Kuzindikira mitundu yosiyanasiyana ya ma nodules a m'mapapo omwe amadziwika ndi computed tomography (CT) kukupitirira kukhala kovuta kwambiri pakuchita zachipatala. Pano, tikuwonetsa metabolome yapadziko lonse ya zitsanzo 480 za seramu, kuphatikizapo zowongolera zathanzi, ma nodules a m'mapapo osapweteka, ndi adenocarcinoma ya m'mapapo ya gawo loyamba. Ma adenocarcinomas ali ndi mawonekedwe apadera a metabolomic, pomwe ma nodules osavulaza ndi anthu athanzi ali ndi kufanana kwakukulu mu ma profiles a metabolomic. Mu gulu lopeza (n = 306), gulu la ma metabolites 27 linazindikirika kuti lisiyanitse pakati pa ma nodules osavulaza ndi owopsa. AUC ya chitsanzo chosiyanitsa mu kutsimikizira kwamkati (n = 104) ndi kutsimikizira kwakunja (n = 111) magulu anali 0.915 ndi 0.945, motsatana. Kusanthula kwa njira kunawonetsa kuchuluka kwa ma metabolites a glycolytic okhudzana ndi kuchepa kwa tryptophan mu seramu ya adenocarcinoma ya m'mapapo poyerekeza ndi ma nodules osavulaza ndi zowongolera zathanzi, ndipo adawonetsa kuti kutengedwa kwa tryptophan kumalimbikitsa glycolysis m'maselo a khansa ya m'mapapo. Kafukufuku wathu akuwonetsa kufunika kwa zizindikiro za metabolite m'magazi poyesa chiopsezo cha timibulu ta m'mapapo tomwe timazindikirika ndi CT.
Kuzindikira msanga ndikofunikira kwambiri kuti odwala khansa apulumuke. Zotsatira za US National Lung Cancer Screening Trial (NLST) ndi European NELSON Study zasonyeza kuti kuyezetsa pogwiritsa ntchito computed tomography (LDCT) yotsika mlingo kungachepetse kwambiri imfa za khansa ya m'mapapo m'magulu omwe ali pachiwopsezo chachikulu1,2,3. Kuyambira pamene LDCT yagwiritsidwa ntchito kwambiri poyezetsa khansa ya m'mapapo, kuchuluka kwa zizindikiro za radiographic za nodules za m'mapapo zomwe sizili ndi zizindikiro kwapitirira kuwonjezeka 4. Nodules za m'mapapo zimatanthauzidwa ngati focal opacities mpaka 3 cm m'mimba mwake 5. Tikukumana ndi zovuta poyesa kuthekera kwa khansa komanso kuthana ndi kuchuluka kwa nodules za m'mapapo zomwe zapezeka mwangozi pa LDCT. Zofooka za CT zingayambitse kuyezetsa pafupipafupi ndi zotsatira zabodza, zomwe zimapangitsa kuti pakhale kulowererapo kosafunikira komanso chithandizo chopitirira muyeso6. Chifukwa chake, pakufunika kupanga ma biomarker odalirika komanso othandiza kuti tizindikire bwino khansa ya m'mapapo pachiyambi ndikusiyanitsa nodules zambiri zabwino pozizindikira koyamba 7.
Kusanthula kwathunthu kwa mamolekyu a magazi (seramu, plasma, maselo a mononuclear a m'magazi), kuphatikizapo majini, proteomics kapena DNA methylation8,9,10, kwapangitsa chidwi chachikulu pakupeza zizindikiro zodziwira khansa ya m'mapapo. Pakadali pano, njira za metabolomics zimayesa zinthu zomwe zimakhudzidwa ndi zochita zamkati ndi zakunja ndipo motero zimagwiritsidwa ntchito kulosera kuyambika kwa matenda ndi zotsatira zake. Liquid chromatography-tandem mass spectrometry (LC-MS) ndi njira yomwe imagwiritsidwa ntchito kwambiri pa maphunziro a metabolomics chifukwa cha kukhudzidwa kwake kwakukulu komanso kuchuluka kwakukulu kwa mphamvu, komwe kumatha kuphimba metabolites ndi zinthu zosiyanasiyana za physicochemical11,12,13. Ngakhale kusanthula kwa metabolomic padziko lonse lapansi kwa plasma/seramu kwagwiritsidwa ntchito kuzindikira zizindikiro zomwe zimagwirizanitsidwa ndi matenda a khansa ya m'mapapo14,15,16,17 ndi mphamvu ya chithandizo, 18 magulu a metabolite a m'magazi kuti asiyanitse pakati pa nodules za m'mapapo zosaopsa ndi zoyipa akadali ophunziridwa kwambiri. - kafukufuku wamkulu.
Adenocarcinoma ndi squamous cell carcinoma ndi mitundu iwiri ikuluikulu ya khansa ya m'mapapo yopanda maselo ang'onoang'ono (NSCLC). Mayeso osiyanasiyana a CT screening akusonyeza kuti adenocarcinoma ndiye mtundu wodziwika bwino wa khansa ya m'mapapo1,19,20,21. Mu kafukufukuyu, tagwiritsa ntchito ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) kuti tichite kusanthula kwa metabolomics pa zitsanzo 695 za seramu, kuphatikiza zowongolera zathanzi, ma nodule a m'mapapo osapweteka, ndi CT-detected ≤3 cm. Kuwunika kwa adenocarcinoma ya m'mapapo ya Gawo I. Tapeza gulu la ma metabolites a m'magazi omwe amasiyanitsa adenocarcinoma ya m'mapapo ndi ma nodule osavulaza komanso zowongolera zathanzi. Kuwunika kwa njira yowonjezera kwawonetsa kuti tryptophan yosazolowereka ndi kagayidwe ka shuga ndi kusintha kofala mu adenocarcinoma ya m'mapapo poyerekeza ndi ma nodule osavulaza komanso zowongolera zathanzi. Pomaliza, tinakhazikitsa ndikutsimikizira kagayidwe ka seramu kamene kali ndi luso lapadera komanso lozindikira kuti tisiyanitse pakati pa timitsempha ta m'mapapo toyipa komanso tosaopsa tomwe timapezeka ndi LDCT, zomwe zingathandize kuzindikira matenda osiyanasiyana koyambirira komanso kuwunika zoopsa.
Mu kafukufuku waposachedwa, zitsanzo za seramu zofanana ndi za amuna ndi akazi komanso zaka zinasonkhanitsidwa kuchokera kwa anthu 174 omwe ali ndi thanzi labwino, odwala 292 omwe ali ndi ziphuphu zopanda vuto m'mapapo, ndi odwala 229 omwe ali ndi khansa ya m'mapapo ya gawo loyamba. Makhalidwe a anthu 695 omwe adapezeka akuwonetsedwa mu Supplementary Table 1.
Monga momwe zasonyezedwera mu Chithunzi 1a, zitsanzo zonse 480 za seramu, kuphatikizapo 174 zowongolera thanzi (HC), 170 benign nodules (BN), ndi zitsanzo 136 za stage I lung adenocarcinoma (LA), zinasonkhanitsidwa ku Sun Yat-sen University Cancer Center. Gulu lofufuza za metabolomic profiling yosayang'aniridwa pogwiritsa ntchito ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Monga momwe zasonyezedwera mu Chithunzi Chowonjezera 1, ma metabolites osiyana pakati pa LA ndi HC, LA ndi BN adadziwika kuti akhazikitse chitsanzo chogawa ndikufufuzanso njira yosiyana. Zitsanzo 104 zomwe zinasonkhanitsidwa ndi Sun Yat-sen University Cancer Center ndi zitsanzo 111 zomwe zinasonkhanitsidwa ndi zipatala zina ziwiri zinayesedwa mkati ndi kunja, motsatana.
a Anthu ofufuza omwe adafufuza metabolomics ya serum padziko lonse lapansi pogwiritsa ntchito ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). b Kusanthula kwa Partial least squares discriminant (PLS-DA) ya metabolome yonse ya zitsanzo 480 za serum kuchokera ku gulu lofufuza, kuphatikiza zowongolera zathanzi (HC, n = 174), ma nodules osalimba (BN, n = 170), ndi stage I lung adenocarcinoma (Los Angeles, n = 136). +ESI, positive electrospray ionization mode, -ESI, negative electrospray ionization mode. c–e Metabolites omwe ali ndi kuchuluka kosiyana kwambiri m'magulu awiri operekedwa (two-tailed Wilcoxon signed rank test, false discovery rate adjusted p value, FDR <0.05) akuwonetsedwa mu red (fold change > 1.2) ndi blue (fold change < 0.83). ) akuwonetsedwa pa volcano graphic. Mapu a kutentha kwa magulu osiyanasiyana omwe akuwonetsa kusiyana kwakukulu pa kuchuluka kwa ma metabolites omwe ali ndi mawu pakati pa LA ndi BN. Deta yochokera imaperekedwa mu mawonekedwe a mafayilo a deta yochokera.
Metabolome yonse ya seramu ya 174 HC, 170 BN ndi 136 LA mu gulu lofufuza idasanthulidwa pogwiritsa ntchito kusanthula kwa UPLC-HRMS. Choyamba tikuwonetsa kuti zitsanzo zowongolera khalidwe (QC) zimasonkhana pakati pa chitsanzo chosayang'aniridwa cha principal component analysis (PCA), kutsimikizira kukhazikika kwa magwiridwe antchito a kafukufukuyu (Chithunzi Chowonjezera 2).
Monga momwe zasonyezedwera mu kusanthula kwa gawo laling'ono kwambiri (PLS-DA) mu Chithunzi 1 b, tapeza kuti panali kusiyana komveka bwino pakati pa LA ndi BN, LA ndi HC mu njira zabwino (+ESI) ndi zoipa (−ESI) za electrospray ionization. Komabe, palibe kusiyana kwakukulu komwe kunapezeka pakati pa BN ndi HC mu mikhalidwe ya +ESI ndi -ESI.
Tapeza zinthu 382 zosiyana pakati pa LA ndi HC, zinthu 231 zosiyana pakati pa LA ndi BN, ndi zinthu 95 zosiyana pakati pa BN ndi HC (Wilcoxon signed rank test, FDR <0.05 ndi multiple change >1.2 kapena <0.83) (Chithunzi .1c-e). . Mapeyala adalembedwanso (Supplementary Data 3) motsutsana ndi database (mzCloud/HMDB/Chemspider library) ndi m/z value, retention time ndi fragmentation mass spectrum search (tsatanetsatane wofotokozedwa mu gawo la Njira) 22. Pomaliza, ma metabolites 33 ndi 38 omwe adalembedwa ndi kusiyana kwakukulu adadziwika kuti ndi LA motsutsana ndi BN (Chithunzi 1f ndi Supplementary Table 2) ndi LA motsutsana ndi HC (Supplementary Picture 3 ndi Supplementary Table 2), motsatana. Mosiyana ndi zimenezi, ma metabolites atatu okha omwe ali ndi kusiyana kwakukulu mu kuchuluka ndi omwe adapezeka mu BN ndi HC (Table Supplementary 2), mogwirizana ndi kugwirizana pakati pa BN ndi HC mu PLS-DA. Ma metabolites osiyanasiyanawa amaphimba mitundu yosiyanasiyana ya biochemicals (Chithunzi Chowonjezera 4). Potengera zonsezi, zotsatirazi zikuwonetsa kusintha kwakukulu mu metabolome ya seramu yomwe ikuwonetsa kusintha koyipa kwa khansa ya m'mapapo yoyambirira poyerekeza ndi ma nodules a m'mapapo kapena anthu athanzi. Pakadali pano, kufanana kwa metabolome ya seramu ya BN ndi HC kukusonyeza kuti ma nodules a m'mapapo osalimba amatha kukhala ndi makhalidwe ambiri achilengedwe ndi anthu athanzi. Popeza kusintha kwa majini a epidermal growth factor receptor (EGFR) ndikofala mu adenocarcinoma subtype 23 ya m'mapapo, tinayesetsa kudziwa momwe kusintha kwa ma driver kukhudzira metabolome ya seramu. Kenako tinasanthula mbiri yonse ya metabolomic ya milandu 72 yokhala ndi EGFR m'gulu la adenocarcinoma ya m'mapapo. Chosangalatsa n'chakuti, tapeza ma profiles ofanana pakati pa odwala omwe ali ndi EGFR mutant (n = 41) ndi odwala omwe ali ndi EGFR wild-type (n = 31) mu PCA analysis (Supplementary Picture 5a). Komabe, tapeza ma metabolites 7 omwe kuchuluka kwawo kunasintha kwambiri mwa odwala omwe ali ndi EGFR mutation poyerekeza ndi odwala omwe ali ndi EGFR wild-type (t test, p < 0.05 ndi fold change > 1.2 kapena < 0.83) (Supplementary Picture 5b). Ambiri mwa ma metabolites awa (5 mwa 7) ndi acylcarnitines, omwe amagwira ntchito yofunika kwambiri mu njira zophikira mafuta acid.
Monga momwe zasonyezedwera mu ntchito yomwe yawonetsedwa pa Chithunzi 2 a, ma biomarker a gulu la nodule adapezeka pogwiritsa ntchito ma operators osachepera kwathunthu a shrinkage ndi kusankha kutengera ma metabolites 33 osiyana omwe adapezeka mu LA (n = 136) ndi BN (n = 170). Kuphatikiza kwabwino kwambiri kwa zosintha (LASSO) - chitsanzo cha binary logistic regression. Kutsimikizika kwa magawo khumi kunagwiritsidwa ntchito kuyesa kudalirika kwa chitsanzocho. Kusankhidwa kosinthika ndi kukhazikika kwa magawo kumasinthidwa ndi chilango cha maximization ndi parameter λ24. Kusanthula kwa metabolomics padziko lonse lapansi kunachitikanso padera m'magulu a internal validation (n = 104) ndi external validation (n = 111) kuti ayesere magwiridwe antchito a mtundu wa discriminant. Zotsatira zake, ma metabolites 27 omwe adapezeka adadziwika kuti ndi chitsanzo chabwino kwambiri chosiyanitsa chokhala ndi AUC value yayikulu kwambiri (Chithunzi 2b), pakati pawo 9 anali ndi ntchito yowonjezereka ndipo 18 ntchito yotsika mu LA poyerekeza ndi BN (Chithunzi 2c).
Kayendedwe ka ntchito yomanga gulu la ma nodule a m'mapapo, kuphatikizapo kusankha gulu labwino kwambiri la ma metabolites a seramu mu seti yopezera pogwiritsa ntchito chitsanzo cha binary logistic regression kudzera mu kutsimikizira kopitilira kakhumi ndi kuwunika magwiridwe antchito oneneratu m'maseti otsimikizira amkati ndi akunja. b Ziwerengero zotsimikizira zodutsa za chitsanzo cha LASSO regression cha kusankha biomarker ya metabolic. Manambala omwe aperekedwa pamwambapa akuyimira chiwerengero chapakati cha ma biomarker osankhidwa pa λ yoperekedwa. Mzere wofiira wokhala ndi madontho umayimira mtengo wapakati wa AUC pa lambda yofanana. Mipiringidzo yolakwika ya imvi imayimira ma AUC ochepa komanso apamwamba kwambiri. Mzere wokhala ndi madontho umasonyeza chitsanzo chabwino kwambiri ndi ma biomarker 27 osankhidwa. AUC, dera lomwe lili pansi pa curve ya receiver operating characteristic (ROC). c Kusintha kwa ma metabolites 27 osankhidwa mu gulu la LA poyerekeza ndi gulu la BN mu gulu lopeza. Mzere wofiira - kuyambitsa. Mzere wabuluu ndi kuchepa. Ma curve a d–f Receiver operating characteristic (ROC) akuwonetsa mphamvu ya chitsanzo chosiyanitsa kutengera kuphatikiza kwa ma metabolite 27 mu ma seti otsimikizira opeza, amkati, ndi akunja. Deta yochokera imaperekedwa mu mawonekedwe a mafayilo a data yochokera.
Chitsanzo cholosera chinapangidwa kutengera ma coefficients olemera a ma metabolites 27 awa (Table Supplementary 3). Kusanthula kwa ROC kutengera ma metabolites 27 awa kunapereka malo omwe ali pansi pa mtengo wa curve (AUC) wa 0.933, kukhudzidwa kwa gulu lopeza kunali 0.868, ndipo kutsimikizika kunali 0.859 (Chithunzi 2d). Pakadali pano, pakati pa ma metabolites 38 odziwika pakati pa LA ndi HC, gulu la ma metabolites 16 linapeza AUC ya 0.902 yokhala ndi kukhudzidwa kwa 0.801 ndi kutsimikizika kwa 0.856 pakusiyanitsa LA ndi HC (Chithunzi Chowonjezera 6a-c). Ma AUC otengera malire osiyanasiyana a kusintha kwa ma fold kwa ma metabolites osiyana nawonso anayerekezeredwa. Tinapeza kuti chitsanzo cha gulu chinachita bwino kwambiri pakusiyanitsa pakati pa LA ndi BN (HC) pamene mulingo wosintha kwa fold unayikidwa pa 1.2 motsutsana ndi 1.5 kapena 2.0 (Chithunzi Chowonjezera 7a,b). Chitsanzo cha magulu, chozikidwa pa magulu 27 a metabolite, chinatsimikiziridwanso m'magulu amkati ndi akunja. AUC inali 0.915 (kukhudzidwa 0.867, kutsimikizika 0.811) kuti itsimikizire mkati ndi 0.945 (kukhudzidwa 0.810, kutsimikizika 0.979) kuti itsimikizire kunja (Chithunzi 2e, f). Kuti tiwone momwe ma labotale amagwirira ntchito, zitsanzo 40 kuchokera ku gulu lakunja zinasanthulidwa mu labotale yakunja monga momwe tafotokozera mu gawo la Njira. Kulondola kwa magulu kunapeza AUC ya 0.925 (Chithunzi Chowonjezera 8). Chifukwa lung squamous cell carcinoma (LUSC) ndi mtundu wachiwiri wofala kwambiri wa khansa ya m'mapapo yopanda maselo ang'onoang'ono (NSCLC) pambuyo pa lung adenocarcinoma (LUAD), tinayesanso kugwiritsa ntchito kotsimikizika kwa ma metabolic profiles. BN ndi milandu 16 ya LUSC. AUC ya kusiyana pakati pa LUSC ndi BN inali 0.776 (Chithunzi Chowonjezera 9), kusonyeza luso lochepa poyerekeza ndi kusiyana pakati pa LUAD ndi BN.
Kafukufuku wasonyeza kuti kukula kwa timibulu pa zithunzi za CT kumagwirizana bwino ndi kuthekera kwa khansa ndipo kumakhalabe chifukwa chachikulu cha chithandizo cha timibulu25,26,27. Kusanthula deta kuchokera ku gulu lalikulu la kafukufuku wofufuza wa NELSON kunawonetsa kuti chiopsezo cha khansa mwa anthu omwe ali ndi timibulu <5 mm chinali chofanana ndi cha anthu omwe alibe timibulu28. Chifukwa chake, kukula kochepa komwe kumafunika kuyang'aniridwa nthawi zonse kwa CT ndi 5 mm, monga momwe bungwe la British Thoracic Society (BTS) linanenera, ndi 6 mm, monga momwe bungwe la Fleischner Society linanenera. Komabe, timibulu tomwe tili ndi timibulu toposa 6 mm komanso topanda zizindikiro zodziwika bwino, zotchedwa indeterminate pulmonary nodules (IPN), tidakali vuto lalikulu pakuwunika ndi kuyang'anira mu ntchito zachipatala30,31. Kenako tinafufuza ngati kukula kwa timibulu kunakhudza zizindikiro za metabolomic pogwiritsa ntchito zitsanzo zosonkhanitsidwa kuchokera ku magulu ofufuza ndi otsimikizira mkati. Poyang'ana kwambiri pa ma biomarker 27 ovomerezeka, choyamba tinayerekeza ma PCA profiles a HC ndi BN sub-6 mm metabolomes. Tapeza kuti mfundo zambiri za deta ya HC ndi BN zinagwirizana, zomwe zikusonyeza kuti milingo ya metabolite ya m'magazi inali yofanana m'magulu onse awiri (Chithunzi 3a). Mapu a mawonekedwe osiyanasiyana amitundu yosiyanasiyana adasungidwabe mu BN ndi LA (Chithunzi 3b, c), pomwe kusiyana kunawonedwa pakati pa nodules zoyipa ndi zosaopsa m'magawo a 6-20 mm (Chithunzi 3d). Gulu ili linali ndi AUC ya 0.927, kudziwika kwa 0.868, ndi kukhudzidwa kwa 0.820 poneneratu khansa ya nodules yolemera 6 mpaka 20 mm (Chithunzi 3e, f). Zotsatira zathu zikuwonetsa kuti gululi limatha kuwona kusintha kwa kagayidwe kachakudya komwe kumachitika chifukwa cha kusintha koyambirira kwa khansa, mosasamala kanthu za kukula kwa nodules.
Kuyerekeza kwa ma profiles a PCA pakati pa magulu otchulidwa kutengera gulu la metabolism la ma metabolites 27. CC ndi BN < 6 mm. b 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 Mzere wa Receiver operating characteristic (ROC) womwe ukuwonetsa magwiridwe antchito a chitsanzo cha ma nodules 6–20 mm. f Ma values otheka adawerengedwa kutengera chitsanzo cha logistic regression cha ma nodules olemera 6–20 mm. Mzere wa imvi wowala ukuyimira mtengo wabwino kwambiri wa cutoff (0.455). Manambala omwe ali pamwambapa akuyimira kuchuluka kwa milandu yomwe ikuyembekezeredwa ku Los Angeles. Gwiritsani ntchito mayeso a t a Student's tailed two-tailed. PCA, kusanthula kwa zigawo zazikulu. Malo a AUC pansi pa curve. Deta yochokera imaperekedwa mu mawonekedwe a mafayilo a deta yochokera.
Zitsanzo zinayi (zazaka 44-61) zokhala ndi kukula kofanana kwa ma nodule a m'mapapo (7-9 mm) zinasankhidwanso kuti ziwonetse momwe chitsanzo cholosera khansa chinagwirira ntchito (Chithunzi 4a, b). Poyamba, Mlandu 1 unaonekera ngati nodule yolimba yokhala ndi calcification, chinthu chogwirizana ndi ubwino, pomwe Mlandu 2 unaonekera ngati nodule yolimba pang'ono yopanda zizindikiro zomveka bwino. Ma CT scan atatu otsatira adawonetsa kuti milandu iyi inakhalabe yokhazikika kwa zaka 4 ndipo motero inkaonedwa ngati ma nodule opanda poizoni (Chithunzi 4a). Poyerekeza ndi kuwunika kwachipatala kwa ma CT scans otsatizana, kusanthula kwa metabolite ya serum imodzi ndi chitsanzo cha current classifier mwachangu komanso molondola kunazindikira ma nodule opanda poizoni kutengera zoletsa zomwe zingachitike (Table 1). Chithunzi 4b mu nkhani 3 chikuwonetsa nodule yokhala ndi zizindikiro za pleural retraction, yomwe nthawi zambiri imagwirizanitsidwa ndi khansa32. Mlandu 4 unaonekera ngati nodule yopanda poizoni yopanda umboni wa chifukwa chosaopsa. Milandu yonseyi inanenedweratu kuti ndi yoyipa malinga ndi chitsanzo cha classifier (Table 1). Kuwunika kwa adenocarcinoma ya m'mapapo kunawonetsedwa powunika histopathological pambuyo pa opaleshoni yochotsa mapapo (Chithunzi 4b). Pa seti yotsimikizira zakunja, kagulu ka kagayidwe kachakudya kananeneratu molondola milandu iwiri ya timibulu ta m'mapapo tosatsimikizika toposa 6 mm (Chithunzi Chowonjezera 10).
Zithunzi za CT za zenera la axial la mapapo a anthu awiri omwe ali ndi timinofu tosaopsa. Mu nkhani yoyamba, CT scan patatha zaka 4 inasonyeza chipolopolo cholimba chokhazikika cha 7 mm chokhala ndi calcium mu lobe yakumanja yapansi. Mu nkhani yachiwiri, CT scan patatha zaka 5 inasonyeza chipolopolo chokhazikika, cholimba pang'ono chokhala ndi mainchesi a 7 mm mu lobe yakumanja yapamwamba. b Zithunzi za CT za window ya axial za mapapo ndi maphunziro ofanana a matenda a milandu iwiri ya adenocarcinoma ya siteji I asanachotsedwe m'mapapo. Nkhani yachitatu inasonyeza chipolopolo chokhala ndi mainchesi a 8 mm mu lobe yakumanja yapamwamba yokhala ndi pleural retraction. Nkhani yachinayi inasonyeza chipolopolo cha galasi lolimba pang'ono cholemera 9 mm mu lobe yakumanzere yapamwamba. Kupaka utoto kwa hematoxylin ndi eosin (H&E) kwa minofu ya mapapo yochotsedwa (scale bar = 50 μm) kusonyeza kukula kwa acinar kwa adenocarcinoma ya m'mapapo. Mivi imasonyeza timinofu tomwe tapezeka pazithunzi za CT. Zithunzi za H&E ndi zithunzi zoyimira minda yambiri (>3) ya microscopic yomwe yafufuzidwa ndi katswiri wa matenda.
Poganizira zonsezi, zotsatira zathu zikuwonetsa kufunika kwa zizindikiro za metabolite m'magazi pakuwunika kosiyana kwa timitsempha ta m'mapapo, zomwe zingakhale zovuta poyesa kuyeza kwa CT.
Kutengera ndi gulu lovomerezeka la metabolite, tinayesetsa kupeza zinthu zogwirizana ndi zamoyo zomwe zimasintha kwambiri kagayidwe kachakudya. Kusanthula kwa KEGG pathway enrichment ndi MetaboAnalyst kunapeza njira 6 zomwe zasintha kwambiri pakati pa magulu awiriwa (LA vs. HC ndi LA vs. BN, kusinthidwa p ≤ 0.001, zotsatira > 0.01). Kusinthaku kunadziwika ndi kusokonezeka kwa kagayidwe ka pyruvate, kagayidwe ka tryptophan, kagayidwe ka niacin ndi nicotinamide, glycolysis, kayendedwe ka TCA, ndi kagayidwe ka purine (Chithunzi 5a). Kenako tinachitanso metabolomics yolunjika kuti titsimikizire kusintha kwakukulu pogwiritsa ntchito kuchuluka kwathunthu. Kuzindikira metabolites wamba m'njira zomwe zimasinthidwa kawirikawiri ndi triple quadrupole mass spectrometry (QQQ) pogwiritsa ntchito miyezo yeniyeni ya metabolite. Makhalidwe a chiwerengero cha anthu omwe ali pachitsanzo cha kafukufuku wa metabolomics akuphatikizidwa mu Supplementary Table 4. Mogwirizana ndi zotsatira zathu za metabolomics padziko lonse lapansi, kusanthula kwa kuchuluka kwa anthu kunatsimikizira kuti hypoxanthine ndi xanthine, pyruvate, ndi lactate zinawonjezeka mu LA poyerekeza ndi BN ndi HC (Chithunzi 5b, c, p <0.05). Komabe, palibe kusiyana kwakukulu pakati pa metabolites awa komwe kunapezeka pakati pa BN ndi HC.
Kusanthula kwa njira ya KEGG yopezera metabolites yosiyana kwambiri mu gulu la LA poyerekeza ndi magulu a BN ndi HC. Kuyesedwa kwa Global-tailed two-tailed kunagwiritsidwa ntchito, ndipo ma p values anasinthidwa pogwiritsa ntchito njira ya Holm-Bonferroni (yosinthidwa p ≤ 0.001 ndi kukula kwa zotsatira > 0.01). b–d Violin plots yowonetsa milingo ya hypoxanthine, xanthine, lactate, pyruvate, ndi tryptophan mu serum HC, BN, ndi LA yotsimikiziridwa ndi LC-MS/MS (n = 70 pa gulu lililonse). Mizere yoyera ndi yakuda yokhala ndi madontho imasonyeza median ndi quartile, motsatana. e Violin plot yowonetsa normalized Log2TPM (zolemba pa miliyoni) mRNA expression ya SLC7A5 ndi QPRT mu lung adenocarcinoma (n = 513) poyerekeza ndi minofu ya mapapo yachibadwa (n = 59) mu dataset ya LUAD-TCGA. Bokosi loyera likuyimira mtundu wa interquartile, mzere wakuda wopingasa pakati ukuyimira wapakati, ndipo mzere wakuda wowongoka wochokera m'bokosi ukuyimira 95% confidence interval (CI). f Pearson correlation plot ya SLC7A5 ndi GAPDH expression mu lung adenocarcinoma (n = 513) ndi normal mapapu tissue (n = 59) mu dataset ya TCGA. Dera loyera likuyimira 95% CI. r, Pearson correlation coefficient. g Ma tryptophan a cell cellular omwe ali mu maselo a A549 omwe amathandizidwa ndi nonspecific shRNA control (NC) ndi shSLC7A5 (Sh1, Sh2) yotsimikiziridwa ndi LC-MS/MS. Kusanthula kwa ziwerengero za zitsanzo zisanu zodziyimira pawokha m'gulu lililonse kumaperekedwa. h Ma cellular levels a NADt (total NAD, kuphatikiza NAD+ ndi NADH) mu maselo a A549 (NC) ndi SLC7A5 knockdown A549 cells (Sh1, Sh2). Kusanthula kwa ziwerengero za zitsanzo zitatu zodziyimira pawokha m'gulu lililonse kumaperekedwa. Ntchito ya Glycolytic ya maselo a A549 isanayambe komanso itatha SLC7A5 kugwetsedwa idayesedwa ndi kuchuluka kwa acidification ya extracellular (ECAR) (n = zitsanzo 4 zodziyimira payokha pagulu lililonse). 2-DG,2-deoxy-D-glucose. Mayeso a Two-tailed Student's t adagwiritsidwa ntchito mu (b–h). Mu (g–i), mipiringidzo yolakwika ikuyimira mean ± SD, kuyesa kulikonse kunachitika katatu padera ndipo zotsatira zake zinali zofanana. Deta yochokera imaperekedwa mu mawonekedwe a mafayilo a deta yochokera.
Poganizira za kusintha kwakukulu kwa kagayidwe ka tryptophan m'gulu la LA, tinayesanso kuchuluka kwa tryptophan m'magazi m'magulu a HC, BN, ndi LA pogwiritsa ntchito QQQ. Tapeza kuti tryptophan m'magazi inachepa mu LA poyerekeza ndi HC kapena BN (p < 0.001, Chithunzi 5d), zomwe zikugwirizana ndi zomwe zapezeka kale kuti kuchuluka kwa tryptophan m'magazi kumakhala kotsika mwa odwala omwe ali ndi khansa ya m'mapapo kuposa m'magulu athanzi ochokera ku gulu lowongolera33,34,35. Kafukufuku wina wogwiritsa ntchito PET/CT tracer 11C-methyl-L-tryptophan adapeza kuti nthawi yosungira chizindikiro cha tryptophan m'maselo a khansa ya m'mapapo inawonjezeka kwambiri poyerekeza ndi zilonda zabwino kapena minofu yachibadwa36. Tikuganiza kuti kuchepa kwa tryptophan mu seramu ya LA kungasonyeze kutenga kwa tryptophan komwe kumachitika ndi maselo a khansa ya m'mapapo.
Zimadziwikanso kuti zotsatira za njira ya kynurenine ya tryptophan catabolism ndi NAD+37,38, yomwe ndi gawo lofunikira kwambiri pakuchita kwa glyceraldehyde-3-phosphate ndi 1,3-bisphosphoglycerate mu glycolysis39. Ngakhale maphunziro am'mbuyomu adayang'ana kwambiri pa ntchito ya tryptophan catabolism pakulamulira chitetezo cha mthupi, tidayesetsa kufotokoza momwe tryptophan dysregulation imagwirira ntchito ndi njira za glycolytic zomwe zawonedwa mu kafukufukuyu. Solute transporter family 7 member 5 (SLC7A5) imadziwika kuti ndi tryptophan transporter43,44,45. Quinolinic acid phosphoribosyltransferase (QPRT) ndi enzyme yomwe ili pansi pa njira ya kynurenine yomwe imasintha quinolinic acid kukhala NAMN46. Kuyang'ana deta ya LUAD TCGA kudawonetsa kuti SLC7A5 ndi QPRT zonse zidakwera kwambiri mu minofu yotupa poyerekeza ndi minofu yachibadwa (Chithunzi 5e). Kuwonjezeka kumeneku kunawonedwa mu magawo I ndi II komanso magawo III ndi IV a adenocarcinoma ya m'mapapo (Chithunzi Chowonjezera 11), zomwe zikusonyeza kusokonekera koyambirira kwa kagayidwe ka tryptophan komwe kumakhudzana ndi kupangika kwa tumori.
Kuphatikiza apo, deta ya LUAD-TCGA inasonyeza mgwirizano wabwino pakati pa SLC7A5 ndi GAPDH mRNA yowonetsedwa m'zitsanzo za odwala khansa (r = 0.45, p = 1.55E-26, Chithunzi 5f). Mosiyana ndi zimenezi, palibe mgwirizano wofunikira womwe unapezeka pakati pa zizindikiro za majini m'minofu ya m'mapapo (r = 0.25, p = 0.06, Chithunzi 5f). Kutsika kwa SLC7A5 (Chithunzi Chowonjezera 12) m'maselo a A549 kunachepetsa kwambiri kuchuluka kwa tryptophan m'maselo ndi NAD(H) (Chithunzi 5g,h), zomwe zimapangitsa kuti ntchito ya glycolytic ichepe monga momwe imayesedwera ndi kuchuluka kwa acidification ya extracellular (ECAR) (Chithunzi 1). 5i). Chifukwa chake, kutengera kusintha kwa kagayidwe kachakudya mu seramu ndi kuzindikira mu vitro, tikuganiza kuti kagayidwe ka tryptophan kangapange NAD+ kudzera mu njira ya kynurenine ndikuchita gawo lofunika kwambiri pakulimbikitsa glycolysis mu khansa ya m'mapapo.
Kafukufuku wasonyeza kuti kuchuluka kwa timitsempha ta m'mapapo tosadziŵika bwino tomwe tapezeka ndi LDCT kungapangitse kuti pakhale kufunikira koyesa kwina monga PET-CT, biopsy ya m'mapapo, ndi chithandizo chopitirira muyeso chifukwa cha kuzindikira kolakwika kwa khansa.31 Monga momwe zasonyezedwera pa Chithunzi 6, kafukufuku wathu adapeza gulu la ma metabolites a m'magazi omwe ali ndi phindu lotha kuzindikira lomwe lingathandize kusintha kugawa kwa chiopsezo ndikuwongolera pambuyo pake timitsempha ta m'mapapo tomwe tapezeka ndi CT.
Ma nodule a m'mapapo amawunikidwa pogwiritsa ntchito computed tomography (LDCT) yotsika mtengo yokhala ndi zithunzi zomwe zikusonyeza zifukwa zabwino kapena zoyipa. Zotsatira zosatsimikizika za ma nodule zimatha kubweretsa maulendo obwerezabwereza, njira zosafunikira, komanso chithandizo chopitirira muyeso. Kuphatikizidwa kwa ma classifiers a metabolic mu seramu omwe ali ndi phindu lozindikira kungathandize kuwunika zoopsa komanso kuyang'anira ma nodule a m'mapapo. PET positron emission tomography.
Deta yochokera ku kafukufuku wa US NLST ndi kafukufuku wa ku Europe wa NELSON ikusonyeza kuti kufufuza magulu omwe ali pachiwopsezo chachikulu pogwiritsa ntchito computed tomography (LDCT) yotsika mtengo kungachepetse imfa za khansa ya m'mapapo1,3. Komabe, kuwunika zoopsa ndi kuyang'anira matenda ambiri a m'mapapo omwe apezeka ndi LDCT kumakhalabe kovuta kwambiri. Cholinga chachikulu ndikukonza magulu oyenera a ma protocol omwe alipo a LDCT pophatikiza ma biomarker odalirika.
Ma biomarker ena a mamolekyu, monga ma metabolites a m'magazi, apezeka poyerekeza khansa ya m'mapapo ndi ma controls athanzi15,17. Mu kafukufuku wapano, tinayang'ana kwambiri pakugwiritsa ntchito kusanthula kwa metabolomics ya seramu kuti tisiyanitse pakati pa ma nodules a m'mapapo osavulaza ndi owopsa omwe amapezeka mwangozi ndi LDCT. Tinayerekeza zitsanzo za seramu metabolome ya healthy control (HC), ma nodules a m'mapapo osavulaza (BN), ndi zitsanzo za phazi loyamba la lung adenocarcinoma (LA) pogwiritsa ntchito kusanthula kwa UPLC-HRMS. Tapeza kuti HC ndi BN zinali ndi ma profiles ofanana a kagayidwe kachakudya, pomwe LA inawonetsa kusintha kwakukulu poyerekeza ndi HC ndi BN. Tinapeza magulu awiri a ma metabolites a m'magazi omwe amasiyanitsa LA ndi HC ndi BN.
Ndondomeko yodziwira matenda a khansa ya m'mapapo yopanda vuto lililonse komanso yoopsa imadalira kwambiri kukula, kuchulukana, mawonekedwe, ndi kukula kwa matenda a khansa pakapita nthawi. Kafukufuku wakale wasonyeza kuti kukula kwa matendawa kumagwirizana kwambiri ndi mwayi wokhala ndi khansa ya m'mapapo. Ngakhale kwa odwala omwe ali pachiwopsezo chachikulu, chiopsezo cha khansa m'magawo a <6 mm ndi <1%. Chiwopsezo cha khansa ya m'magawo apakati pa 6 mpaka 20 mm chimachokera pa 8% mpaka 64%30. Chifukwa chake, Fleischner Society imalimbikitsa kuti m'mimba mwake mukhale ndi 6 mm kuti mufufuze CT nthawi zonse. 29 Komabe, kuwunika zoopsa ndi kasamalidwe ka matenda a mtima osatsimikizika (IPN) opitilira 6 mm sikunachitike mokwanira 31. Kuyang'anira matenda a mtima obadwa nawo nthawi zambiri kumadalira kudikira mosamala ndi kuyang'aniridwa pafupipafupi kwa CT.
Kutengera ndi metabolome yotsimikizika, tinawonetsa koyamba kuti pali kugwirizana kwa zizindikiro za metabolomic pakati pa anthu athanzi ndi timibulu tosalimba <6 mm. Kufanana kwa zamoyo kukugwirizana ndi zomwe zidapezeka kale za CT kuti chiopsezo cha khansa ya timibulu tosalimba <6 mm ndi chotsika ngati cha anthu opanda timibulu.30 Tiyenera kudziwa kuti zotsatira zathu zikuwonetsanso kuti timibulu tosalimba <6 mm ndi ≥6 mm tili ndi kufanana kwakukulu mu metabolomic profiles, zomwe zikusonyeza kuti tanthauzo la ntchito ya benign etiology limagwirizana mosasamala kanthu za kukula kwa timibulu. Chifukwa chake, mapanelo amakono a serum metabolite angapereke mayeso amodzi ngati mayeso osatha pamene timibulu tapezeka koyamba pa CT ndipo mwina timachepetsa kuyang'anira mobwerezabwereza. Nthawi yomweyo, gulu lomwelo la biomarkers la metabolic linasiyanitsa timibulu tosalimba ≥6 mm kukula kuchokera ku timibulu tosalimba ndipo linapereka maulosi olondola a IPN a kukula kofanana komanso mawonekedwe osamveka bwino pazithunzi za CT. Kagulu ka serum metabolism kameneka kachita bwino poneneratu khansa ya timibulu ≥6 mm ndi AUC ya 0.927. Poganizira zonsezi, zotsatira zathu zikusonyeza kuti zizindikiro zapadera za metabolomic m'magazi zimatha kuwonetsa kusintha kwa kagayidwe kachakudya koyambirira komwe kumachitika chifukwa cha chotupa ndipo zitha kukhala ndi phindu ngati zodziwira zoopsa, mosasamala kanthu za kukula kwa nodule.
Chodziwika bwino n'chakuti, khansa ya m'mapapo ya adenocarcinoma (LUAD) ndi khansa ya squamous cell (LUSC) ndi mitundu ikuluikulu ya khansa ya m'mapapo ya non-small cell (NSCLC). Popeza LUSC imagwirizana kwambiri ndi kugwiritsa ntchito fodya47 ndipo LUAD ndiye histology yodziwika bwino ya timibulu ta m'mapapo tomwe timapezeka pa CT screening48, chitsanzo chathu cha classifier chinapangidwira makamaka zitsanzo za adenocarcinoma ya gawo loyamba. Wang ndi anzake adayang'ananso pa LUAD ndipo adapeza zizindikiro zisanu ndi zinayi za lipid pogwiritsa ntchito lipidomics kusiyanitsa khansa ya m'mapapo yoyambirira ndi anthu athanzi17. Tinayesa chitsanzo cha classifier chomwe chilipo pa milandu 16 ya LUSC ya gawo loyamba ndi timibulu 74 tomwe sitingathe kuchiza ndipo tinaona kulondola kochepa kwa LUSC (AUC 0.776), zomwe zikusonyeza kuti LUAD ndi LUSC zitha kukhala ndi zizindikiro zawo za metabolomic. Zoonadi, LUAD ndi LUSC zawonetsedwa kuti zimasiyana malinga ndi chifukwa, chiyambi cha zamoyo komanso kusintha kwa majini49. Chifukwa chake, mitundu ina ya histology iyenera kuphatikizidwa mu maphunziro ophunzitsira kuzindikira khansa ya m'mapapo potengera anthu m'mapulogalamu owunikira.
Apa, tapeza njira zisanu ndi chimodzi zomwe zimasinthidwa kwambiri mu adenocarcinoma ya m'mapapo poyerekeza ndi zowongolera zathanzi komanso ma nodules osavulaza. Xanthine ndi hypoxanthine ndi ma metabolites ofala a njira ya kagayidwe ka purine. Mogwirizana ndi zotsatira zathu, zinthu zapakati zomwe zimagwirizana ndi kagayidwe ka purine zidawonjezeka kwambiri mu seramu kapena minofu ya odwala omwe ali ndi adenocarcinoma ya m'mapapo poyerekeza ndi zowongolera zathanzi kapena odwala omwe ali pa siteji yoyambirira ya 15,50. Kuchuluka kwa xanthine ndi hypoxanthine m'magazi kungawonetse anabolism yomwe imafunika chifukwa cha kuchulukana mwachangu kwa maselo a khansa. Kusagwira bwino ntchito kwa kagayidwe ka shuga ndi chizindikiro chodziwika bwino cha kagayidwe ka khansa51. Pano, tawona kuwonjezeka kwakukulu kwa pyruvate ndi lactate mu gulu la LA poyerekeza ndi gulu la HC ndi BN, zomwe zikugwirizana ndi malipoti am'mbuyomu a zolakwika za njira ya glycolytic mu ma profiles a metabolome a odwala omwe si ang'onoang'ono a khansa ya m'mapapo (NSCLC) ndi owongolera athanzi. Zotsatira zake ndizofanana52,53.
Chofunika kwambiri, tawona mgwirizano wosiyana pakati pa kagayidwe ka pyruvate ndi tryptophan mu seramu ya adenocarcinomas ya m'mapapo. Kuchuluka kwa tryptophan mu seramu kunachepetsedwa mu gulu la LA poyerekeza ndi gulu la HC kapena BN. Chochititsa chidwi n'chakuti, kafukufuku wakale wogwiritsa ntchito gulu loyembekezera adapeza kuti kuchuluka kochepa kwa tryptophan yozungulira kumagwirizanitsidwa ndi chiopsezo chowonjezeka cha khansa ya m'mapapo 54. Tryptophan ndi amino acid yofunika yomwe timapeza kuchokera ku chakudya. Tikuganiza kuti kuchepa kwa tryptophan mu seramu mu adenocarcinoma ya m'mapapo kungasonyeze kuchepa mwachangu kwa metabolite iyi. Ndizodziwika bwino kuti zotsatira za tryptophan catabolism kudzera mu njira ya kynurenine ndiye gwero la kaphatikizidwe katsopano ka NAD+. Chifukwa chakuti NAD+ imapangidwa makamaka kudzera mu njira yopulumutsira, kufunika kwa NAD+ mu kagayidwe ka tryptophan pa thanzi ndi matenda kukadali kofunikira kudziwika46. Kusanthula kwathu kwa database ya TCGA kunawonetsa kuti kufalikira kwa tryptophan transporter solute transporter 7A5 (SLC7A5) kunawonjezeka kwambiri mu adenocarcinoma ya m'mapapo poyerekeza ndi zowongolera zachizolowezi ndipo kunagwirizana bwino ndi kufalikira kwa enzyme ya glycolytic GAPDH. Kafukufuku wakale adayang'ana kwambiri pa ntchito ya tryptophan catabolism pakuletsa chitetezo chamthupi chotsutsana ndi chotupa40,41,42. Apa tikuwonetsa kuti kuletsa kuyamwa kwa tryptophan pochepetsa SLC7A5 m'maselo a khansa ya m'mapapo kumabweretsa kuchepa kwa kuchuluka kwa NAD m'maselo ndi kuchepa kwa ntchito ya glycolytic nthawi imodzi. Mwachidule, kafukufuku wathu amapereka maziko achilengedwe a kusintha kwa kagayidwe ka seramu komwe kumakhudzana ndi kusintha kwa khansa ya m'mapapo.
Kusintha kwa EGFR ndi komwe kumayendetsa kwambiri kusintha kwa odwala omwe ali ndi NSCLC. Mu kafukufuku wathu, tapeza kuti odwala omwe ali ndi kusintha kwa EGFR (n = 41) anali ndi ma metabolomic profiles ofanana ndi odwala omwe ali ndi EGFR yamtundu wachilengedwe (n = 31), ngakhale kuti tapeza kuchepa kwa kuchuluka kwa seramu kwa odwala ena a EGFR mutant mwa odwala acylcarnitine. Ntchito yodziwika bwino ya acylcarnitines ndikunyamula magulu a acyl kuchokera ku cytoplasm kupita ku mitochondrial matrix, zomwe zimapangitsa kuti ma acid acids azisungunuka kuti apange mphamvu 55. Mogwirizana ndi zomwe tapeza, kafukufuku waposachedwapa adazindikiranso ma metabolome ofanana pakati pa EGFR mutant ndi EGFR wild-type tumors pofufuza metabolome yapadziko lonse ya zitsanzo 102 za minofu ya adenocarcinoma ya m'mapapo50. Chochititsa chidwi n'chakuti, kuchuluka kwa acylcarnitine kunapezekanso m'gulu la EGFR mutant. Chifukwa chake, kaya kusintha kwa kuchuluka kwa acylcarnitine kukuwonetsa kusintha kwa kagayidwe kachakudya komwe kumayambitsidwa ndi EGFR ndi njira zama molekyulu zomwe zili pansi pake kungayenere kufufuzidwa kwina.
Pomaliza, kafukufuku wathu wakhazikitsa njira yodziwira matenda osiyanasiyana a m'mapapo ndipo wapereka njira yogwirira ntchito yomwe ingathandize kuwunika zoopsa ndikuthandizira kuyang'anira matenda pogwiritsa ntchito CT scan.
Kafukufukuyu adavomerezedwa ndi Komiti ya Makhalidwe Abwino ya Chipatala cha Khansa cha Sun Yat-sen University, Chipatala Choyamba Chogwirizana cha Sun Yat-sen University, ndi Komiti ya Makhalidwe Abwino ya Chipatala cha Khansa cha Zhengzhou University. M'magulu ofufuza ndi kutsimikizira mkati, ma sera 174 ochokera kwa anthu athanzi ndi ma sera 244 ochokera ku ma nodule osavulaza adasonkhanitsidwa kuchokera kwa anthu omwe amayesedwa pachaka ku Dipatimenti Yoyang'anira ndi Kuteteza Khansa, Sun Yat-sen University Cancer Center, ndi ma nodule 166 osavulaza. seramu. Ma adenocarcinoma a m'mapapo a Stage I adasonkhanitsidwa kuchokera ku Sun Yat-sen University Cancer Center. Mu gulu lotsimikizira lakunja, panali milandu 48 ya ma nodule osavulaza, milandu 39 ya siteji I lung adenocarcinoma kuchokera ku Chipatala Choyamba Chogwirizana cha Sun Yat-sen University, ndi milandu 24 ya siteji I lung adenocarcinoma kuchokera ku Chipatala cha Khansa cha Zhengzhou. Sun Yat-sen University Cancer Center inasonkhanitsanso milandu 16 ya khansa ya m'mapapo ya squamous cell stage I kuti iyese kuzindikira momwe kagayidwe kachakudya kamagawidwira (makhalidwe a wodwala akuwonetsedwa mu Supplementary Table 5). Zitsanzo kuchokera ku gulu lofufuza ndi gulu lotsimikizira mkati zinasonkhanitsidwa pakati pa Januwale 2018 ndi Meyi 2020. Zitsanzo za gulu lotsimikizira kunja zinasonkhanitsidwa pakati pa Ogasiti 2021 ndi Okutobala 2022. Pofuna kuchepetsa tsankho pakati pa amuna ndi akazi, milandu yofanana ya amuna ndi akazi inaperekedwa ku gulu lililonse. Gulu Lofufuza ndi Gulu Lowunikira Pakati. Jenda la ophunzira linatsimikiziridwa kutengera lipoti lodzidziwitsa. Chilolezo chodziwitsidwa chinaperekedwa kuchokera kwa ophunzira onse ndipo palibe malipiro omwe adaperekedwa. Anthu omwe anali ndi timibulu tosalimba anali omwe anali ndi chiŵerengero chokhazikika cha CT scan pazaka 2 mpaka 5 panthawi yofufuza, kupatulapo mlandu umodzi kuchokera ku chitsanzo chotsimikizira kunja, chomwe chinasonkhanitsidwa asanachite opaleshoni ndikupezeka ndi histopathology. Kupatula bronchitis yosatha. Milandu ya adenocarcinoma ya m'mapapo inasonkhanitsidwa asanachotsedwe mapapo ndipo inatsimikiziridwa ndi matenda. Magazi osala kudya anasonkhanitsidwa m'machubu olekanitsa seramu popanda mankhwala oletsa magazi kuundana. Magazi anaundana kwa ola limodzi kutentha kwa chipinda kenako anaikidwa mu centrifuge pa 2851 × g kwa mphindi 10 pa 4°C kuti atenge supernatant ya seramu. Ma aliquot a seramu anazizira pa -80°C mpaka metabolite itachotsedwa. Dipatimenti Yoona za Kupewa Khansa ndi Kufufuza Zachipatala ya Sun Yat-sen University Cancer Center inasonkhanitsa seramu kuchokera kwa opereka 100 athanzi, kuphatikizapo chiwerengero chofanana cha amuna ndi akazi azaka zapakati pa 40 ndi 55. Ma voliyumu ofanana a chitsanzo chilichonse cha opereka anasakanizidwa, dziwe lomwe linatuluka linatengedwa ndikusungidwa pa -80°C. Kusakaniza kwa seramu kunagwiritsidwa ntchito ngati zinthu zowunikira kuwongolera khalidwe ndi kuyika deta muyeso.
Seramu yodziwika bwino ndi zitsanzo zoyesera zinasungunuka ndipo metabolites zinachotsedwa pogwiritsa ntchito njira yophatikizana yochotsera (MTBE/methanol/madzi) 56. Mwachidule, 50 μl ya seramu inasakanizidwa ndi 225 μl ya methanol yozizira kwambiri ndi 750 μl ya methyl tert-butyl ether yozizira kwambiri (MTBE). Sakanizani chisakanizocho ndikuchiyika pa ayezi kwa ola limodzi. Kenako zitsanzozo zinasakanizidwa ndipo vortex inasakanizidwa ndi 188 μl ya madzi a MS-grade okhala ndi miyezo yamkati (13C-lactate, 13C3-pyruvate, 13C-methionine, ndi 13C6-isoleucine, ogulidwa ku Cambridge Isotope Laboratories). Kenako chisakanizocho chinayikidwa mu centrifuge pa 15,000 × g kwa mphindi 10 pa 4 °C, ndipo gawo lotsika linasamutsidwa m'machubu awiri (125 μL iliyonse) kuti ayesedwe mu LC-MS m'njira zabwino ndi zoipa. Pomaliza, chitsanzocho chinasanduka nthunzi kukhala chouma mu chosungira madzi chothamanga kwambiri.
Ma metabolites ouma adapangidwanso mu 120 μl ya 80% acetonitrile, adasinthidwa kwa mphindi 5, ndikuyikidwa centrifuge pa 15,000 × g kwa mphindi 10 pa 4°C. Supernatants adasamutsidwa m'mabotolo agalasi a amber okhala ndi ma microinserts kuti aphunzire metabolomics. Kusanthula kwa metabolomics kosayang'aniridwa pa nsanja ya ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Ma metabolites adalekanitsidwa pogwiritsa ntchito njira ya Dionex Ultimate 3000 UPLC ndi ACQUITY BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters). Mu positive ion mode, magawo oyenda anali 95% (A) ndi 50% acetonitrile (B), iliyonse ili ndi 10 mmol/L ammonium acetate ndi 0.1% formic acid. Mu mawonekedwe oipa, magawo oyenda A ndi B anali ndi 95% ndi 50% acetonitrile, motsatana, magawo onse awiri 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. Mzati unasungidwa pa 40°C ndipo chitsanzocho chinali pa 10°C mu autosampler. Kuthamanga kwa madzi kunali 0.3 ml/min, voliyumu ya jakisoni inali 3 μl. Chipangizo choyezera kuchuluka kwa ma spectrometer cha Q-Exactive Orbitrap (Thermo Fisher Scientific) chokhala ndi electrospray ionization (ESI) chinagwiritsidwa ntchito mu full scan mode ndipo chinalumikizidwa ndi ddMS2 monitoring mode kuti chisonkhanitse deta yambiri. Ma parameter a MS adakhazikitsidwa motere: spray voltage +3.8 kV/- 3.2 kV, capillary temperature 320°C, shielding gas 40 arb, assistant gas 10 arb, probe heater temperature 350°C, scanning range 70–1050 m/h, resolution. 70 000. Deta idapezeka pogwiritsa ntchito Xcalibur 4.1 (Thermo Fisher Scientific).
Kuti aone ubwino wa deta, zitsanzo zosonkhanitsidwa zowongolera khalidwe (QC) zinapangidwa pochotsa ma 10 μL aliquots a supernatant kuchokera ku chitsanzo chilichonse. Majakisoni asanu ndi limodzi a zitsanzo zowongolera khalidwe adasanthulidwa kumayambiriro kwa ndondomeko yowunikira kuti awone kukhazikika kwa dongosolo la UPLC-MS. Zitsanzo zowongolera khalidwe zimayikidwa nthawi ndi nthawi mu gulu. Magulu onse 11 a zitsanzo za seramu mu kafukufukuyu adasanthulidwa ndi LC-MS. Magawo a chisakanizo cha seramu pool kuchokera kwa opereka 100 athanzi adagwiritsidwa ntchito ngati zinthu zofotokozera m'magulu osiyanasiyana kuti ayang'anire njira yochotsera ndikusintha zotsatira za gulu lililonse. Kusanthula kwa metabolomics kosalunjika kwa gulu lopeza, gulu lotsimikizira mkati, ndi gulu lotsimikizira lakunja kunachitika ku Metabolomics Center of Sun Yat-sen University. Laboratory yakunja ya Guangdong University of Technology Analysis and Testing Center idasanthulanso zitsanzo 40 kuchokera ku gulu lakunja kuti ayesere momwe chitsanzo cha classifier chikuyendera.
Pambuyo pochotsa ndi kukonzanso, kuchuluka kwathunthu kwa ma metabolites a m'magazi kunayesedwa pogwiritsa ntchito ultra-high performance liquid chromatography-tandem mass spectrometry (Agilent 6495 triple quadrupole) ndi electrospray ionization (ESI) source mu multiple reaction monitoring (MRM) mode. ACQUITY BEH Amide column (2.1 × 100 mm, 1.7 μm, Waters) idagwiritsidwa ntchito kulekanitsa ma metabolites. Gawo loyenda linali ndi 90% (A) ndi 5% acetonitrile (B) yokhala 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. Mzati unasungidwa pa 40 °C ndipo chitsanzocho chinasungidwa pa 10 °C mu autosampler. Kuthamanga kwa madzi kunali 0.3 mL/min ndipo voliyumu ya jakisoni inali 1 μL. Ma parameter a MS adakhazikitsidwa motere: capillary voltage ±3.5 kV, nebulizer pressure 35 psi, sheath gas flow 12 L/min, sheath gas temperature 350°C, pressure gas temperature 250°C, ndi pressure gas flow 14 l/min. Kusintha kwa MRM kwa tryptophan, pyruvate, lactate, hypoxanthine ndi xanthine kunali 205.0–187.9, 87.0–43.4, 89.0–43.3, 135.0–92.3 ndi 151.0–107. 9 motsatana. Deta idasonkhanitsidwa pogwiritsa ntchito Mass Hunter B.07.00 (Agilent Technologies). Pa zitsanzo za m'magazi, tryptophan, pyruvate, lactate, hypoxanthine, ndi xanthine zinayesedwa pogwiritsa ntchito ma calibration curves a njira zosakaniza zokhazikika. Pa zitsanzo za m'maselo, kuchuluka kwa tryptophan kunasinthidwa kukhala muyezo wamkati ndi kuchuluka kwa mapuloteni a m'maselo.
Kuchotsa kwa Peak (m/z ndi nthawi yosungira (RT)) kunachitika pogwiritsa ntchito Compound Discovery 3.1 ndi TraceFinder 4.0 (Thermo Fisher Scientific). Pofuna kuthetsa kusiyana komwe kulipo pakati pa magulu, chiŵerengero chilichonse cha khalidwe la chitsanzo choyesera chinagawidwa ndi chiŵerengero cha khalidwe cha zinthu zofotokozera kuchokera ku gulu lomwelo kuti mupeze kuchuluka koyerekeza. Kupatuka kwa muyezo woyerekeza kwa miyezo yamkati isanayambe komanso itatha kukhazikika kwawonetsedwa mu Supplementary Table 6. Kusiyana pakati pa magulu awiriwa kunadziwika ndi false discovery rate (FDR<0.05, Wilcoxon signed rank test) ndi fold change (>1.2 kapena <0.83). Raw MS deta ya zinthu zochotsedwa ndi deta ya MS yokonzedwa ndi serum ikuwonetsedwa mu Supplementary Data 1 ndi Supplementary Data 2, motsatana. Peak annotation idachitika kutengera magawo anayi odziwika, kuphatikiza metabolites odziwika, mankhwala olembedwa, magulu a compound odziwika, ndi mankhwala osadziwika 22. Kutengera ndi kusaka kwa database mu Compound Discovery 3.1 (mzCloud, HMDB, Chemspider), mankhwala achilengedwe okhala ndi miyezo yofananira ya MS/MS kapena ma annotation ofanana enieni mu mzCloud (chigoli > 85) kapena Chemspider adasankhidwa kukhala pakati pa metabolome yosiyana. Ma annotations apamwamba pa chinthu chilichonse akuphatikizidwa mu Supplementary Data 3. MetaboAnalyst 5.0 idagwiritsidwa ntchito posanthula univariate ya kuchuluka kwa metabolite komwe kumakhala koyenera. MetaboAnalyst 5.0 idawunikanso kusanthula kwa njira ya KEGG kutengera ma metabolites osiyanasiyana. Kusanthula kwa gawo lalikulu (PCA) ndi kusanthula kwa gawo laling'ono (PLS-DA) zidawunikidwa pogwiritsa ntchito pulogalamu ya ropls (v.1.26.4) yokhala ndi stack normalization ndi autoscaling. Chitsanzo chabwino kwambiri cha metabolite biomarker cholosera khansa ya nodule chidapangidwa pogwiritsa ntchito binary logistic regression yokhala ndi osachepera absolute shrinkage ndi selection operator (LASSO, R package v.4.1-3). Kugwira ntchito kwa chitsanzo chosiyanitsa m'maseti ozindikira ndi kutsimikizira kunadziwika ndi kuyerekeza AUC kutengera kusanthula kwa ROC malinga ndi phukusi la pROC (v.1.18.0.). Kuchepetsa mwayi wabwino kwambiri kunapezeka kutengera chizindikiro chachikulu cha Youden cha chitsanzocho (kukhudzidwa + kutsimikizika - 1). Zitsanzo zokhala ndi mitengo yochepera kapena yayikulu kuposa malire zidzanenedweratu ngati timibulu tosaopsa ndi adenocarcinoma ya m'mapapo, motsatana.
Maselo a A549 (#CCL-185, American Type Culture Collection) adakulitsidwa mu F-12K medium yokhala ndi 10% FBS. Ma sequence a short hairpin RNA (shRNA) omwe amalunjika ku SLC7A5 ndi nontargeting control (NC) adayikidwa mu lentiviral vector pLKO.1-puro. Ma sequence a antisense a shSLC7A5 ndi awa: Sh1 (5′-GGAGAAAACCTGAACAGTT-3′), Sh2 (5′-GCCGTGGACTTCGGGAACTAT-3′). Ma antibodies ku SLC7A5 (#5347) ndi tubulin (#2148) adagulidwa kuchokera ku Cell Signaling Technology. Ma antibodies ku SLC7A5 ndi tubulin adagwiritsidwa ntchito pochepetsa 1:1000 pa Western blot analysis.
Seahorse XF Glycolytic Stress Test imayesa kuchuluka kwa acidification ya extracellular acidification (ECAR). Mu mayesowa, shuga, oligomycin A, ndi 2-DG zinaperekedwa motsatizana kuti ziyese mphamvu ya glycolytic ya maselo monga momwe ECAR imayezera.
Maselo a A549 omwe adalowetsedwa ndi non-targeting control (NC) ndi shSLC7A5 (Sh1, Sh2) adayikidwa usiku wonse m'mbale za mainchesi 10. Ma metabolites a maselo adachotsedwa ndi 1 ml ya methanol yamadzi ozizira kwambiri ya 80%. Maselo omwe ali mu yankho la methanol adachotsedwa, adasonkhanitsidwa mu chubu chatsopano, ndikuyikidwa mu centrifuge pa 15,000 × g kwa mphindi 15 pa 4°C. Sonkhanitsani 800 µl ya supernatant ndikuumitsa pogwiritsa ntchito vacuum concentrator yothamanga kwambiri. Ma pellets a metabolite ouma adawunikidwa kuti awone kuchuluka kwa tryptophan pogwiritsa ntchito LC-MS/MS monga tafotokozera pamwambapa. Ma cell NAD(H) omwe ali m'ma cell a A549 (NC ndi shSLC7A5) adayesedwa pogwiritsa ntchito chida cha quantitative NAD+/NADH colorimetric (#K337, BioVision) malinga ndi malangizo a wopanga. Ma protein omwe ali mu chitsanzo chilichonse adayesedwa kuti azitha kusintha kuchuluka kwa metabolites.
Palibe njira zowerengera zomwe zinagwiritsidwa ntchito poyesa kukula kwa chitsanzo. Kafukufuku wakale wa metabolomics womwe cholinga chake chinali kupeza biomarker15,18 waonedwa ngati miyeso yodziwira kukula, ndipo poyerekeza ndi malipoti awa, chitsanzo chathu chinali chokwanira. Palibe zitsanzo zomwe sizinachotsedwe mu gulu la kafukufuku. Zitsanzo za seramu zinaperekedwa mwachisawawa ku gulu lopeza (milandu 306, 74.6%) ndi gulu lotsimikizira mkati (milandu 104, 25.4%) ya maphunziro a metabolomics osayang'aniridwa. Tinasankhanso mwachisawawa milandu 70 kuchokera ku gulu lililonse kuchokera ku gulu lopeza la maphunziro a metabolomics. Ofufuzawo adachititsidwa khungu kuti asawone kugawidwa kwa gulu panthawi yosonkhanitsa ndi kusanthula deta ya LC-MS. Kusanthula kwa ziwerengero za deta ya metabolomics ndi zoyeserera za maselo kumafotokozedwa m'magawo a Zotsatira, Nthano za Chithunzi, ndi Njira. Kuyeza kwa tryptophan ya maselo, NADT, ndi ntchito ya glycolytic kunachitika katatu padera ndi zotsatira zofanana.
Kuti mudziwe zambiri zokhudza kapangidwe ka kafukufukuyu, onani Chidule cha Lipoti Lachilengedwe logwirizana ndi nkhaniyi.
Deta ya MS ya zinthu zomwe zachotsedwa ndi deta ya MS yokhazikika ya seramu yowunikira zikuwonetsedwa mu Supplementary Data 1 ndi Supplementary Data 2, motsatana. Zolemba zapamwamba za zinthu zosiyana zawonetsedwa mu Supplementary Data 3. Seti ya data ya LUAD TCGA ikhoza kutsitsidwa kuchokera ku https://portal.gdc.cancer.gov/. Deta yolowera yojambulira graph yaperekedwa mu deta yoyambira. Deta yoyambira yaperekedwa munkhaniyi.
Gulu Lofufuza za Kuyezetsa Mapapu la Dziko Lonse, ndi zina zotero. Kuchepetsa imfa za khansa ya m'mapapo pogwiritsa ntchito tomography yocheperako. Northern England. J. Med. 365, 395–409 (2011).
Kramer, BS, Berg, KD, Aberle, DR ndi Prophet, PC Kuyezetsa khansa ya m'mapapo pogwiritsa ntchito CT yotsika mtengo: zotsatira kuchokera ku National Lung Screening Study (NLST). J. Med. Screen 18, 109–111 (2011).
De Koning, HJ, ndi ena. Kuchepetsa imfa za khansa ya m'mapapo pogwiritsa ntchito njira yowunikira ya CT mu kafukufuku wosankhidwa mwachisawawa. Northern England. J. Med. 382, 503–513 (2020).
Nthawi yotumizira: Sep-18-2023