Magungunan rigakafi na rigakafi muhimmin fasali ne na yanayin ƙwayoyin cuta (TME), amma ban da wasu 'yan kaɗan, ba a san asalinsu ba. A nan, mun bincika ciwace-ciwacen da ƙwayoyin T daga ciwace-ciwacen da ascites na marasa lafiya da ke da cutar kansa mai girma (HGSC) don bayyana metabolome na waɗannan sassan TME daban-daban. Ascites da ƙwayoyin ciwon daji suna da bambance-bambancen metabolite masu yawa. Idan aka kwatanta da ascites, ƙwayoyin T masu shiga cikin ciwon suna da wadataccen arziki sosai a cikin 1-methylnicotinamide (MNA). Kodayake matakin MNA a cikin ƙwayoyin T yana da girma, bayyanar nicotinamide N-methyltransferase (enzyme wanda ke haɓaka canja wurin ƙungiyoyin methyl daga S-adenosylmethionine zuwa nicotinamide) yana iyakance ga fibroblasts da ƙwayoyin ciwon daji. A aiki, MNA yana haifar da ƙwayoyin T don fitar da cytokine tumor necrosis factor alpha mai haɓaka ciwon daji. Saboda haka, MNA da aka samo daga TME yana ba da gudummawa ga daidaita garkuwar jiki na ƙwayoyin T kuma yana wakiltar yuwuwar maƙasudin rigakafi don maganin ciwon daji na ɗan adam.
Magungunan da aka samo daga ƙari na iya yin tasiri mai zurfi kan rigakafin ciwon daji, kuma shaidu da yawa sun nuna cewa suna iya zama babban abin da ke haifar da ci gaban cututtuka (1). Baya ga tasirin Warburg, aikin da aka yi kwanan nan ya fara bayyana yanayin metabolism na ƙwayoyin ciwon daji da alaƙarsa da yanayin garkuwar jiki na yanayin ƙwayoyin cuta (TME). Nazarin da aka yi kan samfuran linzamin kwamfuta da ƙwayoyin T na ɗan adam sun nuna cewa metabolism na glutamine (2), metabolism na oxidative (3) da metabolism na glucose (4) na iya yin aiki daban-daban akan ƙungiyoyi daban-daban na ƙwayoyin rigakafi. Da yawa daga cikin metabolites a cikin waɗannan hanyoyin suna hana aikin anti-tumor na ƙwayoyin T. An tabbatar da cewa toshewar coenzyme tetrahydrobiopterin (BH4) na iya lalata yaduwar ƙwayoyin T, kuma ƙaruwar BH4 a cikin jiki na iya haɓaka amsawar rigakafin ciwon daji wanda CD4 da CD8 ke jagoranta. Bugu da ƙari, ana iya ceton tasirin immunosuppressive na kynurenine ta hanyar shan BH4 (5). A cikin maye gurbin isocitrate dehydrogenase (IDH) glioblastoma, fitar da enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) yana hana kunnawa, yaduwa da kuma ayyukan cytolysis na ƙwayoyin T (6). Kwanan nan, an nuna cewa methylglyoxal, wani samfurin glycolysis, ana samar da shi ta hanyar ƙwayoyin hanawa na asalin myeloid, kuma canja wurin ƙwayoyin T na methylglyoxal na iya hana aikin ƙwayoyin T masu tasiri. A cikin magani, rashin daidaituwar methylglyoxal na iya shawo kan ayyukan ƙwayoyin hanawa da aka samo daga myeloid (MDSC) da kuma haɓaka maganin toshewar wuraren bincike a cikin samfuran linzamin kwamfuta (7). Waɗannan nazarin tare suna jaddada muhimmiyar rawar da metabolites da aka samo daga TME ke takawa wajen daidaita aikin da ayyukan ƙwayoyin T.
An ba da rahoton rashin aikin ƙwayoyin T sosai a cikin ciwon daji na ovarian (8). Wannan wani ɓangare ne saboda halayen metabolism da ke tattare da hypoxia da kuma rashin daidaituwar jijiyoyin ciwon daji (9), wanda ke haifar da canza glucose da tryptophan zuwa samfuran da ba a haɗa su ba kamar lactic acid da kynurenine. Yawan lactate na waje yana rage samar da interferon-γ (IFN-γ) kuma yana haifar da bambance-bambancen ƙananan ƙungiyoyin myelosuppressive (10, 11). Amfani da tryptophan yana hana yaduwar ƙwayoyin T kai tsaye kuma yana hana siginar mai karɓar ƙwayoyin T (12-14). Duk da waɗannan abubuwan da aka lura, an gudanar da ayyuka da yawa game da metabolism na garkuwar jiki a cikin al'adar ƙwayoyin T a cikin vitro ta amfani da hanyoyin da aka inganta, ko kuma an iyakance su ga samfuran linzamin kwamfuta iri ɗaya a cikin vivo, babu ɗayan da ke nuna bambancin cutar kansar ɗan adam da muhallin macro da micro na jiki.
Wani abu da ya zama ruwan dare gama gari game da cutar kansar mahaifa shine yaɗuwar peritoneal da kuma bayyanar ascites. Tarin ruwan tantanin halitta a cikin ascites yana da alaƙa da ci gaba da cutar da kuma rashin kyakkyawan hasashen (15). A cewar rahotanni, wannan yanki na musamman yana da hypoxic, yana da matakan girma na jijiyoyin jini (VEGF) da indoleamine 2,3-dioxygenase (IDO), kuma ƙwayoyin T da ƙwayoyin hana myeloid suna shiga ciki (15-18). Yanayin metabolism na ascites na iya bambanta da na ciwon kansa, don haka sake tsara ƙwayoyin T a cikin sararin peritoneal ba a fayyace shi ba. Bugu da ƙari, manyan bambance-bambancen da bambancin da ke tsakanin ascites da metabolites da ke cikin yanayin ciwon na iya hana shigar ƙwayoyin rigakafi da aikinsu akan ciwon daji, kuma ana buƙatar ƙarin bincike.
Domin magance waɗannan matsalolin, mun tsara wata hanyar rabuwar ƙwayoyin halitta mai sauƙi da kuma hanyar chromatography ta ruwa (LC-MS/MS) don nazarin nau'ikan ƙwayoyin halitta daban-daban (gami da ƙwayoyin CD4 + da CD8 + T) da kuma a cikin da tsakanin ƙari. Tarin ƙwayoyin halittarta yana ratsa ƙwayoyin halitta a cikin yanayin ascites da ƙari iri ɗaya na majiyyaci. Muna amfani da wannan hanyar tare da babban girma na kwararar cytometry da siginar RNA ta sel guda ɗaya (scRNA-seq) don samar da hoto mai kyau na yanayin metabolism na waɗannan manyan al'ummomi. Wannan hanyar ta nuna ƙaruwa mai yawa a cikin matakin 1-methylnicotinamide (MNA) a cikin ƙwayoyin tumor T, kuma gwaje-gwajen in vitro sun nuna cewa tasirin immunomodulatory na MNA akan aikin ƙwayoyin T ba a san shi ba a da. Gabaɗaya, wannan hanyar tana bayyana hulɗar metabolism tsakanin ƙari da ƙwayoyin rigakafi, kuma tana ba da fahimta ta musamman game da metabolites na daidaita garkuwar jiki, wanda zai iya zama da amfani ga maganin rigakafi na ciwon daji na kwai na T. Damar magani.
Mun yi amfani da cytometry mai girma-girma don auna yawan shan glucose a lokaci guda [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) da aikin mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) alamomi ne na gefe-gefe waɗanda ke bambanta ƙwayoyin rigakafi da yawan ƙwayoyin ƙari (Tebur S2 da Hoto S1A). Wannan bincike ya nuna cewa idan aka kwatanta da ƙwayoyin T, ascites da ƙwayoyin ƙari suna da matakan shan glucose mafi girma, amma suna da ƙananan bambance-bambance a cikin aikin mitochondrial. Matsakaicin shan glucose na ƙwayoyin ƙari [CD45-EpCAM (EpCAM)+] ya ninka na ƙwayoyin T sau uku zuwa huɗu, kuma matsakaicin shan glucose na ƙwayoyin CD4 + T ya ninka na ƙwayoyin CD8 + T sau 1.2, wanda ke nuna cewa lymphocytes masu shiga cikin tumor (TIL) suna da buƙatun metabolism daban-daban ko da a cikin TME ɗaya (Hoto na 1A). Sabanin haka, aikin mitochondrial a cikin ƙwayoyin ƙari yana kama da na ƙwayoyin CD4 + T, kuma aikin mitochondrial na nau'ikan ƙwayoyin biyu ya fi na ƙwayoyin CD8 + T girma (Hoto na 1B). Gabaɗaya, waɗannan sakamakon suna bayyana matakin metabolism. Ayyukan metabolism na ƙwayoyin ƙari ya fi na ƙwayoyin CD4 + T girma, kuma aikin metabolism na ƙwayoyin CD4 + T ya fi na ƙwayoyin CD8 + T girma. Duk da waɗannan tasirin a cikin nau'ikan ƙwayoyin halitta, babu wani bambanci mai daidaito a cikin yanayin metabolism na ƙwayoyin CD4 + da CD8 + T ko rabon su a cikin ascites idan aka kwatanta da ciwace-ciwacen (Hoto na 1C). Sabanin haka, a cikin ɓangaren ƙwayoyin CD45, rabon ƙwayoyin EpCAM+ a cikin ƙari ya ƙaru idan aka kwatanta da ascites (Hoto na 1D). Mun kuma lura da bambanci bayyananne tsakanin abubuwan EpCAM+ da EpCAM-cell. Kwayoyin EpCAM+ (tumor) suna da yawan shan glucose da aikin mitochondrial fiye da ƙwayoyin EpCAM-, wanda ya fi aikin metabolism na fibroblasts a cikin ƙwayoyin ƙari a cikin TME (Hoto na 1, E da F).
(A da B) Matsakaicin ƙarfin haske (MFI) na shan glucose (2-NBDG) (A) da aikin mitochondrial na ƙwayoyin CD4 + T (MitoTracker ja mai duhu) (B) Jadawalin wakilai (hagu) da bayanai masu tambari (Dama), ƙwayoyin CD8 + T da ƙwayoyin EpCAM + CD45-tumor daga ascites da ƙari. (C) Rabon ƙwayoyin CD4 + da CD8 + (na ƙwayoyin CD3 + T) a cikin ascites da ƙari. (D) Yawancin ƙwayoyin EpCAM + na ƙari a cikin ascites da ƙari (CD45−). (E da F) EpCAM + CD45-tumor da EpCAM-CD45-matrix glucose a cikin ascites da ƙari (2-NBDG) (E) da aikin mitochondrial (MitoTracker ja mai duhu) (F) jadawalin wakilai (hagu) da bayanai masu tambari (Dama) Ascites da ƙwayoyin ƙari. (G) Jadawalin wakilci na bayanin CD25, CD137 da PD1 ta hanyar cytometry mai gudana. (H da I) bayyanar CD25, CD137 da PD1 akan ƙwayoyin CD4 + T (H) da ƙwayoyin CD8 + T (I). (J da K) Naive, ƙwaƙwalwar tsakiya (Tcm), effector (Teff) da ƙwaƙwalwar effector (Tem) bisa ga bayyanar CCR7 da CD45RO. Hotuna masu wakiltar (hagu) da bayanai na tabular (dama) na ƙwayoyin CD4 + T (J) da ƙwayoyin CD8 + T (K) a cikin ascites da ƙari. Ƙimar P an ƙaddara ta hanyar gwajin t-gwajin da aka haɗa (*P<0.05, **P<0.01 da ***P<0.001). Layin yana wakiltar marasa lafiya da suka dace (n = 6). FMO, fluorescence ban da ɗaya; MFI, matsakaicin ƙarfin haske.
Ƙarin bincike ya nuna wasu muhimman bambance-bambance tsakanin yanayin halittar ƙwayoyin T da aka warware sosai. Abubuwan da aka kunna (Hoto na 1, G zuwa I) da ƙwaƙwalwar mai tasiri (Hoto na 1, J da K) a cikin ciwace-ciwacen sun fi yawan ascites (daidaiton ƙwayoyin CD3 + T). Hakazalika, nazarin yanayin ta hanyar bayyanar alamun kunnawa (CD25 da CD137) da alamun raguwar ƙwayoyin halitta [protein mutuwar ƙwayoyin halitta da aka tsara 1 (PD1)] sun nuna cewa kodayake halayen metabolism na waɗannan al'ummomi sun bambanta (Hoto na S1, B zuwa E), Amma babu wani muhimmin bambance-bambancen metabolism da aka lura akai-akai tsakanin ƙananan ƙwayoyin halitta marasa aiki, masu tasiri ko ƙwaƙwalwar ajiya (Hoto na S1, F zuwa I). ​​An tabbatar da waɗannan sakamakon ta amfani da hanyoyin koyon injin don sanya siffofin ƙwayoyin halitta ta atomatik (21), wanda ya ƙara bayyana kasancewar adadi mai yawa na ƙwayoyin ƙashi (CD45 + / CD3- / CD4 + / CD45RO +) a cikin ascites na majiyyaci (Hoto na S2A). Daga cikin dukkan nau'ikan ƙwayoyin halitta da aka gano, wannan yawan ƙwayoyin myeloid sun nuna mafi girman yawan shan glucose da kuma aikin mitochondrial (Hoto na S2, B zuwa G). Waɗannan sakamakon suna nuna bambance-bambancen metabolism masu ƙarfi tsakanin nau'ikan ƙwayoyin halitta da yawa da ake samu a cikin ascites da ƙari a cikin marasa lafiya na HGSC.
Babban ƙalubalen fahimtar halayen metabonomic na TIL shine buƙatar ware samfuran ƙwayoyin T na isasshen tsarki, inganci da yawa daga ƙari. Binciken da aka yi kwanan nan ya nuna cewa hanyoyin rarrabawa da haɓaka beads bisa ga cytometry na kwarara na iya haifar da canje-canje a cikin bayanan metabolite na ƙwayoyin halitta (22-24). Don shawo kan wannan matsalar, mun inganta hanyar haɓaka beads don ware da ware TIL daga ciwon daji na kwai na ɗan adam da aka cire ta hanyar tiyata kafin a yi nazari ta LC-MS/MS (duba Kayan Aiki da Hanyoyi; Hoto na 2A). Domin tantance tasirin wannan yarjejeniya gabaɗaya akan canje-canjen metabolite, mun kwatanta bayanan metabolite na ƙwayoyin T da masu ba da gudummawa masu lafiya ke kunnawa bayan matakin rabuwar bead da ke sama da ƙwayoyin da ba a raba beads ba amma sun kasance akan kankara. Wannan binciken kula da inganci ya gano cewa akwai babban alaƙa tsakanin waɗannan yanayi biyu (r = 0.77), kuma maimaita fasaha na ƙungiyar metabolites 86 yana da babban maimaitawa (Hoto na 2B). Saboda haka, waɗannan hanyoyin za su iya yin cikakken nazarin metabolite a cikin ƙwayoyin da ke samun wadatar nau'in tantanin halitta, don haka suna samar da dandamali na farko mai ƙuduri don gano takamaiman metabolites a cikin HGSC, ta haka ne ke ba mutane damar samun zurfin fahimtar takamaiman tantanin halitta shirin metabolism na jima'i.
(A) Tsarin zane na wadatar da beads na maganadisu. Kafin a yi nazari ta LC-MS/MS, ƙwayoyin za su yi zagaye uku a jere na wadatar da beads na maganadisu ko kuma su ci gaba da zama a kan kankara. (B) Tasirin nau'in wadatarwa akan yawan metabolites. Matsakaicin ma'auni uku ga kowane nau'in wadatarwa ± SE. Layin launin toka yana wakiltar alaƙar 1:1. Haɗin cikin aji (ICC) na ma'auni masu maimaitawa da aka nuna a cikin lakabin axis. NAD, nicotinamide adenine dinucleotide. (C) Tsarin zane na aikin nazarin metabolite na marasa lafiya. Ana tattara ascites ko ƙari daga marasa lafiya kuma ana kiyaye su. An yi nazarin ƙaramin ɓangare na kowane samfurin ta hanyar kwararar cytometry, yayin da sauran samfuran suka yi zagaye uku na wadatarwa don ƙwayoyin CD4+, CD8+ da CD45-. An yi nazarin waɗannan sassan tantanin halitta ta amfani da LC-MS/MS. (D) Taswirar zafi na yawan metabolite da aka daidaita. Dendrogram yana wakiltar tarin Ward na nisan Euclidean tsakanin samfuran. (E) Babban nazarin sassan (PCA) na taswirar metabolite na samfurin, wanda ke nuna kwafi uku na kowane samfurin, samfuran daga majiyyaci ɗaya an haɗa su ta layi. (F) PCA na bayanin metabolite na samfurin da aka sanya wa majiyyaci (watau, ta amfani da ɗan lokaci kaɗan); nau'in samfurin yana da iyaka da ƙushin convex. PC1, babban sashi na 1; PC2, babban sashi na 2.
Na gaba, mun yi amfani da wannan hanyar haɓaka don yin nazarin metabolites guda 99 a cikin sassan ƙwayoyin CD4 +, CD8 + da CD45 a cikin manyan ascites da ƙari na marasa lafiya shida na HGSC (Hoto na 2C, Hoto na S3A da Tebur S3 da S4). Yawan jama'ar da ke sha'awar sun kai kashi 2% zuwa 70% na babban samfurin ƙwayoyin halitta masu rai na asali, kuma rabon ƙwayoyin halitta ya bambanta sosai tsakanin marasa lafiya. Bayan raba beads, ƙaramin ɓangaren sha'awa (CD4+, CD8+ ko CD45-) ya kai fiye da kashi 85% na dukkan ƙwayoyin halitta masu rai a cikin samfurin a matsakaici. Wannan hanyar haɓakawa tana ba mu damar yin nazarin yawan ƙwayoyin halitta daga metabolism na ƙwayoyin cuta na ɗan adam, wanda ba zai yiwu a yi daga manyan samfura ba. Ta amfani da wannan yarjejeniya, mun ƙaddara cewa l-kynurenine da adenosine, waɗannan metabolites guda biyu masu kyau na immunosuppressive sun haɓaka a cikin ƙwayoyin T na tumor ko ƙwayoyin tumor (Hoto na S3, B da C). Saboda haka, waɗannan sakamakon suna nuna aminci da ikon rabuwar ƙwayoyinmu da fasahar mass spectrometry don nemo metabolites masu mahimmanci a cikin kyallen majiyyaci.
Bincikenmu ya kuma nuna rabuwar ƙwayoyin halitta mai ƙarfi a tsakanin marasa lafiya da kuma tsakanin su (Hoto na 2D da Hoto na S4A). Musamman ma, idan aka kwatanta da sauran marasa lafiya, marasa lafiya 70 sun nuna halaye daban-daban na rayuwa (Hoto na 2E da Hoto na S4B), wanda ke nuna cewa akwai yiwuwar samun bambancin rayuwa mai yawa tsakanin marasa lafiya. Ya kamata a lura cewa idan aka kwatanta da sauran marasa lafiya (lita 1.2 zuwa 2; Tebur S1), jimlar adadin ascites da aka tattara a cikin marasa lafiya 70 (80 ml) ya yi ƙanƙanta. Kula da bambancin rayuwa tsakanin marasa lafiya yayin nazarin manyan abubuwan da ke cikin jiki (misali, ta amfani da nazarin sake dawowa na wani ɓangare) yana nuna canje-canje masu daidaito tsakanin nau'ikan ƙwayoyin halitta, kuma nau'ikan ƙwayoyin halitta da/ko yanayin rayuwa mai sauƙi an tattara su a sarari bisa ga bayanin metabolite (Hoto na 2F). Binciken metabolites guda ɗaya ya jaddada waɗannan tasirin kuma ya bayyana manyan bambance-bambance tsakanin nau'ikan ƙwayoyin halitta da yanayin rayuwa mai sauƙi. Ya kamata a lura cewa babban bambanci da aka lura shine MNA, wanda yawanci ana wadatar da shi a cikin ƙwayoyin CD45 da kuma cikin ƙwayoyin CD4+ da CD8+ waɗanda ke shiga cikin ciwon (Hoto na 3A). Ga ƙwayoyin CD4+, wannan tasirin ya fi bayyana, kuma MNA a cikin ƙwayoyin CD8+ suma yana da alaƙa da muhalli. Duk da haka, wannan ba shi da mahimmanci, saboda marasa lafiya uku ne kawai za a iya tantance su don sakamakon ciwon CD8+. Baya ga MNA, a cikin nau'ikan ƙwayoyin cuta daban-daban a cikin ascites da ciwace-ciwacen, sauran metabolites waɗanda ba su da kyau a cikin TIL suma suna da wadata daban-daban (Figures S3 da S4). Saboda haka, waɗannan bayanan sun bayyana wani tsari mai kyau na metabolites na immunomodulatory don ƙarin bincike.
(A) Daidaita abun ciki na MNA a cikin ƙwayoyin CD4+, CD8+ da CD45- daga ascites da ƙari. Tsarin akwatin yana nuna matsakaicin (layi), kewayon interquartile (hinge firam) da kewayon bayanai, har zuwa sau 1.5 kewayon interquartile (wisker firam). Kamar yadda aka bayyana a cikin Kayan Majinyata da Hanyoyin, yi amfani da ƙimar limma na majinyaci don tantance ƙimar P (*P<0.05 da **P<0.01). (B) Zane-zane na tsarin metabolism na 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. Enzymes (kore): 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 na scRNA-seq na ascites (launin toka) da ƙari (ja; n = marasa lafiya 3). (D) Bayyanar NNMT a cikin al'ummomin ƙwayoyin halitta daban-daban da aka gano ta amfani da scRNA-seq. (E) Bayyanar NNMT da AOX1 a cikin SK-OV-3, koda na ɗan adam (HEK) 293T, ƙwayoyin T da ƙwayoyin T da aka yi wa magani da MNA. An nuna bayyanar da aka naɗe dangane da SK-OV-3. An nuna tsarin bayyanar tare da SEM (n = 6 masu ba da gudummawa masu lafiya). Ana ɗaukar ƙimar Ct sama da 35 a matsayin ba za a iya gano ta ba (UD). (F) Bayyanar SLC22A1 da SLC22A2 a cikin ƙwayoyin T-OV-3, HEK293T, ƙwayoyin T da ƙwayoyin T da aka yi wa magani da 8mM MNA. An nuna bayyanar da aka naɗe dangane da SK-OV-3. An nuna tsarin bayyanar tare da SEM (n = masu ba da gudummawa masu lafiya 6). Ana ɗaukar ƙimar Ct sama da 35 a matsayin waɗanda ba za a iya gano su ba (UD). (G) Abubuwan da ke cikin ƙwayoyin MNA na ƙwayoyin halitta a cikin ƙwayoyin T masu ba da gudummawa masu lafiya da aka kunna bayan awanni 72 na haɗuwa da MNA. An nuna tsarin bayyanar tare da SEM (n = masu ba da gudummawa masu lafiya 4).
Ana samar da MNA ta hanyar canja wurin ƙungiyar methyl daga S-adenosyl-1-methionine (SAM) zuwa nicotinamide (NA) ta hanyar nicotinamide N-methyltransferase (NNMT; Hoto na 3B). NNMT yana da yawan bayyana a cikin nau'ikan cututtukan daji na ɗan adam kuma yana da alaƙa da yaduwa, mamayewa da metastasis (25-27). Don fahimtar tushen MNA a cikin ƙwayoyin T a cikin TME, mun yi amfani da scRNA-seq don kwatanta bayyanar NNMT a cikin nau'ikan ƙwayoyin halitta a cikin ascites da ƙari na marasa lafiya uku na HGSC (Tebur S5). Binciken kimanin ƙwayoyin halitta 6,500 ya nuna cewa a cikin yanayin ascites da ƙari, bayyanar NNMT ta takaita ga yawan ƙwayoyin fibroblast da ƙwayoyin ƙari da ake tsammani (Hoto na 3, C da D). Ya kamata a lura cewa babu bayyanar NNMT a bayyane a cikin kowace al'umma da ke nuna PTPRC (CD45 +) (Hoto na 3D da Hoto na S5A), wanda ke nuna cewa an shigar da MNA da aka gano a cikin bakan metabolite cikin ƙwayoyin T. Bayyanar aldehyde oxidase 1 (AOX1) yana canza MNA zuwa 1-methyl-2-pyridone-5-carboxamide (2-PYR) ko 1-methyl-4-pyridone-5-carboxamide (4-PYR); Hoto na 3B kuma an takaita shi ga yawan ƙwayoyin fibroblasts da ke bayyana COL1A1 (Hoto na S5A), waɗanda tare suka nuna cewa ƙwayoyin T ba su da ikon daidaita tsarin MNA na al'ada. An tabbatar da tsarin bayyanar waɗannan kwayoyin halittar da ke da alaƙa da MNA ta amfani da saitin bayanan ƙwayoyin halitta na biyu daga ascites daga marasa lafiya na HGSC (Hoto na S5B; n = 6) (16). Bugu da ƙari, nazarin ƙididdigar polymerase chain reaction (qPCR) na ƙwayoyin T masu ba da gudummawa masu lafiya da aka yi wa magani da MNA ya nuna cewa idan aka kwatanta da ƙwayoyin ciwon ovarian SK-OV-3 masu sarrafawa, NNMT ko AOX1 kusan ba a bayyana ba (Hoto na 3E). Waɗannan sakamakon da ba a zata ba sun nuna cewa ana iya fitar da MNA daga fibroblasts ko ƙari zuwa ƙwayoyin T da ke kusa da TME.
Duk da cewa 'yan takara sun haɗa da dangin masu jigilar cation na halitta 1 zuwa 3 (OCT1, OCT2 da OCT3) waɗanda dangin mai ɗaukar cation mai narkewa 22 (SLC22) (SLC22A1, SLC22A2 da SLC22A3) suka tsara, masu jigilar MNA har yanzu ba a fayyace su ba (28). QPCR na mRNA daga ƙwayoyin T masu ba da gudummawa masu lafiya sun nuna ƙarancin matakan bayyana SLC22A1 amma matakan SLC22A2 da ba a iya gano su ba, wanda ya tabbatar da cewa an riga an ruwaito shi a cikin wallafe-wallafen (Hoto na 3F) (29). Sabanin haka, layin ƙwayoyin cutar kansa na ovarian SK-OV-3 ya nuna manyan matakan masu jigilar duka biyun (Hoto na 3F).
Domin a gwada yiwuwar ƙwayoyin T suna da ikon shan MNA na ƙasashen waje, an yi wa ƙwayoyin T masu ba da gudummawa masu lafiya magani na tsawon awanni 72 a gaban yawan MNA daban-daban. Idan babu MNA na waje, ba za a iya gano abubuwan da ke cikin ƙwayoyin MNA ba (Hoto na 3G). Duk da haka, ƙwayoyin T da aka kunna da aka yi wa magani da MNA na waje sun nuna ƙaruwar abubuwan da ke cikin MNA a cikin ƙwayoyin, har zuwa 6 mM MNA (Hoto na 3G). Wannan sakamakon ya nuna cewa duk da ƙarancin matakin bayyanar mai jigilar kaya da rashin babban enzyme da ke da alhakin metabolism na MNA a cikin ƙwayoyin, TIL har yanzu yana iya ɗaukar MNA.
Bambance-bambancen metabolites a cikin ƙwayoyin T marasa lafiya da gwaje-gwajen sha MNA a cikin vitro suna ƙara yiwuwar cewa fibroblasts masu alaƙa da ciwon daji (CAF) suna fitar da MNA kuma ƙwayoyin ƙari na iya daidaita yanayin TIL da aikin T. Don tantance tasirin MNA akan ƙwayoyin T, an kunna ƙwayoyin T masu ba da gudummawa masu lafiya a cikin vitro a gaban ko babu MNA, kuma an kimanta yawan su da samar da cytokine. Bayan kwana 7 na ƙara MNA a mafi girman allurai, adadin jama'a ya ragu kaɗan, yayin da aka ci gaba da kasancewa da ƙarfi a duk allurai (Hoto na 4A). Bugu da ƙari, maganin MNA na waje ya haifar da ƙaruwa a cikin adadin ƙwayoyin CD4 + da CD8 + T waɗanda ke nuna tumor necrosis factor-α (TNFα; Hoto na 4B). Akasin haka, samar da IFN-γ a cikin ƙwayoyin sel na CD4 + T ya ragu sosai, amma ba a cikin ƙwayoyin CD8 + T ba, kuma babu wani canji mai mahimmanci a cikin interleukin 2 (IL-2; Hoto na 4, C da D). Saboda haka, gwajin immunosorbent na enzyme-linked immunosorbent (ELISA) na supernatants daga waɗannan al'adun ƙwayoyin T da aka yi wa magani da MNA ya nuna ƙaruwa mai yawa a cikin TNFα, raguwa a cikin IFN-γ, kuma babu canji a cikin IL-2 (Hoto na 4, E zuwa G). . Raguwar IFN-γ yana nuna cewa MNA na iya taka rawa wajen hana ayyukan hana ƙari na ƙwayoyin T. Domin kwaikwayon tasirin MNA akan gubar da ke haifar da ƙwayoyin T, ƙwayoyin T (FRα-CAR-T) masu karɓar antigen na chimeric waɗanda ke kai hari ga mai karɓar folate α da CAR-T (GFP) waɗanda ƙwayoyin mononuclear na jini masu lafiya (PBMC) ke sarrafawa. An haɓaka ƙwayoyin CAR-T na tsawon awanni 24 a gaban MNA, sannan aka haɗa su tare da ƙwayoyin ciwon ovarian na ɗan adam SK-OV-3 waɗanda ke bayyana mai karɓar folate α a rabo mai tasiri zuwa manufa na 10:1. Maganin MNA ya haifar da raguwa sosai a cikin ayyukan kashe ƙwayoyin FRα-CAR-T, wanda yayi kama da ƙwayoyin FRα-CAR-T da aka yi wa magani da adenosine (Hoto na 4H).
(A) Jimlar adadin ƙwayoyin halitta masu rai da ninki biyu na yawan jama'a (PD) kai tsaye daga al'ada a rana ta 7. Jadawalin sandar yana wakiltar matsakaicin + SEM na masu ba da gudummawa masu lafiya shida. Yana wakiltar bayanai daga aƙalla gwaje-gwaje masu zaman kansu guda 3. (B zuwa D) an yi amfani da CD3/CD28 da IL-2 don kunna ƙwayoyin T a yawan MNA na su na tsawon kwanaki 7. Kafin a yi nazari, an ƙarfafa ƙwayoyin tare da PMA/ionomycin tare da GolgiStop na tsawon awanni 4. Bayyanar TNFα (B) a cikin ƙwayoyin T. Misalin hoto (hagu) da bayanai na tabular (dama) na bayyanar TNFα a cikin ƙwayoyin halitta masu rai. Bayyanar IFN-γ (C) da IL-2 (D) a cikin ƙwayoyin T. An auna bayyanar cytokines ta hanyar cytometry mai gudana. Jadawalin sandar yana wakiltar matsakaicin (n = masu ba da gudummawa masu lafiya 6) + SEM. Yi amfani da nazarin bambance-bambancen hanya ɗaya da maimaita ma'auni (*P<0.05 da **P<0.01) don tantance ƙimar P. Yana wakiltar bayanai daga aƙalla gwaje-gwaje masu zaman kansu guda 3. An yi amfani da CD3/CD28 da IL-2 don kunna ƙwayoyin T a yawan MNA da suka samu na tsawon kwanaki 7. An tattara maganin kafin da kuma bayan awanni 4 na motsa PMA/ionomycin. An auna yawan TNFα (E), IFN-γ (F) da IL-2 (G) ta hanyar ELISA. Jadawalin sandar yana wakiltar matsakaicin (n = masu ba da gudummawa 5 masu lafiya) + SEM. An ƙayyade ƙimar P ta amfani da nazarin bambanci na hanya ɗaya da ma'auni mai maimaitawa (*P<0.05). Layin da aka yi wa alama yana nuna iyakar ganowa. (H) Gwajin lysis na ƙwayoyin halitta. An daidaita ƙwayoyin FRα-CAR-T ko GFP-CAR-T tare da adenosine (250μM) ko MNA (10 mM) na tsawon awanni 24, ko kuma an bar su ba tare da magani ba (Ctrl). An auna kashi na kashe ƙwayoyin SK-OV-3. An ƙayyade ƙimar P ta hanyar gwajin Welch t (*P<0.5 da **P<0.01).
Domin samun fahimtar tsarin sarrafa TNFα da ke dogara da MNA, an kimanta canje-canje a cikin TNFα mRNA na ƙwayoyin T da aka yi wa magani da MNA (Hoto na 5A). Kwayoyin T masu ba da gudummawa masu lafiya da aka yi wa magani da MNA sun nuna ƙaruwa sau biyu a cikin matakan fassarar TNFα, wanda ke nuna cewa MNA ya dogara da tsarin fassarar TNFα. Don bincika wannan tsarin sarrafawa mai yiwuwa, an kimanta abubuwan da aka sani guda biyu waɗanda ke daidaita TNFα, wato abubuwan da ke kunna TNFα na nukiliya (NFAT) da takamaiman furotin 1 (Sp1), a martanin ɗaure MNA ga mai haɓaka TNFα na kusa (30). Mai haɓaka TNFα ya ƙunshi wuraren ɗaure NFAT guda 6 da aka gano da kuma wuraren ɗaure Sp1 guda 2, suna haɗuwa a wuri ɗaya [-55 nau'i-nau'i na tushe (bp) daga 5'cap] (30). Chromatin immunoprecipitation (ChIP) ya nuna cewa lokacin da aka yi wa magani da MNA, ɗaure Sp1 ga mai haɓaka TNFα ya ninka sau uku. Haɗa NFAT kuma ya ƙara girma kuma ya kusanci mahimmanci (Hoto na 5B). Waɗannan bayanai sun nuna cewa MNA tana daidaita bayyanar TNFα ta hanyar rubutun Sp1, kuma zuwa ƙaramin matakin bayyanar NFAT.
(A) Idan aka kwatanta da ƙwayoyin T da aka haɓaka ba tare da MNA ba, canjin naɗewar bayyanar TNFα a cikin ƙwayoyin T da aka yi wa magani da MNA. An nuna tsarin bayyanar tare da SEM (n = masu ba da gudummawa 5 masu lafiya). Yana wakiltar bayanai daga aƙalla gwaje-gwaje 3 masu zaman kansu. (B) Mai haɓaka TNFα na ƙwayoyin T da aka yi wa magani da ko ba tare da 8 mM MNA bayan NFAT da Sp1 an haɗa su da motsa jiki (Ctrl) da PMA/ionomycin na tsawon awanni 4. An yi amfani da Immunoglobulin G (IgG) da H3 azaman sarrafawa mara kyau da tabbatacce don rigakafi, bi da bi. Ƙimar ChIP ta nuna cewa ɗaurewar Sp1 da NFAT ga mai haɓaka TNFα a cikin ƙwayoyin da aka yi wa magani da MNA ya ƙaru sau da yawa idan aka kwatanta da sarrafawa. Yana wakiltar bayanai daga aƙalla n = gwaje-gwaje 3 masu zaman kansu. Ƙimar P da aka ƙaddara ta gwaje-gwajen t da yawa (*** P <0.01). (C) Idan aka kwatanta da ascites na HGSC, ƙwayoyin T (marasa guba) sun nuna ƙaruwar bayyanar TNF a cikin ƙari. Launuka suna wakiltar marasa lafiya daban-daban. An ɗauki samfurin ƙwayoyin da aka nuna bazuwar zuwa 300 kuma an yi musu jitter don iyakance yawan zubar da jini (** Padj = 0.0076). (D) Tsarin MNA da aka gabatar don ciwon daji na ovarian. Ana samar da MNA a cikin ƙwayoyin ƙari da fibroblasts a cikin TME kuma ƙwayoyin T suna ɗaukar su. MNA yana ƙara ɗaure Sp1 zuwa mai haɓaka TNFα, wanda ke haifar da ƙaruwar kwafi na TNFα da samar da cytokine na TNFα. MNA kuma yana haifar da raguwar IFN-γ. Hana aikin ƙwayoyin T yana haifar da raguwar ikon kashewa da haɓaka ciwace-ciwacen.
A cewar rahotanni, TNFα yana da tasirin hana ciwace-ciwace da hana ciwace-ciwace da suka dogara da gaba da baya, amma yana da rawar da ya shahara wajen haɓaka girma da kuma yaduwar cutar kansar ovarian (31-33). A cewar rahotanni, yawan TNFα a cikin ascites da kyallen ƙari a cikin marasa lafiya da cutar kansar ovarian ya fi na kyallen da ba su da kyau (34-36). Dangane da tsari, TNFα na iya daidaita kunnawa, aiki da yaduwar ƙwayoyin farin jini, da kuma canza yanayin ƙwayoyin cutar kansa (37, 38). Daidai da waɗannan binciken, nazarin bayyanar kwayoyin halitta daban-daban ya nuna cewa an ƙara yawan TNF a cikin ƙwayoyin T a cikin kyallen ƙari idan aka kwatanta da ascites (Hoto na 5C). Ƙara yawan bayyanar TNF ya bayyana ne kawai a cikin yawan ƙwayoyin T tare da yanayin da ba shi da guba (Hoto na S5A). A taƙaice, waɗannan bayanan suna goyon bayan ra'ayin cewa MNA yana da tasirin hana garkuwar jiki da haɓaka ciwace-ciwace biyu a cikin HGSC.
Lakabi mai haske bisa ga tsarin kwararar jini ya zama babban hanyar nazarin metabolism na TIL. Waɗannan nazarin sun nuna cewa idan aka kwatanta da lymphocytes na jini na gefe ko ƙwayoyin T daga gabobin lymphoid na sakandare, murine da TIL na ɗan adam suna da babban yanayin ɗaukar glucose (4, 39) da kuma asarar aikin mitochondrial a hankali (19, 40). Kodayake mun lura da irin wannan sakamako a cikin wannan binciken, babban ci gaban shine kwatanta metabolism na ƙwayoyin ƙari da TIL daga ƙwayar ƙari iri ɗaya da aka cire. Daidai da wasu daga cikin waɗannan rahotannin da suka gabata, ƙwayoyin ƙari (CD45-EpCAM +) daga ascites da ƙari suna da yawan shan glucose fiye da ƙwayoyin CD8 + da CD4 + T, suna tallafawa cewa yawan shan glucose na ƙwayoyin ƙari za a iya kwatanta shi da ƙwayoyin T. Manufar gasar ƙwayoyin T. TME. Duk da haka, aikin mitochondrial na ƙwayoyin ƙari ya fi na ƙwayoyin CD8 + T, amma aikin mitochondrial yayi kama da na ƙwayoyin CD4 + T. Waɗannan sakamakon suna ƙarfafa jigon da ke fitowa cewa metabolism na oxidative yana da mahimmanci ga ƙwayoyin ƙari (41, 42). Sun kuma ba da shawarar cewa ƙwayoyin CD8 + T na iya zama mafi sauƙin kamuwa da matsalar oxidative fiye da ƙwayoyin CD4 + T, ko kuma ƙwayoyin CD4 + T na iya amfani da tushen carbon banda glucose don ci gaba da aikin mitochondrial (43, 44). Ya kamata a lura cewa ba mu ga wani bambanci a cikin ɗaukar glucose ko aikin mitochondrial tsakanin masu tasirin CD4 + T, ƙwaƙwalwar T effector da ƙwayoyin ƙwaƙwalwar T na tsakiya a cikin ascites ba. Hakazalika, yanayin bambance-bambancen ƙwayoyin CD8 + T a cikin ciwace-ciwacen ba shi da alaƙa da canje-canje a cikin ɗaukar glucose, yana nuna babban bambanci tsakanin ƙwayoyin T da aka haɓaka a cikin vitro da TIL na ɗan adam a cikin vivo (22). An kuma tabbatar da waɗannan abubuwan lura ta hanyar amfani da rarraba yawan ƙwayoyin ta atomatik mara son kai, wanda ya ƙara bayyana cewa ƙwayoyin CD45 + / CD3- / CD4 + / CD45RO + waɗanda ke da yawan ɗaukar glucose da aikin mitochondrial fiye da ƙwayoyin ƙari suna da yawa amma suna da yawan ƙwayoyin halitta masu aiki. Wannan yawan jama'a na iya wakiltar ƙaramin adadin ƙwayoyin myeloid suppressor ko ƙwayoyin dendritic plasmacytoid da aka gano a cikin binciken scRNA-seq. Duk da cewa an ruwaito dukkan waɗannan a cikin ciwon daji na mahaifa na ɗan adam [45], har yanzu suna buƙatar ƙarin aiki shine a bayyana wannan ƙaramin yawan ƙwayoyin cuta na myeloid.
Duk da cewa hanyoyin da suka dogara da kwararar cytometry na iya fayyace bambance-bambancen gabaɗaya a cikin metabolism na glucose da oxidative tsakanin nau'ikan tantanin halitta, ainihin metabolites da glucose ko wasu tushen carbon ke samarwa don metabolism na mitochondrial a cikin TME ba a tantance su ba tukuna. Sanya kasancewar ko rashin metabolites ga wani rukunin TIL yana buƙatar tsarkake yawan tantanin halitta daga ƙwayar da aka cire. Saboda haka, hanyar haɓaka tantanin halitta tare da mass spectrometry na iya samar da fahimta game da metabolites waɗanda aka wadatar da su daban-daban a cikin ƙwayoyin T da yawan ƙwayoyin ƙari a cikin samfuran marasa lafiya masu dacewa. Kodayake wannan hanyar tana da fa'idodi fiye da rarraba tantanin halitta da aka kunna ta hanyar fluorescence, wasu ɗakunan karatu na metabolite na iya shafar saboda kwanciyar hankali da/ko saurin juyawa (22). Duk da haka, hanyarmu ta sami damar gano metabolites guda biyu da aka gane suna da kariya daga garkuwar jiki, adenosine da kynurenine, saboda sun bambanta sosai tsakanin nau'ikan samfura.
Binciken metabonomic ɗinmu game da ciwace-ciwacen da ƙananan nau'ikan TIL yana ba da ƙarin haske game da rawar da metabolites ke takawa a cikin TME na ovarian. Da farko, ta amfani da cytometry na kwarara, mun gano cewa babu wani bambanci a cikin aikin mitochondrial tsakanin ciwace-ciwacen da ƙwayoyin CD4 + T. Duk da haka, binciken LC-MS/MS ya bayyana manyan canje-canje a cikin yawan metabolites tsakanin waɗannan al'ummomi, yana nuna cewa ƙarshe game da metabolism na TIL da aikin metabolism gabaɗaya yana buƙatar fassarar da aka yi da kyau. Na biyu, MNA shine metabolite tare da babban bambanci tsakanin ƙwayoyin CD45 da ƙwayoyin T a cikin ascites, ba ciwace-ciwacen ba. Saboda haka, rarrabuwa da wurin ciwon na iya samun tasiri daban-daban akan metabolism na TIL, wanda ke nuna yiwuwar bambance-bambancen a cikin wani yanayi na micro. Na uku, bayyanar enzyme mai samar da MNA NNMT galibi yana iyakance ga CAF, wanda shine ƙwayoyin ciwace-ciwacen zuwa ƙaramin mataki, amma ana lura da matakan MNA da ake iya ganowa a cikin ƙwayoyin T da aka samo daga ciwace-ciwacen. Yawan bayyanar NNMT a cikin CAF na ovarian yana da tasirin da aka sani na haɓaka ciwon daji, wani ɓangare saboda haɓaka metabolism na CAF, mamayewar ƙari da metastasis (27). Duk da cewa matakin TIL gabaɗaya matsakaici ne, bayyanar NNMT a cikin CAF yana da alaƙa da nau'in mesenchymal na Cancer Genome Atlas (TCGA), wanda ke da alaƙa da rashin kyakkyawan hasashen (27, 46, 47). A ƙarshe, bayyanar enzyme AOX1 da ke da alhakin lalata MNA shi ma yana iyakance ga yawan CAF, wanda ke nuna cewa ƙwayoyin T ba su da ikon daidaita MNA. Waɗannan sakamakon suna goyon bayan ra'ayin cewa kodayake ana buƙatar ƙarin aiki don tabbatar da wannan binciken, babban matakan MNA a cikin ƙwayoyin T na iya nuna kasancewar yanayin CAF mai hana garkuwar jiki.
Ganin ƙarancin matakin bayyanawa na masu jigilar MNA da kuma matakan furotin masu mahimmanci da ba a iya gano su ba da ke cikin metabolism na MNA, kasancewar MNA a cikin ƙwayoyin T ba abin mamaki ba ne. Ba za a iya gano NNMT ko AOX1 ta hanyar nazarin scRNA-seq da kuma niyya qPCR na ƙungiyoyi biyu masu zaman kansu ba. Waɗannan sakamakon sun nuna cewa ƙwayoyin T ba a haɗa MNA ba, amma ana sha daga TME da ke kewaye. Gwaje-gwajen in vitro sun nuna cewa ƙwayoyin T suna yawan tara MNA na waje.
Nazarinmu a cikin vitro ya nuna cewa MNA na waje yana haifar da bayyanar TNFα a cikin ƙwayoyin T kuma yana haɓaka ɗaurewar Sp1 ga mai haɓaka TNFα. Kodayake TNFα yana da ayyukan hana ƙari da hana ƙari, a cikin ciwon daji na ovarian, TNFα na iya haɓaka haɓakar ciwon daji na ovarian (31-33). Rage TNFα a cikin al'adar ƙwayoyin ƙari na ovarian ko kawar da siginar TNFα a cikin samfuran linzamin kwamfuta na iya inganta samar da cytokine mai kumburi da TNFα ke haifarwa da hana haɓakar ƙari (32, 35). Saboda haka, a wannan yanayin, MNA da aka samo daga TME na iya aiki azaman metabolite mai hana kumburi ta hanyar hanyar da ta dogara da TNFα ta hanyar madauki na autocrine, don haka yana haɓaka faruwa da yaɗuwar ciwon daji na ovarian (31). Dangane da wannan yuwuwar, ana nazarin toshewar TNFα a matsayin mai yuwuwar maganin cutar kansar ovarian (37, 48, 49). Bugu da ƙari, MNA yana lalata gubar ƙwayoyin CAR-T zuwa ƙwayoyin ƙari na ovarian, yana ba da ƙarin shaida don hana garkuwar jiki ta MNA. Gabaɗaya, waɗannan sakamakon suna nuna samfurin da ciwace-ciwacen da ƙwayoyin CAF ke fitarwa MNA zuwa cikin TME na waje. Ta hanyar (i) ƙarfafa haɓakar ciwon daji na kwai da TNF ke haifarwa da kuma (ii) hana ayyukan ƙwayoyin T da MNA ke haifarwa, wannan na iya samun tasirin ciwace-ciwace guda biyu (Hoto na 5D).
A ƙarshe, ta hanyar amfani da haɗin haɓaka ƙwayoyin halitta cikin sauri, jerin ƙwayoyin halitta guda ɗaya da kuma bayanin yanayin rayuwa, wannan binciken ya bayyana babban bambance-bambancen immunometabolomic tsakanin ciwace-ciwacen da ƙwayoyin ascites a cikin marasa lafiya na HGSC. Wannan cikakken bincike ya nuna cewa akwai bambance-bambance a cikin ɗaukar glucose da aikin mitochondrial tsakanin ƙwayoyin T, kuma ya gano MNA a matsayin metabolite mai sarrafa garkuwar jiki wanda ba na tantanin halitta ba. Waɗannan bayanai suna da tasiri kan yadda TME ke shafar metabolism na ƙwayoyin T a cikin ciwon daji na ɗan adam. Kodayake an bayar da rahoton gasa kai tsaye don abubuwan gina jiki tsakanin ƙwayoyin T da ƙwayoyin cutar kansa, metabolites kuma suna iya aiki a matsayin masu kula da kai tsaye don haɓaka ci gaban ƙari da kuma yiwuwar danne martanin garkuwar jiki na ciki. Ƙarin bayanin aikin waɗannan metabolites masu sarrafawa na iya buɗe wasu dabarun don haɓaka martanin rigakafi na hana ƙari.
An samo samfuran marasa lafiya da bayanan asibiti ta hanyar ma'ajiyar ƙwayoyin cutar kansa ta BC wadda Cibiyar Kula da Tissue ta Kanada ta ba da takardar shaidarta. Dangane da yarjejeniyar da Kwamitin Ɗabi'a na Binciken Ciwon daji na BC da Jami'ar British Columbia (H07-00463) suka amince da ita, duk samfuran marasa lafiya da bayanan asibiti sun sami izini a rubuce ko kuma sun yi watsi da amincewarsu a hukumance. Ana adana samfuran a cikin BioBank mai takardar shaidar (BRC-00290). An nuna cikakkun halayen marasa lafiya a cikin Tebur S1 da S5. Don cryopreservation, ana amfani da scalpel don narkar da samfurin ciwon mara ta hanyar injiniya sannan a tura shi ta hanyar matatar 100-micron don samun dakatarwar ƙwayoyin halitta guda ɗaya. An sanya ascites na majiyyaci a 1500 rpm na minti 10 a zafin jiki na 4°C don cire ƙwayoyin da ke cikin su da kuma cire su. An adana ƙwayoyin da aka samo daga ƙari da ascites a cikin kashi 50% na sinadarin AB na ɗan adam wanda ba shi da zafi (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) da 10% na dimethyl sulfoxide. An narke waɗannan dakatarwar ƙwayoyin halitta guda ɗaya da aka kiyaye kuma an yi amfani da su don tantance metabolomics da metabolite da aka bayyana a ƙasa.
Cikakken matsakaiciyar ta ƙunshi tace 0.22 μm RPMI 50:50 mai ƙarin RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) wanda aka ƙara da kashi 10% na sinadarin AB na ɗan adam wanda ba shi da zafi (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x maganin Penicillin Streptomycin (PenStrep) (Thermo Fisher Scientific) da 50 μMB-mercaptoethanol. An ƙara AimV (Invitrogen) da 20 mM Hepes (Thermo Fisher Scientific) da 2 mM l-glutamine (Thermo Fisher Scientific). Ma'aunin tabo na cytometer ya ƙunshi 0.22μm mai tace phosphate buffered saline (PBS; Invitrogen) wanda aka ƙara da 3% na sinadarin AB na ɗan adam wanda ba shi da zafi (Sigma). Ma'aunin haɓaka ƙwayoyin halitta ya ƙunshi PBS mai tace 0.22μm kuma an ƙara masa 0.5% na sinadarin AB na ɗan adam wanda ba shi da ƙarfi a jiki (Sigma-Aldrich).
A cikin matsakaicin zafin jiki na 37°C, an yi wa ƙwayoyin fenti da 10 nM MT DR da 100 μM 2-NBDG na tsawon mintuna 30. Na gaba, an yi wa ƙwayoyin fenti da fenti mai rai eF506 a 4°C na tsawon mintuna 15. A sake sanya ƙwayoyin a cikin FC Block (eBioscience) da Brilliant Stain Buffer (BD Biosciences), a narkar da su a cikin ma'aunin ... An daidaita ƙarfin hasken haske (MFI) na 2-NBDG da MT DR ta hanyar log, sannan aka yi amfani da gwajin t mai haɗin gwiwa don nazarin ƙididdiga don la'akari da marasa lafiya da suka yi daidai. Cire duk yawan jama'a da ke da abubuwan da suka faru ƙasa da 40 daga binciken; shigar da ƙimar MFI na 1 don duk wani ƙima mara kyau kafin yin nazarin ƙididdiga da hangen nesa na bayanai.
Domin ƙara wa dabarun ƙofa hannu na kwamitin aiwatarwa da ke sama, mun yi amfani da cikakken bayanin da bishiyar takaita siffar (FAUST) (21) ta yi don sanya ƙwayoyin halitta ta atomatik ga yawan jama'a bayan kawar da ƙwayoyin da suka mutu a cikin FlowJo. Muna sarrafa fitarwa da hannu don haɗa yawan jama'a waɗanda suka yi kama da ba a rarraba su daidai ba (haɗa PD1+ da ƙwayoyin ƙari na PD1) da kuma yawan jama'a da aka riƙe. Kowane samfurin ya ƙunshi matsakaicin ƙwayoyin halitta sama da 2%, ga jimillar yawan jama'a 11.
An yi amfani da centrifugation na Ficoll gradient density centrifugation don raba PBMC da samfuran rabuwar leukocyte (STEMCELL Technologies). An ware ƙwayoyin CD8 + T daga PBMC ta amfani da CD8 MicroBeads (Miltenyi) kuma an faɗaɗa su a cikin cikakken matsakaici ta amfani da TransAct (Miltenyi) na tsawon makonni 2 bisa ga umarnin masana'anta. An bar ƙwayoyin su tsaya na tsawon kwanaki 5 a cikin cikakken matsakaici wanda ke ɗauke da IL-7 (10 ng/ml; PeproTech), sannan aka sake motsa su da TransAct. A rana ta 7, bisa ga umarnin masana'anta, an yi amfani da CD45 MicroBeads na ɗan adam (Miltenyi) don wadatar da ƙwayoyin halitta a zagaye uku a jere. An ware ƙwayoyin don nazarin kwararar cytometry (kamar yadda aka bayyana a sama), kuma an ware ƙwayoyin miliyan ɗaya sau uku don nazarin LC-MS/MS. An sarrafa samfuran ta hanyar LC-MS/MS kamar yadda aka bayyana a ƙasa. Mun kiyasta ƙimar metabolite da ta ɓace tare da adadin ion na 1,000. Ana daidaita kowane samfurin ta hanyar jimlar lambar ion (TIC), ana canza shi ta hanyar logarithmically kuma ana daidaita shi ta atomatik a cikin MetaboAnalystR kafin a yi nazari.
An narke daurin kwayar halitta guda ɗaya na kowane majiyyaci kuma an tace ta ta hanyar matattarar 40 μm zuwa matsakaici cikakke (kamar yadda aka bayyana a sama). Dangane da ka'idar masana'anta, an yi amfani da zagaye uku na zaɓi mai kyau ta hanyar raba beads mai maganadisu ta amfani da MicroBeads (Miltenyi) don wadatar da samfuran ƙwayoyin CD8+, CD4+ da CD45- (a kan kankara). A takaice, ana sake haɗa ƙwayoyin a cikin ma'ajiyar haɓaka ƙwayoyin halitta (kamar yadda aka bayyana a sama) kuma ana ƙirga su. An haɗa ƙwayoyin da beads na CD8 na ɗan adam, beads na CD4 na ɗan adam ko beads na CD45 na ɗan adam (Miltenyi) a zafin jiki na 4°C na mintuna 15, sannan a wanke su da ma'ajiyar haɓaka ƙwayoyin halitta. An wuce samfurin ta cikin ginshiƙin LS (Miltenyi), kuma an tattara ɓangarorin masu kyau da marasa kyau. Domin rage tsawon lokaci da kuma haɓaka matakin dawo da ƙwayoyin halitta, ana amfani da ma'ajiyar CD8 don zagaye na biyu na haɓaka CD4+, kuma ana amfani da ma'ajiyar CD44 don haɓaka CD45 na gaba. Ajiye maganin a kan kankara a duk lokacin aikin rabuwa.
Domin shirya samfuran da za a yi amfani da su wajen nazarin sinadaran, an wanke ƙwayoyin halitta sau ɗaya da ruwan gishiri mai sanyi, sannan aka ƙara 1 ml na methanol 80% a kowane samfurin, sannan aka murƙushe shi aka daskare shi a cikin ruwa mai nitrogen. An yi amfani da samfuran sau uku a lokacin daskare-narkewa kuma aka sanya su a cikin 14,000 rpm na tsawon mintuna 15 a zafin jiki na 4°C. Ana fitar da sinadarin da ke ɗauke da sinadaran har sai ya bushe. An sake narkar da sinadaran a cikin 50 μl na 0.03% formic acid, aka murƙushe su don haɗawa, sannan aka sanya su a cikin centrifuge don cire tarkace.
Cire metabolites kamar yadda aka bayyana a sama. A mayar da supernatant zuwa kwalbar ruwa mai aiki mai yawa don binciken metabolomics. Yi amfani da tsarin magani na bazuwar don magance kowane samfurin da adadin ƙwayoyin halitta iri ɗaya don hana tasirin rukuni. Mun yi kimantawa mai inganci na metabolites na duniya da aka buga a baya akan AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). An yi nazarin Chromatographic da haɗin yankin kololuwa ta amfani da software na MultiQuant version 2.1 (Applied Biosystems SCIEX).
An yi amfani da ƙidayar ion na 1000 don kimanta ƙimar metabolite da ta ɓace, kuma an yi amfani da TIC na kowane samfurin don ƙididdige yankin kololuwar da aka daidaita na kowane metabolite da aka gano don gyara canje-canjen da aka gabatar ta hanyar nazarin kayan aiki daga sarrafa samfurin. Bayan an daidaita TIC, ana amfani da MetaboAnalystR(51) (sigogi na tsoho) don juyawa logarithmic da sikelin layi na atomatik. Mun yi amfani da PCA tare da fakitin vegan R don yin bincike na bincike na bambance-bambancen metabolome tsakanin nau'ikan samfura, kuma mun yi amfani da nazarin redundancy na ɓangare don bincika marasa lafiya. Yi amfani da hanyar Ward don gina dendrogram na taswirar zafi don haɗa nisan Euclidean tsakanin samfura. Mun yi amfani da limma (52) akan yawan metabolite da aka daidaita don gano metabolites masu yawa a cikin dukkan nau'in tantanin halitta da ƙananan yanayi. Domin sauƙaƙe bayanin, muna amfani da ma'aunin matsakaicin rukuni don ƙayyade samfurin, kuma muna la'akari da nau'ikan tantanin halitta a cikin ƙananan yanayi kamar kowane rukuni (n = ƙungiyoyi 6); Don gwajin mahimmanci, mun yi maimaita ma'auni sau uku ga kowane metabolite. Domin gujewa kwafi na ƙarya, an haɗa majiyyaci a matsayin cikas a cikin ƙirar limma. Domin duba bambance-bambancen metabolites tsakanin marasa lafiya daban-daban, mun daidaita samfurin limma har da marasa lafiya ta hanyar da aka ƙayyade. Muna ba da rahoton mahimmancin bambancin da aka riga aka ƙayyade tsakanin nau'in tantanin halitta da yanayin Padj <0.05 (gyaran Benjamin-Hochberg).
Bayan ƙara ƙarfin kuzari ta amfani da Kayan Cire Kwayoyin Halittar Miltenyi (>80% na rayuwa), an yi jerin kwayoyin halitta guda ɗaya akan jimlar ƙwayoyin halitta da aka daskare da kuma samfuran ƙari ta amfani da tsarin bayyana kwayoyin halitta na 10x 5′. An yi nazarin shari'o'i biyar tare da ƙari da ascites masu dacewa, kodayake ƙarancin yuwuwar samfurin ƙari ɗaya ya hana shigar da shi. Domin cimma zaɓen marasa lafiya da yawa, mun haɗa samfuran kowane majiyyaci a cikin layin mai sarrafa chromium 10x, kuma muka bincika wuraren ascites da ƙari daban-daban. Bayan jerin [Illumina HiSeq 4000 28×98 bp paired end (PE), kwayar halittar Quebec; Matsakaicin karatu 73,488 da 41,378 a kowace tantanin halitta don ƙari da ascites bi da bi]], mun yi amfani da CellSNP da Vireo (53) (bisa ga CellSNP kamar yadda SNP na ɗan adam na gama gari (VCF) wanda GRCh38 ya bayar an sanya shi asalin mai bayarwa. Muna amfani da SNPRelate don gano asalin da ya fi kusa (IBS) na yanayin kwayar halittar majiyyaci (IBS), ban da ƙwayoyin halitta da ƙwayoyin halitta da ba a sanya su a matsayin duplexes ba da kuma masu ba da gudummawa masu daidaitawa tsakanin ascites da samfuran ƙari (54). Dangane da wannan aikin, mun riƙe shari'o'i uku tare da wakilcin tantanin halitta mai yawa a cikin ƙari da ascites don nazarin ƙasa. Bayan yin matakin tacewa mai yawa a cikin marufi na scater (55) da scran (56) BioConductor, wannan ya haifar da ƙwayoyin halitta 6975 (ƙwayoyin halitta 2792 da 4183 daga ƙari da ascites, bi da bi) don bincike. Muna amfani da rukunin igraph (57) na cibiyar sadarwa ta maƙwabta mafi kusa (SNN) bisa ga nisan Jaccard zuwa ga ƙwayoyin rukuni ta hanyar bayyanawa. An yi wa ƙungiyoyin bayanin da hannu zuwa nau'ikan ƙwayoyin halitta bisa ga bayyanar kwayar halitta mai alama kuma an nuna su da t-SNE. Ana bayyana ƙwayoyin T na cytotoxic ta hanyar bayyanar CD8A da GZMA, ban da ƙananan rukuni masu ƙarancin bayyanar furotin na ribosomal. Mun sami damar shiga bayanan da aka buga na Izar et al. (16), gami da haɗarsu da t-SNE, na iya sarrafa haɗuwar bayyanar tsakanin alamun ƙwayoyin rigakafi da bayyanar NNMT.
An raba PBMC daga samfuran rabuwar leukocyte (STEMCELL Technologies) ta hanyar amfani da centrifugation na Ficoll gradient density centrifugation. An ware ƙwayoyin CD3 + daga PBMC ta amfani da beads na CD3 (Miltenyi). A gaban ko babu MNA, an kunna ƙwayoyin CD3+ tare da CD3 mai ɗaure da farantin (5μg/ml), CD28 mai narkewa (3μg/ml) da IL-2 (300 U/ml; Proleukin). A ranar ƙarshe ta faɗaɗawa, an kimanta wanzuwar (Fixable Viability Dye eFluor450, eBioscience) da yaduwa (123count eBeads, Thermo Fisher Scientific) ta hanyar kwararar cytometry. Kimanta aikin tasirin ta hanyar ƙarfafa ƙwayoyin halitta tare da PMA (20 ng/ml) da ionomycin (1μg/ml) tare da GolgiStop na tsawon awanni 4, sannan a sa ido kan CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) da TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). A ƙarfafa ƙwayoyin qPCR da ChIP tare da PMA (20 ng/ml) da ionomycin (1μg/ml) na tsawon awanni 4. An tattara supernatant na ELISA kafin da bayan ƙarfafawa tare da PMA (20 ng/ml) da ionomycin (1 μg/ml) na tsawon awanni 4.
Bi tsarin masana'anta don ware RNA ta amfani da RNeasy Plus Mini Kit (QIAGEN). Yi amfani da QIAshredder (QIAGEN) don daidaita samfurin. Yi amfani da babban ƙarfin RNA zuwa kayan aikin cDNA (Thermo Fisher Scientific) don haɗa DNA mai dacewa (cDNA). Yi amfani da TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) don ƙididdige bayyanar kwayar halitta (bisa ga tsarin masana'anta) tare da waɗannan binciken: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] da Hs01010726_m1 (SLC22A2). An gudanar da samfuran a kan tsarin PCR na StepOnePlus na ainihin lokaci (Applied Biosystems) (Applied Biosystems) a cikin farantin amsawar rijiyoyin MicroAmp mai sauri mai 96 (Applied Biosystems) tare da fim ɗin MicroAmp. Duk wani ƙimar Ct da ya wuce 35 ana ɗaukarsa a matsayin sama da matakin ganowa kuma ana yi masa alama a matsayin wanda ba za a iya gano shi ba.
Yi ChIP kamar yadda aka bayyana a baya (58). A takaice, an yi wa ƙwayoyin magani da formaldehyde (ƙarshen yawan 1.42%) kuma an saka su a cikin zafin ɗaki na minti 10. Yi amfani da ƙarin buffer buffer (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl da 0.1% NP-40) a kan kankara na minti 10, sannan a sake dakatar da su a cikin buffer immunoprecipitation kamar yadda aka bayyana (58). Daga nan aka haɗa samfurin da waɗannan zagayowar: zagayowar 10 (ƙwanƙwasa 20 na daƙiƙa 1) da kuma lokacin daƙiƙa 40. Haɗa ChIP-grade immunoglobulin G (Fasahar Siginar Kwayoyin Halitta; 1μl), histone H3 (Fasahar Siginar Kwayoyin Halitta; 3μl), NFAT (Invitrogen; 3μl) da SP1 (Fasahar Siginar Kwayoyin Halitta; 3μl) tare da samfurin a 4°CC girgiza cikin dare. A saka ƙwai mai gina jiki (Thermo Fisher Scientific) a cikin samfurin a zafin 4°C tare da girgiza a hankali na tsawon awa 1, sannan a yi amfani da ƙwai mai siffar chelex (Bio-Rad) don ƙara wa DNA ƙarfi, sannan a yi amfani da proteinase K (Thermo Fisher) don narkewar furotin. An gano mai haɓaka TNFα ta hanyar PCR: gaba, GGG TAT CCT TGA TGC TTG TGT; akasin haka, GTG CCA ACA ACT GCC TTT ATA TG (samfurin 207-bp). Image Lab (Bio-Rad) ne ya samar da hotunan kuma aka auna su ta amfani da software na ImageJ.
An tattara sinadarin halittar ƙwayoyin halitta kamar yadda aka bayyana a sama. An gudanar da tantancewar bisa ga tsarin masana'anta na kayan aikin TNFα ELISA na ɗan adam (Invitrogen), kayan aikin ELISA na ɗan adam (Invitrogen) da kayan aikin ELISA na ɗan adam (Abcam). Dangane da ka'idar masana'anta, an narkar da sinadarin a cikin ruwa 1:100 don gano TNFα da IL-2, da kuma 1:3 don gano IFN-γ. Yi amfani da EnVision 2104 Multilabel Reader (PerkinElmer) don auna sha a 450 nm.
An raba PBMC daga samfuran rabuwar leukocyte (STEMCELL Technologies) ta hanyar amfani da centrifugation na Ficoll gradient density centrifugation. An ware ƙwayoyin CD3 + daga PBMC ta amfani da beads na CD3 (Miltenyi). A gaban ko babu MNA, an kunna ƙwayoyin CD3+ tare da CD3 mai ɗaure da farantin (5μg/ml), CD28 mai narkewa (3μg/ml) da IL-2 (300 U/ml; Proleukin) na tsawon kwana 3. Bayan kwana 3, an tattara ƙwayoyin kuma an wanke su da ruwan gishiri 0.9%, kuma an daskare pellet ɗin. An yi amfani da cytometry na kwarara (Cytek Aurora; 3L-16V-14B-8R configuration) ta amfani da eBeads 123count.
Cire metabolites kamar yadda aka bayyana a sama. An sake haɗa busasshen cirewar a yawan ƙwayoyin halitta 4000/μl. Yi nazarin samfurin ta hanyar amfani da chromatography na lokaci-lokaci (1290 Infinity II, Agilent Technologies, Santa Clara, CA) da kuma CORTECS T3 column (2.1×150 mm, girman barbashi 1.6-μm, girman rami 120-Å; #186008500, Waters). Na'urar auna yawan polar mass spectrometer (6470, Agilent), inda electrospray ionization ke aiki a yanayin da ya dace. Matakin wayar hannu A shine 0.1% formic acid (a cikin H2O), mataki na wayar hannu B shine 90% acetonitrile, 0.1% formic acid. Matsakaicin LC shine mintuna 0 zuwa 2 na 100% A, mintuna 2 zuwa 7.1 na 99% B, da mintuna 7.1 zuwa 8 na 99% B. Sannan a sake daidaita ginshiƙin da matakin motsi na A a ƙimar kwararar 0.6 ml/min na tsawon mintuna 3. . Yawan kwararar shine 0.4ml/min, kuma ana dumama ɗakin ginshiƙin zuwa 50°C. Yi amfani da ma'aunin sinadarai na MNA (M320995, Kamfanin Masana'antar Bincike na Toronto, North York, Ontario, Kanada) don saita lokacin riƙewa (RT) da canji (RT = mintuna 0.882, canji 1 = 137→94.1, canji 2 = 137→92, Juyawa 3 = 137→78). Lokacin da dukkan canje-canje uku suka faru a daidai lokacin riƙewa, ana amfani da canji 1 don ƙididdigewa don tabbatar da takamaiman bayani. An samar da daidaitaccen lanƙwasa na MNA (Kamfanin Masana'antu na Toronto) ta hanyar haɗa sinadarin mai guda shida (1 mg/ml) don samun ma'aunin 0.1, 1.0, 10 da 100 ng/ml da 1.0 da 10μg/ml bi da bi ruwa. Iyakar ganowa ita ce 1 ng/ml, kuma amsawar layi tana tsakanin 10 ng/ml da 10 μg/ml. Kowace allurar microliters biyu na samfur da misali ana amfani da ita don nazarin LC/MS, kuma ana gudanar da samfurin sarrafa inganci gauraye a kowace allura takwas don tabbatar da daidaiton dandamalin bincike. Amsoshin MNA na duk samfuran ƙwayoyin halitta da aka yi wa magani da MNA suna cikin kewayon layi na gwajin. An yi nazarin bayanai ta amfani da software na nazarin adadi na MassHunter (v9.0, Agilent).
An ɗauko ginin αFR-CAR na ƙarni na biyu daga Song et al. (59). A takaice, ginin ya ƙunshi abubuwan da ke ciki: jerin jagororin CD8a, guntu mai canza sarka ɗaya na ɗan adam na αFR, yankin hinge na CD8a da transmembrane, yankin ƙwayoyin halitta na CD27 da yankin ƙwayoyin halitta na CD3z. GenScript ne ya haɗa cikakken jerin CAR, sannan aka yi kwafi zuwa vector na bayyanar lentiviral na ƙarni na biyu a sama da kaset ɗin bayyanar GFP da aka yi amfani da shi don kimanta ingancin watsawa.
Ana samar da cutar Lentivirus ta hanyar transfection na ƙwayoyin HEK293T [American Type Culture Collection (ATCC); an girma a cikin maganin Eagle da aka gyara na Dulbecco wanda ke ɗauke da kashi 10% na serum na shanu na tayi (FBS) da 1% PenStrep, kuma an yi amfani da vector na CAR-GFP kuma plasmids ɗin da aka marufi (psPAX2 da pMD2.G, Addene) suna amfani da lipofection amine (Sigma-Aldrich). An tattara supernatant mai ɗauke da kwayar cutar awanni 48 da 72 bayan transfection, an tace, kuma an tattara ta ta hanyar ultracentrifugation. Ajiye supernatant mai ɗauke da kwayar cutar a -80°C har sai transfuction ya ƙare.
Ana raba PBMC daga samfuran rabuwar leukocyte masu lafiya (STEMCELL Technologies) ta hanyar amfani da centrifugation na Ficoll gradient density centrifugation. Yi amfani da zaɓi mai kyau na CD8 microbeads (Miltenyi) don ware ƙwayoyin CD8+ daga PBMC. Ƙara ƙwayoyin T tare da TransAct (Miltenyi) da kuma a cikin matsakaici na TexMACS [Miltenyi; an ƙara shi da kashi 3% na serum na ɗan adam wanda ba shi da zafi, 1% PenStrep da IL-2 (300 U/ml)]. Awa ashirin da huɗu bayan ƙarfafawa, an ƙara ƙwayoyin T tare da lentivirus (10 μl mai yawan ƙwayar cuta a kowace ƙwayoyin 106). Kwanaki 1 zuwa 3 bayan an canza Cytek Aurora (a kan FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), kimanta bayyanar GFP na ƙwayoyin don nuna ingancin transduction na akalla 30%.
An yi wa ƙwayoyin CAR-T magani na tsawon awanni 24 a cikin Immunocult (STEMCELL Technologies; an ƙara musu 1% PenStrep) a ƙarƙashin waɗannan yanayi: ba a yi musu magani ba, an yi musu magani da adenosine 250 μM ko 10 mM MNA. Bayan an yi musu magani kafin a fara magani, an wanke ƙwayoyin CAR-T da PBS kuma an haɗa su da ƙwayoyin SK-OV-3 20,000 [ATCC; a cikin McCoy 5A matsakaici (Sigma-Aldrich) an ƙara musu 10% FBS da 1% PenStrep a 10: An ƙara yawan tasirin sakamako zuwa manufa na 1 a cikin sau uku a cikin ƙarin maganin Immunocult. An yi amfani da ƙwayoyin SK-OV-3 da ƙwayoyin SK-OV-3 da aka haɗa da saponin dijitalis (0.5mg/ml; Sigma-Aldrich) a matsayin masu sarrafawa marasa kyau da masu kyau, bi da bi. Bayan awanni 24 na noma tare, an tattara ruwan da ke cikin ƙasa kuma an auna lactate dehydrogenase (LDH) bisa ga umarnin masana'anta (LDH Glo Cytotoxicity Assay Kit, Promega). An narkar da ruwan da ke cikin ƙasa da kashi 1:50 a cikin ma'aunin LDH. An auna kashi na kisa ta amfani da dabarar da ke ƙasa: kashi na kisa = kashi na gyara / matsakaicin adadin kisa x 100%, inda kashi na gyara = ƙwayoyin T na al'ada kawai, da matsakaicin adadin kisa = tabbataccen iko-korau.
Kamar yadda aka bayyana a cikin rubutu ko kayan aiki da hanyoyin, yi amfani da GraphPad Prism 8, Microsoft Excel ko R v3.6.0 don nazarin ƙididdiga. Idan an tattara samfura da yawa daga majiyyaci ɗaya (kamar ascites da ƙari), muna amfani da gwajin t mai haɗin gwiwa ko kuma mu haɗa majiyyaci a matsayin sakamako bazuwar a cikin samfurin layi ko na gabaɗaya kamar yadda ya dace. Don nazarin metabolomics, ana yin gwajin mahimmanci a cikin sau uku.
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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 Bell J.dinni Grar Jones (R.ph Nelson) Jones), Phineas T. Hamilton (Phineas T.
MNA tana ba da gudummawa ga danne garkuwar jiki na ƙwayoyin T kuma tana wakiltar wata manufa ta rigakafi don magance cutar kansar ɗan adam.
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 Bell J.dinni Grar Jones (R.ph Nelson) Jones), Phineas T. Hamilton (Phineas T.
MNA tana ba da gudummawa ga danne garkuwar jiki na ƙwayoyin T kuma tana wakiltar wata manufa ta rigakafi don magance cutar kansar ɗan adam.
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