Ma metabolites oteteza thupi ku matenda ndi chinthu chofunikira kwambiri pa malo ozungulira khansa (TME), koma kupatulapo ochepa, mayina awo sakudziwika bwino. Pano, tafufuza zotupa ndi maselo a T ochokera ku zotupa ndi ascites a odwala omwe ali ndi khansa ya serous carcinoma yapamwamba (HGSC) kuti tipeze metabolome ya magawo osiyanasiyana a TME. Ma Ascites ndi maselo a chotupa ali ndi kusiyana kwakukulu kwa metabolite. Poyerekeza ndi ascites, maselo a T omwe amalowa m'thupi amakhala ndi 1-methylnicotinamide (MNA). Ngakhale kuti kuchuluka kwa MNA m'maselo a T kumakwera, mawonekedwe a nicotinamide N-methyltransferase (enzyme yomwe imayambitsa kusamutsa magulu a methyl kuchokera ku S-adenosylmethionine kupita ku nicotinamide) amangopezeka ku fibroblasts ndi maselo a chotupa. Mwanjira ina, MNA imalimbikitsa maselo a T kutulutsa cytokine tumor necrosis factor alpha yomwe imalimbikitsa chotupa. Chifukwa chake, MNA yochokera ku TME imathandizira pakulamulira chitetezo cha mthupi kwa maselo a T ndipo imayimira cholinga cha immunotherapy chochizira khansa ya anthu.
Ma metabolites ochokera ku chotupa amatha kukhala ndi mphamvu yoletsa kwambiri chitetezo cha mthupi cholimbana ndi chotupa, ndipo umboni wochulukirapo ukuwonetsa kuti amathanso kukhala mphamvu yofunikira kwambiri yopititsira patsogolo matenda (1). Kuphatikiza pa Warburg effect, ntchito yaposachedwa yayamba kufotokoza momwe maselo a chotupa amagwirira ntchito komanso ubale wake ndi chitetezo cha mthupi cha chotupa microenvironment (TME). Kafukufuku pa zitsanzo za mbewa ndi maselo a T a anthu awonetsa kuti kagayidwe ka glutamine (2), kagayidwe ka okosijeni (3) ndi kagayidwe ka shuga (4) zimatha kugwira ntchito pawokha pamagulu osiyanasiyana a maselo a chitetezo cha mthupi. Ma metabolites angapo m'njira izi amaletsa ntchito yolimbana ndi chotupa ya maselo a T. Zatsimikiziridwa kuti kutsekeka kwa coenzyme tetrahydrobiopterin (BH4) kumatha kuwononga kuchuluka kwa maselo a T, ndipo kuwonjezeka kwa BH4 m'thupi kumatha kuwonjezera chitetezo cha mthupi cholimbana ndi chotupa chomwe chimayendetsedwa ndi CD4 ndi CD8. Kuphatikiza apo, mphamvu yoletsa chitetezo cha mthupi ya kynurenine ikhoza kupulumutsidwa ndi kupereka BH4 (5). Mu isocitrate dehydrogenase (IDH) mutant glioblastoma, kutulutsa kwa enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kumaletsa kuyambitsa, kuchulukana ndi ntchito ya cytolysis ya maselo a T (6). Posachedwapa, zawonetsedwa kuti methylglyoxal, yomwe imachokera ku glycolysis, imapangidwa ndi maselo oletsa omwe amachokera ku myeloid, ndipo kusamutsa maselo a T kwa methylglyoxal kumatha kuletsa ntchito ya maselo a T. Pa chithandizo, kuletsa kwa methylglyoxal kumatha kugonjetsa ntchito ya maselo oletsa omwe amachokera ku myeloid (MDSC) ndikuwonjezera chithandizo cha checkpoint blockade mu zitsanzo za mbewa (7). Maphunziro awa pamodzi akugogomezera gawo lofunika la metabolites yochokera ku TME pakulamulira ntchito ndi ntchito ya maselo a T.
Kusagwira bwino ntchito kwa maselo a T kwafotokozedwa kwambiri mu khansa ya m'chiberekero (8). Izi zimachitika chifukwa cha makhalidwe a kagayidwe kachakudya omwe amapezeka mu hypoxia ndi mitsempha yamagazi yolakwika (9), zomwe zimapangitsa kuti shuga ndi tryptophan zisinthe kukhala zinthu zina monga lactic acid ndi kynurenine. Kuchuluka kwa lactate yakunja kwa thupi kumachepetsa kupanga kwa interferon-γ (IFN-γ) ndipo kumapangitsa kusiyana kwa magulu ang'onoang'ono a myelosuppressive (10, 11). Kugwiritsa ntchito tryptophan kumaletsa mwachindunji kuchuluka kwa maselo a T ndikuletsa chizindikiro cha maselo a T (12-14). Ngakhale kuti izi zawonedwa, ntchito yambiri yokhudzana ndi kagayidwe ka chitetezo cha mthupi idachitika mu in vitro T cell culture pogwiritsa ntchito media yabwino, kapena kungokhala pa zitsanzo za mbewa zofanana mu vivo, zomwe sizikuwonetsa kwathunthu kusiyana kwa khansa ya anthu ndi chilengedwe cha macro ndi micro.
Chizindikiro chofala cha khansa ya m'chiberekero ndi kufalikira kwa peritoneal ndi kuwoneka kwa ascites. Kuchulukana kwa madzi m'maselo mu ascites kumalumikizidwa ndi matenda opita patsogolo komanso kusakhala bwino kwa matenda (15). Malinga ndi malipoti, gawo lapaderali ndi lopanda poizoni, lili ndi milingo yayikulu ya vascular endothelial growth factor (VEGF) ndi indoleamine 2,3-dioxygenase (IDO), ndipo limalowetsedwa ndi maselo olamulira a T ndi maselo oletsa myeloid (15-18). Malo ogwiritsira ntchito kagayidwe kachakudya a ascites akhoza kukhala osiyana ndi a chotupacho, kotero kukonzanso maselo a T m'malo ozungulira sikudziwika bwino. Kuphatikiza apo, kusiyana kwakukulu ndi kusiyana pakati pa ascites ndi metabolites zomwe zili m'malo otupa zitha kulepheretsa kulowa kwa maselo a chitetezo chamthupi ndi ntchito yawo pa zotupa, ndipo kafukufuku wowonjezereka ukufunika.
Pofuna kuthetsa mavutowa, tinapanga njira yodziwira kusiyana kwa maselo ndi madzi a chromatography tandem mass spectrometry (LC-MS/MS) kuti tiphunzire mitundu yosiyanasiyana ya maselo (kuphatikiza maselo a CD4 + ndi CD8 + T) komanso mkati ndi pakati pa zotupa. Ma metabolites ake amazungulira maselo omwewo m'malo omwewo a ascites ndi malo otupa a wodwala. Timagwiritsa ntchito njira iyi limodzi ndi high-dimensional flow cytometry ndi single-cell RNA sequencing (scRNA-seq) kuti tipereke chithunzi chotsimikizika cha momwe kagayidwe kachakudya ka anthu ofunikirawa alili. Njirayi idawonetsa kuwonjezeka kwakukulu kwa kuchuluka kwa 1-methylnicotinamide (MNA) m'maselo a T otupa, ndipo zoyeserera za in vitro zidawonetsa kuti mphamvu ya MNA yowongolera chitetezo chamthupi pa ntchito ya maselo a T sinali yodziwika kale. Kawirikawiri, njira iyi ikuwonetsa kuyanjana kwa kagayidwe kachakudya pakati pa zotupa ndi maselo oteteza chitetezo chamthupi, ndipo imapereka chidziwitso chapadera cha ma metabolites olamulira chitetezo chamthupi, omwe angakhale othandiza pochiza khansa ya ovarian yochokera ku maselo a T.
Tinagwiritsa ntchito njira yoyezera kuchuluka kwa shuga m'magazi kuti nthawi imodzi tiyese kuchuluka kwa shuga m'magazi [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) ndi ntchito ya mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) ndi zizindikiro zomwe zimasiyanitsa maselo oteteza thupi ndi maselo otupa (Table S2 ndi Chithunzi S1A). Kusanthula kumeneku kunawonetsa kuti poyerekeza ndi maselo a T, ascites ndi maselo otupa ali ndi kuchuluka kwa shuga m'magazi, koma ali ndi kusiyana kochepa mu ntchito ya mitochondrial. Kuchuluka kwa shuga m'magazi [CD45-EpCAM (EpCAM)+] ndi katatu kapena kanayi kuposa maselo a T, ndipo kuchuluka kwa shuga m'magazi a CD4 + T ndi nthawi 1.2 kuposa maselo a CD8 + T, zomwe zikusonyeza kuti ma lymphocyte olowa m'magazi (TIL) ali ndi zofunikira zosiyanasiyana za kagayidwe kachakudya ngakhale mu TME yomweyo (Chithunzi 1A). Mosiyana ndi zimenezi, ntchito ya mitochondrial m'maselo a chotupa ndi yofanana ndi ya maselo a CD4 + T, ndipo ntchito ya mitochondrial ya mitundu yonse iwiri ya maselo ndi yokwera kuposa ya maselo a CD8 + T (Chithunzi 1B). Kawirikawiri, zotsatirazi zikuwonetsa kuchuluka kwa kagayidwe kachakudya. Ntchito ya kagayidwe kachakudya ya maselo a chotupa ndi yokwera kuposa ya maselo a CD4 + T, ndipo ntchito ya kagayidwe kachakudya ya maselo a CD4 + T ndi yokwera kuposa ya maselo a CD8 + T. Ngakhale kuti zotsatirazi zimachitika m'mitundu yonse ya maselo, palibe kusiyana kokhazikika pa momwe maselo a CD4 + ndi CD8 + T amagwirira ntchito kapena kuchuluka kwawo mu ascites poyerekeza ndi zotupa (Chithunzi 1C). Mosiyana ndi zimenezi, mu gawo la maselo a CD45, kuchuluka kwa maselo a EpCAM + mu chotupacho kunawonjezeka poyerekeza ndi ascites (Chithunzi 1D). Tinaonanso kusiyana komveka bwino kwa kagayidwe kachakudya pakati pa zigawo za maselo a EpCAM+ ndi EpCAM. Maselo a EpCAM+ (chotupa) ali ndi shuga wambiri komanso ntchito ya mitochondrial kuposa maselo a EpCAM, zomwe ndi zapamwamba kwambiri kuposa ntchito ya kagayidwe kachakudya ya ma fibroblast m'maselo a chotupa mu TME (Chithunzi 1, E ndi F).
(A ndi B) Mphamvu yapakati ya kuwala kwa glucose (MFI) ya kuchuluka kwa shuga m'magazi (2-NBDG) (A) ndi ntchito ya mitochondrial ya maselo a CD4 + T (MitoTracker wofiira wakuda) (B) Ma graph oyimira (kumanzere) ndi deta yolembedwa (Kumanja), maselo a CD8 + T ndi maselo a EpCAM + CD45-chotupa ochokera ku ascites ndi chotupa. (C) Chiŵerengero cha maselo a CD4 + ndi CD8 + (a maselo a CD3 + T) mu ascites ndi chotupa. (D) Kuchuluka kwa maselo a chotupa a EpCAM + mu ascites ndi chotupa (CD45−). (E ndi F) EpCAM + CD45-chotupa ndi EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) ndi ntchito ya mitochondrial (MitoTracker wofiira wakuda) (F) ma graph oyimira (kumanzere) ndi deta yolembedwa (Kumanja) Ma Ascites ndi maselo a chotupa. (G) Ma graph oyimira a ma CD25, CD137 ndi PD1 expression ndi flow cytometry. (H ndi I) Kuwonetsedwa kwa CD25, CD137 ndi PD1 pa maselo a CD4 + T (H) ndi maselo a CD8 + T (I). (J ndi K) Mitundu ya Naive, memory central (Tcm), effector (Teff) ndi effector memory (Tem) kutengera kuwonetsedwa kwa CCR7 ndi CD45RO. Zithunzi zoyimira (kumanzere) ndi deta ya tabular (kumanja) ya maselo a CD4 + T (J) ndi maselo a CD8 + T (K) mu ascites ndi zotupa. Ma P values ​​​​amatsimikiziridwa ndi mayeso a t-paired (*P<0.05, **P<0.01 ndi ***P<0.001). Mzerewu umayimira odwala ofanana (n = 6). FMO, fluorescence minus one; MFI, mphamvu yapakati ya fluorescence.
Kusanthula kwina kwawonetsa kusiyana kwina kwakukulu pakati pa mkhalidwe wa maselo a T omwe ali ndi vuto lalikulu. Kukumbukira koyambitsidwa (Chithunzi 1, G mpaka I) ndi kukumbukira kwa effector (Chithunzi 1, J ndi K) m'matenda kumachitika pafupipafupi kuposa ascites (chiwerengero cha maselo a CD3 + T). Mofananamo, kusanthula phenotype ndi kuwonetsa zizindikiro zoyambitsa (CD25 ndi CD137) ndi zizindikiro zochepetsera [puloteni ya kufa kwa maselo yokonzedwa 1 (PD1)] kunawonetsa kuti ngakhale makhalidwe a kagayidwe kachakudya a anthuwa ndi osiyana (Chithunzi S1, B mpaka E), Koma palibe kusiyana kwakukulu kwa kagayidwe kachakudya komwe kunawonedwa nthawi zonse pakati pa magulu a naive, effector kapena memory (Chithunzi S1, F mpaka I). ​​Zotsatirazi zinatsimikiziridwa pogwiritsa ntchito njira zophunzirira makina kuti zigawire maselo okha (21), zomwe zinawonetsanso kupezeka kwa maselo ambiri a m'mafupa (CD45 + / CD3- / CD4 + / CD45RO +) mu ascites a wodwalayo (Chithunzi S2A). Pakati pa mitundu yonse ya maselo omwe adadziwika, kuchuluka kwa maselo a myeloid awa kunawonetsa kuchuluka kwa shuga m'magazi komanso ntchito ya mitochondrial (Chithunzi S2, B mpaka G). Zotsatirazi zikuwonetsa kusiyana kwakukulu kwa kagayidwe kachakudya pakati pa mitundu yambiri ya maselo yomwe imapezeka mu ascites ndi zotupa mwa odwala a HGSC.
Vuto lalikulu pakumvetsetsa mawonekedwe a TIL ndikufunika kopatula zitsanzo za maselo a T omwe ali oyera mokwanira, abwino komanso ambiri kuchokera ku zotupa. Kafukufuku waposachedwapa wasonyeza kuti njira zosankhidwira ndi kukulitsa mikanda kutengera flow cytometry zitha kubweretsa kusintha kwa ma metabolite a maselo (22-24). Kuti tithetse vutoli, takonza njira yowonjezerera mikanda kuti tipewe ndikupatula TIL kuchokera ku khansa ya ovarian ya anthu yochotsedwa opaleshoni isanayesedwe ndi LC-MS/MS (onani Zipangizo ndi Njira; Chithunzi 2A). Kuti tiwone momwe protocol iyi imakhudzira kusintha kwa metabolite, tinayerekeza ma metabolite a maselo a T omwe amayendetsedwa ndi opereka athanzi pambuyo pa sitepe yolekanitsa mikanda pamwambapa ndi maselo omwe sanalekanitsidwe mikanda koma anakhalabe pa ayezi. Kusanthula kowongolera khalidwe kumeneku kunapeza kuti pali mgwirizano waukulu pakati pa zinthu ziwirizi (r = 0.77), ndipo kubwerezabwereza kwaukadaulo kwa gulu la ma metabolite 86 kuli ndi kubwerezabwereza kwakukulu (Chithunzi 2B). Choncho, njira zimenezi zimatha kuchita kusanthula kolondola kwa metabolite m'maselo omwe akuchulukitsidwa ndi mtundu wa maselo, motero kupereka njira yoyamba yodziwira metabolite inayake mu HGSC, motero zimathandiza anthu kumvetsetsa bwino za kapangidwe ka maselo. Pulogalamu ya kagayidwe ka kugonana.
(A) Chithunzi cha schematic cha magnetic bead enrichment. LC-MS/MS isanasanthule, maselo adzadutsa maulendo atatu otsatizana a magnetic bead enrichment kapena adzakhalabe pa ayezi. (B) Zotsatira za mtundu wa schematic pa kuchuluka kwa metabolites. Avereji ya miyeso itatu ya mtundu uliwonse wa schematic ± SE. Mzere wa imvi ukuyimira ubale wa 1:1. Inter-class correlation (ICC) ya miyeso yobwerezabwereza yomwe yawonetsedwa mu axis label. NAD, nicotinamide adenine dinucleotide. (C) Chithunzi cha schematic cha kayendedwe ka metabolite ya wodwala. Ascites kapena zotupa zimasonkhanitsidwa kuchokera kwa odwala ndikusungidwa. Gawo laling'ono la chitsanzo chilichonse linasanthulidwa ndi flow cytometry, pomwe zitsanzo zotsalazo zinadutsa maulendo atatu a schematic a CD4+, CD8+ ndi CD45- cells. Magawo a maselo awa adasanthulidwa pogwiritsa ntchito LC-MS/MS. (D) Mapu otentha a kuchuluka kwa metabolite kokhazikika. Dendrogram ikuyimira magulu a Ward a mtunda wa Euclidean pakati pa zitsanzo. (E) Kusanthula kwakukulu kwa zigawo (PCA) za mapu a metabolite, kuwonetsa makope atatu a chitsanzo chilichonse, zitsanzo kuchokera kwa wodwala yemweyo zimalumikizidwa ndi mzere. (F) PCA ya mbiri ya metabolite ya chitsanzocho yokhazikika pa wodwalayo (mwachitsanzo, pogwiritsa ntchito kuchulukitsa pang'ono); mtundu wa chitsanzo umachepetsedwa ndi chipolopolo chozungulira. PC1, gawo lalikulu 1; PC2, gawo lalikulu 2.
Kenako, tinagwiritsa ntchito njira yowonjezerayi pofufuza ma metabolites 99 mu maselo a CD4+, CD8+ ndi CD45 m'maselo oyambira a ascites ndi zotupa za odwala asanu ndi mmodzi a HGSC (Chithunzi 2C, Chithunzi S3A ndi Table S3 ndi S4). Chiwerengero cha anthu omwe ali ndi chidwi chimawerengera 2% mpaka 70% ya zitsanzo zazikulu zoyambirira za maselo amoyo, ndipo kuchuluka kwa maselo kumasiyana kwambiri pakati pa odwala. Pambuyo polekanitsa mikanda, gawo lowonjezera la chidwi (CD4+, CD8+ kapena CD45-) limawerengera oposa 85% ya maselo onse amoyo mu chitsanzocho pa avareji. Njira yowonjezerayi imatithandiza kusanthula kuchuluka kwa maselo kuchokera ku kagayidwe ka minofu ya chotupa cha anthu, zomwe sizingatheke kuchokera ku zitsanzo zazikulu. Pogwiritsa ntchito njira iyi, tinapeza kuti l-kynurenine ndi adenosine, ma metabolites awiriwa odziwika bwino oletsa chitetezo chamthupi anali okwera m'maselo a T otupa kapena maselo otupa (Chithunzi S3, B ndi C). Chifukwa chake, zotsatirazi zikuwonetsa kukhulupirika ndi kuthekera kwa ukadaulo wathu wolekanitsa maselo ndi mass spectrometry kuti tipeze ma metabolites ofunikira m'thupi m'maselo a odwala.
Kusanthula kwathu kunawonetsanso kulekanitsidwa kwakukulu kwa kagayidwe ka maselo mkati ndi pakati pa odwala (Chithunzi 2D ndi Chithunzi S4A). Makamaka, poyerekeza ndi odwala ena, wodwala 70 anasonyeza makhalidwe osiyanasiyana a kagayidwe ka maselo (Chithunzi 2E ndi Chithunzi S4B), zomwe zikusonyeza kuti pakhoza kukhala kusiyana kwakukulu kwa kagayidwe ka maselo pakati pa odwala. Ndikofunikira kudziwa kuti poyerekeza ndi odwala ena (malita 1.2 mpaka 2; Table S1), kuchuluka konse kwa ascites komwe kunasonkhanitsidwa mwa wodwala 70 (80 ml) kunali kochepa. Kulamulira kusiyana kwa odwala pakati pa odwala panthawi yosanthula zigawo zazikulu (mwachitsanzo, pogwiritsa ntchito kusanthula pang'ono kwa kuchuluka kwa maselo) kumasonyeza kusintha kosalekeza pakati pa mitundu ya maselo, ndipo mitundu ya maselo ndi/kapena malo ozungulira maselo amaphatikizidwa bwino malinga ndi mawonekedwe a metabolite (Chithunzi 2F). Kusanthula kwa ma metabolite amodzi kunagogomezera zotsatirazi ndipo kunawonetsa kusiyana kwakukulu pakati pa mitundu ya maselo ndi malo ozungulira maselo. Ndikofunikira kudziwa kuti kusiyana kwakukulu komwe kumawoneka ndi MNA, yomwe nthawi zambiri imakhala ndi ma CD45- maselo ndi ma CD4+ ndi ma CD8+ maselo omwe amalowa m'chotupa (Chithunzi 3A). Kwa maselo a CD4 +, izi ndizodziwikiratu, ndipo MNA m'maselo a CD8 + ikuwoneka kuti imakhudzidwa kwambiri ndi chilengedwe. Komabe, izi sizofunikira, chifukwa odwala atatu okha mwa asanu ndi mmodzi ndi omwe angayesedwe kuti apeze kuchuluka kwa CD8+ ya chotupa. Kuphatikiza pa MNA, m'mitundu yosiyanasiyana ya maselo mu ascites ndi zotupa, ma metabolites ena omwe samadziwika bwino mu TIL nawonso ndi olemera mosiyana (Zithunzi S3 ndi S4). Chifukwa chake, deta iyi ikuwonetsa gulu lodalirika la ma metabolites owongolera chitetezo chamthupi kuti afufuze kwambiri.
(A) Kuchuluka kwa MNA mu maselo a CD4+, CD8+ ndi CD45- kuchokera ku ascites ndi chotupa. Chithunzi cha bokosilo chikuwonetsa pakati (mzere), interquartile range (frame hinge) ndi data range, mpaka kuwirikiza 1.5 kuposa interquartile range (frame whisker). Monga tafotokozera mu Patient Materials and Methods, gwiritsani ntchito limma value ya wodwalayo kuti mudziwe P value (*P<0.05 ndi **P<0.01). (B) Schematic diagram ya kagayidwe ka MNA (60). Metabolites: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl-2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Ma Enzymes (obiriwira): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins; NAMPT, nicotinamide phosphoribosyl transferase; AOX1, aldehyde oxidase 1; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase; Pnp1, purine nucleoside phosphorylase. (C) t-SNE ya scRNA-seq ya ascites (imvi) ndi chotupa (chofiira; n = odwala 3). (D) Kuwonetsedwa kwa NNMT m'magulu osiyanasiyana a maselo omwe apezeka pogwiritsa ntchito scRNA-seq. (E) Kuwonetsedwa kwa NNMT ndi AOX1 mu SK-OV-3, impso ya mwana wosabadwayo (HEK) 293T, maselo a T ndi maselo a T omwe amathandizidwa ndi MNA. Kuwonetsedwa kopindika kukuwonetsedwa poyerekeza ndi SK-OV-3. Kapangidwe ka mawu ndi SEM kakuwonetsedwa (n = opereka 6 athanzi). Ma Ct opitilira 35 amaonedwa kuti ndi osapezeka (UD). (F) Kuwonekera kwa SLC22A1 ndi SLC22A2 mu SK-OV-3, HEK293T, maselo a T ndi maselo a T omwe amathandizidwa ndi 8mM MNA. Kuwonekera kopindika kukuwonetsedwa poyerekeza ndi SK-OV-3. Kapangidwe ka mawonekedwe ndi SEM kakuwonetsedwa (n = 6 opereka thanzi). Ma Ct opitilira 35 amaonedwa kuti ndi osawoneka (UD). (G) Kuchuluka kwa MNA m'maselo a T opereka thanzi omwe ayambitsidwa pambuyo pa maola 72 ophatikizidwa ndi MNA. Kapangidwe ka mawonekedwe ndi SEM kakuwonetsedwa (n = 4 opereka thanzi).
MNA imapangidwa mwa kusamutsa gulu la methyl kuchokera ku S-adenosyl-1-methionine (SAM) kupita ku nicotinamide (NA) ndi nicotinamide N-methyltransferase (NNMT; Chithunzi 3B). NNMT imafalikira kwambiri m'mitundu yosiyanasiyana ya khansa ya anthu ndipo imagwirizanitsidwa ndi kuchulukana, kulowa m'thupi komanso kufalikira kwa maselo (25-27). Kuti timvetse bwino komwe MNA imachokera m'maselo a T mu TME, tidagwiritsa ntchito scRNA-seq kuti tifotokoze momwe NNMT imaonekera m'mitundu yonse ya maselo mu ascites ndi zotupa za odwala atatu a HGSC (Table S5). Kusanthula kwa maselo pafupifupi 6,500 kunawonetsa kuti m'malo otupa ndi m'malo otupa, kufotokozera kwa NNMT kunali kokha kwa anthu omwe akuganiziridwa kuti ndi fibroblast ndi maselo otupa (Chithunzi 3, C ndi D). Ndikofunikira kudziwa kuti palibe kufotokozera kwa NNMT koonekeratu mu gulu lililonse lomwe limafotokoza PTPRC (CD45 +) (Chithunzi 3D ndi Chithunzi S5A), zomwe zikusonyeza kuti MNA yomwe yapezeka mu metabolite spectrum yalowetsedwa m'maselo a T. Kufotokozedwa kwa aldehyde oxidase 1 (AOX1) kumasintha MNA kukhala 1-methyl-2-pyridone-5-carboxamide (2-PYR) kapena 1-methyl-4-pyridone-5-carboxamide (4-PYR); Chithunzi 3B) chimangokhala pa kuchuluka kwa ma fibroblast omwe amatulutsa COL1A1 (Chithunzi S5A), zomwe pamodzi zimasonyeza kuti maselo a T alibe mphamvu ya kagayidwe ka MNA kachibadwa. Kapangidwe ka majeremusi okhudzana ndi MNA awa adatsimikiziridwa pogwiritsa ntchito deta yachiwiri yodziyimira payokha ya maselo kuchokera ku ascites kuchokera kwa odwala a HGSC (Chithunzi S5B; n = 6) (16). Kuphatikiza apo, kusanthula kwa kuchuluka kwa polymerase chain reaction (qPCR) kwa maselo athanzi a T omwe adalandira MNA kunawonetsa kuti poyerekeza ndi maselo oletsa khansa ya ovarian a SK-OV-3, NNMT kapena AOX1 sizinatchulidwe (Chithunzi 3E). Zotsatira zosayembekezereka izi zikusonyeza kuti MNA ikhoza kutulutsidwa kuchokera ku ma fibroblast kapena zotupa kupita ku maselo a T oyandikana nawo mu TME.
Ngakhale kuti anthu omwe akufuna kuyesedwa akuphatikizapo banja la anthu onyamula ma organic cation 1 mpaka 3 (OCT1, OCT2 ndi OCT3) omwe amalembedwa ndi banja la soluble carrier 22 (SLC22) (SLC22A1, SLC22A2 ndi SLC22A3), anthu onyamula ma MNA akadali osadziwika (28). QPCR ya mRNA kuchokera ku maselo a T opereka athanzi inawonetsa kuchuluka kochepa kwa SLC22A1 koma kuchuluka kosawoneka kwa SLC22A2, komwe kunatsimikizira kuti idanenedwa kale m'mabuku (Chithunzi 3F) (29). Mosiyana ndi zimenezi, mzere wa maselo a khansa ya ovarian tumor wa SK-OV-3 unawonetsa kuchuluka kwakukulu kwa anthu onyamula ma ovarian onse awiri (Chithunzi 3F).
Pofuna kuyesa kuthekera kwa maselo a T kukhala ndi mphamvu yoyamwa MNA yachilendo, maselo a T opereka athanzi adakulitsidwa kwa maola 72 pamaso pa kuchuluka kosiyanasiyana kwa MNA. Popanda MNA yakunja, kuchuluka kwa MNA m'maselo sikungathe kuzindikirika (Chithunzi 3G). Komabe, maselo a T omwe adathandizidwa ndi MNA yakunja adawonetsa kuwonjezeka kwa kuchuluka kwa MNA m'maselo kutengera mlingo, mpaka 6 mM MNA (Chithunzi 3G). Zotsatirazi zikusonyeza kuti ngakhale kuti pali kuchepa kwa transporter expression komanso kusowa kwa enzyme yayikulu yomwe imayambitsa kagayidwe ka MNA m'maselo, TIL ikhozabe kutenga MNA.
Kuchuluka kwa ma metabolites m'maselo a T a odwala komanso kuyesa kwa MNA mu vitro kumawonjezera mwayi woti ma fibroblasts (CAF) omwe amagwirizana ndi khansa amatulutsa MNA ndipo maselo otupa amatha kuwongolera mawonekedwe ndi ntchito ya TIL. Kuti adziwe momwe MNA imakhudzira maselo a T, maselo a T opereka athanzi adayambitsidwa mu vitro pomwe MNA ilipo kapena palibe, ndipo kuchulukana kwawo ndi kupanga kwa cytokine zidayesedwa. Pambuyo pa masiku 7 owonjezera MNA pa mlingo wapamwamba kwambiri, kuchuluka kwa anthu kawiri kunachepetsedwa pang'ono, pomwe mphamvu idasungidwa pamlingo wonse (Chithunzi 4A). Kuphatikiza apo, chithandizo cha MNA yakunja chinapangitsa kuti kuchuluka kwa maselo a CD4 + ndi CD8 + T omwe akuwonetsa chotupa necrosis factor-α (TNFα; Chithunzi 4B). Mosiyana ndi izi, kupanga kwa IFN-γ mkati mwa maselo kunachepa kwambiri m'maselo a CD4 + T, koma osati m'maselo a CD8 + T, ndipo panalibe kusintha kwakukulu mu interleukin 2 (IL-2; Chithunzi 4, C ndi D). Chifukwa chake, kuyesa kwa immunosorbent komwe kumalumikizidwa ndi enzyme (ELISA) kwa supernatants kuchokera ku ma cell a T omwe amathandizidwa ndi MNA kunawonetsa kuwonjezeka kwakukulu kwa TNFα, kuchepa kwa IFN-γ, komanso kusasintha kwa IL-2 (Chithunzi 4, E mpaka G). . Kuchepa kwa IFN-γ kukuwonetsa kuti MNA ikhoza kukhala ndi gawo loletsa ntchito yolimbana ndi chotupa ya maselo a T. Pofuna kutsanzira zotsatira za MNA pa cytotoxicity yothandizidwa ndi maselo a T, maselo a chimeric antigen receptor T (FRα-CAR-T) omwe amalunjika ku folate receptor α ndi CAR-T (GFP) omwe amayendetsedwa ndi maselo a green fluorescent protein (GFP) -CAR-T) amapangidwa ndi maselo athanzi a mononuclear amagazi operekedwa ndi donor (PBMC). Maselo a CAR-T adakulitsidwa kwa maola 24 pamaso pa MNA, kenako adakulitsidwa pamodzi ndi maselo a khansa ya ovarian a SK-OV-3 omwe akuwonetsa folate receptor α pa chiŵerengero cha effector to target cha 10:1. Chithandizo cha MNA chinapangitsa kuti ntchito yopha maselo a FRα-CAR-T ichepe kwambiri, zomwe zinali zofanana ndi maselo a FRα-CAR-T omwe adalandira adenosine (Chithunzi 4H).
(A) Chiwerengero chonse cha maselo otheka komanso kuchuluka kwa anthu (PD) mwachindunji kuchokera ku chikhalidwe pa tsiku la 7. Grafu ya bar ikuyimira avereji + SEM ya opereka asanu ndi mmodzi athanzi. Imayimira deta kuchokera ku zoyeserera zosachepera n = 3 zodziyimira pawokha. (B mpaka D) CD3/CD28 ndi IL-2 zidagwiritsidwa ntchito kuyambitsa maselo a T pamlingo wawo wa MNA kwa masiku 7. Asanayesedwe, maselo adalimbikitsidwa ndi PMA/ionomycin ndi GolgiStop kwa maola 4. Kuwonetsedwa kwa TNFα (B) m'maselo a T. Chithunzi chachitsanzo (kumanzere) ndi deta ya tabular (kumanja) ya kuwonetsedwa kwa TNFα m'maselo amoyo. Kuwonetsedwa kwa IFN-γ (C) ndi IL-2 (D) m'maselo a T. Kuwonetsedwa kwa ma cytokines kunayesedwa ndi flow cytometry. Grafu ya bar ikuyimira avereji (n = 6 opereka athanzi) + SEM. Gwiritsani ntchito kusanthula kwa njira imodzi kwa kusiyana ndi miyeso yobwerezabwereza (*P<0.05 ndi **P<0.01) kuti mudziwe mtengo wa P. Imayimira deta kuchokera ku zoyeserera zosachepera n = 3 zodziyimira pawokha. (E mpaka G) CD3/CD28 ndi IL-2 zinagwiritsidwa ntchito kuyambitsa maselo a T pa kuchuluka kwawo kwa MNA kwa masiku 7. Chomeracho chinasonkhanitsidwa maola 4 asanayambe komanso atatha kusonkhezera PMA/ionomycin. Kuchuluka kwa TNFα (E), IFN-γ (F) ndi IL-2 (G) kunayesedwa ndi ELISA. Grafu ya bar ikuyimira avareji (n = 5 opereka thanzi labwino) + SEM. P value idatsimikiziridwa pogwiritsa ntchito kusanthula kwa njira imodzi kwa kusiyana ndi kuyeza mobwerezabwereza (*P<0.05). Mzere wokhala ndi madontho umasonyeza malire ozindikira a kuzindikira. (H) Kuyesa kwa Cell lysis. Maselo a FRα-CAR-T kapena GFP-CAR-T adasinthidwa ndi adenosine (250μM) kapena MNA (10 mM) kwa maola 24, kapena osachiritsidwa (Ctrl). Kupha kwa maselo a SK-OV-3 kunayesedwa. P value idatsimikiziridwa ndi mayeso a Welch t (*P<0.5 ndi **P<0.01).
Pofuna kumvetsetsa bwino momwe TNFα imagwirira ntchito mogwirizana ndi MNA, kusintha kwa TNFα mRNA ya maselo a T omwe amathandizidwa ndi MNA kunayesedwa (Chithunzi 5A). Maselo a T omwe amapereka chithandizo chathanzi omwe amathandizidwa ndi MNA adawonetsa kuwonjezeka kawiri kwa kuchuluka kwa TNFα transcription, zomwe zikusonyeza kuti MNA imadalira TNFα transcriptional regulation. Kuti afufuze njira yowongolera iyi, zinthu ziwiri zodziwika bwino zomwe zimalamulira TNFα, zomwe ndi activated T cell nuclear factor (NFAT) ndi protein 1 (Sp1), zidawunikidwa poyankha MNA yomangirira ku proximal TNFα promoter (30). TNFα promoter ili ndi malo 6 odziwika a NFAT binding ndi malo awiri omangira a Sp1, omwe amalumikizana pamalo amodzi [-55 base pairs (bp) kuchokera ku 5'cap] (30). Chromatin immunoprecipitation (ChIP) idawonetsa kuti ikalandira chithandizo ndi MNA, kumangirira kwa Sp1 ku TNFα promoter kunawonjezeka katatu. Kuphatikizidwa kwa NFAT kunakulanso ndi kufunika koyandikira (Chithunzi 5B). Deta iyi ikusonyeza kuti MNA imayang'anira momwe TNFα imagwiritsidwira ntchito kudzera mu Sp1 transcription, ndipo pang'ono momwe NFAT imagwiritsidwira ntchito.
(A) Poyerekeza ndi maselo a T omwe adakulitsidwa popanda MNA, kusintha kwa mawonekedwe a TNFα m'maselo a T omwe adachiritsidwa ndi MNA. Kapangidwe ka mawonekedwe ndi SEM kakuwonetsedwa (n = 5 opereka athanzi). Kuyimira deta kuchokera ku zoyeserera zosachepera n = 3 zodziyimira pawokha. (B) Chothandizira TNFα cha maselo a T chomwe chidachiritsidwa ndi kapena popanda 8 mM MNA pambuyo pa NFAT ndi Sp1 zidaphatikizidwa ndi (Ctrl) ndi PMA/ionomycin stimulation kwa maola 4. Immunoglobulin G (IgG) ndi H3 zidagwiritsidwa ntchito ngati zowongolera zoyipa komanso zabwino za immunoprecipitation, motsatana. Kuyeza kwa ChIP kunawonetsa kuti kumangirira kwa Sp1 ndi NFAT ku chothandizira TNFα m'maselo omwe adachiritsidwa ndi MNA kudakwera kangapo poyerekeza ndi chowongolera. Kuyimira deta kuchokera ku zoyeserera zosachepera n = 3 zodziyimira pawokha. P value yodziwika ndi mayeso angapo a t (*** P <0.01). (C) Poyerekeza ndi ascites a HGSC, maselo a T (osakhala ndi cytotoxic) adawonetsa kuwonjezeka kwa mawonekedwe a TNF mu chotupacho. Mitunduyo imayimira odwala osiyanasiyana. Maselo omwe awonetsedwa ayesedwa mwachisawawa kufika pa 300 ndipo agwedezeka kuti achepetse kuchuluka kwa maselo omwe amatengedwa (** Padj = 0.0076). (D) Chitsanzo cha MNA chomwe chaperekedwa cha khansa ya m'chiberekero. MNA imapangidwa m'maselo a chotupa ndi ma fibroblast mu TME ndipo imatengedwa ndi maselo a T. MNA imawonjezera kumangirira kwa Sp1 ku chothandizira cha TNFα, zomwe zimapangitsa kuti TNFα itulutsidwe komanso kupanga TNFα cytokine. MNA imayambitsanso kuchepa kwa IFN-γ. Kuletsa ntchito ya maselo a T kumabweretsa kuchepa kwa mphamvu yopha komanso kukula kwa chotupa mwachangu.
Malinga ndi malipoti, TNFα ili ndi zotsatira zotsutsana ndi chotupa kutsogolo ndi kumbuyo, koma ili ndi gawo lodziwika bwino pakulimbikitsa kukula ndi kufalikira kwa khansa ya m'chiberekero (31-33). Malinga ndi malipoti, kuchuluka kwa TNFα mu ascites ndi minofu ya chotupa mwa odwala khansa ya m'chiberekero ndi kokwera kuposa komwe kumakhala mu minofu yopanda poizoni (34-36). Ponena za njira, TNFα imatha kuwongolera kuyambitsa, kugwira ntchito ndi kuchuluka kwa maselo oyera amagazi, ndikusintha mtundu wa maselo a khansa (37, 38). Mogwirizana ndi zomwe zapezekazi, kusanthula kwa majini osiyanasiyana kunawonetsa kuti TNF inali yokwera kwambiri mu maselo a T mu minofu ya chotupa poyerekeza ndi ascites (Chithunzi 5C). Kuwonjezeka kwa mawonekedwe a TNF kunaonekera kokha mu magulu a maselo a T omwe ali ndi phenotype yosakhala ya cytotoxic (Chithunzi S5A). Mwachidule, deta iyi ikuthandizira lingaliro lakuti MNA ili ndi zotsatira ziwiri zoletsa chitetezo chamthupi komanso zolimbikitsa chotupa mu HGSC.
Kulemba zilembo za fluorescent kutengera flow cytometry kwakhala njira yayikulu yophunzirira kagayidwe ka TIL. Kafukufukuyu wasonyeza kuti poyerekeza ndi ma lymphocyte am'magazi kapena ma T cell ochokera ku ziwalo zachiwiri za lymphoid, ma TIL a mucosa ndi anthu ali ndi chizolowezi chachikulu chotenga shuga (4, 39) komanso kuchepa pang'onopang'ono kwa ntchito ya mitochondrial (19, 40). Ngakhale tawona zotsatira zofanana mu kafukufukuyu, chitukuko chofunikira ndikuyerekeza kagayidwe ka maselo a chotupa ndi TIL kuchokera ku minofu yofanana ya chotupa. Mogwirizana ndi malipoti ena am'mbuyomu, maselo a chotupa (CD45-EpCAM +) ochokera ku ascites ndi zotupa ali ndi glucose wambiri wotengedwa kuposa maselo a CD8 + ndi CD4 + T, zomwe zikuthandizira kuti glucose wambiri wotengedwa m'maselo a chotupa ukhoza kuyerekezeredwa ndi maselo a T. Lingaliro la mpikisano wa maselo a T. TME. Komabe, ntchito ya mitochondrial ya maselo a chotupa ndi yayikulu kuposa ya maselo a CD8 + T, koma ntchito ya mitochondrial ndi yofanana ndi ya maselo a CD4 + T. Zotsatirazi zimalimbikitsa mutu womwe ukubwera wakuti kagayidwe ka okosijeni ndi kofunikira kwa maselo a chotupa (41, 42). Amanenanso kuti maselo a CD8 + T akhoza kukhala osavuta kugwidwa ndi vuto la okosijeni kuposa maselo a CD4 + T, kapena kuti maselo a CD4 + T angagwiritse ntchito magwero a kaboni kupatula shuga kuti asunge ntchito ya mitochondrial (43, 44). Tiyenera kudziwa kuti sitinaone kusiyana kulikonse pakutengedwa kwa shuga kapena ntchito ya mitochondrial pakati pa ma CD4 + T effectors, T effector memory ndi maselo a T central memory mu ascites. Mofananamo, kusiyana kwa maselo a CD8 + T m'matenda sikukugwirizana ndi kusintha kwa kutengedwa kwa shuga, zomwe zikuwonetsa kusiyana kwakukulu pakati pa maselo a T omwe amalimidwa mu vitro ndi anthu TIL mu vivo (22). Izi zatsimikiziridwanso pogwiritsa ntchito kugawidwa kwa maselo odziyimira okha, komwe kunawonetsanso kuti maselo a CD45 + / CD3- / CD4 + / CD45RO + omwe ali ndi shuga wambiri komanso ntchito ya mitochondrial kuposa maselo a tumor ndi ambiri koma ali ndi maselo ogwira ntchito a Metabolic. Chiwerengerochi chikhoza kuyimira kuchuluka kwa maselo a myeloid suppressor kapena maselo a plasmacytoid dendritic omwe apezeka mu kusanthula kwa scRNA-seq. Ngakhale zonsezi zanenedwa kuti zachitika m'matenda a m'chiberekero mwa anthu [45], zimafunikabe ntchito yowonjezera ndikufotokozera kuchuluka kwa myeloid kumeneku.
Ngakhale njira zogwiritsira ntchito flow cytometry zitha kufotokoza kusiyana kwakukulu kwa shuga ndi kagayidwe ka okosijeni pakati pa mitundu ya maselo, ma metabolites enieni opangidwa ndi shuga kapena magwero ena a kaboni kuti agwiritsidwe ntchito mu mitochondrial metabolism mu TME sanadziwikebe. Kugawa kukhalapo kapena kusakhalapo kwa ma metabolites ku gawo la TIL loperekedwa kumafuna kuyeretsa kuchuluka kwa maselo kuchokera ku minofu yochotsedwa. Chifukwa chake, njira yathu yowonjezera maselo pamodzi ndi mass spectrometry ingapereke chidziwitso cha ma metabolites omwe amachulukitsidwa mosiyana mu maselo a T ndi kuchuluka kwa maselo otupa m'zitsanzo zofanana za odwala. Ngakhale njira iyi ili ndi ubwino kuposa kusandutsa maselo oyambitsidwa ndi fluorescence, malaibulale ena a metabolite angakhudzidwe chifukwa cha kukhazikika kwachilengedwe ndi/kapena kuchuluka kwa kusintha kwachangu (22). Komabe, njira yathu idatha kuzindikira ma metabolites awiri odziwika bwino oletsa chitetezo chamthupi, adenosine ndi kynurenine, chifukwa amasiyana kwambiri pakati pa mitundu ya zitsanzo.
Kusanthula kwathu kagayidwe ka maselo a khansa ndi mitundu ya TIL kumapereka chidziwitso chochulukirapo pa ntchito ya metabolites mu ovarian TME. Choyamba, pogwiritsa ntchito flow cytometry, tinapeza kuti panalibe kusiyana pakati pa ntchito ya mitochondrial pakati pa zotupa ndi maselo a CD4 + T. Komabe, kusanthula kwa LC-MS/MS kunavumbula kusintha kwakukulu pa kuchuluka kwa metabolites pakati pa anthuwa, zomwe zikusonyeza kuti mfundo zokhudzana ndi kagayidwe ka TIL ndi ntchito yake yonse ya kagayidwe ka maselo zimafunika kutanthauzira mosamala. Kachiwiri, MNA ndiye metabolite yokhala ndi kusiyana kwakukulu pakati pa maselo a CD45 ndi maselo a T mu ascites, osati zotupa. Chifukwa chake, kugawikana ndi malo a chotupa zitha kukhala ndi zotsatira zosiyana pa kagayidwe ka TIL, zomwe zikuwonetsa kusiyana komwe kungachitike mu microenvironment yoperekedwa. Chachitatu, kuwonetsedwa kwa enzyme yopanga MNA NNMT makamaka kumangokhala ku CAF, yomwe ndi maselo a chotupa pang'ono, koma milingo ya MNA yomwe imapezeka imapezeka mu maselo a T ochokera ku chotupa. Kuchuluka kwa NNMT mu ovarian CAF kuli ndi zotsatira zodziwika bwino zolimbikitsa khansa, chifukwa cha kulimbikitsa kagayidwe ka CAF, kulowa kwa chotupa ndi kufalikira kwa maselo (27). Ngakhale kuti mulingo wonse wa TIL ndi wochepa, kufotokozera kwa NNMT mu CAF kumagwirizana kwambiri ndi mtundu wa mesenchymal wa Cancer Genome Atlas (TCGA), womwe umagwirizanitsidwa ndi kutsika kwa kuwunika kwa matenda (27, 46, 47). Pomaliza, kufotokozera kwa enzyme AOX1 yomwe imayambitsa kuwonongeka kwa MNA kumangokhala kokha kwa anthu a CAF, zomwe zikusonyeza kuti maselo a T alibe mphamvu yogwiritsira ntchito MNA. Zotsatirazi zikuthandizira lingaliro lakuti ngakhale kuti pakufunika ntchito yowonjezera kuti zitsimikizire izi, kuchuluka kwa MNA m'maselo a T kungasonyeze kukhalapo kwa CAF microenvironment yoletsa chitetezo cha mthupi.
Popeza kuchuluka kochepa kwa ma transporter a MNA komanso kuchuluka kosawoneka kwa mapuloteni ofunikira omwe amakhudzidwa ndi kagayidwe ka MNA, kupezeka kwa MNA m'maselo a T sikunadziwike mosayembekezereka. NNMT kapena AOX1 sizinadziwike mwa kusanthula kwa scRNA-seq ndi qPCR yolunjika ya magulu awiri odziyimira pawokha. Zotsatirazi zikusonyeza kuti MNA simapangidwa ndi maselo a T, koma imatengedwa kuchokera ku TME yozungulira. Kuyesera kwa in vitro kukuwonetsa kuti maselo a T amakonda kusonkhanitsa MNA yochokera kunja.
Kafukufuku wathu wa in vitro wasonyeza kuti MNA yakunja imayambitsa kuwonekera kwa TNFα mu maselo a T ndikuwonjezera kumangirira kwa Sp1 ku chothandizira cha TNFα. Ngakhale TNFα ili ndi ntchito zotsutsana ndi chotupa komanso zotsutsana ndi chotupa, mu khansa ya ovarian, TNFα imatha kulimbikitsa kukula kwa khansa ya ovarian (31-33). Kuchepetsa TNFα mu chikhalidwe cha maselo a chotupa cha ovarian kapena kuchotsa chizindikiro cha TNFα mu zitsanzo za mbewa kungathandize kupanga cytokine yotupa yomwe imayendetsedwa ndi TNFα ndikuletsa kukula kwa chotupa (32, 35). Chifukwa chake, pankhaniyi, MNA yochokera ku TME ikhoza kugwira ntchito ngati metabolite yoyambitsa kutupa kudzera mu njira yodalira TNFα kudzera mu autocrine loop, motero imalimbikitsa kupezeka ndi kufalikira kwa khansa ya ovarian (31). Kutengera kuthekera kumeneku, kutsekeka kwa TNFα kukuphunziridwa ngati mankhwala ochiritsira khansa ya ovarian (37, 48, 49). Kuphatikiza apo, MNA imasokoneza kuopsa kwa maselo a CAR-T ku maselo a chotupa cha ovarian, kupereka umboni wina wokhudza kuletsa chitetezo chamthupi komwe kumayendetsedwa ndi MNA. Zonsezi pamodzi, zotsatirazi zikusonyeza chitsanzo chomwe zotupa ndi maselo a CAF amatulutsira MNA mu TME yakunja. Kudzera mu (i) kukulitsa kwa khansa ya ovarian yomwe imayambitsa TNF komanso (ii) kuletsa ntchito ya cytotoxic ya maselo a T yomwe imayambitsa MNA, izi zitha kukhala ndi zotsatira ziwiri za chotupa (Chithunzi 5D).
Pomaliza, pogwiritsa ntchito kuphatikiza kwa kukulitsa maselo mwachangu, kutsata kwa maselo amodzi ndi kusanthula kagayidwe kachakudya, kafukufukuyu adawulula kusiyana kwakukulu kwa immunometabolomic pakati pa zotupa ndi maselo a ascites mwa odwala a HGSC. Kusanthula kwathunthu kumeneku kunawonetsa kuti pali kusiyana kwa kuyamwa kwa shuga ndi ntchito ya mitochondrial pakati pa maselo a T, ndipo kunazindikira MNA ngati metabolite yodziyimira payokha yolamulira chitetezo chamthupi. Deta iyi imakhudza momwe TME imakhudzira kagayidwe ka maselo a T mu khansa ya anthu. Ngakhale mpikisano wachindunji wa michere pakati pa maselo a T ndi maselo a khansa wanenedwa, metabolites imathanso kugwira ntchito ngati owongolera osalunjika kuti alimbikitse kupita patsogolo kwa chotupa komanso mwina kuletsa mayankho a chitetezo chamthupi. Kufotokozera kwina kwa ntchito ya metabolites olamulira awa kungatsegule njira zina zowonjezera mayankho a chitetezo chamthupi chotsutsana ndi chotupa.
Zitsanzo za odwala ndi zambiri zachipatala zinapezedwa kudzera mu malo osungira minofu ya khansa ya BC omwe anavomerezedwa ndi Canadian Tissue Repository Network. Malinga ndi ndondomeko yovomerezedwa ndi BC Cancer Research Ethics Committee ndi University of British Columbia (H07-00463), zitsanzo zonse za odwala ndi zambiri zachipatala zinapeza chilolezo cholembedwa chodziwitsidwa kapena mwalamulo zinasiya chilolezo chawo. Zitsanzozo zimasungidwa mu BioBank yovomerezeka (BRC-00290). Makhalidwe a wodwala akuwonetsedwa mu Matebulo S1 ndi S5. Poteteza cryopreservation, scalpel imagwiritsidwa ntchito kuwononga chitsanzo cha chotupa cha wodwalayo kenako ndikuchikankhira kudzera mu fyuluta ya 100-micron kuti apeze kuyimitsidwa kwa selo limodzi. Ma ascites a wodwalayo adayikidwa mu centrifuge pa 1500 rpm kwa mphindi 10 pa 4°C kuti achotse maselowo ndikuchotsa supernatant. Maselo opezeka kuchokera ku chotupa ndi ascites adasungidwa mu 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) ndi 10% dimethyl sulfoxide. Ma suspension a single cell osungidwawa adasungunuka ndikugwiritsidwa ntchito pa metabolomics ndi metabolite determining zomwe zafotokozedwa pansipa.
Chomera chonsecho chimakhala ndi 0.22 μm yosefedwa 50:50 yowonjezera RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) yowonjezeredwa ndi 10% heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Penicillin Streptomycin (PenStrep) solution (Thermo Fisher Scientific) ndi 50 μMB-mercaptoethanol. AimV (Invitrogen) imawonjezeredwa ndi 20 mM Hepes (Thermo Fisher Scientific) ndi 2 mM l-glutamine (Thermo Fisher Scientific). Flow cytometer staining buffer inali ndi 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) yowonjezeredwa ndi 3% heat-inactivated AB human serum (Sigma). Chosungiramo zinthu zolimbitsa maselo chimapangidwa ndi 0.22μm filtered PBS ndipo chimawonjezeredwa ndi 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
Mu 37°C yonse, maselo adapakidwa utoto wa 10 nM MT DR ndi 100 μM 2-NBDG kwa mphindi 30. Kenako, maselo adapakidwa utoto wa viability eF506 pa 4°C kwa mphindi 15. Bwezeretsani maselo mu FC Block (eBioscience) ndi Brilliant Stain Buffer (BD Biosciences), sungunulani mu flow cytometer staining buffer (malinga ndi malangizo a wopanga), ndikuyika mu incubator kwa mphindi 10 kutentha kwa chipinda. Ikani ma antibodies mu maselo ndi gulu la ma antibodies (Table S2) mu flow cytometry staining buffer pa 4°C kwa mphindi 20. Bwezeretsani maselo mu flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) musanayese. Gwiritsani ntchito SpectroFlo ndi FlowJo V10 kuti mufufuze deta ya kuchuluka kwa maselo, ndikugwiritsa ntchito GraphPad Prism 8 kuti mupange deta. Mphamvu yapakati ya kuwala kwa 2-NBDG ndi MT DR idasinthidwa kukhala yofanana, kenako mayeso a t ophatikizidwa adagwiritsidwa ntchito poyesa ziwerengero kuti awerengere odwala omwe akugwirizana. Chotsani anthu onse omwe ali ndi zochitika zosakwana 40 kuchokera ku kusanthula; lowetsani mtengo wa MFI wa 1 pa ziwerengero zilizonse zoyipa musanayambe kusanthula ziwerengero ndikuwonetsa deta.
Pofuna kuwonjezera njira yogwiritsira ntchito magating pamanja ya gulu la ndondomeko pamwambapa, tagwiritsa ntchito mawu onse olembedwa ndi mtengo woletsa mawonekedwe (FAUST) (21) kuti tigawire maselo kwa anthu onse titachotsa maselo akufa mu FlowJo. Timayendetsa pamanja zotsatira zake kuti tiphatikize anthu omwe akuwoneka kuti agawidwa molakwika (kuphatikiza PD1+ ndi maselo a PD1-chotupa) ndi anthu omwe asungidwa. Chitsanzo chilichonse chili ndi avareji ya maselo opitilira 2%, kwa anthu onse 11.
Kuchepetsa kuchuluka kwa ma cell a Ficoll gradient density kunagwiritsidwa ntchito kulekanitsa PBMC ndi zinthu zolekanitsa ma leukocyte (STEMCELL Technologies). Maselo a CD8 + T anachotsedwa ku PBMC pogwiritsa ntchito CD8 MicroBeads (Miltenyi) ndipo anakulitsidwa mu medium yonse pogwiritsa ntchito TransAct (Miltenyi) kwa milungu iwiri malinga ndi malangizo a wopanga. Maselowo analoledwa kuyima kwa masiku 5 mu medium yonse yokhala ndi IL-7 (10 ng/ml; PeproTech), kenako anakonzedwanso ndi TransAct. Pa tsiku la 7, malinga ndi malangizo a wopanga, ma CD45 MicroBeads a anthu (Miltenyi) anagwiritsidwa ntchito kukulitsa maselo m'magawo atatu otsatizana. Maselowo anasankhidwa kuti ayesedwe kuti azitha kusanthula ma flow cytometry (monga tafotokozera pamwambapa), ndipo maselo miliyoni imodzi anasankhidwa katatu kuti ayesedwe ku LC-MS/MS. Zitsanzozo zinakonzedwa ndi LC-MS/MS monga tafotokozera pansipa. Tinayesa kuchuluka kwa metabolite komwe kukusowa ndi nambala ya ion ya 1,000. Chitsanzo chilichonse chimasinthidwa ndi chiwerengero chonse cha ma ion (TIC), chimasinthidwa ndi logarithmically ndikusinthidwa zokha mu MetaboAnalystR musanayese kusanthula.
Kuyimitsidwa kwa selo limodzi la wodwala aliyense kunasungunuka ndikusefedwa kudzera mu fyuluta ya 40 μm kukhala cholumikizira chonse (monga tafotokozera pamwambapa). Malinga ndi njira ya wopanga, magawo atatu otsatizana a kusankha kwabwino pogwiritsa ntchito kulekanitsa mikanda ya maginito pogwiritsa ntchito MicroBeads (Miltenyi) adagwiritsidwa ntchito kuonjezera zitsanzo za maselo a CD8+, CD4+ ndi CD45 (pa ayezi). Mwachidule, maselowo amabwezeretsedwanso mu buffer yolimbitsa maselo (monga tafotokozera pamwambapa) ndikuwerengedwa. Maselowo adayikidwa ndi mikanda ya CD8 ya anthu, mikanda ya CD4 ya anthu kapena mikanda ya CD45 ya anthu (Miltenyi) pa 4°C kwa mphindi 15, kenako adatsukidwa ndi buffer yolimbitsa maselo. Chitsanzocho chimadutsa mu LS column (Miltenyi), ndipo magawo abwino ndi oipa amasonkhanitsidwa. Kuti achepetse nthawi ndikuwonjezera gawo lobwezeretsa maselo, gawo la CD8 limagwiritsidwa ntchito pa gawo lachiwiri la kukulitsa CD4+, ndipo gawo la CD4 limagwiritsidwa ntchito pa kukulitsa CD45. Sungani yankho pa ayezi panthawi yonse yolekanitsa.
Pofuna kukonzekera zitsanzo zowunikira metabolite, maselowo anatsukidwa kamodzi ndi madzi ozizira a mchere, ndipo 1 ml ya 80% methanol inawonjezedwa ku chitsanzo chilichonse, kenako n’kuchotsedwa vortex ndikuzizidwa mu nayitrogeni yamadzimadzi. Zitsanzozo zinachitidwa maulendo atatu ozizira ndi kusungunuka ndi centrifuge pa 14,000 rpm kwa mphindi 15 pa 4°C. Supernatant yokhala ndi metabolite imasanduka nthunzi mpaka itauma. Ma metabolitewo anasungunukanso mu 50 μl ya 0.03% formic acid, kusungunuka vortex kuti isakanizidwe, kenako n’kusungunuka kuti zinyalala zichotsedwe.
Chotsani ma metabolites monga tafotokozera pamwambapa. Samutsani supernatant ku botolo lamadzimadzi lochita bwino kwambiri kuti mufufuze za metabolomics. Gwiritsani ntchito njira yochiritsira mwachisawawa kuti muchiritse chitsanzo chilichonse ndi maselo ofanana kuti mupewe zotsatira za batch. Tinachita kuwunika kwabwino kwa ma metabolites apadziko lonse omwe adasindikizidwa kale pa AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Kusanthula kwa chromatographic ndi kuphatikiza kwa malo oyambira zidachitika pogwiritsa ntchito pulogalamu ya MultiQuant version 2.1 (Applied Biosystems SCIEX).
Chiwerengero cha ma ion cha 1000 chinagwiritsidwa ntchito poyesa kuchuluka kwa metabolite komwe kulibe, ndipo TIC ya chitsanzo chilichonse inagwiritsidwa ntchito kuwerengera dera lokhazikika la metabolite iliyonse yopezeka kuti ikonze kusintha komwe kwayambitsidwa ndi kusanthula kwa zida kuchokera pakukonza zitsanzo. Pambuyo poti TIC yakhazikika, MetaboAnalystR(51) (parameter yokhazikika) imagwiritsidwa ntchito potembenuza logarithmic ndi automatic norm line scaling. Tinagwiritsa ntchito PCA yokhala ndi vegan R package kuti tichite kusanthula kofufuza kwa kusiyana kwa metabolome pakati pa mitundu ya zitsanzo, ndipo tinagwiritsa ntchito kusanthula pang'ono kwa redundancy kuti tiwunike odwala. Gwiritsani ntchito njira ya Ward kuti mupange dendrogram ya kutentha kuti musonkhanitse mtunda wa Euclidean pakati pa zitsanzo. Tinagwiritsa ntchito limma (52) pa kuchuluka kwa metabolite kokhazikika kuti tizindikire metabolites wochuluka mosiyanasiyana pamtundu wonse wa selo ndi microenvironment. Kuti tichepetse kufotokozera, timagwiritsa ntchito parameter ya gulu kuti titchule chitsanzo, ndikuganizira mitundu ya maselo mu microenvironment ngati gulu lililonse (n = magulu 6); Pa mayeso ofunikira, tinayesa katatu mobwerezabwereza pa metabolite iliyonse. Pofuna kupewa kubwerezabwereza kolakwika, wodwalayo anaphatikizidwa ngati chopinga pakupanga limma. Pofuna kuwona kusiyana kwa metabolites pakati pa odwala osiyanasiyana, tinasintha chitsanzo cha limma kuphatikiza odwala mwanjira yokhazikika. Tinanena za kufunika kwa kusiyana komwe kunanenedwa kale pakati pa mtundu wa selo ndi malo ozungulira a Padj <0.05 (Benjamini-Hochberg correction).
Pambuyo powonjezera mphamvu pogwiritsa ntchito Miltenyi Dead Cell Removal Kit (>80% viability), kusanthula kwa transcriptome ya selo limodzi kunachitika pa ascites yonse yamoyo yozizira ndi zitsanzo za chotupa pogwiritsa ntchito njira ya 10x 5′gene expression. Milandu isanu yokhala ndi zotupa zofanana ndi ascites idawunikidwa, ngakhale kuti kuchepa kwa moyo kuchokera ku chitsanzo chimodzi cha chotupa kudalepheretsa kuphatikizidwa. Kuti tipeze kusankha kosiyanasiyana kwa odwala, tidaphatikiza zitsanzo za wodwala aliyense m'mizere ya 10x chromium controller, ndikusanthula ascites ndi malo a chotupa padera. Pambuyo pofufuza [Illumina HiSeq 4000 28×98 bp paired end (PE), genome ya Quebec; Pa avareji ya kuwerenga 73,488 ndi 41,378 pa selo iliyonse ya chotupa ndi ascites motsatana], tinagwiritsa ntchito CellSNP ndi Vireo (53) (kutengera CellSNP monga SNP ya munthu wamba (VCF) yoperekedwa ndi GRCh38 imapatsidwa chizindikiritso cha wopereka. Timagwiritsa ntchito SNPRelate kuti tidziwe chizindikiritso chapafupi kwambiri (IBS) cha mkhalidwe wa genotype wa wodwalayo (IBS), kupatula maselo ndi maselo osaperekedwa omwe amadziwika ngati ma duplexes ndi opereka ofanana pakati pa ascites ndi zitsanzo za chotupa (54). Pamaziko a ntchitoyi, tinasunga milandu itatu yokhala ndi mawonekedwe ambiri a maselo mu chotupa ndi ascites kuti tiwunikenso pansi. Titachita gawo losefera mu scater (55) ndi scran (56) BioConductor packaging, izi zinapereka maselo 6975 (maselo 2792 ndi 4183 kuchokera ku chotupa ndi ascites, motsatana) kuti tiwunike. Timagwiritsa ntchito igraph's (57) Louvain clustering ya shared nearest neighbor network (SNN) kutengera Jaccard mtunda wa maselo a magulu malinga ndi momwe amafotokozera. Maguluwa adalembedwa pamanja kuti agwirizane ndi mitundu ya maselo oyerekeza kutengera momwe majini amafotokozera ndipo adawonetsedwa ndi t-SNE. Maselo a T omwe ali ndi poizoni amatanthauzidwa ndi momwe CD8A ndi GZMA zimafotokozera, kupatula magulu ang'onoang'ono omwe ali ndi mapuloteni otsika a ribosomal. Tinapeza deta yofalitsidwa ya Izar et al. (16), kuphatikizapo t-SNE embedding yawo, imatha kuwongolera momwe mafotokozedwe amagwirizanirana pakati pa ma immune cell markers ndi momwe NNMT imafotokozera.
Ma PBMC adalekanitsidwa ndi zinthu zolekanitsa maselo a leukocyte (STEMCELL Technologies) pogwiritsa ntchito Ficoll gradient density centrifugation. Ma CD3 + cells adachotsedwa ku PBMC pogwiritsa ntchito ma CD3 beads (Miltenyi). Pakakhala kapena kusakhalapo kwa MNA, ma CD3 + cells adayatsidwa ndi plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) ndi IL-2 (300 U/ml; Proleukin). Patsiku lomaliza la kukula, viability (Fixable Viability Dye eFluor450, eBioscience) ndi proliferation (123count eBeads, Thermo Fisher Scientific) zidayesedwa pogwiritsa ntchito flow cytometry. Yesani ntchito ya effector poyambitsa maselo okhala ndi PMA (20 ng/ml) ndi ionomycin (1μg/ml) ndi GolgiStop kwa maola 4, ndikuyang'anira CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) ndi TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Yambitsani maselo a qPCR ndi ChIP okhala ndi PMA (20 ng/ml) ndi ionomycin (1μg/ml) kwa maola 4. ELISA supernatant inasonkhanitsidwa isanayambe komanso itatha kusonkhezeredwa ndi PMA (20 ng/ml) ndi ionomycin (1 μg/ml) kwa maola 4.
Tsatirani njira ya wopanga kuti mulekanitse RNA pogwiritsa ntchito RNeasy Plus Mini Kit (QIAGEN). Gwiritsani ntchito QIAshredder (QIAGEN) kuti musinthe chitsanzocho kukhala chofanana. Gwiritsani ntchito RNA yokhala ndi mphamvu zambiri (Thermo Fisher Scientific) kuti mupange DNA yowonjezera (cDNA). Gwiritsani ntchito TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) kuti muyese kuchuluka kwa majini (malinga ndi njira ya wopanga) ndi ma probes otsatirawa: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] ndi Hs01010726_m1 (SLC22A2). Zitsanzozo zinayendetsedwa pa StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) mu MicroAmp fast optical 96-well reaction plate (Applied Biosystems) yokhala ndi MicroAmp optical film. Mtengo uliwonse wa Ct woposa 35 umaonedwa kuti uli pamwamba pa malire ozindikira ndipo umalembedwa kuti sungathe kuzindikirika.
Chitani ChIP monga momwe tafotokozera kale (58). Mwachidule, maselowo adachiritsidwa ndi formaldehyde (kuchuluka komaliza 1.42%) ndipo adayikidwa kutentha kwa chipinda kwa mphindi 10. Gwiritsani ntchito buffer yowonjezera yotupa (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl ndi 0.1% NP-40) pa ayezi kwa mphindi 10, kenako muyimitsenso mu buffer ya immunoprecipitation monga momwe tafotokozera (58). Kenako chitsanzocho chinayikidwa ndi sonicated ndi machitidwe otsatirawa: machitidwe 10 (20 1-second pulses) ndi nthawi yokhazikika ya masekondi 40. Ikani ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) ndi SP1 (Cell Signaling Technology; 3μl) ma antibodies ndi chitsanzocho pa 4°CC shake usiku wonse. Ikani mikanda ya protein A (Thermo Fisher Scientific) ndi chitsanzo pa 4°C ndikugwedeza pang'ono kwa ola limodzi, kenako gwiritsani ntchito mikanda ya chelex (Bio-Rad) kuti muwonjezere DNA, ndikugwiritsa ntchito proteinase K (Thermo Fisher) kuti mugaye mapuloteni. Chothandizira cha TNFα chinapezeka ndi PCR: forward, GGG TAT CCT TGA TGC TTG TGT; m'malo mwake, GTG CCA ACA ACT GCC TTT ATA TG (207-bp product). Zithunzizo zinapangidwa ndi Image Lab (Bio-Rad) ndipo zinayesedwa pogwiritsa ntchito pulogalamu ya ImageJ.
Kapangidwe ka maselo kameneka kanasonkhanitsidwa monga tafotokozera pamwambapa. Kuzindikira kunachitika motsatira njira zomwe wopanga amagwiritsa ntchito pa human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) ndi human IFN-γ ELISA kit (Abcam). Malinga ndi njira ya wopanga, kapangidwe kameneka kanachepetsedwa 1:100 kuti kazindikire TNFα ndi IL-2, ndi 1:3 kuti kazindikire IFN-γ. Gwiritsani ntchito EnVision 2104 Multilabel Reader (PerkinElmer) kuti muyese kuyamwa kwa madzi pa 450 nm.
Ma PBMC adalekanitsidwa ndi zinthu zolekanitsa maselo a leukocyte (STEMCELL Technologies) pogwiritsa ntchito Ficoll gradient density centrifugation. Ma CD3 + cells adachotsedwa ku PBMC pogwiritsa ntchito ma CD3 beads (Miltenyi). Pakakhala kapena kusakhalapo kwa MNA, ma CD3+ cells adayatsidwa ndi plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) ndi IL-2 (300 U/ml; Proleukin) kwa masiku atatu. Patatha masiku atatu, ma cells adasonkhanitsidwa ndikutsukidwa ndi 0.9% saline, ndipo pellet idazimitsidwa. Kuchuluka kwa ma cell kunachitika pogwiritsa ntchito flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) pogwiritsa ntchito 123count eBeads.
Ma metabolites ochotsedwa monga tafotokozera pamwambapa. Chotsitsa choumacho chinakonzedwanso pamlingo wa ma cell equivalents 4000/μl. Unikani chitsanzocho pogwiritsa ntchito reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) ndi CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), momwe electrospray ionization imagwirira ntchito bwino. Mobile phase A ndi 0.1% formic acid (mu H2O), mobile phase B ndi 90% acetonitrile, 0.1% formic acid. Kuchuluka kwa LC ndi mphindi 0 mpaka 2 pa 100% A, mphindi 2 mpaka 7.1 pa 99% B, ndi mphindi 7.1 mpaka 8 pa 99% B. Kenako gwirizanitsaninso mzati ndi gawo loyenda A pamlingo wa 0.6 ml/min kwa mphindi 3. . Kuchuluka kwa mayendedwe ndi 0.4ml/min, ndipo chipinda cha mzati chimatenthedwa kufika 50°C. Gwiritsani ntchito muyezo wa mankhwala oyera a MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) kuti mukhazikitse nthawi yosungira (RT) ndi kusintha (RT = mphindi 0.882, kusintha 1 = 137→94.1, kusintha 2 = 137→92, Kusintha 3 = 137→78). Kusintha konseku kukakhala nthawi yolondola yosungira, kusintha 1 kumagwiritsidwa ntchito poyesa kuchuluka kuti zitsimikizire kutsimikizika. Mzere wokhazikika wa MNA (Toronto Research Chemical Company) unapangidwa pogwiritsa ntchito njira zisanu ndi chimodzi zochepetsera shuga (1 mg/ml) kuti mupeze miyezo ya 0.1, 1.0, 10 ndi 100 ng/ml ndi 1.0 ndi 10μg/ml motsatana. Malire ozindikira ndi 1 ng/ml, ndipo yankho lolunjika lili pakati pa 10 ng/ml ndi 10μg/ml. Jakisoni iliyonse ya ma microliters awiri a chitsanzo ndi muyezo imagwiritsidwa ntchito poyesa LC/MS, ndipo chitsanzo chosakanikirana chowongolera khalidwe chimayendetsedwa jakisoni iliyonse isanu ndi itatu kuti zitsimikizire kukhazikika kwa nsanja yowunikira. Mayankho a MNA a zitsanzo zonse za maselo ochiritsidwa ndi MNA anali mkati mwa mzere woyesera. Kusanthula deta kunachitika pogwiritsa ntchito pulogalamu yowunikira kuchuluka kwa MassHunter (v9.0, Agilent).
Kapangidwe ka αFR-CAR ka m'badwo wachiwiri kanatengedwa kuchokera kwa Song et al. (59). Mwachidule, kapangidwe kake kali ndi izi: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge ndi transmembrane region, CD27 intracellular domain ndi CD3z intracellular domain. Kapangidwe ka CAR konse kanapangidwa ndi GenScript, kenako kanapangidwa mu lentiviral expression vector ya m'badwo wachiwiri pamwamba pa kaseti ya GFP expression yomwe imagwiritsidwa ntchito poyesa momwe transduction imagwirira ntchito.
Lentivirus imapangidwa ndi transfection ya maselo a HEK293T [American Type Culture Collection (ATCC); imakula mu Dulbecco's modified Eagle medium yokhala ndi 10% fetal bovine serum (FBS) ndi 1% PenStrep, ndipo imagwiritsidwa ntchito CAR-GFP vector ndi The packaging plasmids (psPAX2 ndi pMD2.G, Addgene) pogwiritsa ntchito lipofection amine (Sigma-Aldrich). Supernatant yokhala ndi kachilomboka idasonkhanitsidwa maola 48 ndi 72 mutapatsidwa transfection, yosefedwa, ndikuyikiridwa ndi ultracentrifugation. Sungani supernatant yoyikiridwa pa -80°C mpaka mutapatsidwa transfection.
Ma PBMC amalekanitsidwa ndi zinthu zopatukana za leukocyte zathanzi (STEMCELL Technologies) pogwiritsa ntchito Ficoll gradient density centrifugation. Gwiritsani ntchito ma CD8 microbeads (Miltenyi) kuti mulekanitse maselo a CD8+ kuchokera ku PBMC. Konzani maselo a T ndi TransAct (Miltenyi) ndi TexMACS medium [Miltenyi; yowonjezera ndi 3% heat-inactivated human serum, 1% PenStrep ndi IL-2 (300 U/ml)]. Maola makumi awiri ndi anayi mutatha kukondoweza, maselo a T adalowetsedwa ndi lentivirus (10 μl concentrated virus supernatant pa maselo 106). Patatha masiku 1 mpaka 3 mutatha kukondoweza pa Cytek Aurora (pa FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), fufuzani momwe GFP imaonekera m'maselo kuti muwonetse kuti transduction ikugwira ntchito bwino kwa osachepera 30%.
Maselo a CAR-T adalimidwa kwa maola 24 mu Immunocult (STEMCELL Technologies; yowonjezera ndi 1% PenStrep) pansi pa mikhalidwe iyi: osachiritsidwa, ochiritsidwa ndi 250 μM adenosine kapena 10 mM MNA. Pambuyo pa chithandizo choyambirira, maselo a CAR-T adatsukidwa ndi PBS ndipo adaphatikizidwa ndi maselo 20,000 a SK-OV-3 [ATCC; mu McCoy 5A medium (Sigma-Aldrich) owonjezeredwa ndi 10% FBS ndi 1% PenStrep pa 10: Chiŵerengero cha effector to target cha 1 chidakulitsidwa mu katatu mu Immunocult medium yowonjezera. Maselo a SK-OV-3 ndi maselo a SK-OV-3 odzazidwa ndi digitalis saponin (0.5mg/ml; Sigma-Aldrich) adagwiritsidwa ntchito ngati zowongolera zoyipa ndi zabwino, motsatana. Pambuyo pa maola 24 akulima pamodzi, supernatant inasonkhanitsidwa ndipo lactate dehydrogenase (LDH) inayesedwa motsatira malangizo a wopanga (LDH Glo Cytotoxicity Assay Kit, Promega). Supernatant ya LDH inachepetsedwa 1:50 mu LDH buffer. Chiŵerengero cha kuphedwa chinayesedwa pogwiritsa ntchito njira iyi: chiŵerengero cha kuphedwa = chiŵerengero cha kukonzedwa / chiŵerengero chachikulu cha kuphedwa x 100%, pomwe chiŵerengero cha kukonzedwa = maselo a co-culture-T okha, ndipo chiŵerengero chachikulu cha kuphedwa = chiŵerengero chabwino cha kulamulira-negative.
Monga tafotokozera m'malemba kapena zida ndi njira, gwiritsani ntchito GraphPad Prism 8, Microsoft Excel kapena R v3.6.0 pofufuza ziwerengero. Ngati zitsanzo zingapo zasonkhanitsidwa kuchokera kwa wodwala yemweyo (monga ascites ndi chotupa), timagwiritsa ntchito mayeso a t ophatikizidwa kapena kuphatikiza wodwalayo ngati zotsatira zosasinthika mu chitsanzo cholunjika kapena chophatikizana momwe ziyenera kukhalira. Pa kusanthula kwa metabolomics, mayeso ofunikira amachitidwa katatu.
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Funso ili limagwiritsidwa ntchito poyesa ngati ndinu mlendo ndikuletsa kutumiza sipamu yokha.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellar Jones (Russell. G. Jones), Phineas T. Hamilton (Phineas T.
MNA imathandizira kuletsa chitetezo cha mthupi cha maselo a T ndipo imayimira cholinga cha chithandizo cha immunotherapy chochizira khansa ya anthu.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellar Jones (Russell. G. Jones), Phineas T. Hamilton (Phineas T.
MNA imathandizira kuletsa chitetezo cha mthupi cha maselo a T ndipo imayimira cholinga cha chithandizo cha immunotherapy chochizira khansa ya anthu.
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