulethe i-95% kunye ne-50% ye-acetonitrile, ngokulandelanayo, zombini izigaba ziqulethe i-10 mmol / L ammonium acetate, pH = 9. Inkqubo ye-gradient yayilolu hlobo lulandelayo: 0-0.5 min, 2% B;0.5–12 min, 2–50% B;12–14 imiz, 50–98% B;14–16 imiz, 98% B;16–16.1.imiz, 98 –2% B;I-16.1-20 min, i-2% B. Ikholomu igcinwe kwi-40 ° C kunye nesampuli kwi-10 ° C kwi-autosampler.Umlinganiselo wokuhamba wawuyi-0.3 ml / min, umthamo we-injection wawuyi-3 μl.I-Q-Exactive Orbitrap mass spectrometer (i-Thermo Fisher Scientific) ene-electrospray ionization (ESI) umthombo yasetyenziswa kwimodi yokuskena ngokupheleleyo kwaye idityaniswe ne-ddMS2 imo yokubeka iliso ukuqokelela umthamo omkhulu wedatha.Iiparamitha ze-MS zibekwe ngolu hlobo lulandelayo: i-voltage yokutshiza +3.8 kV/- 3.2 kV, iqondo lobushushu le-capillary 320°C, i-gas yokukhusela i-40 arb, i-gas encedisayo ye-10 arb, i-probe heater ye-350 ° C, i-scanning range 70-1050 m / h, isisombululo.70 000. Idatha yafunyanwa kusetyenziswa i-Xcalibur 4.1 (i-Thermo Fisher Scientific).
Ukuvavanya umgangatho wedatha, iisampuli zolawulo lomgangatho odibeneyo (QC) zenziwe ngokususa i-10 μL ye-aliquots ye-supernatant kwisampulu nganye.Iinaliti zesampulu zokulawula umgangatho ezintandathu zahlalutywa ekuqaleni kokulandelelana kokuhlalutya ukuvavanya ukuzinza kwenkqubo ye-UPLC-MS.Iisampulu zolawulo lomgangatho zingeniswa ngamaxesha athile kwibhetshi.Zonke iibhetshi ze-11 zeesampulu ze-serum kolu phononongo zahlalutywa yi-LC-MS.Ii-aliquots zomxube we-serum pool ezisuka kwi-100 labanikeli abasempilweni ziye zasetyenziswa njengesixhobo sokubhekisela kwiibhetshi ezilandelelanayo ukujonga inkqubo yotsalo kunye nokulungelelanisa iziphumo zebhetshi ukuya kwibhetshi.Uhlalutyo olungajoliswanga lwe-metabolomics lweqela lokufumanisa, iqela lokuqinisekisa lwangaphakathi, kunye neqela lokuqinisekisa langaphandle lwenziwa kwiZiko leMetabolomics yeYunivesithi yaseSun Yat-sen.Ilabhoratri yangaphandle yeYunivesithi yaseGuangdong yoHlalutyo lweThekhnoloji kunye neZiko loVavanyo liphinde lahlalutya iisampulu ze-40 ezivela kwiqela langaphandle ukuvavanya ukusebenza kwemodeli yokuhlelwa.
Emva kokutsalwa kunye nokuhlengahlengiswa, ubungakanani obupheleleyo be-serum metabolites bulinganiswe kusetyenziswa i-ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) kunye ne-electrospray ionization (ESI) umthombo kwimowudi yokujonga ukuphendula ngokuphindaphindiweyo (MRM).Ikholomu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Amanzi) yayisetyenziselwa ukwahlula i-metabolites.Isigaba esihambahambayo sasiquka i-90% (A) kunye ne-5% ye-acetonitrile (B) kunye ne-10 mmol / L ammonium acetate kunye ne-0.1% isisombululo se-ammonia.Iprogram yegradient yaba ngolu hlobo lulandelayo: 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.Ikholomu igcinwe kwi-40 ° C kunye nesampuli kwi-10 ° C kwi-autosampler.Umyinge wokuhamba wawuyi-0.3 mL / min kwaye umthamo wenaliti wawuyi-1 μL.Iiparamitha ze-MS zibekwe ngolu hlobo lulandelayo: i-capillary voltage ± 3.5 kV, i-nebulizer yoxinzelelo lwe-35 psi, i-sheath gas flow 12 L / min, ukushisa kwegesi ye-sheath 350 ° C, ukushisa kwegesi yokumisa i-250 ° C, kunye nokumisa ukuhamba kwegesi 14 l / min.Ukuguqulwa kwe-MRM ye-tryptophan, i-pyruvate, i-lactate, i-hypoxanthine kunye ne-xanthine yayiyi-205.0-187.9, 87.0-43.4, 89.0-43.3, 135.0-92.3 kunye ne-151.0-107.9 ngokulandelelanayo.Idatha yaqokelelwa kusetyenziswa i-Mass Hunter B.07.00 (Agilent Technologies).Kwiisampulu zeserum, i-tryptophan, i-pyruvate, i-lactate, i-hypoxanthine, kunye ne-xanthine ziye zalinganiswa kusetyenziswa i-calibration curves ye-standard mix mix solutions.Kwiisampuli zeeseli, umxholo we-tryptophan wawuqhelekile kumgangatho wangaphakathi kunye nobunzima beprotheni yeseli.
I-Peak extraction (m / z kunye nexesha lokugcinwa (RT)) yenziwe ngokusebenzisa i-Compound Discovery 3.1 kunye ne-TraceFinder 4.0 (i-Thermo Fisher Scientific).Ukuphelisa iyantlukwano enokubakho phakathi kweebhetshi, incopho nganye yeempawu zesampulu yovavanyo yahlulwe ngencopho yophawu lwemathiriyeli yereferensi ukusuka kwibhetshi enye ukufumana ubuninzi obunxulumeneyo.Ukutenxa komgangatho ohambelanayo wemigangatho yangaphakathi ngaphambi nangemva komgangatho uboniswe kwiTheyibhile eyoNgezelelweyo 6. Umahluko phakathi kwamaqela amabini ubonakaliswe ngeqondo lokufumanisa ubuxoki (FDR <0.05, uvavanyo lwenqanaba olusayinwe nguWilcoxon) kunye nokuguqulwa kokutshintsha (> 1.2 okanye <0.83).Idatha ye-MS ekrwada yeempawu ezikhutshiweyo kunye nedatha ye-MS elungiswe kwiserum iboniswa kwiDatha eyoNgezelelweyo 1 kunye neDatha eyoNgezelelweyo yesi-2, ngokulandelelanayo.I-Peak annotation yenziwe ngokusekelwe kumanqanaba amane achaziweyo okuchongwa, kubandakanywa i-metabolites echongiweyo, i-puatively annotated compounds, i- putatively characterized classes, kunye ne-compounds engaziwayo i-22.Ngokusekelwe kuphando lwedatha kwi-Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), iikhompawundi ze-biological kunye ne-MS / MS ehambelana nemigangatho eqinisekisiweyo okanye inkcazo yomdlalo ochanekileyo kwi-mzCloud (amanqaku> 85) okanye i-Chemspider ekugqibeleni yakhethwa njengabaphakathi phakathi kwe-metabolome eyahlukileyo.Incopho yenkcazo yenqaku ngalinye ibandakanyiwe kwiDatha eyoNgezelelweyo 3. I-MetaboAnalyst 5.0 isetyenziselwe uhlalutyo olungaguqukiyo lwe-sum-normalized metabolite abundance.I-MetaboAnalyst 5.0 iphinde yavavanya uhlalutyo lwendlela ye-KEGG yokutyebisa ngokusekelwe kwiimetabolites ezahlukeneyo kakhulu.Uhlalutyo lwecandelo eliphambili (PCA) kunye ne-partial least squares discriminant analysis (PLS-DA) zahlalutywa kusetyenziswa iphakheji yesoftware ye-ropls (v.1.26.4) kunye ne-stack normalization kunye ne-autoscaling.Imodeli ye-biomarker ye-metabolite efanelekileyo yokuqikelela ubugwenxa be-nodule yenziwe kusetyenziswa ukuguqulwa kwe-binary logistic kunye nokunciphisa okuncinci kunye nomqhubi wokukhetha (i-LASSO, iphakheji ye-R v.4.1-3).Ukusebenza kwemodeli yocalucalulo ekufumaneni nasekuqinisekiseni iisethi zibonakaliswe ngokuqikelela i-AUC ngokusekelwe kuhlalutyo lwe-ROC ngokwephakheji ye-pROC (v.1.18.0.).I-cutoff ye-optimal probability cutoff ifunyenwe ngokusekelwe kwi-index ye-Youden ephezulu yomzekelo (ubuntununtunu + obucacileyo - 1).Iisampulu ezinamaxabiso angaphantsi okanye amakhulu kunomqobo ziya kuqikelelwa njengeeqhuqhuva ezinobungozi kunye ne-lung adenocarcinoma, ngokulandelelanayo.
Iiseli ze-A549 (#CCL-185, Iqoqo leNkcubeko yeNkcubeko yaseMelika) zakhuliswa kwi-F-12K medium equkethe i-10% ye-FBS.Ulandelelwano lwe-hairpin olufutshane lwe-RNA (shRNA) olujolise kwi-SLC7A5 kunye nolawulo olungajolisiyo (NC) lufakwe kwi-lentiviral vector pLKO.1-puro.Ukulandelelana kwe-antisense ye-shSLC7A5 ngolu hlobo lulandelayo: Sh1 (5′-GGAGAAACCTGATGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGGAACTAT-3′).Ii-Antibodies ukuya kwi-SLC7A5 (#5347) kunye ne-tubulin (#2148) zathengwa kwiTekhnoloji yokuSayina kweCell.Ii-Antibodies ukuya kwi-SLC7A5 kunye ne-tubulin zisetyenziswe kwi-dilution ye-1: 1000 kuhlalutyo lwe-blot yaseNtshona.
I-Seahorse XF Glycolytic Stress Test ilinganisa amanqanaba e-extracellular acidification (ECAR).Kwi-assay, i-glucose, i-oligomycin A, kunye ne-2-DG zalawulwa ngokulandelelana ukuvavanya umthamo we-cellular glycolytic njengoko ulinganiswe yi-ECAR.
Iiseli ze-A549 ezidluliselwe ngolawulo olungajoliswanga (NC) kunye ne-shSLC7A5 (Sh1, Sh2) zifakwe ubusuku bonke kwiitya ze-10 cm ububanzi.I-metabolites yeeseli ikhutshwe nge-1 ml ye-ice-cold 80% ye-methanol enamanzi.Iiseli kwisisombululo se-methanol zachithwa, ziqokelelwa kwi-tube entsha, kwaye i-centrifuged kwi-15,000 × g ye-15 min kwi-4 ° C.Qokelela i-800 µl ye-supernatant kwaye yome usebenzisa isigxininisi sevacuum esinesantya esiphezulu.Iipelisi ze-metabolite ezomileyo zaye zahlalutywa kumanqanaba e-tryptophan usebenzisa i-LC-MS / MS njengoko kuchazwe ngasentla.Amanqanaba e-Cellular NAD (H) kwiiseli ze-A549 (NC kunye ne-shSLC7A5) zilinganiswe ngokusebenzisa ikiti yombala we-NAD +/NADH (#K337, BioVision) ngokwemiyalelo yomenzi.Amanqanaba eeprotheyini alinganiswa kwisampulu nganye ukulungelelanisa inani le-metabolites.
Akukho ndlela zamanani zisetyenzisiweyo ukumisela kwangaphambili ubungakanani besampulu.Izifundo zangaphambili ze-metabolomics ezijolise ekufumaneni i-biomarker15,18 ziye zathathwa njengemilinganiselo yokumisela ubungakanani, kwaye xa kuthelekiswa nezi ngxelo, isampuli yethu yayanele.Akukho zisampuli zikhutshiweyo kwiqela lophononongo.Iisampulu zeSerum zabelwa ngokungaqhelekanga kwiqela lokufumanisa (amatyala e-306, i-74.6%) kunye neqela langaphakathi lokuqinisekisa (amatyala e-104, i-25.4%) kwizifundo ze-metabolomics ezingajoliswanga.Siphinde sikhethe ngokungacwangciswanga iimeko ze-70 kwiqela ngalinye ukusuka ekufumaneni okumiselweyo kwizifundo ezijoliswe kwi-metabolomics.Abaphandi baphuphuthekiswa kwisabelo seqela ngexesha lokuqokelela idatha ye-LC-MS kunye nohlalutyo.Uhlalutyo lweenkcukacha-manani lwedatha ye-metabolomics kunye novavanyo lweeseli luchazwe kwiZiphumo ezifanelekileyo, iiNgcaciso zeMifanekiso, kunye neeNdlela zeNdlela.Ubungakanani be-tryptophan yeselula, i-NADT, kunye nomsebenzi we-glycolytic wenziwa kathathu ngokuzimeleyo kunye neziphumo ezifanayo.
Ngolwazi oluthe vetshe malunga noyilo lophononongo, bona i-Natural Portfolio Report Abstract ehambelana neli nqaku.
Idatha ye-MS ekrwada yeempawu ezikhutshiweyo kunye nedatha ye-MS yesiqhelo ye-serum yereferensi iboniswe kwiDatha eyoNgezelelweyo 1 kunye neDatha eyoNgezelelweyo yesi-2, ngokulandelanayo.Incopho yezichasiselo zeempawu zokwahluka zithiwe thaca kwiDatha eyoNgezelelweyo yesi-3. Iseti yedatha ye-LUAD TCGA inokukhutshelwa ku-https://portal.gdc.cancer.gov/.Idatha yegalelo yokucwangcisa igrafu inikwe kwidatha yomthombo.Idatha yomthombo inikwe eli nqaku.
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UDe Koning, HJ, et al.Ukunciphisa ukufa komhlaza wemiphunga kunye nokuhlolwa kwe-CT yevolumetric kulingo olungenamkhethe.kuMntla weNgilani.J. Med.382, 503–513 (2020).
Ixesha lokuposa: Sep-18-2023