Ukuxilongwa okuhlukile kwamaqhubu e-pulmonary okutholwe yi-computed tomography (CT) kusalokhu kuyinselele ekusebenzeni kwezokwelapha. Lapha, sichaza i-metabolome yomhlaba wonke yamasampula e-serum angu-480, okuhlanganisa izilawuli ezinempilo, amaqhubu e-lung angenabungozi, kanye ne-stage I lung adenocarcinoma. Ama-Adenocarcinoma abonisa amaphrofayili e-metabolomic ahlukile, kuyilapho amaqhubu angenabungozi kanye nabantu abanempilo benokufana okuphezulu kumaphrofayili e-metabolomic. Eqenjini lokutholwa (n = 306), kwatholakala isethi yama-metabolites angu-27 ukuze kuhlukaniswe phakathi kwamaqhubu angenabungozi kanye nalawo anobungozi. I-AUC yemodeli yokuhlukanisa ekuqinisekisweni kwangaphakathi (n = 104) kanye nokuqinisekiswa kwangaphandle (n = 111) amaqembu ayengu-0.915 kanye no-0.945, ngokulandelana. Ukuhlaziywa kwendlela kwembule ukwanda kwama-metabolites e-glycolytic ahlobene nokuncipha kwe-tryptophan ku-serum ye-lung adenocarcinoma uma kuqhathaniswa namaqhubu angenabungozi kanye nezilawuli ezinempilo, futhi kwasikisela ukuthi ukuthathwa kwe-tryptophan kukhuthaza i-glycolysis kumaseli omdlavuza wamaphaphu. Ucwaningo lwethu luqokomisa ukubaluleka kwama-biomarker e-serum metabolite ekuhloleni ingozi yamaqhubu e-pulmonary atholwe yi-CT.
Ukuxilongwa kusenesikhathi kubalulekile ekuthuthukiseni amazinga okusinda kweziguli ezinomdlavuza. Imiphumela evela ku-US National Lung Cancer Screening Trial (NLST) kanye ne-European NELSON Study ikhombisile ukuthi ukuhlolwa nge-computed tomography (LDCT) yomthamo ophansi kunganciphisa kakhulu ukufa komdlavuza wamaphaphu emaqenjini asengozini enkulu1,2,3. Kusukela ukusetshenziswa kabanzi kwe-LDCT ekuhlolweni komdlavuza wamaphaphu, ukwanda kokutholwa kwe-x-ray okungazelelwe kwama-nodules e-pulmonary angenazimpawu kuqhubeke nokwanda 4. Ama-nodules e-pulmonary achazwa njenge-focal opacities afinyelela ku-3 cm ububanzi 5. Sibhekene nobunzima ekuhloleni amathuba okuba nomdlavuza kanye nokubhekana nenani elikhulu lama-nodules e-pulmonary atholakale ngengozi ku-LDCT. Ukulinganiselwa kwe-CT kungaholela ekuhlolweni okulandela njalo kanye nemiphumela engalungile, okuholela ekungeneleleni okungadingekile kanye nokwelashwa ngokweqile6. Ngakho-ke, kunesidingo sokuthuthukisa ama-biomarker athembekile futhi awusizo ukuze kutholakale kahle umdlavuza wamaphaphu ezigabeni zokuqala futhi kuhlukaniswe ama-nodules amaningi angenabungozi ekutholakaleni kokuqala 7.
Ukuhlaziywa okuphelele kwegazi (i-serum, i-plasma, amaseli egazi angaphandle kwe-mononuclear), okuhlanganisa i-genomics, i-proteomics noma i-DNA methylation8,9,10, kuholele ekusaseni elikhulayo ekutholakaleni kwe-biomarkers zokuxilonga umdlavuza wamaphaphu. Okwamanje, izindlela ze-metabolomics zilinganisa imikhiqizo yokugcina yamaseli ethonywa yizenzo zangaphandle nezangaphandle ngakho-ke zisetshenziswa ukubikezela ukuqala kwesifo kanye nomphumela. I-Liquid chromatography-tandem mass spectrometry (LC-MS) iyindlela esetshenziswa kabanzi yezifundo ze-metabolomics ngenxa yokuzwela kwayo okuphezulu kanye nobubanzi obukhulu be-dynamic, obungamboza ama-metabolites anezakhiwo ezahlukene ze-physicochemical11,12,13. Nakuba ukuhlaziywa kwe-metabolomic yomhlaba wonke kwe-plasma/serum kusetshenziswe ukuhlonza ama-biomarkers ahlobene nokuxilongwa komdlavuza wamaphaphu14,15,16,17 kanye nokusebenza kahle kokwelashwa, izigaba ze-metabolite ze-serum ezingu-18 zokuhlukanisa phakathi kwama-nodule amaphaphu angenabungozi nalawo anobungozi kusazofundwa kakhulu. -ucwaningo olukhulu.
I-Adenocarcinoma kanye ne-squamous cell carcinoma yizinhlobo ezimbili eziyinhloko zomdlavuza wamaphaphu ongewona omncane (i-NSCLC). Ukuhlolwa okuhlukahlukene kwe-CT screening kubonisa ukuthi i-adenocarcinoma uhlobo oluvame kakhulu lwe-histological lomdlavuza wamaphaphu1,19,20,21. Kulolu cwaningo, sisebenzise i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) ukwenza ukuhlaziywa kwe-metabolomics kumasampula e-serum angu-695, kufaka phakathi izilawuli ezinempilo, ama-nodules e-pulmonary angenabungozi, kanye ne-CT-detected ≤3 cm. Ukuhlolwa kwe-Stage I lung adenocarcinoma. Sithole iphaneli yama-metabolites e-serum ahlukanisa i-lung adenocarcinoma kuma-nodules angenabungozi kanye nezilawuli ezinempilo. Ukuhlaziywa kokucebisa indlela kwembule ukuthi i-tryptophan engavamile kanye ne-glucose metabolism kuyizinguquko ezivamile ku-lung adenocarcinoma uma kuqhathaniswa nama-nodules angenabungozi kanye nezilawuli ezinempilo. Ekugcineni, sasungula futhi saqinisekisa i-serum metabolic classifier enokucaca okuphezulu kanye nokuzwela ukuhlukanisa phakathi kwamaqhubu amaphaphu anobungozi nangenabungozi atholwe yi-LDCT, okungasiza ekuxilongweni okuhlukile kokuqala kanye nokuhlolwa kwengozi.
Esifundweni samanje, amasampula e-serum ahambisana nobulili nobudala aqoqwe emuva kubantu abangu-174 abaphilile, iziguli ezingu-292 ezinezinhlayiya ze-pulmonary ezingengozini, kanye neziguli ezingu-229 ezine-lung adenocarcinoma yesigaba sokuqala. Izici zabantu abangu-695 ziboniswe kuThebula Elingeziwe 1.
Njengoba kuboniswe kuMfanekiso 1a, amasampula e-serum angu-480, kufaka phakathi amasampula angu-174 e-healthy control (HC), ama-benign nodules angu-170 (BN), kanye namasampula angu-136 e-stage I lung adenocarcinoma (LA), aqoqwe eSun Yat-sen University Cancer Center. Iqembu lokutholwa kwe-metabolomic profiles engahlosiwe kusetshenziswa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Njengoba kuboniswe kuMfanekiso Ongeziwe 1, ama-metabolite ahlukene phakathi kwe-LA ne-HC, i-LA ne-BN atholakale ukuze kusungulwe imodeli yokuhlukanisa futhi kuhlolwe kabanzi ukuhlaziywa kwendlela yokuhlukanisa. Amasampula angu-104 aqoqwe yiSun Yat-sen University Cancer Center kanye namasampula angu-111 aqoqwe ezinye izibhedlela ezimbili ahlolwa ngaphakathi nangaphandle, ngokulandelana.
a Inani labantu abacwaningwayo eqenjini lokutholakala abahlolwe nge-metabolomics ye-serum yomhlaba wonke besebenzisa i-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). b Ukuhlaziywa kwe-Partial least squares discriminant analysis (PLS-DA) ye-metabolome iyonke yamasampula e-serum angu-480 avela eqenjini lokucwaninga, okuhlanganisa nezilawuli ezinempilo (HC, n = 174), ama-nodule angenabungozi (BN, n = 170), kanye ne-stage I lung adenocarcinoma (Los Angeles, n = 136). +ESI, imodi ye-electrospray ionization ye-positive electrospray, -ESI, imodi ye-electrospray ionization engemihle. c–e Ama-Metabolites anobuningi obuhlukene kakhulu emaqenjini amabili anikeziwe (ukuhlolwa kwe-rank ye-Wilcoxon esayinwe ngemisila emibili, inani le-p elilungisiwe le-false discovery rate, i-FDR <0.05) aboniswe ngokubomvu (ushintsho olugoqekile > 1.2) kanye nohlaza okwesibhakabhaka (ushintsho olugoqekile < 0.83). ) aboniswe ku-volcano graphic. f Imephu yokushisa yokuhlanganisa amaqoqo ngokulandelana kwezigaba ekhombisa umehluko obalulekile enanini lama-metabolites anezichasiselo phakathi kwe-LA ne-BN. Idatha yomthombo inikezwa ngesimo samafayela edatha yomthombo.
I-metabolome ephelele ye-serum ye-174 HC, i-170 BN kanye ne-136 LA eqenjini lokutholwa ihlaziywe kusetshenziswa ukuhlaziywa kwe-UPLC-HRMS. Okokuqala sibonisa ukuthi amasampula okulawula ikhwalithi (i-QC) ahlangana kahle phakathi kwemodeli yokuhlaziywa kwengxenye eyinhloko engagadiwe (i-PCA), okuqinisekisa ukuzinza kokusebenza kocwaningo lwamanje (Isithombe Esingeziwe 2).
Njengoba kuboniswe ku-partial least squares-discriminant analysis (PLS-DA) ku-Figure 1 b, sithole ukuthi kwakukhona umehluko ocacile phakathi kwe-LA ne-BN, i-LA ne-HC kumamodi e-electrospray ionization a-positive (+ESI) kanye ne-negative (−ESI). Kodwa-ke, akukho mehluko obalulekile otholakale phakathi kwe-BN ne-HC ezimweni ze-+ESI kanye ne--ESI.
Sithole izici zomehluko ezingu-382 phakathi kwe-LA ne-HC, izici zomehluko ezingu-231 phakathi kwe-LA ne-BN, kanye nezici zomehluko ezingu-95 phakathi kwe-BN ne-HC (ukuhlolwa kwesikhundla esisayinwe yi-Wilcoxon, i-FDR <0.05 kanye noshintsho oluningi >1.2 noma <0.83) (Umfanekiso .1c-e). . Ama-peaks aphinde achazwa (Idatha Engeziwe 3) ngokumelene nesizindalwazi (umtapo wezincwadi we-mzCloud/HMDB/Chemspider) ngenani le-m/z, isikhathi sokugcina kanye nokusesha kwe-mass spectrum yokuqhekeka (imininingwane echazwe esigabeni sezindlela) 22. Ekugcineni, ama-metabolite angu-33 no-38 anezincazelo ezinomehluko omkhulu ngobuningi atholakala ku-LA ngokumelene ne-BN (Umfanekiso 1f kanye neThebula Engeziwe 2) kanye ne-LA ngokumelene ne-HC (Umfanekiso Engeziwe 3 kanye neThebula Engeziwe 2), ngokulandelana. Ngokuphambene nalokho, kutholakale ama-metabolite amathathu kuphela anomehluko omkhulu ngobuningi ku-BN kanye ne-HC (Ithebula Elingeziwe 2), okuhambisana nokufana phakathi kwe-BN kanye ne-HC ku-PLS-DA. Lawa ma-metabolite ahlukene amboza ububanzi be-biochemicals (Isithombe Esingeziwe 4). Uma kuhlanganiswa, le miphumela ikhombisa izinguquko ezibalulekile ku-serum metabolome ezibonisa ukuguqulwa okulimazayo komdlavuza wamaphaphu wesigaba sokuqala uma kuqhathaniswa nama-nodule amaphaphu angenabungozi noma abantu abanempilo. Okwamanje, ukufana kwe-serum metabolome ye-BN kanye ne-HC kusikisela ukuthi ama-nodule amaphaphu angenabungozi angabelana ngezici eziningi zebhayoloji nabantu abanempilo. Njengoba ukuguqulwa kwezakhi zofuzo ze-epidermal growth factor receptor (EGFR) kuvamile ku-lung adenocarcinoma subtype 23, sifuna ukuthola umthelela wokuguqulwa komshayeli ku-serum metabolome. Sabe sesihlaziya iphrofayili iyonke ye-metabolomic yamacala angu-72 anesimo se-EGFR eqenjini le-lung adenocarcinoma. Ngokuthakazelisayo, sithole amaphrofayili afanayo phakathi kweziguli eziguqukile ze-EGFR (n = 41) kanye neziguli zohlobo lwe-EGFR zasendle (n = 31) ekuhlaziyweni kwe-PCA (Isithombe Esingeziwe 5a). Kodwa-ke, sithole ama-metabolite angu-7 anenani elishintshile kakhulu ezigulini ezine-EGFR mutation uma kuqhathaniswa neziguli ezine-EGFR yohlobo lwe-wild (t test, p < 0.05 kanye ne-fold change > 1.2 noma < 0.83) (Isithombe Esingeziwe 5b). Iningi lala ma-metabolite (5 kwangu-7) yi-acylcarnitines, adlala indima ebalulekile ezindleleni zokuxiliswa kwe-fatty acid.
Njengoba kuboniswe emsebenzini oboniswe kuMfanekiso 2 a, ama-biomarker okuhlukaniswa kwamaqhubu atholwe kusetshenziswa ama-operator angama-least absolute shrinkage kanye nokukhetha okusekelwe kuma-metabolite angu-33 ahlukene atholwe ku-LA (n = 136) kanye ne-BN (n = 170). Inhlanganisela engcono kakhulu yezinguquko (i-LASSO) - imodeli yokuguqulwa kwe-binary logistic. Ukuqinisekiswa kwe-cross-validation okuphindwe kayishumi kusetshenziswe ukuhlola ukuthembeka kwemodeli. Ukukhethwa kwe-variable kanye nokuhlelwa kwepharamitha kulungiswa ngesijeziso sokukhulisa amathuba ngepharamitha engu-λ24. Ukuhlaziywa kwe-metabolomics yomhlaba wonke kwenziwe ngokuzimela kumaqembu okuqinisekiswa kwangaphakathi (n = 104) kanye nokuqinisekiswa kwangaphandle (n = 111) ukuhlola ukusebenza kwesigaba semodeli yokuhlukanisa. Ngenxa yalokho, ama-metabolite angu-27 kusethi yokuthola atholwe njengemodeli yokuhlukanisa engcono kakhulu enenani elikhulu kakhulu le-AUC (Umfanekiso 2b), phakathi kwawo angu-9 ayenomsebenzi owandisiwe kanye nomsebenzi onciphile ongu-18 ku-LA uma kuqhathaniswa ne-BN (Umfanekiso 2c).
Ukuhamba komsebenzi wokwakha i-pulmonary nodule classifier, kufaka phakathi ukukhetha iphaneli engcono kakhulu yama-serum metabolites kusethi yokuthola kusetshenziswa imodeli yokubuyela emuva kwe-binary logistic ngokusebenzisa ukuqinisekiswa okuphindwe kayishumi kanye nokuhlola ukusebenza kokubikezela kumasethi wokuqinisekisa wangaphakathi nangaphandle. b Izibalo zokuqinisekiswa okuphambene kwemodeli yokubuyela emuva ye-LASSO yokukhethwa kwe-biomarker ye-metabolic. Izinombolo ezinikezwe ngenhla zimele inani elimaphakathi lama-biomarker akhethiwe ku-λ enikeziwe. Umugqa onamachashazi abomvu umele inani elimaphakathi le-AUC ku-lambda ehambisanayo. Amabha amaphutha ampunga amelela amanani amancane kanye naphezulu e-AUC. Umugqa onamachashazi ukhombisa imodeli engcono kakhulu ngama-biomarker akhethiwe angu-27. I-AUC, indawo engaphansi kwejika lesici sokusebenza se-receiver (ROC). c Izinguquko ezigoqekayo zama-metabolites akhethiwe angu-27 eqenjini le-LA uma kuqhathaniswa neqembu le-BN eqenjini lokuthola. Ikholomu ebomvu - ukusebenza. Ikholomu eluhlaza okwesibhakabhaka iwukwehla. Ama-curve e-d–f Receiver operating characteristic (ROC) abonisa amandla emodeli yokuhlukanisa ngokusekelwe ekuhlanganisweni kwama-metabolite angu-27 kumasethi wokuqinisekisa okutholakalayo, angaphakathi, nangaphandle. Idatha yomthombo inikezwa ngesimo samafayela edatha yomthombo.
Imodeli yokubikezela yadalwa ngokusekelwe kuma-coefficients okubuyela emuva anesisindo alawa ma-metabolite angu-27 (Ithebula Elingeziwe 3). Ukuhlaziywa kwe-ROC okusekelwe kulawa ma-metabolite angu-27 kuveze indawo engaphansi kwenani le-curve (AUC) elingu-0.933, ukuzwela kweqembu lokutholakala kwakungu-0.868, kanti ukucaciswa kwakungu-0.859 (Isithombe 2d). Phakathi naleso sikhathi, phakathi kwama-metabolite angu-38 ahlukanisiwe phakathi kwe-LA ne-HC, isethi yama-metabolite angu-16 ithole i-AUC engu-0.902 enobuzwe obungu-0.801 kanye nokucaciswa okungu-0.856 ekuhlukaniseni i-LA ne-HC (Isithombe Esingeziwe 6a-c). Amanani e-AUC asekelwe emikhawulweni ehlukene yokushintsha kokugoba yama-metabolite ahlukile nawo aqhathaniswa. Sithole ukuthi imodeli yokuhlela isebenze kahle kakhulu ekuhlukaniseni phakathi kwe-LA ne-BN (HC) lapho izinga lokushintsha kokugoba libekwe ku-1.2 uma kuqhathaniswa no-1.5 noma u-2.0 (Isithombe Esingeziwe 7a,b). Imodeli yokuhlela, esekelwe emaqenjini angu-27 e-metabolite, yaqinisekiswa ngokwengeziwe kuma-cohort angaphakathi nangaphandle. I-AUC yayingu-0.915 (ukuzwela 0.867, ukucaciswa 0.811) kokuqinisekiswa kwangaphakathi kanye no-0.945 (ukuzwela 0.810, ukucaciswa 0.979) kokuqinisekiswa kwangaphandle (Isithombe 2e, f). Ukuze kuhlolwe ukusebenza kahle kwe-interlaboratory, amasampula angu-40 avela eqenjini langaphandle ahlaziywa elabhorethri langaphandle njengoba kuchaziwe esigabeni seMethods. Ukunemba kokuhlela kufinyelele i-AUC engu-0.925 (Isithombe Esingeziwe 8). Ngenxa yokuthi i-lung squamous cell carcinoma (LUSC) iwuhlobo lwesibili oluvame kakhulu lomdlavuza wamaphaphu ongewona amaseli amancane (NSCLC) ngemuva kwe-lung adenocarcinoma (LUAD), siphinde sahlola ukusetshenziswa okungenzeka okuqinisekisiwe kwamaphrofayili e-metabolic. I-BN kanye namacala angu-16 e-LUSC. I-AUC yokubandlulula phakathi kwe-LUSC ne-BN yayingu-0.776 (Isithombe Esengeziwe 9), okubonisa ikhono eliphansi uma kuqhathaniswa nokubandlulula phakathi kwe-LUAD ne-BN.
Izifundo zibonise ukuthi usayizi wamaqhubu ezithombeni ze-CT uhlobene kahle nethuba lomdlavuza futhi uhlala uyisici esiyinhloko sokwelashwa kwamaqhubu25,26,27. Ukuhlaziywa kwedatha evela eqenjini elikhulu locwaningo lokuhlola i-NELSON kubonise ukuthi ingozi yomdlavuza kubantu abanamaqhubu angaphansi kuka-5 mm yayifana naleyo yabantu abangenamaqhubu28. Ngakho-ke, usayizi omncane odinga ukubhekwa njalo kwe-CT ngu-5 mm, njengoba kunconywe yi-British Thoracic Society (BTS), kanye no-6 mm, njengoba kunconywe yi-Fleischner Society 29. Kodwa-ke, amaqhubu amakhulu kuno-6 mm futhi angenazo izici ezisobala ezingabonakali, abizwa ngokuthi amaqhubu amaqhubu amaqhubu (i-IPN), ahlala eyinselele enkulu ekuhlolweni nasekuphathweni emtholampilo30,31. Okulandelayo sihlole ukuthi usayizi wamaqhubu uthonye yini izimpawu ze-metabolomic sisebenzisa amasampula ahlanganisiwe avela kumaqoqo okutholwa kanye nokuqinisekiswa kwangaphakathi. Sigxile kuma-biomarker angu-27 aqinisekisiwe, siqale saqhathanisa amaphrofayili e-PCA e-HC kanye ne-BN sub-6 mm metabolomes. Sithole ukuthi amaphuzu amaningi edatha e-HC kanye ne-BN ahambisana, okubonisa ukuthi amazinga e-serum metabolite ayefana kuwo womabili amaqembu (Isithombe 3a). Amamephu esici kuzo zonke izinhlobo zobukhulu obuhlukene agcinwe ku-BN kanye ne-LA (Isithombe 3b, c), kanti ukuhlukaniswa kwabonwa phakathi kwamaqhubu amabi kanye namaqhubu angenabungozi ebangeni eliyi-6-20 mm (Isithombe 3d). Leli qembu lalino-AUC ongu-0.927, ukucaciswa okungu-0.868, kanye nokuzwela okungu-0.820 kokubikezela umdlavuza wamaqhubu olinganiselwa ku-6 kuya ku-20 mm (Isithombe 3e, f). Imiphumela yethu ikhombisa ukuthi i-classifier ingakwazi ukuthola izinguquko ze-metabolic ezibangelwa ukuguqulwa kokuqala kwe-malignant, kungakhathaliseki ukuthi usayizi wamaqhubu ungakanani.
Isikhangiso Ukuqhathaniswa kwamaphrofayili e-PCA phakathi kwamaqembu acacisiwe ngokusekelwe ku-metabolic classifier yama-metabolites angu-27. I-CC ne-BN < 6 mm. b I-BN < 6 mm vs I-BN 6–20 mm. ku-LA 6–20 mm vs I-LA 20–30 mm. g I-BN 6–20 mm kanye ne-LA 6–20 mm. I-GC, n = 174; I-BN < 6 mm, n = 153; I-BN 6–20 mm, n = 91; I-LA 6–20 mm, n = 89; I-LA 20–30 mm, n = 77. e Ijika lesici sokusebenza se-Receiver (ROC) elibonisa ukusebenza kwemodeli okuhlukile kwama-nodules angu-6–20 mm. f Amanani okunokwenzeka abalwe ngokusekelwe kumodeli yokubuyela emuva kwe-logistic yama-nodules alinganisa u-6–20 mm. Umugqa ompunga onamachashazi umele inani elifanele kakhulu lokusika (0.455). Izinombolo ezingenhla zimele iphesenti lamacala alindelwe eLos Angeles. Sebenzisa ukuhlolwa kwe-t komfundi okunemisila emibili. I-PCA, ukuhlaziywa kwengxenye eyinhloko. Indawo ye-AUC ngaphansi kwejika. Idatha yomthombo inikezwa ngesimo samafayela edatha yomthombo.
Amasampula amane (aneminyaka engu-44-61) anobukhulu obufanayo bamaqhubu ephaphu (7-9 mm) akhethwe kabanzi ukuze abonise ukusebenza kwemodeli yokubikezela umdlavuza ehlongozwayo (Isithombe 4a, b). Ekuhlolweni kokuqala, iCase 1 lavezwa njengeqhubu eliqinile eline-calcification, isici esihlotshaniswa nokuba mnene, kanti iCase 2 lavezwa njengeqhubu eliqinile elingacacile elingenazo izici ezicacile ezinobungozi. Izigameko ezintathu zokulandelela ze-CT scan zibonise ukuthi la macala ahlala eqinile esikhathini seminyaka engu-4 futhi ngenxa yalokho abhekwa njengamaqhubu angenalutho (Isithombe 4a). Uma kuqhathaniswa nokuhlolwa kwemitholampilo kwama-CT scan alandelanayo, ukuhlaziywa kwe-serum metabolite ye-single-shot ngemodeli yamanje yokuhlukanisa kwahlonza ngokushesha nangokufanele la maqhubu angenalutho ngokusekelwe ezinkingeni ezingaba khona (Ithebula 1). Isibalo 4b esimweni 3 sibonisa iqhubu elinezimpawu zokuhoxa kwe-pleural, okuvame ukuhlotshaniswa nomdlavuza32. Icala 4 lavezwa njengeqhubu eliqinile elingacacile elingenabo ubufakazi bembangela engathandeki. Wonke la macala abikezelwe njengelibi ngokwemodeli yokuhlukanisa (Ithebula 1). Ukuhlolwa kwe-lung adenocarcinoma kwaboniswa ngokuhlolwa kwe-histopathological ngemuva kokuhlinzwa kokususwa kwamaphaphu (Isithombe 4b). Kwisethi yokuqinisekiswa kwangaphandle, i-metabolic classifier yabikezela ngokunembile amacala amabili amaqhubu amaphaphu angacaci amakhulu kuno-6 mm (Isithombe Esingeziwe 10).
Izithombe ze-CT zefasitela le-axial lamaphaphu ezigameko ezimbili zamaqhubu angenabungozi. Esimweni soku-1, i-CT scan ngemva kweminyaka emi-4 ibonise iqhubu eliqinile eliqinile elingama-7 mm eline-calcification ku-lobe engezansi kwesokudla. Esimweni sesi-2, i-CT scan ngemva kweminyaka emi-5 iveze iqhubu eliqinile, eliqinile kancane elinobubanzi obuyi-7 mm ku-lobe engenhla kwesokudla. b Ifasitela le-Axial Izithombe ze-CT zamaphaphu kanye nezifundo ze-pathological ezihambisanayo zamacala amabili e-stage I adenocarcinoma ngaphambi kokususwa kwamaphaphu. Icala lesi-3 liveze iqhubu elinobubanzi obuyi-8 mm ku-lobe engenhla kwesokudla elinokubuyiselwa kwe-pleural. Icala lesi-4 liveze iqhubu eliqinile kancane elingilazi yomhlaba elinobubanzi obuyi-9 mm ku-lobe engenhla kwesobunxele. Ukufakwa kwe-Hematoxylin kanye ne-eosin (H&E) kwezicubu zamaphaphu ezisusiwe (ibha yesikali = 50 μm) okubonisa iphethini yokukhula kwe-acinar ye-lung adenocarcinoma. Imicibisholo ikhombisa amaqhubu atholwe ezithombeni ze-CT. Izithombe ze-H&E ziyizithombe ezimele amasimu amaningi amancane (>3) ahlolwe yi-pathologist.
Uma sibheka ndawonye, imiphumela yethu ikhombisa inani elingaba khona lama-biomarker e-serum metabolite ekuxilongweni okuhlukile kwama-nodules e-pulmonary, okungaba yinselele lapho kuhlolwa ukuhlolwa kwe-CT.
Ngokusekelwe kuphaneli ye-metabolite ehlukanisiwe eqinisekisiwe, sifuna ukuthola izixhumanisi zezinto eziphilayo zezinguquko ezinkulu ze-metabolic. Ukuhlaziywa kokucebisa indlela ye-KEGG yi-MetaboAnalyst kuthole izindlela ezi-6 ezivamile ezishintshiwe kakhulu phakathi kwamaqembu amabili anikeziwe (i-LA vs. HC kanye ne-LA vs. BN, kulungisiwe p ≤ 0.001, umphumela > 0.01). Lezi zinguquko zibonakaliswe ukuphazamiseka kwe-metabolism ye-pyruvate, i-tryptophan metabolism, i-niacin kanye ne-nicotinamide metabolism, i-glycolysis, umjikelezo we-TCA, kanye ne-purine metabolism (Isithombe 5a). Sabe sesenza i-metabolomics eqondiwe ukuqinisekisa izinguquko ezinkulu sisebenzisa i-absolute quantification. Ukunqunywa kwama-metabolite avamile ezindleleni eziguquliwe ezivamile yi-triple quadrupole mass spectrometry (QQQ) sisebenzisa amazinga e-metabolite angempela. Izici zezibalo zesampula eqondiwe yocwaningo lwe-metabolomics zifakiwe kuThebula Elingeziwe 4. Ngokuhambisana nemiphumela yethu yomhlaba wonke ye-metabolomics, ukuhlaziywa kwenani kuqinisekisile ukuthi i-hypoxanthine kanye ne-xanthine, i-pyruvate, kanye ne-lactate kwandisiwe ku-LA uma kuqhathaniswa ne-BN kanye ne-HC (Isithombe 5b, c, p <0.05). Kodwa-ke, akukho mehluko obalulekile kulawa ma-metabolites owatholakala phakathi kwe-BN kanye ne-HC.
Ukuhlaziywa kokunongwa kwendlela ye-KEGG kwama-metabolite ahlukene kakhulu eqenjini le-LA uma kuqhathaniswa namaqembu e-BN kanye ne-HC. Kusetshenziswe i-Globaltest enemisila emibili, futhi amanani e-p alungiswa kusetshenziswa indlela ye-Holm-Bonferroni (ilungisiwe i-p ≤ 0.001 kanye nosayizi womphumela > 0.01). b–d Ama-Violin plots abonisa amazinga e-hypoxanthine, i-xanthine, i-lactate, i-pyruvate, kanye ne-tryptophan ku-serum HC, i-BN, kanye ne-LA enqunywe yi-LC-MS/MS (n = 70 eqenjini ngalinye). Imigqa emhlophe nemnyama enamachashazi ikhombisa i-median kanye ne-quartile, ngokulandelana. e I-Violin plot ekhombisa ukubonakaliswa kwe-mRNA ye-Log2TPM evamile (ama-transcripts ngesigidi) ye-SLC7A5 kanye ne-QPRT ku-adenocarcinoma yamaphaphu (n = 513) uma kuqhathaniswa nezicubu zamaphaphu ezivamile (n = 59) kusethi yedatha ye-LUAD-TCGA. Ibhokisi elimhlophe limelela ububanzi be-interquartile, umugqa omnyama ovundlile ophakathi nendawo umelela i-median, kanti umugqa omnyama oqondile osuka ebhokisini umelela isikhawu sokuqiniseka esingu-95% (CI). f I-Pearson correlation plot ye-SLC7A5 kanye nokubonakaliswa kwe-GAPDH ku-lung adenocarcinoma (n = 513) kanye nezicubu zamaphaphu ezivamile (n = 59) kusethi yedatha ye-TCGA. Indawo empunga imelela i-95% CI. r, i-Pearson correlation coefficient. g Amazinga e-tryptophan yeselula ajwayelekile kumaseli e-A549 adluliselwe ngokulawulwa kwe-shRNA okungeyona i-specific (NC) kanye ne-shSLC7A5 (Sh1, Sh2) enqunywe yi-LC-MS/MS. Ukuhlaziywa kwezibalo kwamasampula amahlanu azimele ngokwezinto eziphilayo eqenjini ngalinye kuyethulwa. h Amazinga eselula e-NADt (i-NAD iyonke, kufaka phakathi i-NAD+ kanye ne-NADH) kumaseli e-A549 (NC) kanye namaseli e-SLC7A5 knockdown A549 (Sh1, Sh2). Ukuhlaziywa kwezibalo kwamasampula amathathu azimele ngokwezinto eziphilayo eqenjini ngalinye kuyethulwa. Umsebenzi we-Glycolytic wamaseli e-A549 ngaphambi nangemva kokudilizwa kwe-SLC7A5 ulinganiswe ngesilinganiso se-extracellular acidification (ECAR) (n = amasampula angu-4 azimele ngokwezinto eziphilayo ngeqembu ngalinye). I-2-DG,2-deoxy-D-glucose. Ukuhlolwa kwe-t komfundi okunemisila emibili kusetshenziswe ku-(b–h). Ku-(g–i), amabha amaphutha amelela isilinganiso ± SD, ukuhlolwa ngakunye kwenziwa kathathu ngokuzimela futhi imiphumela yayifana. Idatha yomthombo inikezwa ngesimo samafayela edatha yomthombo.
Uma sibheka umthelela obalulekile wokuguqulwa kwe-metabolism ye-tryptophan eqenjini le-LA, sihlole namazinga e-serum tryptophan emaqenjini e-HC, BN, kanye ne-LA sisebenzisa i-QQQ. Sithole ukuthi i-serum tryptophan yehlisiwe ku-LA uma kuqhathaniswa ne-HC noma i-BN (p < 0.001, Umfanekiso 5d), okuhambisana nokutholakele kwangaphambilini kokuthi amazinga e-tryptophan ajikelezayo aphansi ezigulini ezinomdlavuza wamaphaphu kunasezilawulini ezinempilo ezivela eqenjini lokulawula33,34,35. Olunye ucwaningo olusebenzisa i-PET/CT tracer 11C-methyl-L-tryptophan luthole ukuthi isikhathi sokugcina isignali ye-tryptophan ezicutshini zomdlavuza wamaphaphu sanda kakhulu uma kuqhathaniswa nezilonda ezingengozini noma izicubu ezivamile36. Sicabanga ukuthi ukwehla kwe-tryptophan ku-serum ye-LA kungabonisa ukumuncwa kwe-tryptophan esebenzayo ngamaseli omdlavuza wamaphaphu.
Kuyaziwa futhi ukuthi umkhiqizo wokugcina wendlela ye-kynurenine ye-tryptophan catabolism yi-NAD+37,38, okuyi-substrate ebalulekile yokusabela kwe-glyceraldehyde-3-phosphate ne-1,3-bisphosphoglycerate ku-glycolysis39. Ngenkathi izifundo zangaphambilini zigxile endimeni ye-tryptophan catabolism ekulawulweni komzimba, sifuna ukucacisa ukusebenzisana phakathi kokungasebenzi kahle kwe-tryptophan kanye nezindlela ze-glycolytic ezibonwe ocwaningweni lwamanje. Ilungu lesi-5 lomndeni we-Solute transporter 7 (SLC7A5) laziwa njenge-tryptophan transporter43,44,45. I-Quinolinic acid phosphoribosyltransferase (QPRT) iyi-enzyme etholakala phansi kwendlela ye-kynurenine eguqula i-quinolinic acid ibe yi-NAMN46. Ukuhlolwa kwesethi yedatha ye-LUAD TCGA kwembule ukuthi kokubili i-SLC7A5 kanye ne-QPRT kwanda kakhulu ezicutshini zesimila uma kuqhathaniswa nezicubu ezivamile (Isithombe 5e). Lokhu kwanda kubonwe ezigabeni I no-II kanye nasezigabeni III no-IV ze-lung adenocarcinoma (Isithombe Esingeziwe 11), okubonisa ukuphazamiseka kwasekuqaleni kwe-metabolism ye-tryptophan ehlotshaniswa ne-tumorigenesis.
Ngaphezu kwalokho, isethi yedatha ye-LUAD-TCGA ibonise ubudlelwano obuhle phakathi kokubonakaliswa kwe-SLC7A5 kanye ne-GAPDH mRNA kumasampula eziguli ezinomdlavuza (r = 0.45, p = 1.55E-26, Umfanekiso 5f). Ngokuphambene nalokho, akukho ubudlelwano obubalulekile obutholakale phakathi kwezisayini zezakhi zofuzo ezinjalo ezicutshini zamaphaphu ezivamile (r = 0.25, p = 0.06, Umfanekiso 5f). Ukwehla kwe-SLC7A5 (Isithombe Esingeziwe 12) kumaseli e-A549 kunciphisa kakhulu amazinga e-tryptophan yeselula kanye ne-NAD(H) (Isithombe 5g,h), okuholela emsebenzini we-glycolytic oncishisiwe njengoba kulinganiswa ngesilinganiso se-extracellular acidification (ECAR) (Isithombe 1). 5i). Ngakho-ke, ngokusekelwe ezinguqukweni ze-metabolic ku-serum kanye nokutholwa kwe-in vitro, sicabanga ukuthi i-tryptophan metabolism ingakhiqiza i-NAD+ ngendlela ye-kynurenine futhi idlale indima ebalulekile ekukhuthazeni i-glycolysis kumdlavuza wamaphaphu.
Izifundo zibonise ukuthi inani elikhulu lamaqhubu angenasisekelo atholakala yi-LDCT lingaholela esidingweni sokuhlolwa okwengeziwe njenge-PET-CT, i-biopsy yamaphaphu, kanye nokwelashwa ngokweqile ngenxa yokuxilongwa okungelona iqiniso kwesifo somdlavuza.31 Njengoba kuboniswe kuMfanekiso 6, ucwaningo lwethu luthole iphaneli yama-metabolites e-serum enenani lokuxilonga elingathuthukisa ukwahlukaniswa kwengozi kanye nokuphathwa okulandelayo kwamaqhubu amaphaphu atholakala yi-CT.
Amaqhubu ephaphu ahlolwa kusetshenziswa i-computed tomography (LDCT) esezingeni eliphansi ngezici zezithombe ezibonisa izimbangela ezingengozi noma ezimbi. Umphumela ongaqiniseki wamaqhubu ungaholela ekuvakasheni okulandelela njalo, ukungenelela okungadingekile, kanye nokwelashwa ngokweqile. Ukufakwa kwezinhlobo ze-serum metabolic classifiers ezinenani lokuxilonga kungathuthukisa ukuhlolwa kwengozi kanye nokuphathwa okulandelayo kwamaqhubu ephaphu. I-PET positron emission tomography.
Idatha evela ocwaningweni lwe-US NLST kanye nocwaningo lwe-European NELSON iphakamisa ukuthi ukuhlola amaqembu asengozini enkulu nge-computed tomography (LDCT) yomthamo ophansi kunganciphisa ukufa komdlavuza wamaphaphu1,3. Kodwa-ke, ukuhlolwa kwengozi kanye nokuphathwa kwezokwelapha okulandelayo kwenani elikhulu lamaqhubu amaphaphu atholwe yi-LDCT kusalokhu kuyinselele kakhulu. Umgomo oyinhloko ukwenza ngcono ukuhlukaniswa okufanele kwezinqubo ezikhona ezisekelwe ku-LDCT ngokufaka ama-biomarker athembekile.
Ama-biomarker athile ama-molecule, njenge-metabolites yegazi, atholakale ngokuqhathanisa umdlavuza wamaphaphu nezilawuli ezinempilo15,17. Esifundweni samanje, sigxile ekusetshenzisweni kokuhlaziywa kwe-serum metabolomics ukuhlukanisa phakathi kwama-nodules e-pulmonary angenabungozi nalawo anobungozi atholakale ngengozi yi-LDCT. Siqhathanise isampula ye-serum metabolome yomhlaba wonke yokulawula okunempilo (HC), ama-nodules e-lung angenabungozi (BN), kanye namasampula e-lung adenocarcinoma (LA) esigaba sokuqala sisebenzisa ukuhlaziywa kwe-UPLC-HRMS. Sithole ukuthi i-HC ne-BN zinephrofayili efanayo ye-metabolic, kanti i-LA ibonise izinguquko ezibalulekile uma kuqhathaniswa ne-HC ne-BN. Sithole amasethi amabili e-serum metabolites ahlukanisa i-LA ne-HC ne-BN.
Uhlelo lwamanje lokuhlonza amaqhubu angenabungozi nanomdlavuza lusekelwe kakhulu kusayizi, ubuningi, isimo kanye nezinga lokukhula kwamaqhubu ngokuhamba kwesikhathi. Izifundo zangaphambilini zikhombisile ukuthi ubukhulu bamaqhubu buhlobene eduze nethuba lomdlavuza wamaphaphu. Ngisho nasezigulini ezisengozini enkulu, ingozi yomdlavuza kumaqhubu angaphansi kuka-6 mm i-<1%. Ingozi yomdlavuza kumaqhubu aphakathi kuka-6 kuya ku-20 mm isukela ku-8% kuya ku-64%30. Ngakho-ke, i-Fleischner Society itusa ububanzi obungama-cutoff obungu-6 mm ukuze kulandelwe i-CT njalo. 29 Kodwa-ke, ukuhlolwa kwengozi kanye nokuphathwa kwamaqhubu angenasifo samaphaphu (i-IPN) amakhulu kuno-6 mm akwenziwanga ngokwanele 31. Ukuphathwa kwamanje kwesifo senhliziyo esizalwa naso ngokuvamile kusekelwe ekulindeni okuqaphile ngokuqapha njalo kwe-CT.
Ngokusekelwe ku-metabolome eqinisekisiwe, sibonise okokuqala ukufana kwezimpawu ze-metabolomic phakathi kwabantu abaphilile kanye nama-nodule angenabungozi <6 mm. Ukufana kwezinto eziphilayo kuhambisana nokutholakele kwangaphambilini kwe-CT ukuthi ingozi yomdlavuza wama-nodule <6 mm iphansi njengakubantu abangenawo ama-nodule.30 Kufanele kuqashelwe ukuthi imiphumela yethu ikhombisa nokuthi ama-nodule angenabungozi <6 mm kanye no-≥6 mm afana kakhulu kumaphrofayili e-metabolomic, okuphakamisa ukuthi incazelo yokusebenza ye-etiology engathandeki iyahambisana kungakhathaliseki usayizi wama-nodule. Ngakho-ke, amaphaneli e-serum metabolite okuxilonga anamuhla anganikeza ukuhlolwa okukodwa njengokuhlolwa kokungabikho lapho ama-nodule etholakala okokuqala ku-CT futhi kungenzeka anciphise ukuqapha okulandelanayo. Ngesikhathi esifanayo, iphaneli efanayo yama-biomarker e-metabolic yahlukanisa ama-nodule anobungozi ≥6 mm ngobukhulu kuma-nodule angenabungozi futhi yanikeza izibikezelo ezinembile zama-IPN anobukhulu obufanayo kanye nezici ze-morphological ezingacacile ezithombeni ze-CT. Lesi sigaba se-serum metabolism senze kahle ekubikezeleni umdlavuza wama-nodule ≥6 mm nge-AUC engu-0.927. Uma sibheka ndawonye, imiphumela yethu ikhombisa ukuthi izimpawu ze-metabolomic ze-serum ezihlukile zingabonisa ngqo izinguquko ze-metabolic ezibangelwa umdlavuza futhi zibe nenani elingase libe yizibikezelo zengozi, kungakhathaliseki ukuthi amaqhubu angakanani.
Okuphawulekayo ukuthi i-lung adenocarcinoma (LUAD) kanye ne-squamous cell carcinoma (LUSC) yizinhlobo eziyinhloko zomdlavuza wamaphaphu ongewona omncane (NSCLC). Njengoba i-LUSC ihlotshaniswa kakhulu nokusetshenziswa kukagwayi47 futhi i-LUAD iyi-histology evame kakhulu yamaqhubu amaphaphu atholakale ekuhlolweni kwe-CT48, imodeli yethu yokuhlukanisa yakhelwe ngqo amasampula e-stage I adenocarcinoma. UWang nozakwabo bagxile ku-LUAD futhi bathola izimpawu ze-lipid eziyisishiyagalolunye besebenzisa ama-lipidomics ukuhlukanisa umdlavuza wamaphaphu wesigaba sokuqala kubantu abaphilile17. Sihlole imodeli yamanje yokuhlukanisa emacaleni ayi-16 e-stage I LUSC kanye namaqhubu angama-74 angenabungozi futhi sabona ukunemba okuphansi kokubikezela kwe-LUSC (AUC 0.776), okuphakamisa ukuthi i-LUAD ne-LUSC zingaba nezimpawu zazo ze-metabolomic. Ngempela, i-LUAD ne-LUSC kuboniswe ukuthi ziyahlukahluka ngemvelaphi, imvelaphi yezinto eziphilayo kanye nokuphazamiseka kwezakhi zofuzo49. Ngakho-ke, ezinye izinhlobo ze-histology kufanele zifakwe kumamodeli okuqeqesha okutholakala komdlavuza wamaphaphu ngokusekelwe kubantu ezinhlelweni zokuhlola.
Lapha, sithole izindlela eziyisithupha ezivame ukuguqulwa kakhulu ku-lung adenocarcinoma uma kuqhathaniswa nezilawuli ezinempilo kanye nama-nodule angenabungozi. I-Xanthine kanye ne-hypoxanthine kuyizinto ezivamile ze-metabolite ye-purine metabolic pathway. Ngokuhambisana nemiphumela yethu, ama-intermediate ahlobene ne-purine metabolism akhuliswe kakhulu ku-serum noma ezicutshini zeziguli ezine-lung adenocarcinoma uma kuqhathaniswa nezilawuli ezinempilo noma iziguli ezisezingeni lokuqala lokuhlasela15,50. Amazinga aphezulu e-serum xanthine kanye ne-hypoxanthine angabonisa i-anabolism edingekayo ngamaseli omdlavuza akhula ngokushesha. Ukuphazamiseka kwe-glucose metabolism kuyisici esaziwayo se-cancer metabolism51. Lapha, sibone ukwanda okukhulu kwe-pyruvate kanye ne-lactate eqenjini le-LA uma kuqhathaniswa neqembu le-HC kanye ne-BN, okuhambisana nemibiko yangaphambilini yokuphazamiseka kwendlela ye-glycolytic kumaphrofayili e-serum metabolome ezigulini ezingezona ezincane zomdlavuza wamaphaphu (NSCLC) kanye nezilawuli ezinempilo. imiphumela iyahambisana52,53.
Okubalulekile, sibone ukuhlangana okuphambene phakathi kwe-pyruvate kanye ne-tryptophan metabolism ku-serum yama-lung adenocarcinomas. Amazinga e-serum tryptophan ancishisiwe eqenjini le-LA uma kuqhathaniswa neqembu le-HC noma le-BN. Ngokuthakazelisayo, ucwaningo olukhulu lwangaphambilini olusebenzisa iqembu elilindelekile luthole ukuthi amazinga aphansi e-tryptophan ajikelezayo ahlotshaniswa nengozi eyengeziwe yomdlavuza wamaphaphu 54. I-Tryptophan iyi-amino acid ebalulekile esiyithola ngokuphelele ekudleni. Siphetha ngokuthi ukuncipha kwe-serum tryptophan ku-lung adenocarcinoma kungabonisa ukuncipha okusheshayo kwale metabolite. Kwaziwa kahle ukuthi umkhiqizo wokugcina we-tryptophan catabolism ngendlela ye-kynurenine ungumthombo wokwenziwa kwe-de novo NAD+. Ngenxa yokuthi i-NAD+ ikhiqizwa ngokuyinhloko ngendlela yokusindisa, ukubaluleka kwe-NAD+ ku-tryptophan metabolism empilweni nasezifweni kusazonqunywa46. Ukuhlaziywa kwethu kwesizindalwazi se-TCGA kukhombisile ukuthi ukuvezwa kwe-tryptophan transporter solute transporter 7A5 (SLC7A5) kwanda kakhulu ku-lung adenocarcinoma uma kuqhathaniswa nokulawula okuvamile futhi kwahlotshaniswa kahle nokuvezwa kwe-glycolytic enzyme GAPDH. Izifundo zangaphambilini zigxile kakhulu endimeni ye-tryptophan catabolism ekucindezeleni impendulo yokuzivikela komzimba yokulwa nomdlavuza40,41,42. Lapha sibonisa ukuthi ukuvinjelwa kokuthathwa kwe-tryptophan ngokuqothulwa kwe-SLC7A5 kumaseli omdlavuza wamaphaphu kuholela ekwehleni okulandelayo kwamazinga e-NAD yeselula kanye nokuncipha komsebenzi we-glycolytic ngesikhathi esifanayo. Ngamafuphi, ucwaningo lwethu luhlinzeka ngesisekelo sebhayoloji sezinguquko ku-serum metabolism ehlotshaniswa nokuguqulwa okubi kwe-lung adenocarcinoma.
Ukuguqulwa kwe-EGFR yikona okudala kakhulu ukuguquka kwezakhi zofuzo ezigulini ezine-NSCLC. Esifundweni sethu, sithole ukuthi iziguli ezine-EGFR mutation (n = 41) zinephrofayili ye-metabolomic efana neziguli ezine-EGFR yohlobo lwe-wild-type (n = 31), yize sithole amazinga e-serum aphansi kwezinye iziguli ezine-EGFR mutant ezigulini ze-acylcarnitine. Umsebenzi osunguliwe we-acylcarnitines ukuhambisa amaqembu e-acyl kusuka ku-cytoplasm kuya ku-mitochondrial matrix, okuholela ekufakweni kwe-oxidation kwama-fatty acids ukukhiqiza amandla 55. Ngokuvumelana nokutholakele kwethu, ucwaningo lwamuva nje luphinde lwabona amaphrofayili e-metabolome afanayo phakathi kwe-EGFR mutant kanye ne-EGFR wild-type tumors ngokuhlaziya i-metabolome yomhlaba wonke yamasampula ezicubu ze-adenocarcinoma angu-102. Ngokuthakazelisayo, okuqukethwe kwe-acylcarnitine nakho kwatholakala eqenjini le-EGFR mutant. Ngakho-ke, ukuthi izinguquko emazingeni e-acylcarnitine zibonisa izinguquko ze-metabolic ezibangelwa yi-EGFR kanye nezindlela zama-molecule ezingaphansi zingadinga ukufundwa okwengeziwe.
Ekuphetheni, ucwaningo lwethu lusungula i-serum metabolic classifier yokuxilongwa okuhlukile kwamaqhubu ephaphu futhi luphakamisa indlela yokusebenza engenza ngcono ukuhlolwa kwengozi futhi inikeze lula ukuphathwa kwemitholampilo ngokusekelwe ekuhlolweni kwe-CT scan.
Lolu cwaningo luvunyiwe yiKomidi Lokuziphatha leSibhedlela Somdlavuza saseSun Yat-sen University, iSibhedlela Sokuqala Esihlangene saseSun Yat-sen University, kanye neKomidi Lokuziphatha leSibhedlela Somdlavuza saseZhengzhou University. Emaqenjini okuthola nokuqinisekisa kwangaphakathi, ama-sera angu-174 kubantu abaphilile kanye nama-sera angu-244 avela kuma-nodule angenabungozi aqoqwe kubantu abahlolwa minyaka yonke eMnyango Wokulawula Nokuvimbela Umdlavuza, iSikhungo Somdlavuza saseSun Yat-sen University, kanye nama-nodule angenabungozi angu-166. I-serum. Ama-adenocarcinoma amaphaphu eSigaba I aqoqwe eSikhungweni Somdlavuza saseSun Yat-sen University. Eqenjini lokuqinisekisa langaphandle, kwakukhona amacala angu-48 ama-nodule angenabungozi, amacala angu-39 e-adenocarcinoma yamaphaphu esigaba I avela eSibhedlela Sokuqala Esihlangene saseSun Yat-sen University, kanye namacala angu-24 e-adenocarcinoma yamaphaphu esigaba I avela eSibhedlela Somdlavuza saseZhengzhou. I-Sun Yat-sen University Cancer Center iphinde yaqoqa amacala angu-16 omdlavuza wamaphaphu we-squamous cell wesigaba sokuqala ukuze kuhlolwe ikhono lokuxilonga le-metabolic classifier esungulwe (izici zesiguli ziboniswe kuThebula Elingeziwe 5). Amasampula avela eqenjini lokutholwa kanye neqembu lokuqinisekiswa kwangaphakathi aqoqwe phakathi kukaJanuwari 2018 noMeyi 2020. Amasampula eqembu lokuqinisekiswa kwangaphandle aqoqwe phakathi kuka-Agasti 2021 no-Okthoba 2022. Ukuze kuncishiswe ukucwaswa ngokobulili, cishe inani elilinganayo lamacala abesilisa nabesifazane labelwa eqenjini ngalinye. Ithimba Lokuthola kanye neQembu Lokubuyekeza Kwangaphakathi. Ubulili babahlanganyeli bunqunywe ngokusekelwe embikweni wabo. Imvume enolwazi yatholakala kubo bonke ababambiqhaza futhi akukho sinxephezelo esanikezwa. Abantu ababenamaqhubu angenabungozi kwakuyilabo ababene-CT scan score ezinzile eminyakeni engu-2 kuya kwemi-5 ngesikhathi sokuhlaziywa, ngaphandle kwecala elilodwa elivela kusampula yokuqinisekiswa kwangaphandle, elaqoqwa ngaphambi kokuhlinzwa futhi lahlolwa nge-histopathology. Ngaphandle kwe-bronchitis engapheli. Amacala e-lung adenocarcinoma aqoqwe ngaphambi kokususwa kwamaphaphu futhi aqinisekiswa ngokuxilongwa kwe-pathological. Amasampula egazi lokuzila ukudla aqoqwe ngamapayipi okuhlukanisa i-serum ngaphandle kwama-anticoagulant. Amasampula egazi aqina ihora eli-1 ekushiseni kwegumbi bese efakwa ku-centrifuge ku-2851 × g imizuzu eyi-10 ku-4°C ukuze kuqoqwe i-serum supernatant. Ama-aliquot e-serum aqandisiwe ku--80°C kuze kube yilapho kukhishwe i-metabolite. UMnyango Wokuvimbela Umdlavuza Nokuhlolwa Kwezokwelapha we-Sun Yat-sen University Cancer Center waqoqa ichibi le-serum kubanikeli abayi-100 abanempilo, okuhlanganisa nenani elilinganayo lamadoda nabesifazane abaneminyaka engu-40 kuya kwengu-55. Amavolumu alinganayo esampula ngayinye yabanikeli axutshwe, ichibi eliphumayo lahlanganiswa futhi lagcinwa ku--80°C. Ingxube ye-serum yasetshenziswa njengezinto zokubhekisela zokulawula ikhwalithi kanye nokulinganisa idatha.
Amasampula e-serum kanye nokuhlolwa okubhekiselwe kuwo ancibilikiswa futhi ama-metabolites akhishwa kusetshenziswa indlela yokukhipha ehlanganisiwe (MTBE/methanol/water) 56. Kafushane, ama-50 μl e-serum axutshwe nama-225 μl e-methanol ebandayo kanye nama-750 μl e-methyl tert-butyl ether (MTBE) ebandayo. Hlanganisa ingxube bese uyibeka eqhweni ihora eli-1. Amasampula abe esexutshwa bese i-vortex ixutshwa nama-188 μl amanzi e-MS-grade aqukethe amazinga angaphakathi (13C-lactate, 13C3-pyruvate, 13C-methionine, kanye ne-13C6-isoleucine, athengwe eCambridge Isotope Laboratories). Ingxube yabe isifakwa ku-centrifuge ku-15,000 × g imizuzu eyi-10 ku-4 °C, kanti isigaba esingezansi sadluliselwa kumashubhu amabili (125 μL ngalinye) ukuze kuhlaziywe i-LC-MS ngezindlela ezinhle nezimbi. Ekugcineni, isampula yashiswa yaba yomile ku-vacuum concentrator enesivinini esikhulu.
Ama-metabolite omisiwe aphinde ahlanganiswa ku-120 μl we-acetonitrile engu-80%, ahlushwa umoya imizuzu emi-5, futhi afakwa ku-centrifuge ku-15,000 × g imizuzu eyi-10 ku-4°C. Ama-Supernatant adluliselwe kuma-vial engilazi anombala ophuzi ane-microinserts yezifundo ze-metabolomics. Ukuhlaziywa kwe-metabolomics okungahlosiwe ku-platform ye-ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Ama-Metabolites ahlukaniswe kusetshenziswa uhlelo lwe-Dionex Ultimate 3000 UPLC kanye nekholomu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Waters). Kumodi ye-ion enhle, izigaba ezihambayo zaziyi-95% (A) kanye ne-50% acetonitrile (B), ngayinye iqukethe i-10 mmol/L ammonium acetate kanye ne-0.1% formic acid. Kumodi engalungile, izigaba eziphathwayo A kanye no-B zaziqukethe i-acetonitrile engu-95% kanye no-50%, ngokulandelana, zombili izigaba zaziqukethe i-10 mmol/L ammonium acetate, i-pH = 9. Uhlelo lwe-gradient lwalunjengolulandelayo: 0–0.5 min, 2% B; 0.5–12 min, 2–50% B; 12–14 min, 50–98% B; 14–16 min, 98% B; 16–16.1. min, 98–2% B; 16.1–20 min, 2% B. Ikholomu igcinwe ku-40°C kanye nesampula ku-10°C ku-autosampler. Izinga lokugeleza lalingu-0.3 ml/min, ivolumu yomjovo yayingu-3 μl. I-Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) enomthombo we-electrospray ionization (ESI) isetshenziswe kwimodi yokuskena egcwele futhi ihlanganiswe nemodi yokuqapha ye-ddMS2 ukuze kuqoqwe amanani amakhulu edatha. Amapharamitha e-MS abekwe kanje: i-spray voltage +3.8 kV/- 3.2 kV, izinga lokushisa le-capillary 320°C, igesi yokuvikela engu-40 arb, igesi elisizayo engu-10 arb, izinga lokushisa le-probe heater engu-350°C, ibanga lokuskena elingu-70–1050 m/h, isixazululo. 70 000. Idatha yatholakala kusetshenziswa i-Xcalibur 4.1 (Thermo Fisher Scientific).
Ukuze kuhlolwe ikhwalithi yedatha, amasampula okulawula ikhwalithi ahlanganisiwe (QC) akhiqizwe ngokususa ama-aliquot angu-10 μL e-supernatant kusampula ngayinye. Imijovo yesampula yokulawula ikhwalithi eyisithupha yahlaziywa ekuqaleni kochungechunge lokuhlaziya ukuze kuhlolwe ukuzinza kohlelo lwe-UPLC-MS. Amasampula okulawula ikhwalithi abe esefakwa njalo eqoqweni. Wonke amaqoqo angu-11 amasampula e-serum kulolu cwaningo ahlaziywa yi-LC-MS. Ama-aliquot engxube ye-serum pool evela kubanikeli abayi-100 abanempilo asetshenziswe njengezinto zokubhekisela kumaqoqo afanele ukuqapha inqubo yokukhipha nokulungisa imiphumela ye-batch-to-batch. Ukuhlaziywa kwe-metabolomics okungahlosiwe kweqembu lokutholwa, iqembu lokuqinisekisa langaphakathi, kanye neqembu lokuqinisekisa langaphandle kwenziwa eMetabolomics Center of Sun Yat-sen University. Ilabhorethri yangaphandle yeGuangdong University of Technology Analysis and Testing Center nayo ihlaziye amasampula angu-40 avela eqenjini langaphandle ukuze kuhlolwe ukusebenza kwemodeli yokuhlukanisa.
Ngemva kokukhipha nokwakha kabusha, ukulinganisa okuphelele kwama-metabolites e-serum kwalinganiswa kusetshenziswa i-ultra-high performance liquid chromatography-tandem mass spectrometry (i-Agilent 6495 triple quadrupole) enomthombo we-electrospray ionization (ESI) kumodi yokuqapha ukusabela okuningi (MRM). Ikholomu ye-ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Amanzi) yasetshenziswa ukuhlukanisa ama-metabolites. Isigaba esihambayo sasine-90% (A) kanye ne-5% acetonitrile (B) ene-10 mmol/L ammonium acetate kanye nesisombululo se-0.1% ammonia. Uhlelo lwe-gradient lwalunjengolulandelayo: 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 ku-40 °C kanye nesampula ku-10 °C ku-autosampler. Izinga lokugeleza lalingu-0.3 mL/min kanti ivolumu yomjovo yayingu-1 μL. Amapharamitha e-MS abekwe kanje: i-capillary voltage ±3.5 kV, ingcindezi ye-nebulizer engu-35 psi, ukugeleza kwegesi ye-sheath engu-12 L/min, izinga lokushisa legesi ye-sheath engu-350°C, izinga lokushisa legesi yokomisa elingu-250°C, kanye nokugeleza kwegesi yokomisa okungu-14 l/min. Ukuguqulwa kwe-MRM kwe-tryptophan, i-pyruvate, i-lactate, i-hypoxanthine kanye ne-xanthine kwakungama-205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 kanye no-151.0–107. 9 ngokulandelana. Idatha yaqoqwa kusetshenziswa i-Mass Hunter B.07.00 (Agilent Technologies). Kumasampula e-serum, i-tryptophan, i-pyruvate, i-lactate, i-hypoxanthine, ne-xanthine kwalinganiswa kusetshenziswa ama-calibration curves ezixazululo ezijwayelekile zengxube. Kumasampula eseli, okuqukethwe kwe-tryptophan kwalinganiswa kuya ezingeni langaphakathi kanye nesisindo seprotheyini yeseli.
Ukukhishwa kwe-peak (m/z kanye nesikhathi sokugcina (RT)) kwenziwa kusetshenziswa i-Compound Discovery 3.1 kanye ne-TraceFinder 4.0 (Thermo Fisher Scientific). Ukuze kuqedwe umehluko ongaba khona phakathi kwamaqoqo, i-peak ngayinye yesici sesampula yokuhlola yahlukaniswa yi-peak yesici sezinto zokubhekisela ezivela ku-batch efanayo ukuze kutholakale ubuningi obuhlobene. Ukuphambuka okujwayelekile okuhlobene kwezindinganiso zangaphakathi ngaphambi nangemva kokumiswa kuboniswe kuThebula Elingeziwe 6. Umehluko phakathi kwamaqembu amabili wabonakaliswa yizinga lokutholwa okungamanga (i-FDR<0.05, i-Wilcoxon signed rank test) kanye noshintsho lokugoqa (>1.2 noma <0.83). Idatha ye-MS eluhlaza yezici ezikhishwe kanye nedatha ye-MS elungisiwe nge-serum iboniswe ku-Supplementary Data 1 kanye ne-Supplementary Data 2, ngokulandelana. I-peak annotation yenziwe ngokusekelwe emazingeni amane achazwe okuhlonza, kufaka phakathi ama-metabolite ahlonziwe, ama-compound achazwe ngokusobala, amakilasi e-compound achazwe ngokusobala, kanye nama-compound angaziwa 22. Ngokusekelwe ekusesheni kwesizindalwazi ku-Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), ama-biological compounds anezindinganiso eziqinisekisiwe zokufanisa i-MS/MS noma ama-equite match annotations ku-mzCloud (amaphuzu > 85) noma i-Chemspider ekugcineni akhethwe njengabaxhumanisi phakathi kwe-differential metabolome. Ama-peak annotations esici ngasinye afakiwe ku-Supplementary Data 3. I-MetaboAnalyst 5.0 yasetshenziselwa ukuhlaziywa kwe-univariate kwe-sum-normalized metabolite largeness. I-MetaboAnalyst 5.0 iphinde yahlola ukuhlaziywa kwe-KEGG pathway enrichment ngokusekelwe kuma-metabolites ahlukene kakhulu. Ukuhlaziywa kwe-Principal component (PCA) kanye ne-partial least squares discriminant analysis (PLS-DA) kwahlaziywa kusetshenziswa i-ropls software package (v.1.26.4) nge-stack normalization kanye ne-autoscaling. Imodeli ye-biomarker ye-metabolite engcono kakhulu yokubikezela i-nodule mondancy yakhiqizwa kusetshenziswa i-binary logistic regression ene-least absolute shrinkage kanye ne-selection operator (LASSO, R package v.4.1-3). Ukusebenza kwemodeli yokuhlukanisa kumasethi okuthola nokuqinisekisa kwabonakala ngokulinganisa i-AUC ngokusekelwe ekuhlaziyweni kwe-ROC ngokuya ngephakheji ye-pROC (v.1.18.0.). Ukunqunywa kwamathuba okuhle kakhulu kutholakale ngokusekelwe kunkomba ephezulu ye-Youden yemodeli (ukuzwela + ukucaciswa - 1). Amasampula anamanani angaphansi noma amakhulu kunomkhawulo azobikezelwa njengama-nodule angenabungozi kanye ne-lung adenocarcinoma, ngokulandelana.
Amaseli e-A549 (#CCL-185, Iqoqo Lamasiko Ohlobo LwaseMelika) akhuliswe endaweni ephakathi ye-F-12K equkethe i-10% FBS. Uchungechunge lwe-RNA (shRNA) olufushane oluqondiswe ku-SLC7A5 kanye nokulawula okungaqondisi (NC) kwafakwa ku-vector ye-lentiviral pLKO.1-puro. Uchungechunge lwe-antisense lwe-shSLC7A5 lulandelayo: Sh1 (5′-GGAGAAACCTGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGGAACTAT-3′). Ama-antibodies ku-SLC7A5 (#5347) kanye ne-tubulin (#2148) athengwe ku-Cell Signaling Technology. Ama-antibodies ku-SLC7A5 kanye ne-tubulin asetshenziswe ekuxubeni okungu-1:1000 kokuhlaziywa kwe-Western blot.
Ukuhlolwa Kokucindezeleka Kwe-Seahorse XF Glycolytic kukalwa amazinga e-extracellular acidification (ECAR). Ekuhlolweni, i-glucose, i-oligomycin A, kanye ne-2-DG kwanikezwa ngokulandelana ukuze kuhlolwe umthamo we-glycolytic weseli njengoba kulinganiswa yi-ECAR.
Amaseli e-A549 adluliselwe nge-non-targeting control (NC) kanye ne-shSLC7A5 (Sh1, Sh2) afakwe ubusuku bonke ezitsheni ezinobubanzi obuyi-10 cm. Ama-metabolite eseli akhishwe nge-1 ml ye-methanol enamanzi ebanda kakhulu engu-80%. Amaseli asesixazululweni se-methanol akhuhlwa, aqoqwa afakwa epayipini elisha, futhi afakwa ku-centrifuge ku-15,000 × g imizuzu eyi-15 ku-4°C. Qoqa u-800 µl we-supernatant bese womisa kusetshenziswa i-vacuum concentrator enesivinini esikhulu. Ama-pellet e-metabolite omisiwe abe esehlaziywa amazinga e-tryptophan kusetshenziswa i-LC-MS/MS njengoba kuchaziwe ngenhla. Amazinga e-NAD(H) eseli kumaseli e-A549 (NC kanye ne-shSLC7A5) alinganiswa kusetshenziswa ikhithi ye-NAD+/NADH colorimetric (#K337, BioVision) ngokwemiyalelo yomenzi. Amazinga e-protein alinganiswa ngesampula ngayinye ukuze kulungiswe inani lama-metabolite.
Azikho izindlela zezibalo ezisetshenzisiwe ukunquma usayizi wesampula kusengaphambili. Izifundo zangaphambilini ze-metabolomics ezihloselwe ukutholakala kwe-biomarker15,18 zibhekwe njengezilinganiso zokunquma usayizi, futhi uma kuqhathaniswa nale mibiko, isampula yethu yayanele. Azikho izibonelo ezikhishwe eqenjini locwaningo. Amasampula e-serum anikezwa ngokungahleliwe eqenjini lokutholwa (amacala angu-306, 74.6%) kanye neqembu lokuqinisekiswa kwangaphakathi (amacala angu-104, 25.4%) ezifundweni ze-metabolomics ezingahlosiwe. Siphinde sakhetha ngokungahleliwe amacala angu-70 eqenjini ngalinye kusukela kusethi yokutholwa yezifundo ze-metabolomics ezihlosiwe. Abaphenyi baphuphuthekiswa ekunikezelweni kweqembu ngesikhathi sokuqoqwa nokuhlaziywa kwedatha ye-LC-MS. Ukuhlaziywa kwezibalo kwedatha ye-metabolomics kanye nokuhlolwa kweseli kuchazwa ezigabeni ze-Results, Figure Legends, kanye ne-Methods. Ukulinganiswa kwe-tryptophan yeselula, i-NADT, kanye nomsebenzi we-glycolytic kwenziwa kathathu ngokuzimela ngemiphumela efanayo.
Ukuze uthole ulwazi olwengeziwe mayelana nomklamo wocwaningo, bheka i-Natural Portfolio Report Abstract ehlobene nalesi sihloko.
Idatha ye-MS eluhlaza yezici ezikhishwe kanye nedatha ye-MS ejwayelekile ye-serum yokubhekisela iboniswa ku-Supplementary Data 1 kanye ne-Supplementary Data 2, ngokulandelana. Izichasiselo eziphezulu zezici ezihlukile zethulwe ku-Supplementary Data 3. Isethi yedatha ye-LUAD TCGA ingalandwa ku-https://portal.gdc.cancer.gov/. Idatha yokufaka yokudweba igrafu inikezwa kudatha yomthombo. Idatha yomthombo inikezwe kulesi sihloko.
Iqembu Likazwelonke Lokuhlola Amaphaphu, njll. Ukunciphisa ukufa komdlavuza wamaphaphu nge-computed tomography ephansi. INyakatho yeNgilandi. J. Med. 365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR kanye noMprofethi, PC Ukuhlolwa komdlavuza wamaphaphu kusetshenziswa i-CT esezingeni eliphansi: imiphumela evela ku-National Lung Screening Study (NLST). J. Med. Screen 18, 109–111 (2011).
UDe Koning, HJ, nabanye. Ukunciphisa ukufa komdlavuza wamaphaphu ngokuhlolwa kwe-CT ngobuningi esivivinyweni esingahleliwe. INyakatho yeNgilandi. J. Med. 382, 503–513 (2020).
Isikhathi sokuthunyelwe: Septhemba 18-2023