Kuzindikira kosiyana kwa ma pulmonary nodules odziwika ndi computed tomography (CT) kumakhalabe kovuta m'zachipatala.Apa, tikuwonetsa metabolome yapadziko lonse ya zitsanzo za seramu 480, kuphatikiza zowongolera zathanzi, tinthu tating'onoting'ono ta m'mapapo, ndi gawo I lung adenocarcinoma.Adenocarcinomas amawonetsa mawonekedwe apadera a metabolomic, pomwe tinthu tating'onoting'ono komanso anthu athanzi amafanana kwambiri ndi mbiri ya metabolomic.Mu gulu lotulukira (n = 306), seti ya 27 metabolites idazindikirika kuti isiyanitse ma nodule owopsa ndi oyipa.AUC yachitsanzo chatsankho pakutsimikizira kwamkati (n = 104) ndi kutsimikizika kwakunja (n = 111) magulu anali 0.915 ndi 0.945, motsatana.Kuwunika kwa njira kunawonetsa kuchuluka kwa glycolytic metabolites komwe kumalumikizidwa ndi kuchepa kwa tryptophan m'mapapo adenocarcinoma seramu poyerekeza ndi tinthu tating'onoting'ono komanso kuwongolera kwathanzi, ndipo adati kutenga tryptophan kumalimbikitsa glycolysis m'maselo a khansa ya m'mapapo.Kafukufuku wathu akuwunikira kufunika kwa serum metabolite biomarkers powunika kuopsa kwa ma nodule am'mapapo omwe amapezeka ndi CT.
Kuzindikira koyambirira ndikofunikira kuti pakhale moyo wabwino kwa odwala khansa.Zotsatira zochokera ku US National Lung Cancer Screening Trial (NLST) ndi European NELSON Study zasonyeza kuti kuyang'ana ndi mlingo wochepa wa computed tomography (LDCT) kungachepetse kwambiri imfa ya khansa ya m'mapapo m'magulu omwe ali pachiopsezo chachikulu1,2,3.Popeza kufala kwa LDCT poyezetsa khansa ya m'mapapo, kuchuluka kwa zochitika zodziwikiratu za radiographic za asymptomatic pulmonary nodules zapitilira kuwonjezeka 4.Mitsempha ya m'mapapo imatchedwa focal opacities mpaka 3 cm m'mimba mwake 5 .Timakumana ndi zovuta poyesa kuthekera kwa zilonda zam'mimba komanso kuthana ndi kuchuluka kwa ma pulmonary nodule omwe amapezeka mwangozi pa LDCT.Kulephera kwa CT kungayambitse kuyesedwa kotsatiridwa pafupipafupi ndi zotsatira zabodza, zomwe zimatsogolera kulowererapo kosafunikira komanso kuchiritsa mopitirira muyeso6.Choncho, pakufunika kupanga zizindikiro zodalirika komanso zothandiza kuti zizindikire khansa ya m'mapapo kumayambiriro koyambirira ndikusiyanitsa ma nodule ambiri abwino pozindikira koyamba 7.
Kusanthula kwathunthu kwamagazi (seramu, plasma, zotumphukira zamagazi a mononuclear cell), kuphatikiza ma genomics, proteomics kapena DNA methylation8,9,10, kwadzetsa chidwi chokulirapo pakupezeka kwa zozindikiritsa za khansa ya m'mapapo.Pakadali pano, njira zama metabolomics zimayezera zinthu zomwe zimatengera ma cell omwe amakhudzidwa ndi zochitika zakunja komanso zakunja ndipo amagwiritsidwa ntchito kulosera kuyambika kwa matenda ndi zotsatira zake.Liquid chromatography-tandem mass spectrometry (LC-MS) ndi njira yogwiritsidwa ntchito kwambiri pa maphunziro a metabolomics chifukwa cha kukhudzidwa kwake kwakukulu komanso kusinthasintha kwakukulu, komwe kungathe kuphimba ma metabolites okhala ndi physicochemical properties11,12,13.Ngakhale kusanthula kwa metabolomic kwa plasma / seramu kwagwiritsidwa ntchito pozindikira zizindikiro zokhudzana ndi matenda a khansa ya m'mapapo14,15,16,17 komanso chithandizo chamankhwala, 18 serum metabolite classifiers kusiyanitsa pakati pa ma nodule owopsa ndi oyipa a m'mapapo amakhalabe kuti aphunzire zambiri.-kufufuza kwakukulu.
Adenocarcinoma ndi squamous cell carcinoma ndi mitundu iwiri ikuluikulu ya khansa ya m'mapapo yosakhala yaying'ono (NSCLC).Mayesero osiyanasiyana a CT amawonetsa kuti adenocarcinoma ndi mtundu wodziwika kwambiri wa khansa ya m'mapapo1,19,20,21.Mu phunziroli, tidagwiritsa ntchito ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) kuti tifufuze metabolomics pa zitsanzo zonse za 695 za seramu, kuphatikizapo kulamulira kwathanzi, ma pulmonary nodules, ndi CT-yodziwika ≤3 cm.Kuwunika kwa Stage I lung adenocarcinoma.Tidazindikira gulu la seramu metabolites lomwe limasiyanitsa mapapu adenocarcinoma ndi ma nodule owopsa komanso zowongolera zathanzi.Kusanthula kwapang'onopang'ono kwa njira kunawonetsa kuti tryptophan ndi glucose metabolism ndikusintha kofala m'mapapo adenocarcinoma poyerekeza ndi ma nodule owopsa komanso kuwongolera kwathanzi.Pomaliza, tidakhazikitsa ndikutsimikizira kagayidwe ka seramu kagayidwe kazakudya kamene kamakhala kodziwika bwino komanso kumveka bwino kuti tisiyanitse pakati pa zilonda zam'mapapo zam'mimba zopezeka ndi LDCT, zomwe zingathandize kuzindikira koyambirira komanso kuwunika kowopsa.
Pakafukufuku wapano, zitsanzo za seramu zakugonana ndi zaka zofananira zidasonkhanitsidwa motsatira kuchokera ku maulamuliro athanzi a 174, odwala 292 okhala ndi tinthu tating'onoting'ono ta m'mapapo, ndi odwala 229 omwe ali ndi siteji ya I lung adenocarcinoma.Makhalidwe a chiwerengero cha anthu 695 akuwonetsedwa mu Supplementary Table 1.
Monga momwe chithunzi 1a, okwana 480 zitsanzo seramu, kuphatikizapo 174 kulamulira wathanzi (HC), 170 chosaopsa tinatake tozungulira (BN), ndi 136 siteji I m'mapapo adenocarcinoma (LA) zitsanzo, anasonkhanitsidwa pa Sun Yat-sen University Cancer Center.Gulu la Discovery la mbiri yosadziwika ya metabolomic pogwiritsa ntchito ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Monga momwe tawonetsera mu Supplementary Figure 1, ma metabolites osiyana pakati pa LA ndi HC, LA ndi BN adadziwika kuti akhazikitse chitsanzo chamagulu ndikufufuzanso kusanthula njira zosiyana.Zitsanzo za 104 zomwe zinasonkhanitsidwa ndi Sun Yat-sen University Cancer Center ndi zitsanzo za 111 zomwe zinasonkhanitsidwa ndi zipatala zina ziwiri zinatsimikiziridwa mkati ndi kunja, motsatira.
Chiwerengero cha anthu ochita kafukufuku mgulu la otulukira omwe adasanthula ma serum metabolomics padziko lonse lapansi pogwiritsa ntchito ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).b Kusanthula kwapang'ono kocheperako (PLS-DA) kwa metabolome yonse ya 480 seramu zitsanzo kuchokera ku gulu la kafukufuku, kuphatikiza zowongolera zathanzi (HC, n = 174), tinthu tating'onoting'ono (BN, n = 170), ndi gawo I lung adenocarcinoma (Los Angeles, n = 136).+ ESI, njira yabwino yopangira ionization ya electrospray, -ESI, mawonekedwe a ionization a electrospray.c-e Ma metabolites okhala ndi kuchuluka kosiyana kwambiri m'magulu awiri opatsidwa (mayeso amtundu wa Wilcoxon wokhala ndi michira iwiri, kuchuluka kwabodza komwe adasinthidwa p value, FDR <0.05) akuwonetsedwa ofiira (kusintha pindani> 1.2) ndi buluu (kusintha pindani <0.83) .) zowonetsedwa pazithunzi za phirili.f Mapu a kutentha kwa magulu osiyanasiyana omwe amasonyeza kusiyana kwakukulu kwa chiwerengero cha ma metabolites otchulidwa pakati pa LA ndi BN.Dongosolo lachidziwitso limaperekedwa ngati mafayilo oyambira.
Seramu yonse ya metabolome ya 174 HC, 170 BN ndi 136 LA mu gulu lotulukira idawunikidwa pogwiritsa ntchito kusanthula kwa UPLC-HRMS.Choyamba timasonyeza kuti zitsanzo za khalidwe labwino (QC) zimagwirizanitsa mwamphamvu pakatikati pa chitsanzo chosayang'aniridwa ndi principal component analysis (PCA), kutsimikizira kukhazikika kwa momwe phunziroli likuyendera (Supplementary Figure 2).
Monga momwe zasonyezedwera mu magawo ang'onoang'ono-kusankha (PLS-DA) mu Chithunzi 1 b, tapeza kuti panali kusiyana koonekeratu pakati pa LA ndi BN, LA ndi HC mu njira zabwino (+ESI) ndi zoipa (−ESI) electrospray ionization modes. .akutali.Komabe, palibe kusiyana kwakukulu komwe kunapezeka pakati pa BN ndi HC mu +ESI ndi -ESI mikhalidwe.
Tinapeza zinthu 382 zosiyana pakati pa LA ndi HC, 231 kusiyana pakati pa LA ndi BN, ndi 95 kusiyana pakati pa BN ndi HC (Wilcoxon saina udindo mayeso, FDR <0.05 ndi angapo kusintha> 1.2 kapena <0.83) (Figure .1c-e) )..Misozi inafotokozedwanso (Supplementary Data 3) motsutsana ndi database (mzCloud/HMDB/Chemspider library) ndi mtengo wa m/z, nthawi yosungira ndi kufufuza kwamitundu yosiyanasiyana (zambiri zofotokozedwa mu gawo la Njira) 22.Potsirizira pake, 33 ndi 38 otchulidwa metabolites ndi kusiyana kwakukulu kwa kuchuluka kwachuluka kunadziwika kwa LA motsutsana ndi BN (Chithunzi 1f ndi Supplementary Table 2) ndi LA motsutsana ndi HC (Supplementary Figure 3 ndi Supplementary Table 2), motero.Mosiyana ndi izi, ma metabolites a 3 okha omwe ali ndi kusiyana kwakukulu kwa kuchuluka kwake adadziwika mu BN ndi HC (Supplementary Table 2), mogwirizana ndi kuphatikizika pakati pa BN ndi HC mu PLS-DA.Ma metabolites osiyanitsa awa amaphimba mitundu yambiri yamankhwala achilengedwe (Supplementary Figure 4).Kuphatikizidwa pamodzi, zotsatirazi zikuwonetsa kusintha kwakukulu mu seramu metabolome komwe kumawonetsa kusintha koyipa kwa khansa ya m'mapapo yoyambilira poyerekeza ndi tinthu tating'onoting'ono ta m'mapapo kapena anthu athanzi.Pakadali pano, kufanana kwa seramu metabolome ya BN ndi HC kukuwonetsa kuti tinthu tating'onoting'ono ta m'mapapo mwanga titha kugawana zambiri zamoyo ndi anthu athanzi.Popeza kuti kusintha kwa majini a epidermal growth factor receptor (EGFR) kumakhala kofala mu lung adenocarcinoma subtype 23, tinayesetsa kudziwa momwe kusintha kwa madalaivala pa seramu metabolome.Kenako tidasanthula mbiri yonse ya metabolomic ya milandu 72 yokhala ndi EGFR mu gulu la lung adenocarcinoma.Chochititsa chidwi n'chakuti, tinapeza mbiri zofanana pakati pa odwala EGFR mutant (n = 41) ndi EGFR odwala amtundu wamtundu (n = 31) mu kusanthula kwa PCA (Supplementary Figure 5a).Komabe, tinazindikira ma metabolites a 7 omwe kuchuluka kwawo kunasinthidwa kwambiri kwa odwala omwe ali ndi kusintha kwa EGFR poyerekeza ndi odwala omwe ali ndi EGFR zakutchire (t test, p <0.05 ndi fold change> 1.2 kapena <0.83) (Supplementary Figure 5b).Ambiri mwa metabolites (5 mwa 7) ndi ma acylcarnitines, omwe amagwira ntchito yofunika kwambiri panjira zamakutidwe ndi okosijeni wamafuta.
Monga momwe zikuwonetsedwera mu Chithunzi 2 a, ma biomarker a magulu a nodule adapezedwa pogwiritsa ntchito ochepera ocheperako komanso osankhidwa motengera ma metabolites 33 odziwika mu LA (n = 136) ndi BN (n = 170).Kuphatikizika kwabwino kwamitundu yosiyanasiyana (LASSO) - chitsanzo cha binary logistic regression model.Kutsimikizika kwa magawo khumi kunagwiritsidwa ntchito kuyesa kudalirika kwa chitsanzocho.Kusankha kosinthika ndi kukhazikika kwa magawo kumasinthidwa ndi chiwongola dzanja chowonjezereka ndi chizindikiro λ24.Kusanthula kwa metabolomics kwapadziko lonse kunachitidwanso mopanda kutsimikiziridwa kwamkati (n = 104) ndi kutsimikizira kwakunja (n = 111) magulu kuti ayese ntchito yamagulu amtundu wa tsankho.Zotsatira zake, ma metabolites a 27 muzofukufukuwo adadziwika kuti ndi njira yabwino kwambiri yotsatsirana ndi chiwerengero chachikulu cha AUC (mkuyu. 2b), pakati pawo 9 anali ndi ntchito yowonjezereka ndipo 18 inachepa ntchito ku LA poyerekeza ndi BN (mkuyu 2c).
Mayendedwe a ntchito pomanga gulu la pulmonary nodule classifier, kuphatikiza kusankha gulu labwino kwambiri la seramu metabolites muzopezeka pogwiritsa ntchito njira ya binary Logistic regression moduli khumi ndikuwunika magwiridwe antchito olosera m'maseti ovomerezeka amkati ndi akunja.b Ziwerengero zotsimikizika zamtundu wa LASSO regression model pakusankha kwa metabolic biomarker.Manambala omwe aperekedwa pamwambapa akuyimira avereji ya ma biomarkers osankhidwa pa λ.Mzere wamadontho ofiyira umayimira pafupifupi mtengo wa AUC pa lambda yofananira.Zolemba zolakwika zotuwa zimayimira zochepa komanso zochulukirapo za AUC.Mzere wamadontho ukuwonetsa mtundu wabwino kwambiri wokhala ndi ma biomarkers 27 osankhidwa.AUC, dera lomwe lili pansi pa curve ya wolandila (ROC).c Pindani zosintha za 27 zosankhidwa za metabolites mu gulu la LA poyerekeza ndi gulu la BN mu gulu lotulukira.Mzere wofiira - kuyambitsa.Mzere wa buluu ndi kuchepa.d-f Receiver operating character (ROC) yokhotakhota yosonyeza mphamvu ya chitsanzo cha tsankho kutengera kuphatikizika kwa metabolite 27 pakupeza, mkati, ndi kunja kovomerezeka.Dongosolo lachidziwitso limaperekedwa ngati mafayilo oyambira.
Chitsanzo cholosera chinapangidwa kutengera ma coefficients olemetsa a metabolites 27 awa (Table Supplementary 3).Kusanthula kwa ROC kutengera ma metabolites awa a 27 kunapereka malo omwe ali pansi pa curve (AUC) mtengo wa 0.933, kuzindikira kwa gulu lodziwika kunali 0.868, ndipo kutsimikizika kunali 0.859 (mkuyu 2d).Pakadali pano, pakati pa ma metabolites 38 odziwika bwino pakati pa LA ndi HC, ma metabolites 16 adapeza AUC ya 0.902 ndi chidwi cha 0.801 komanso kutsimikizika kwa 0.856 pakusankha LA kuchokera ku HC (Chithunzi chowonjezera 6a-c).Makhalidwe a AUC kutengera magawo osiyanasiyana akusintha kwa ma metabolites osiyanasiyana adafananizidwanso.Tidapeza kuti mtundu wamaguluwo udachita bwino kwambiri pakusankha pakati pa LA ndi BN (HC) pomwe mulingo wosintha umayikidwa kukhala 1.2 motsutsana ndi 1.5 kapena 2.0 (Supplementary Figure 7a,b).Chitsanzo chamagulu, chochokera kumagulu a metabolite a 27, chinatsimikiziridwanso m'magulu amkati ndi kunja.AUC inali 0.915 (sensitivity 0.867, specificity 0.811) kuti itsimikizidwe mkati ndi 0.945 (sensitivity 0.810, specificity 0.979) kuti itsimikizidwe kunja (mkuyu 2e, f).Kuti muwone bwino ntchito ya interlaboratory, zitsanzo za 40 kuchokera ku gulu lakunja zidawunikidwa mu labotale yakunja monga momwe tafotokozera mu gawo la Njira.Kulondola kwamagulu kunapeza AUC ya 0.925 (Chithunzi Chowonjezera 8).Chifukwa lung squamous cell carcinoma (LUSC) ndiye mtundu wachiwiri wodziwika bwino wa khansa ya m'mapapo yopanda maselo ang'onoang'ono (NSCLC) pambuyo pa lung adenocarcinoma (LUAD), tidayesanso kugwiritsa ntchito kovomerezeka kwa mbiri ya metabolic.BN ndi milandu 16 ya LUSC.AUC ya tsankho pakati pa LUSC ndi BN inali 0.776 (Chithunzi Chowonjezera 9), kuwonetsa kuthekera kosauka poyerekeza ndi tsankho pakati pa LUAD ndi BN.
Kafukufuku wasonyeza kuti kukula kwa tinthu tating'onoting'ono pazithunzi za CT kumagwirizana bwino ndi mwayi wowopsa ndipo kumakhalabe chidziwitso chachikulu cha chithandizo cha nodule25,26,27.Kufufuza kwa deta kuchokera ku gulu lalikulu la kafukufuku wowunika wa NELSON kunasonyeza kuti chiopsezo cha matenda opweteka m'magulu omwe ali ndi node <5 mm chinali chofanana ndi cha maphunziro opanda node 28.Choncho, kukula kochepa komwe kumafunikira kuyang'anitsitsa nthawi zonse kwa CT ndi 5 mm, monga momwe akulangizidwa ndi British Thoracic Society (BTS), ndi 6 mm, monga momwe Fleischner Society 29 inalimbikitsa.Komabe, tinthu tating'onoting'ono tokulirapo kuposa 6 mm komanso wopanda mawonekedwe owoneka bwino, otchedwa indeterminate pulmonary nodules (IPN), amakhalabe vuto lalikulu pakuwunika ndi kuyang'anira muzochita zachipatala30,31.Kenako tidawona ngati kukula kwa nodule kudakhudza siginecha ya metabolomic pogwiritsa ntchito zitsanzo zophatikizidwa kuchokera kumagulu opezeka ndi ovomerezeka amkati.Poyang'ana ma biomarkers ovomerezeka a 27, tidayamba kuyerekeza mbiri ya PCA ya HC ndi BN sub-6 mm metabolomes.Tinapeza kuti mfundo zambiri za deta za HC ndi BN zinadutsana, kusonyeza kuti seramu metabolite milingo anali ofanana m'magulu onsewa (mkuyu 3a).Mapu a mapu pamagulu osiyanasiyana a kukula adasungidwa mu BN ndi LA (mkuyu 3b, c), pamene kupatukana kunkawoneka pakati pa tizilombo toyambitsa matenda ndi tizilombo toyambitsa matenda mu 6-20 mm (mkuyu 3d).Gulu ili linali ndi AUC ya 0.927, yodziwika bwino ya 0.868, ndi kukhudzika kwa 0.820 podziwiratu kuipa kwa tinthu tating'onoting'ono toyeza 6 mpaka 20 mm (mkuyu 3e, f).Zotsatira zathu zikuwonetsa kuti wowerengera amatha kujambula kusintha kwa metabolic komwe kumachitika chifukwa cha kusintha koyipa koyambirira, mosasamala kanthu za kukula kwa nodule.
ad Kuyerekeza kwa mbiri ya PCA pakati pamagulu odziwika kutengera gulu la metabolic la 27 metabolites.CC ndi BN <6 mm.BN <6 mm vs BN 6-20 mm.mu LA 6-20 mm motsutsana ndi LA 20-30 mm.g BN 6-20 mm ndi LA 6-20 mm.GC, n = 174;BN <6 mm, n = 153;BN 6-20 mm, n = 91;LA 6-20 mm, n = 89;LA 20-30 mm, n = 77. e Receiver opareting character (ROC) curve yowonetsa machitidwe atsankho a nodule 6-20 mm.f Miyezo yotheka idawerengedwa kutengera mtundu wosinthika wa ma nodule a 6-20 mm.Mzere wa madontho otuwa umayimira mtengo wokwanira wodulira (0.455).Manambala omwe ali pamwambapa akuyimira kuchuluka kwa milandu yomwe ikuyembekezeredwa ku Los Angeles.Gwiritsani ntchito mayeso a t a michira iwiri.PCA, chigawo chachikulu kusanthula.Malo a AUC pansi pa kupindika.Dongosolo lachidziwitso limaperekedwa ngati mafayilo oyambira.
Zitsanzo zinayi (zaka 44-61 zaka) zokhala ndi kukula kwa pulmonary nodule (7-9 mm) zinasankhidwanso kuti ziwonetsere momwe machitidwe amaneneratu zoipa (mkuyu 4a, b).Pakuwunika koyambirira, Mlandu Woyamba udawonetsedwa ngati tinthu tating'onoting'ono tomwe timawerengera, zomwe zimalumikizidwa ndi kufatsa, pomwe Mlandu Wachiwiri udawonetsedwa ngati tinthu tating'ono tomwe timakhazikika popanda zowoneka bwino.Maulendo atatu otsatizana a CT scans adawonetsa kuti milanduyi inakhalabe yokhazikika pazaka za 4 ndipo motero amaonedwa ngati ma benign nodules (mkuyu 4a).Poyerekeza ndi kuwunika kwachipatala kwa ma serial CT scans, kusanthula kwa metabolite ya seramu imodzi ndi mtundu waposachedwa wa classifier mwachangu komanso molondola kuzindikiritsa tinthu tating'onoting'ono tomwe timakhala ndi zovuta zomwe zingachitike (Table 1).Chithunzi 4b ngati 3 ikuwonetsa nodule yokhala ndi zizindikiro za kubwereza kwa pleural, zomwe nthawi zambiri zimagwirizanitsidwa ndi malignancy32.Mlandu 4 woperekedwa ngati nodule yolimba pang'ono yopanda umboni wa chifukwa chabwino.Milandu yonseyi idanenedweratu kuti ndi yoyipa molingana ndi mtundu wamagulu (Table 1).Kuwunika kwa mapapu adenocarcinoma kunasonyezedwa ndi kufufuza kwa histopathological pambuyo pa opaleshoni yochotsa mapapo (mkuyu 4b).Pazovomerezeka zakunja, wowerengera kagayidwe kachakudya adaneneratu molondola milandu iwiri ya mapapu osakhazikika akulu kuposa 6 mm (Chithunzi Chowonjezera 10).
Zithunzi za CT za zenera la axial m'mapapo amilandu iwiri ya nodule za benign.Ngati 1, CT scan pambuyo pa zaka 4 imasonyeza khola lolimba lolimba la 7 mm ndi calcification mu lobe yapansi yoyenera.Ngati 2, CT scan pambuyo pa zaka 5 idavumbulutsa chokhazikika chokhazikika, cholimba pang'ono chokhala ndi mainchesi 7 kumtunda wakumanja.b Axial zenera CT zithunzi za mapapo ndi lolingana pathological maphunziro a milandu iwiri ya siteji I adenocarcinoma pamaso resection mapapo.Mlandu wa 3 udavumbulutsa tinthu tating'onoting'ono tomwe timakhala ndi mainchesi 8 kumtunda wakumanja komwe kumakhala ndi pleural retraction.Mlandu 4 udavumbulutsa tinthu tating'ono tagalasi tolimba pang'ono tokhala ndi mamilimita 9 kumanzere chakumanzere.Kudetsa kwa hematoxylin ndi eosin (H&E) kwa minyewa ya m'mapapo yopangidwanso (mulingo = 50 μm) kuwonetsa mawonekedwe akukula kwa lung adenocarcinoma.Mivi imawonetsa ma nodule omwe apezeka pazithunzi za CT.Zithunzi za H&E ndi zithunzi zoyimira zamagulu angapo (> 3) osawoneka bwino omwe amawunikiridwa ndi katswiri wamankhwala.
Kuphatikizidwa, zotsatira zathu zikuwonetsa kufunikira kwa ma seramu metabolite biomarkers pakuzindikiritsa kosiyana kwa ma pulmonary nodule, omwe angayambitse zovuta pakuwunika kuwunika kwa CT.
Kutengera ndi gulu lovomerezeka la metabolite, tidafuna kudziwa zolumikizana ndi biological zosintha zazikulu za metabolic.KEGG njira yolemeretsa kusanthula ndi MetaboAnalyst adazindikira njira za 6 zomwe zimasinthidwa kwambiri pakati pa magulu awiriwa (LA vs. HC ndi LA vs. BN, kusinthidwa p ≤ 0.001, zotsatira> 0.01).Kusintha kumeneku kunkadziwika ndi kusokonezeka kwa pyruvate metabolism, tryptophan metabolism, niacin ndi nicotinamide metabolism, glycolysis, TCA cycle, ndi purine metabolism (Mkuyu 5a).Kenako tidachitanso ma metabolomics kuti titsimikizire kusintha kwakukulu pogwiritsa ntchito kuchuluka kwathunthu.Kutsimikiza kwa metabolites wamba m'njira zomwe zimasinthidwa pafupipafupi ndi triple quadrupole mass spectrometry (QQQ) pogwiritsa ntchito miyezo yeniyeni ya metabolite.Makhalidwe a chiwerengero cha zitsanzo za kafukufuku wa metabolomics akuphatikizidwa mu Supplementary Table 4. Mogwirizana ndi zotsatira zathu zapadziko lonse za metabolomics, kusanthula kwachulukidwe kunatsimikizira kuti hypoxanthine ndi xanthine, pyruvate, ndi lactate zinawonjezeka mu LA poyerekeza ndi BN ndi HC (Mkuyu 5b, c, pa <0.05).Komabe, palibe kusiyana kwakukulu mu metabolites awa komwe kunapezeka pakati pa BN ndi HC.
KEGG njira yolemeretsa kusanthula kwa metabolites yosiyana kwambiri mu gulu la LA poyerekeza ndi magulu a BN ndi HC.Globaltest yokhala ndi michira iwiri idagwiritsidwa ntchito, ndipo ma p adasinthidwa pogwiritsa ntchito njira ya Holm-Bonferroni (yosinthidwa p ≤ 0.001 ndi kukula kwake> 0.01).b-d Violin ziwembu zosonyeza hypoxanthine, xanthine, lactate, pyruvate, ndi milingo ya tryptophan mu seramu HC, BN, ndi LA yotsimikiziridwa ndi LC-MS/MS (n = 70 pagulu).Mizere yoyera ndi yakuda ya madontho imasonyeza pakati ndi quartile, motsatira.e Chiwembu cha violin chowonetsa Log2TPM yokhazikika (zolemba pa miliyoni) mawu a mRNA a SLC7A5 ndi QPRT mu lung adenocarcinoma (n = 513) poyerekeza ndi minofu yamba ya m'mapapo (n = 59) mu dataset ya LUAD-TCGA.Bokosi loyera limayimira mtundu wa interquartile, mzere wakuda wopingasa pakati ukuyimira wapakatikati, ndipo mzere wakuda woyimirira wotuluka m'bokosi ukuyimira 95% confidence interval (CI).f Pearson correlation plot of SLC7A5 and GAPDH expression in lung adenocarcinoma (n = 513) ndi mapapu abwinobwino (n = 59) mu dataset ya TCGA.Malo otuwa akuyimira 95% CI.r, Pearson corelation coefficient.g Miyezo yamtundu wa tryptophan yama cell m'maselo a A549 osinthidwa ndi shRNA control (NC) ndi shSLC7A5 (Sh1, Sh2) yotsimikiziridwa ndi LC-MS/MS.Kusanthula kwachiwerengero kwa zitsanzo zisanu zodziyimira pawokha pagulu lililonse zimaperekedwa.h Ma cell a NADt (chiwerengero cha NAD, kuphatikiza NAD+ ndi NADH) m'maselo a A549 (NC) ndi SLC7A5 kugwetsa ma cell a A549 (Sh1, Sh2).Kusanthula kwachiwerengero kwa zitsanzo zitatu zodziyimira pawokha pagulu lililonse zimaperekedwa.i Glycolytic ntchito ya ma cell a A549 isanachitike komanso itatha SLC7A5 kugwetsa idayesedwa ndi extracellular acidification rate (ECAR) (n = 4 zitsanzo zodziyimira pawokha pagulu).2-DG, 2-deoxy-D-glucose.Mayeso a Wophunzira a michira iwiri adagwiritsidwa ntchito mu (b–h).Mu (g-i), mipiringidzo yolakwika imayimira tanthauzo la ± SD, kuyesa kulikonse kunachitika katatu paokha ndipo zotsatira zake zinali zofanana.Dongosolo lachidziwitso limaperekedwa ngati mafayilo oyambira.
Poganizira za kukhudzidwa kwakukulu kwa kusintha kwa tryptophan metabolism mu gulu la LA, tidawunikanso milingo ya seramu tryptophan m'magulu a HC, BN, ndi LA pogwiritsa ntchito QQQ.Tidapeza kuti seramu tryptophan idachepetsedwa ku LA poyerekeza ndi HC kapena BN (p <0.001, Chithunzi 5d), zomwe zikugwirizana ndi zomwe zapezeka m'mbuyomu kuti kuchuluka kwa tryptophan komwe kumazungulira kumakhala kotsika kwa odwala omwe ali ndi khansa ya m'mapapo kuposa momwe amawongolera athanzi kuchokera ku gulu lolamulira33,34 , 35 .Kafukufuku wina wogwiritsa ntchito PET/CT tracer 11C-methyl-L-tryptophan adapeza kuti nthawi yosungira chizindikiro cha tryptophan m'minofu ya khansa ya m'mapapo idakwera kwambiri poyerekeza ndi zotupa za benign kapena minofu yabwinobwino36.Timalingalira kuti kuchepa kwa tryptophan mu LA seramu kumatha kuwonetsa kutengeka kwa tryptophan ndi maselo a khansa ya m'mapapo.
Amadziwikanso kuti chomaliza cha njira ya kynurenine ya tryptophan catabolism ndi NAD+37,38, yomwe ndi gawo lofunikira pakuchita kwa glyceraldehyde-3-phosphate ndi 1,3-bisphosphoglycerate mu glycolysis39.Ngakhale maphunziro am'mbuyomu adayang'ana kwambiri gawo la tryptophan catabolism pakuwongolera chitetezo chamthupi, tidafuna kufotokozera kuyanjana pakati pa tryptophan dysregulation ndi njira za glycolytic zomwe zawonedwa mu kafukufuku wapano.Banja la Solute transporter 7 membala 5 (SLC7A5) amadziwika kuti ndi tryptophan transporter43,44,45.Quinolinic acid phosphoribosyltransferase (QPRT) ndi puloteni yomwe ili kumunsi kwa njira ya kynurenine yomwe imasintha quinolinic acid kukhala NAMN46.Kuyang'ana kwa deta ya LUAD TCGA kunawonetsa kuti SLC7A5 ndi QPRT zinali zoyendetsedwa kwambiri mu minofu ya chotupa poyerekeza ndi minofu yachibadwa (Fig. 5e).Kuwonjezeka kumeneku kunawonedwa mu magawo I ndi II komanso magawo III ndi IV a lung adenocarcinoma (Supplementary Figure 11), kusonyeza kusokonezeka koyambirira kwa tryptophan metabolism yokhudzana ndi tumorigenesis.
Kuphatikiza apo, deta ya LUAD-TCGA idawonetsa kulumikizana kwabwino pakati pa SLC7A5 ndi GAPDH mRNA mawu mu zitsanzo za odwala khansa (r = 0.45, p = 1.55E-26, Chithunzi 5f).Mosiyana ndi izi, palibe kulumikizana kwakukulu komwe kunapezeka pakati pa ma siginecha amtundu wotere m'mapapo abwinobwino (r = 0.25, p = 0.06, Chithunzi 5f).Kugunda kwa SLC7A5 (Supplementary Figure 12) m'maselo a A549 kunachepetsa kwambiri ma tryptophan a cell ndi NAD (H) (Chithunzi 5g, h), zomwe zimapangitsa kuti ntchito ya glycolytic ikhale yochepetsetsa monga momwe imayesedwera ndi extracellular acidification rate (ECAR) (Chithunzi 1).5 ndi).Chifukwa chake, kutengera kusintha kwa metabolic mu seramu ndi kuzindikira kwa vitro, timaganiza kuti tryptophan metabolism imatha kupanga NAD + kudzera munjira ya kynurenine ndikuchita gawo lofunikira polimbikitsa glycolysis mu khansa ya m'mapapo.
Kafukufuku wasonyeza kuti chiwerengero chachikulu cha mitsempha ya m'mapapo yosadziwika bwino yomwe imapezeka ndi LDCT ingapangitse kuti pakhale kufunikira koyesa kowonjezereka monga PET-CT, mapapu a m'mapapo, ndi kuwonjezereka chifukwa cha matenda osokoneza bongo.31 Monga momwe tawonetsera pa Chithunzi 6. kafukufuku wathu adapeza gulu la metabolites la seramu lomwe lingakhale ndi phindu lodziwikiratu lomwe lingapangitse kuwongolera kwachiwopsezo komanso kuyang'anira kotsatira kwa ma pulmonary nodules omwe amapezeka ndi CT.
Mitsempha ya m'mapapo imawunikidwa pogwiritsa ntchito mlingo wochepa wa computed tomography (LDCT) wokhala ndi zithunzi zosonyeza zomwe zimayambitsa zabwino kapena zoipa.Zotsatira zosadziwika za nodule zingayambitse maulendo obwereza mobwerezabwereza, kulowerera kosafunikira, ndi kuchiritsidwa mopitirira muyeso.Kuphatikizika kwa magulu a serum metabolic omwe ali ndi mtengo wowunikira kungapangitse kuwunika kwachiwopsezo ndikuwongolera kotsatira kwa ma nodule a pulmonary.PET positron emission tomography.
Zambiri kuchokera ku kafukufuku wa US NLST ndi kafukufuku waku Europe wa NELSON zikuwonetsa kuti kuyang'ana magulu omwe ali pachiwopsezo chachikulu ndi mlingo wochepa wa computed tomography (LDCT) kungachepetse kufa kwa khansa ya m'mapapo1,3.Komabe, kuwunika kwachiwopsezo komanso kuyang'anira kwakanthawi kotsatira zachipatala kwa ma nodule ambiri am'mapapo omwe amapezeka ndi LDCT ndizovuta kwambiri.Cholinga chachikulu ndikukwaniritsa magawo olondola a ma protocol omwe alipo a LDCT pophatikiza ma biomarker odalirika.
Ma biomarker ena a mamolekyulu, monga ma metabolites amagazi, adadziwika poyerekeza khansa ya m'mapapo ndi zowongolera zathanzi15,17.Mu kafukufuku wapano, tidayang'ana pakugwiritsa ntchito kusanthula kwa serum metabolomics kuti tisiyanitse pakati pa ma nodule owopsa komanso oyipa omwe adapezeka mwangozi ndi LDCT.Tidafanizira metabolome ya seramu yapadziko lonse yaumoyo wabwino (HC), ma benign lung nodules (BN), ndi magawo a I lung adenocarcinoma (LA) pogwiritsa ntchito kusanthula kwa UPLC-HRMS.Tidapeza kuti HC ndi BN zinali ndi mbiri yofananira ya metabolic, pomwe LA idawonetsa kusintha kwakukulu poyerekeza ndi HC ndi BN.Tidazindikira magawo awiri a seramu metabolites omwe amasiyanitsa LA kuchokera ku HC ndi BN.
Dongosolo lapano la LDCT lozindikiritsa ma nodule owopsa komanso oyipa makamaka amadalira kukula, kachulukidwe, kapangidwe kazinthu komanso kukula kwa tinthu tating'onoting'ono pakapita nthawi30.Kafukufuku wam'mbuyomu adawonetsa kuti kukula kwa tinthu tating'onoting'ono timagwirizana kwambiri ndi kuthekera kwa khansa ya m'mapapo.Ngakhale odwala omwe ali pachiwopsezo chachikulu, chiwopsezo cha zilonda zam'mimba mu node <6 mm ndi <1%.Chiwopsezo cha zilonda zam'mimba zolemera 6 mpaka 20 mm zimachokera ku 8% mpaka 64% 30.Chifukwa chake, Fleischner Society imalimbikitsa kudulidwa kwa mita ya 6 mm pakutsata kwanthawi zonse kwa CT.29 Komabe, kufufuza ndi kuyang'anira chiopsezo cha indeterminate pulmonary nodules (IPN) zazikulu kuposa 6 mm sizinachitike mokwanira 31.Ulamuliro wamakono wa matenda a mtima wobadwa nawo nthawi zambiri umachokera ku kuyembekezera mwachidwi ndi kuyang'anira pafupipafupi kwa CT.
Kutengera ndi metabolome yovomerezeka, tidawonetsa kwa nthawi yoyamba kuphatikizika kwa siginecha ya metabolomic pakati pa anthu athanzi ndi ma nodule ochepera <6 mm.Kufanana kwachilengedwe kumagwirizana ndi zomwe CT yapeza kale kuti chiopsezo cha zilonda zam'mimba <6 mm ndi chochepa ngati maphunziro opanda node.30 Tiyenera kuzindikira kuti zotsatira zathu zimasonyezanso kuti ma nodules a benign <6 mm ndi ≥6 mm ali ndipamwamba. kufanana mu mbiri ya metabolomic, kutanthauza kuti kutanthauzira kogwira ntchito kwa benign etiology ndikofanana mosasamala kanthu za kukula kwa nodule.Chifukwa chake, mapanelo amakono a seramu metabolite atha kupereka mayeso amodzi ngati mayeso oyeserera pamene tinthu tating'onoting'ono tapezeka pa CT ndipo zitha kuchepetsa kuwunika kwa serial.Panthawi imodzimodziyo, gulu lomwelo la zizindikiro za kagayidwe kachakudya limasiyanitsa tinthu tating'onoting'ono towopsa ≥6 mm kukula kwake kuchokera ku tinthu tating'onoting'ono tating'onoting'ono ndipo anapereka maulosi olondola a IPN a kukula kofanana ndi mawonekedwe osadziwika bwino a morphological pazithunzi za CT.Kagulu ka seramu kagayidwe kachakudya kameneka kanachita bwino poneneratu kuipa kwa tinthu tating'onoting'ono ≥6 mm ndi AUC ya 0.927.Kutengera pamodzi, zotsatira zathu zikuwonetsa kuti ma signature apadera a seramu metabolomic amatha kuwonetsa kusintha kwa kagayidwe kachakudya koyambilira koyambitsa chotupa ndipo kungakhale kothandiza ngati zolosera zachiwopsezo, osadalira kukula kwa nodule.
Makamaka, lung adenocarcinoma (LUAD) ndi squamous cell carcinoma (LUSC) ndi mitundu ikuluikulu ya khansa yosakhala yaying'ono ya m'mapapo (NSCLC).Poganizira kuti LUSC imalumikizidwa kwambiri ndi kugwiritsa ntchito fodya47 ndipo LUAD ndiye mbiri yodziwika bwino ya tinthu tating'ono ta m'mapapo tapezeka pa CT screening48, mtundu wathu wa classifier udapangidwira zitsanzo za siteji yoyamba ya adenocarcinoma.Wang ndi anzawo adayang'ananso pa LUAD ndipo adazindikira siginecha zisanu ndi zinayi za lipid pogwiritsa ntchito lipidomics kusiyanitsa khansa ya m'mapapo yoyambirira ndi anthu athanzi17.Tidayesa mtundu waposachedwa pamilandu 16 ya siteji yoyamba ya LUSC ndi ma nodule 74 ndikuwona zolosera zochepa za LUSC (AUC 0.776), ndikuwonetsa kuti LUAD ndi LUSC zitha kukhala ndi siginecha yawo ya metabolomic.Zowonadi, LUAD ndi LUSC zawonetsedwa kuti zimasiyana mu etiology, biological origin and genetic aberrations49.Chifukwa chake, mitundu ina ya histology iyenera kuphatikizidwa m'machitidwe ophunzitsira kuti azindikire khansa ya m'mapapo potengera kuchuluka kwa anthu pamapulogalamu owunika.
Apa, tidazindikira njira zisanu ndi imodzi zomwe zimasinthidwa pafupipafupi m'mapapo adenocarcinoma poyerekeza ndi zowongolera zathanzi komanso ma nodule owopsa.Xanthine ndi hypoxanthine ndi metabolites wamba wa purine metabolic pathway.Mogwirizana ndi zotsatira zathu, zapakati zomwe zimagwirizanitsidwa ndi purine metabolism zinawonjezeka kwambiri mu seramu kapena minofu ya odwala omwe ali ndi mapapu adenocarcinoma poyerekeza ndi maulamuliro athanzi kapena odwala pa preinvasive stage15,50.Kukwera kwa seramu xanthine ndi hypoxanthine kumatha kuwonetsa anabolism yofunikira pakuchulukirachulukira kwa ma cell a khansa.Kusokoneza kagayidwe ka shuga ndi chizindikiro chodziwika bwino cha kagayidwe kazakudya51.Apa, tawona kuwonjezeka kwakukulu kwa pyruvate ndi lactate mu gulu la LA poyerekeza ndi gulu la HC ndi BN, zomwe zimagwirizana ndi malipoti am'mbuyomu a glycolytic pathway abnormalities mu serum metabolome profiles of non-small cell lung cancer (NSCLC) ndi amazilamulira wathanzi.zotsatira zake ndi zofanana52,53.
Chofunika kwambiri, tidawona kulumikizana kosiyana pakati pa pyruvate ndi tryptophan metabolism mu seramu ya lung adenocarcinomas.Miyezo ya tryptophan ya seramu idachepetsedwa mu gulu la LA poyerekeza ndi gulu la HC kapena BN.Chochititsa chidwi n'chakuti, kafukufuku wamkulu wam'mbuyomo pogwiritsa ntchito gulu loyembekezera adapeza kuti kuchepa kwa tryptophan yozungulira kunagwirizanitsidwa ndi chiopsezo chowonjezeka cha khansa ya m'mapapo 54.Tryptophan ndi amino acid yofunikira yomwe timapeza kuchokera ku chakudya.Titha kunena kuti kuchepa kwa seramu tryptophan m'mapapo adenocarcinoma kumatha kuwonetsa kuchepa kwa metabolite iyi mwachangu.Ndizodziwika bwino kuti chomaliza cha tryptophan catabolism kudzera munjira ya kynurenine ndiye gwero la de novo NAD + synthesis.Chifukwa NAD + imapangidwa makamaka kudzera mu njira yopulumutsira, kufunikira kwa NAD + mu tryptophan metabolism muumoyo ndi matenda sikudziwika46.Kusanthula kwathu kwa nkhokwe ya TCGA kunawonetsa kuti mawu a tryptophan transporter solute transporter 7A5 (SLC7A5) adachulukirachulukira m'mapapo adenocarcinoma poyerekeza ndi zowongolera wamba ndipo adalumikizidwa bwino ndi mawu a glycolytic enzyme GAPDH.Kafukufuku wam'mbuyomu adayang'ana kwambiri gawo la tryptophan catabolism popondereza chitetezo cha mthupi cha antitumor40,41,42.Apa tikuwonetsa kuti kuletsa kwa tryptophan kutengedwa mwa kugwetsa kwa SLC7A5 m'maselo a khansa ya m'mapapo kumabweretsa kutsika kwapang'onopang'ono kwa ma cell a NAD komanso kuchepetsedwa kofanana kwa ntchito ya glycolytic.Mwachidule, phunziro lathu limapereka maziko achilengedwe a kusintha kwa seramu metabolism yokhudzana ndi kusintha koyipa kwa lung adenocarcinoma.
Zosintha za EGFR ndizofala kwambiri kwa odwala omwe ali ndi NSCLC.Mu kafukufuku wathu, tidapeza kuti odwala omwe ali ndi kusintha kwa EGFR (n = 41) anali ndi mbiri yonse ya metabolomic yofanana ndi odwala omwe ali ndi mtundu wa EGFR (n = 31), ngakhale kuti tinapeza kuchepa kwa seramu ya odwala ena a EGFR mutant odwala acylcarnitine.Ntchito yokhazikitsidwa ya acylcarnitines ndiyo kunyamula magulu a acyl kuchokera ku cytoplasm kupita ku matrix a mitochondrial, zomwe zimatsogolera ku okosijeni wa mafuta acids kuti apange mphamvu 55.Mogwirizana ndi zomwe tapeza, kafukufuku waposachedwa adapezanso mbiri yofananira ya metabolome pakati pa zotupa zamtundu wa EGFR ndi EGFR zakuthengo posanthula metabolome yapadziko lonse ya 102 lung adenocarcinoma tissue samples50.Chochititsa chidwi n'chakuti, zinthu za acylcarnitine zinapezekanso mu gulu la EGFR mutant.Chifukwa chake, ngati kusintha kwa ma acylcarnitine kumawonetsa kusintha kwa kagayidwe kachakudya ka EGFR komanso njira zoyambira zamamolekyu zitha kuyenera kuphunziridwanso.
Pomaliza, kafukufuku wathu amakhazikitsa gulu la seramu la metabolic kuti athe kusiyanitsa ma nodule am'mapapo ndikupereka njira yoyendetsera ntchito yomwe ingathe kuwongolera kuwunika kwachiwopsezo ndikuwongolera kasamalidwe kachipatala potengera kuwunika kwa CT scan.
Kafukufukuyu adavomerezedwa ndi Ethics Committee ya Sun Yat-sen University Cancer Hospital, First Affiliated Hospital of Sun Yat-sen University, and Ethics Committee of Zhengzhou University Cancer Hospital.M'magulu opezeka ndi ovomerezeka amkati, ma sera 174 ochokera kwa anthu athanzi ndi ma sera 244 ochokera m'mafupa abwino adasonkhanitsidwa kuchokera kwa anthu omwe amayesedwa pachaka ku dipatimenti ya Cancer Control and Prevention, Sun Yat-sen University Cancer Center, ndi ma nodule 166.seramu.Stage I lung adenocarcinomas anatengedwa kuchokera ku Sun Yat-sen University Cancer Center.M'gulu lovomerezeka lakunja, panali milandu 48 ya tinthu tating'onoting'ono toyipa, 39 milandu ya siteji I lung adenocarcinoma kuchokera ku First Affiliated Hospital ya Sun Yat-sen University, ndi milandu 24 ya stage I lung adenocarcinoma kuchokera ku Zhengzhou Cancer Hospital.Sun Yat-sen University Cancer Center inasonkhanitsanso milandu 16 ya khansa ya m'mapapo ya squamous cell ya squamous kuti ayese luso la kuzindikira kagayidwe ka metabolic (makhalidwe a odwala akuwonetsedwa mu Supplementary Table 5).Zitsanzo zochokera kugulu la zodziwikiratu komanso zotsimikizira zamkati zidasonkhanitsidwa pakati pa Januware 2018 ndi Meyi 2020. Zitsanzo za gulu lovomerezeka lakunja zidasonkhanitsidwa pakati pa Ogasiti 2021 ndi Okutobala 2022. Kuti achepetse kukondera, pafupifupi chiwerengero chofanana cha milandu ya amuna ndi akazi idaperekedwa gulu.Gulu la Discovery and Internal Review Team.Jenda wotenga nawo mbali adatsimikiziridwa potengera lipoti layekha.Chilolezo chodziwitsidwa chinapezedwa kwa onse omwe adatenga nawo gawo ndipo palibe chipukuta misozi chomwe chinaperekedwa.Mitu yokhala ndi ma benign nodules inali yomwe ili ndi chiwerengero chokhazikika cha CT scan pa 2 kwa zaka 5 panthawi yofufuza, kupatulapo mlandu wa 1 kuchokera ku chitsanzo chovomerezeka chakunja, chomwe chinasonkhanitsidwa chisanachitike ndikuzindikiridwa ndi histopathology.Kupatulapo matenda a bronchitis.Matenda a m'mapapo adenocarcinoma adasonkhanitsidwa asanatuluke m'mapapo ndikutsimikiziridwa ndi matenda a pathological.Zitsanzo zamagazi osala kudya zidasonkhanitsidwa m'machubu olekanitsa seramu popanda anticoagulants.Zitsanzo za magazi anali clotted kwa ola 1 kutentha firiji ndiyeno centrifuged pa 2851 × g kwa mphindi 10 pa 4 ° C kusonkhanitsa seramu supernatant.Seramu aliquots anali atazizira pa -80 ° C mpaka metabolite m'zigawo.Dipatimenti Yoteteza Khansa ndi Kufufuza Zamankhwala ku Sun Yat-sen University Cancer Center inasonkhanitsa dziwe la seramu kuchokera kwa opereka 100 athanzi, kuphatikizapo chiwerengero chofanana cha amuna ndi akazi a zaka 40 mpaka 55.Ma voliyumu ofanana a zitsanzo za opereka adasakanizidwa, dziwe lomwe lidatsatiridwalo lidatengedwa ndikusungidwa pa -80 ° C.Kusakaniza kwa seramu kumagwiritsidwa ntchito ngati zowunikira pakuwongolera zabwino komanso kukhazikika kwa data.
Seramu zowunikira ndi zitsanzo zoyesa zidasungunuka ndipo metabolites adachotsedwa pogwiritsa ntchito njira yophatikizira (MTBE / methanol / madzi) 56.Mwachidule, 50 μl ya seramu idasakanizidwa ndi 225 μl ya methanol yozizira kwambiri ndi 750 μl ya ice-cold methyl tert-butyl ether (MTBE).Sakanizani osakaniza ndikuumirira pa ayezi kwa ola limodzi.Zitsanzozo zinasakanizidwa ndi vortex kusakaniza ndi 188 μl ya madzi a MS-grade omwe ali ndi miyezo yamkati (13C-lactate, 13C3-pyruvate, 13C-methionine, ndi 13C6-isoleucine, yogulidwa ku Cambridge Isotope Laboratories).Chosakanizacho chinali centrifuged pa 15,000 × g kwa 10 min pa 4 ° C, ndipo gawo lapansi linasamutsidwa mu machubu awiri (125 μL iliyonse) kwa LC-MS kusanthula mu njira zabwino ndi zoipa.Pomaliza, chitsanzocho chinasinthidwa kukhala nthunzi kuti chiume mu chotengera chothamanga kwambiri cha vacuum.
Ma metabolites owuma adapangidwanso mu 120 μl ya 80% acetonitrile, vortexed kwa 5 min, ndi centrifuged pa 15,000 × g kwa 10 min pa 4 ° C.Ma supernatants adasamutsidwa mu mbale zagalasi za amber zokhala ndi ma microinsets ophunzirira za metabolomics.Kusanthula kwa metabolomics kopanda cholinga pa nsanja ya Ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS).Ma metabolites adalekanitsidwa pogwiritsa ntchito dongosolo la Dionex Ultimate 3000 UPLC ndi gawo la ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Madzi).Mu njira yabwino ya ion, magawo a mafoni anali 95% (A) ndi 50% acetonitrile (B), iliyonse ili ndi 10 mmol / L ammonium acetate ndi 0.1% formic acid.Mumayendedwe oipa, magawo a mafoni A ndi B anali ndi 95% ndi 50% acetonitrile, motero, magawo onsewa anali ndi 10 mmol / L ammonium acetate, pH = 9. Pulogalamu ya gradient inali motere: 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. Chigawocho chinasungidwa pa 40 ° C ndi chitsanzo pa 10 ° C mu autosampler.Kuthamanga kunali 0.3 ml / min, voliyumu ya jekeseni inali 3 μl.Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) yokhala ndi gwero la electrospray ionization (ESI) idagwiritsidwa ntchito ndi scan yathunthu ndikuphatikizidwa ndi ddMS2 yowunikira kuti asonkhanitse kuchuluka kwa data.Magawo a MS adayikidwa motere: magetsi opopera + 3.8 kV/- 3.2 kV, kutentha kwa capillary 320 ° C, kutchingira mpweya 40 arb, gasi wothandizira 10 arb, kutentha kwa heater 350 ° C, kupanga sikani 70-1050 m / h, kuthetsa.70 000. Deta inapezedwa pogwiritsa ntchito Xcalibur 4.1 (Thermo Fisher Scientific).
Kuti muwunikire mtundu wa data, zitsanzo zowongolera bwino (QC) zidapangidwa pochotsa ma 10 μL amphamvu kwambiri pazatsanzo zilizonse.Majekeseni asanu ndi limodzi owongolera khalidwe adawunikidwa koyambirira kwa kuwunika kowunika kuti awone kukhazikika kwa dongosolo la UPLC-MS.Zitsanzo zowongolera zabwino zimaperekedwa nthawi ndi nthawi mu batch.Magulu onse 11 a zitsanzo za seramu mu kafukufukuyu adawunikidwa ndi LC-MS.Ma Aliquots a seramu pool osakaniza kuchokera kwa opereka athanzi 100 adagwiritsidwa ntchito ngati zolozera m'magulu osiyanasiyana kuti aziyang'anira kachulukidwe kake ndikusintha zotsatira za batch-to-batch.Kusanthula kwa metabolomics kosagwirizana ndi gulu lotulukira, gulu lovomerezeka lamkati, ndi gulu lovomerezeka lakunja kunachitika ku Metabolomics Center ya Sun Yat-sen University.Laboratory yakunja ya Guangdong University of Technology Analysis and Testing Center idasanthulanso zitsanzo 40 kuchokera ku gulu lakunja kuti liyese ntchito ya mtundu wamagulu.
Pambuyo pochotsa ndi kukonzanso, kuchuluka kwathunthu kwa metabolites ya seramu kunayesedwa pogwiritsa ntchito ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) yokhala ndi electrospray ionization (ESI) gwero mu multiple reaction monitoring (MRM) mode.Chigawo cha ACQUITY BEH Amide (2.1 × 100 mm, 1.7 μm, Madzi) chinagwiritsidwa ntchito kulekanitsa metabolites.Gawo la mafoni linali 90% (A) ndi 5% acetonitrile (B) ndi 10 mmol / L ammonium acetate ndi 0.1% ammonia solution.Pulogalamu ya gradient inali motere: 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.Mzerewu unasungidwa pa 40 ° C ndi chitsanzo pa 10 ° C mu autosampler.Kuthamanga kunali 0.3 mL / min ndipo voliyumu ya jekeseni inali 1 μL.Magawo a MS adayikidwa motere: capillary voltage ± 3.5 kV, nebulizer pressure 35 psi, mpweya wotuluka m'chimake 12 L / min, kutentha kwa gasi wa m'chimake 350 ° C, kutentha kwa gasi 250 ° C, ndi kuyanika gasi kutuluka kwa 14 l / min.Matembenuzidwe a MRM a tryptophan, pyruvate, lactate, hypoxanthine ndi xanthine anali 205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 ndi 151.0–107.9 motsatana.Deta inasonkhanitsidwa pogwiritsa ntchito Mass Hunter B.07.00 (Agilent Technologies).Pamiyeso ya seramu, tryptophan, pyruvate, lactate, hypoxanthine, ndi xanthine adayesedwa pogwiritsa ntchito ma curve oyeserera a njira zosakanikirana zosakanikirana.Kwa zitsanzo zama cell, zomwe zili ndi tryptophan zidasinthidwa kukhala muyezo wamkati ndi mapuloteni a cell.
Kuchotsa pachimake (m/z ndi nthawi yosungira (RT)) kunachitika pogwiritsa ntchito Compound Discovery 3.1 ndi TraceFinder 4.0 (Thermo Fisher Scientific).Kuti athetse kusiyana komwe kungakhalepo pakati pa magulu, chiwongola dzanja chilichonse chazoyeserera chinagawidwa ndi chiwongola dzanja chazomwe zili mumgulu womwewo kuti tipeze kuchuluka kwake.Kusiyanitsa kwapakati pamiyezo yamkati isanayambe komanso itatha kukhazikitsidwa ikuwonetsedwa mu Supplementary Table 6. Kusiyana pakati pa magulu awiriwa kumadziwika ndi chiwerengero chabodza (FDR <0.05, Wilcoxon chizindikiro chosaina) ndi kusintha kosintha (> 1.2 kapena <0.83).Deta ya Raw ya MS ya zinthu zochotsedwa ndi data yowongolera seramu ya MS ikuwonetsedwa mu Supplementary Data 1 ndi Supplementary Data 2, motsatana.Kufotokozera kwapamwamba kunachitidwa potengera milingo inayi yodziwika bwino, kuphatikiza ma metabolites odziwika, ma putatively annotated compounds, putatively characterized compound class, ndi mankhwala osadziwika 22.Kutengera kusaka kwa database mu Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), ma biological compounds okhala ndi MS/MS ofananira ndi miyezo yovomerezeka kapena matchulidwe enieni a mzCloud (score> 85) kapena Chemspider pomaliza adasankhidwa kukhala opatsirana pakati pa metabolome yosiyana.Ndemanga zapamwamba za gawo lililonse zikuphatikizidwa mu Supplementary Data 3. MetaboAnalyst 5.0 idagwiritsidwa ntchito powunika mosasintha kuchuluka kwa metabolite yokhazikika.MetaboAnalyst 5.0 idawunikanso kusanthula kwa njira za KEGG kutengera ma metabolites osiyanasiyana.Principal component analysis (PCA) ndi partial least squares discriminant analysis (PLS-DA) adawunikidwa pogwiritsa ntchito pulogalamu ya ropls (v.1.26.4) yokhala ndi stack normalization ndi autoscaling.The mulingo woyenera kwambiri metabolite biomarker chitsanzo kulosera zilonda za nodule anapangidwa pogwiritsa ntchito bayinare logistic regression ndi osachepera mtheradi shrinkage ndi kusankha woyendetsa (LASSO, R phukusi v.4.1-3).Kuchita kwachitsanzo chatsankho pazidziwitso ndi kutsimikizira zidadziwika ndi kuyerekezera AUC kutengera kusanthula kwa ROC malinga ndi phukusi la pROC (v.1.18.0.).Kuthekera kocheperako kunapezedwa kutengera index yayikulu ya Youden yachitsanzo (sensitivity + specificity - 1).Zitsanzo zokhala ndi zotsika kapena zokulirapo kuposa zomwe zikuyembekezeka zidzanenedweratu ngati tinthu tating'onoting'ono komanso mapapu adenocarcinoma, motsatana.
Maselo a A549 (#CCL-185, American Type Culture Collection) anakulira mu F-12K sing'anga yomwe ili ndi 10% FBS.Njira zazifupi za RNA (shRNA) zotsatizana ndi SLC7A5 ndi nontargeting control (NC) zidayikidwa mu lentiviral vector pLKO.1-puro.Zotsatira za antisense za shSLC7A5 ndi izi: Sh1 (5'-GGAGAAACCTGATGAACAGTT-3′), Sh2 (5'-GCCGTGGACTTCGGGAACTAT-3′).Ma antibodies kupita ku SLC7A5 (#5347) ndi tubulin (#2148) adagulidwa kuchokera ku Cell Signaling Technology.Ma antibodies kupita ku SLC7A5 ndi tubulin adagwiritsidwa ntchito pa dilution ya 1: 1000 pakuwunika kwa blot yaku Western.
Mayeso a Seahorse XF Glycolytic Stress Test amayesa milingo ya extracellular acidification (ECAR).Mu kuyesa, shuga, oligomycin A, ndi 2-DG adayendetsedwa motsatizana kuyesa mphamvu ya glycolytic yama cell monga momwe amayezera ndi ECAR.
Maselo a A549 omwe adasinthidwa ndi osayang'anira (NC) ndi shSLC7A5 (Sh1, Sh2) adayikidwa usiku wonse mu mbale za 10 cm.Ma cell metabolites adatulutsidwa ndi 1 ml ya ayezi-ozizira 80% amadzimadzi methanol.Maselo a methanol solution adachotsedwa, ndikusonkhanitsidwa mu chubu chatsopano, ndikuyikidwa pakati pa 15,000 × g kwa mphindi 15 pa 4 ° C.Sungani 800 µl zamphamvu kwambiri ndi zowuma pogwiritsa ntchito cholumikizira chothamanga kwambiri.Ma pellets owuma a metabolite adawunikidwa pa milingo ya tryptophan pogwiritsa ntchito LC-MS/MS monga tafotokozera pamwambapa.Maselo a NAD(H) m'maselo a A549 (NC ndi shSLC7A5) adayesedwa pogwiritsa ntchito zida zamtundu wa NAD+/NADH (#K337, BioVision) molingana ndi malangizo a wopanga.Mapuloteni amayesedwa pa chitsanzo chilichonse kuti asinthe kuchuluka kwa metabolites.
Palibe njira zowerengera zomwe zidagwiritsidwa ntchito kuti zitsimikizire kukula kwachitsanzo.Maphunziro am'mbuyomu a metabolomics okhudzana ndi kupezeka kwa biomarker15,18 adawonedwa ngati ma benchmarks kuti adziwe kukula, ndipo poyerekeza ndi malipoti awa, zitsanzo zathu zinali zokwanira.Palibe zitsanzo zomwe zidachotsedwa ku gulu la maphunziro.Zitsanzo za seramu zinaperekedwa mwachisawawa ku gulu lotulukira (milandu ya 306, 74.6%) ndi gulu lovomerezeka lamkati (milandu ya 104, 25.4%) ya maphunziro osadziwika a metabolomics.Tidasankhanso mwachisawawa milandu 70 kuchokera kugulu lililonse kuchokera pazopezeka zomwe zakhazikitsidwa pamaphunziro omwe amayang'aniridwa ndi metabolomics.Ofufuzawo adachititsidwa khungu kuti asagwiritse ntchito gulu panthawi yosonkhanitsa ndi kusanthula deta ya LC-MS.Kusanthula kwachiwerengero cha data ya metabolomics ndi kuyesa kwa ma cell kumafotokozedwa mugawo la Zotsatira, Nthano za Zithunzi, ndi Njira.Kuchulukitsa kwa tryptophan yama cell, NADT, ndi glycolytic ntchito kunachitika katatu paokha ndi zotsatira zofanana.
Kuti mumve zambiri za kapangidwe ka kafukufukuyu, onani Natural Portfolio Report Abstract yokhudzana ndi nkhaniyi.
Deta yaiwisi ya MS yazinthu zochotsedwa ndi data yanthawi zonse ya MS ya seramu yolozera ikuwonetsedwa mu Supplementary Data 1 ndi Supplementary Data 2, motsatana.Ndemanga zapamwamba zamitundu yosiyanasiyana zikuperekedwa mu Supplementary Data 3. Zolemba za LUAD TCGA zitha kutsitsidwa kuchokera ku https://portal.gdc.cancer.gov/.Deta yolowera pokonza ma graph imaperekedwa mu data source.Zomwe zachokera m'nkhaniyi zaperekedwa.
National Lung Screening Study Group, etc. Kuchepetsa kufa kwa khansa ya m'mapapo ndi mlingo wochepa wa computed tomography.Northern England.J. Med.365, 395-409 (2011).
Kramer, BS, Berg, KD, Aberle, DR ndi Prophet, PC Kuyeza khansa ya m'mapapo pogwiritsa ntchito mlingo wochepa wa helical CT: zotsatira za National Lung Screening Study (NLST).J. Med.Screen 18, 109-111 (2011).
De Koning, HJ, et al.Kuchepetsa kufa kwa khansa ya m'mapapo ndi kuwunika kwa volumetric CT pakuyesa kosasintha.Northern England.J. Med.382, 503–513 (2020).
Nthawi yotumiza: Sep-18-2023