Immunomodulatory metabolites cu tumor microenvironment (TME) ah a biapi bikmi sining an si, asinain a tlawmpal te lawng dah ti lo ahcun, an sining cu theih khawh a si rih lo. Hika ah, hi TME tthen dangdang hna i metabolome langhter awkah a sangmi serous carcinoma (HGSC) a ngeimi mizaw hna i an hma le an ascites chungin hma le T cells pawl kha kan hlathlai hna. Ascites le tumor cells nih hin metabolite aa dannak tampi an ngei. Ascites he tahchunh ahcun, tumor-infiltrating T cells cu 1-methylnicotinamide (MNA) in an rum ngaingai. T cells chung i MNA dat cu a sang ko nain, nicotinamide N-methyltransferase (S-adenosylmethionine in nicotinamide ah methyl phu thlennak a bawmtu enzyme) langhternak cu fibroblasts le tumor cells lawng ah a si. Riantuan ning in, MNA nih T cells kha tumor-promoting cytokine tumor necrosis factor alpha chuahter dingin a forh hna. Cucaah, TME-in chuahmi MNA nih T cells hna i an pumsa ralvennak uknak ah a bawmh i minung cancer thlopnak caah a si khomi immunotherapy target a aiawh.
Tumor-derived metabolites nih hin anti-tumor immunity ah a thukmi khamnak an ngei kho, cun tehte tampi nih zawtnak karhternak caah a biapi bikmi thazaang an si khawh ti kha a langhter (1). Warburg nih a chuahpimi thil pin ah, naite riantuannak nih hmawm cells hna i an thlennak dirhmun le hmawm microenvironment (TME) i a ralvennak dirhmun he pehtlaihnak a langhter. Zu model le minung T cells cung i hlathlainak nih a langhter mi cu glutamine metabolism (2), oxidative metabolism (3) le glucose metabolism (4) nih hin immune cell subgroup phunphun ah mah tein rian an ttuan khawh. Hi lam hna ah hin metabolites zeimawzat nih T cells hna i an anti-tumor riantuannak kha an kham. Coenzyme tetrahydrobiopterin (BH4) khamnak nih T cells karhternak a hrawh khawh ti cu a fiang cang, cun pum chung i BH4 a karh ahcun CD4 le CD8 nih a hruaimi anti-tumor immune response a ṭhanchoter khawh. Cun, kynurenine nih a chuahpimi zawtnak khamnak thazaang kha BH4 (5) peknak in khamh khawh a si. Isocitrate dehydrogenase (IDH) mutant glioblastoma ah, enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) chuahnak nih T cell riantuannak, karhternak le cytolysis cawlcanghnak a kham (6). Nai ah, glycolysis in a chuakmi methylglyoxal cu myeloid hramthawknak suppressor cells nih an chuahter ti a si, cun methylglyoxal T cell thlennak nih effector T cell riantuannak a kham khawh. Thlopnak ah, methylglyoxal tlawmternak nih myeloid-derived suppressor cells (MDSC) cawlcanghnak kha a tei khawh i mouse model ah checkpoint blockade thlopnak kha hmunkhat ah a ṭhanchoter khawh (7). Hi hlathlainak hna nih hin TME-in a chuakmi thilri hna nih T cell riantuannak le cawlcanghnak uknak ah a biapi bikmi rian an ttuanmi kha an langhter.
T cell riantuan ningcang lo cu ovarian cancer ah tampi an langhter cang (8). Mah cu a cheu cu hypoxia le a ttha lomi tumor vasculature (9) ah a ummi metabolic sining ruangah a si, cu nih cun glucose le tryptophan kha lactic acid le kynurenine tibantuk a chuakmi thilri ah a thlen. A tam tukmi extracellular lactate nih interferon-γ (IFN-γ) chuahternak a zorter i myelosuppressive subgroups thleidannak a chuahter (10, 11). Tryptophan ei nih T cell karhternak kha direct in a kham i T cell receptor signaling kha a kham (12-14). Hi thil hmuhmi hna um ko hmanh sehlaw, pumsa ralvennak kong he pehtlai in rian tampi cu in vitro T cell cinnak ah a ttha bikmi media hmangin tuah a si, asiloah in vivo ah aa khatmi vok model lawng ah rikhiah a si, cu hna lakah pakhat hmanh nih minung cancer le pumsa lei macro le micro pawngkam aa khat lonak kha tling tein a langhter lo.
Ovarian cancer a langh tawnmi cu pawpi chung a karh i ascites a lang. Ascites chung i cell ti a khonmi cu a zual chin lengmangmi zawtnak le a ttha lomi zawtnak he aa pehtlai (15). Report ning in, hi aa dangmi tthen cu hypoxic a si, vascular endothelial growth factor (VEGF) le indoleamine 2,3-dioxygenase (IDO) a sang, cun T regulatory cells le myeloid inhibitory cells nih a luhhnawh (15-18). Ascites i a thlennak pawngkam cu hmawm hrimhrim he aa dang kho, cucaah peritoneal hmun ah T cells pawl an i remh ning cu a fiang lo. Cun, ascites le tumor pawngkam ah a ummi metabolites karlak ah a biapi mi aa dannak le aa khat lonak nih hin immune cells luh khawhnak le tumor cung i an riantuannak kha a kham khawh, cun hlathlainak tuah chap a herh.
Hi buainak hna hi tawlrel khawhnak ding caah, cell phun dangdang (CD4 + le CD8 + T cells telh chih in) le tumor chung le karlak ah hlathlainak tuah khawhnak ding caah sensitive cell separation le liquid chromatography tandem mass spectrometry (LC-MS/MS) phunglam kan suai. Hi lam hi a sangmi flow cytometry le single-cell RNA sequencing (scRNA-seq) he fonh in kan hman i hi a biapi mi mibu hna i an thilri thlennak dirhmun kha a ttha ngaimi hmanthlak in kan hmuh khawh. Hi lam nih hin tumor T cells ah 1-methylnicotinamide (MNA) a karh ngaingai ti a langhter, cun in vitro hneksaknak nih MNA nih T cell riantuannak ah a chuahpimi immunomodulatory effect cu a hlan ah theih a si lo ti a langhter. A tlangpi in, hi lam nih hin hmawm le pumsa ralvennak cells karlak ah aa pehtlaimi thlennak a langhter, cun ralvennak uknak metabolites kongah a dang tein hmuhnak a pek, cucu T cell-based ovarian cancer immunotherapy thlopnak caah a ttha khomi a si.
Glucose laknak [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) le mitochondrial cawlcanghnak [MitoTracker Deep Red (MT DR)] (2017) kha aa khat tein tahnak caah high-dimensional flow cytometry kan hman zawtnak khamnak cells le tumor cells milu a thleidangtu hmelchunhnak (Table S2 le Figure S1A). Hi hlathlainak nih a langhter mi cu T cells he tahchunh ahcun, ascites le tumor cells nih hin glucose laknak a sang deuh, asinain mitochondrial cawlcanghnak ah aa dannak a tlawm deuh. Tumor cells [CD45-EpCAM (EpCAM)+] nih a lakmi glucose cu T cells nakin a let thum in a let li tiang a si, cun CD4 + T cells nih a lakmi glucose cu CD8 + T cells nakin a let 1.2 a si, cucu Tumor infiltrating lymphocytes (TIL) nih TME hmanthlak aa khatmi hmanh ah metabolic herhmi aa dangmi an ngei ti a langhter (Figure 1). Cu he aa dang in, tumor cells chung i mitochondrial cawlcanghnak cu CD4 + T cells he aa lo, cun cell phun hnih hna i mitochondrial cawlcanghnak cu CD8 + T cells nakin a sang deuh (Figure 1B). A tlangpi in, hi hmuhmi nih hin metabolic level a langhter. Tumor cells hna i an thlennak cawlcanghnak cu CD4 + T cells nakin a sang deuh, cun CD4 + T cells hna i an thlennak cawlcanghnak cu CD8 + T cells nakin a sang deuh. Cell phun kip ah hi thil sining hna um ko hmanh sehlaw, CD4 + le CD8 + T cells hna i an thlennak dirhmun asiloah ascites ah an i tluknak ah tumor he tahchunh tikah aa khatmi thleidannak a um lo (Figure 1C). Cu he aa dang in, CD45-cell tthencheunak ah, tumor chung i EpCAM+ cells zat cu ascites he tahchunh tikah a karh (Figure 1D). EpCAM+ le EpCAM- cell thilri hna karlak ah a fiangmi thlennak zong kan hmuh. EpCAM+ (tumor) cells nih EpCAM- cells nakin glucose laknak le mitochondrial cawlcanghnak a sang deuh, cucu TME chung i tumor cells chung i fibroblasts metabolic cawlcanghnak nakin a sang deuh ngaingai (Figure 1, E le F).
(A le B) Glucose laknak (2-NBDG) (A) le CD4 + T cells (MitoTracker a senmi) mitochondrial cawlcanghnak (B) Aiawhtu graph (orhlei) le tabulated data (orhlei), CD8 + T cells le EpCAM + CD45-tumor cells le tumor cites in an chuahmi (2-NBDG) (A) le mitochondrial cawlcanghnak a tlawm bik a tthawnnak (MFI). (C) Ascites le tumor ah CD4 + le CD8 + cells (CD3 + T cells) zat. (D) Ascites le tumor (CD45−) ah EpCAM + tumor cells zat. (E le F) EpCAM + CD45-tumor le EpCAM-CD45-matrix glucose laknak (2-NBDG) (E) le mitochondrial cawlcanghnak (MitoTracker a senmi) (F) aiawhtu graph (orhlei) le tabulated data ( Orhlei) Ascites le tumor cells. (G) Flow cytometry hmangin CD25, CD137 le PD1 langhternak aiawhtu hmanthlak hna. (H le I) CD25, CD137 le PD1 langhternak cu CD4 + T cells (H) le CD8 + T cells (I) ah a si. (J le K) CCR7 le CD45RO langhternak cung ah hram bunh in naive, central memory (Tcm), effector (Teff) le effector memory (Tem) phenotypes. Ascites le tumor ah CD4 + T cells (J) le CD8 + T cells (K) aiawhtu hmanthlak (orhlei) le tabular data (orhlei). P man hna cu t-test (*P<0.05, **P<0.01 le ***P<0.001) in tuakmi an si. Line nih aa tlakmi mizaw (n = 6) a aiawh. FMO, ceunak minus pakhat; MFI, a tlawmbik ceunak thazaang.
Hlathlainak tuah chapnak nih a ttha ngaimi T cell phenotypic dirhmun karlak ah a dang a biapi mi thleidannak a langhter. Tumor chung i a cawlcangmi (Figure 1, G in I) le effector memory (Figure 1, J le K) cu ascites (CD3 + T cells zat) nakin a tam deuh. Cu bantuk cun, riantuannak hmelchunhnak (CD25 le CD137) le tlawmternak hmelchunhnak [programmed cell thihnak protein 1 (PD1)] langhternak in phenotype hlathlainak nih a langhter mi cu, hi mibu hna i an thilri thlennak sining cu aa dang ko nain (Figure S1, B in E tiang), Asinain naive, asiloah cinkennak thlennak hna karlak ah a biapi mi thlennak a um lo (Figure S1, F1 KEIMAH). Hi hmuhmi hna cu cell phenotypes (21) kha amah tein chiah khawhnak ding caah seh cawnnak lam hmannak in fehter a si, cu nih cun mizaw ascites ah ruh thli cells (CD45 + / CD3- / CD4 + / CD45RO +) tampi a um ti kha a langhter chin (Figure S2A ). Hngalhmi cell phun vialte lakah, hi myeloid cell milu nih hin glucose laknak le mitochondrial cawlcanghnak a sang bik a langhter (Figure S2, B in G tiang). Hi hmuhmi nih hin HGSC mizaw hna i ascites le tumors ah hmuhmi cell phun tampi karlak ah a fekmi metabolic thleidannak a langhter.
TIL i metabolomic sining hngalhthiamnak ah a biapi bikmi harnak cu a tlingmi thianhlimnak, tthatnak le tamnak T cell sample pawl kha hma chungin thleidan a herhnak hi a si. Nai ah tuahmi hlathlainak nih a langhter ning ahcun, flow cytometry cung ah hram bunh in thleidannak le bead tthanternak lam nih cellular metabolite profile ah thlennak a chuahpi khawh (22-24). Hi buainak tei khawhnak ding caah, LC-MS/MS in hlathlainak tuah hlan ah operation in thlehmi minung hnuk cancer in TIL kha thleidan le thleidan khawhnak ding caah bead enrichment method kha kan remh (Materials and Methods zoh; Figure 2A). Hi protocol nih hin metabolite thlennak cungah a chuahpimi thil vialte tuaktan khawhnak ding caah, a cunglei bead tthennak step hnu ah a ngandammi bawmchantu hna nih an cawlcangtermi T cells metabolite profile kha bead tthen lomi nain tikhor cung ah a ummi cells he kan tahchunh hna. Hi tthatnak zohfelnak hlathlainak nih hi thil sining pahnih karlak ah pehtlaihnak sang a um ti a hmuh (r = 0.77), cun 86 metabolites phu i thiamnak lei in tuah tthan khawhnak a sang (Figure 2B). Cucaah, hi lam hna nih hin cell phun tthanchonak a tuahmi cells ah a dikmi metabolite hlathlainak an tuah khawh, cucaah HGSC chung i a hleiin metabolite pawl hngalh khawhnak caah a hmasa bik a sangmi hmunhma a pek khawh, cucaah mi nih cell thleidannak Sexual metabolism program kong thukpi in theihthiamnak an hmuh khawh.
(A) Magnetic bead tthanchonak hmanthlak. LC-MS/MS in hlathlainak tuah hlanah, cells pawl cu a pehzulhmi magnetic bead tthanchonak voithum an tuah lai asiloah tikhor cungah an um lai. (B) Metabolites tamnak cungah tthanchonak phun nih a chuahpimi thil. Tthanchonak phunkhat cio caah tahnak pathum i a tlangpi ± SE. A senmi hri nih 1:1 pehtlaihnak a aiawh. Axis min cazin ah langhtermi tahnak tuah tthanmi hna i tthencheunak chung pehtlaihnak (ICC). NAD, nicotinamide adenine dinucleotide. (C) Mizaw metabolite hlathlainak riantuannak kalning hmanthlak. Ascites asiloah hma pawl cu mizaw hna sin in an lak hna i lumternak ah an chiah hna. Sample pakhat cio chungin tlawmpal cu flow cytometry in hlathlainak tuah a si, a tangmi sample pawl cu CD4+, CD8+ le CD45- cells caah tthanchonak voi thum an tuah. Hi cell cheukhat hna cu LC-MS/MS hmangin hlathlainak an tuah hna. (D) A tlarimi metabolite tamnak lumnak map. Dendrogram nih Ward nih sample karlak ah Euclidean hlatnak a fonhmi a aiawh. (E) Sample metabolite map i a biapi bikmi thilri hlathlainak (PCA), sample pakhat cio i aa khatmi pathum a langhtermi, mizaw pakhat chungin sample pawl cu line in an i pehtlaih. (F) Mizaw cungah aa hngatmi sample i metabolite profile PCA (cucu, a cheu tlawmpal hman); zohchunhmi phun cu a tlongmi tilawng nih a kham. PC1, a biapi bikmi thilri 1; PC2, a biapi bikmi thilri 2.
A hnu ah, hi rumternak lam hi HGSC mizaw paruk hna i an ascites le an hma a hrampi ah CD4 +, CD8 + le CD45-cell tthencheunak 99 metabolites hlathlainak caah kan hman (Figure 2C, Figure S3A le Table S3 le S4). Lungthawhnak a ngeimi milu cu a nungmi cells tampi lak i 2% in 70% tiang a si, cun cells zat cu mizaw pakhat le pakhat karlak ah aa dang ngai. Beads pawl tthen hnu ah, a tthawntermi tthencheunak (CD4+, CD8+ asiloah CD45-) nih a tlangpi in sample chung i a nungmi cells vialte lakah 85% nak tam a lak. Hi tthanchonak lam nih hin minung hmawm tissue metabolism chungin cell milu kha hlathlainak tuah khawhnak a kan pek, cucu sample nganpi in tuah khawh a si lo. Hi protocol hmangin, l-kynurenine le adenosine, hi ttha tein langhtermi immunosuppressive metabolites pahnih cu tumor T cells asiloah tumor cells ah an kai ti kha kan hngalh (Figure S3, B le C). Cucaah, hi hmuhmi nih hin kan cell tthennak le mass spectrometry thiamnak nih mizaw vun chung i nunnak lei in a biapi mi thilri pawl hmuh khawhnak zumhawktlak sinak le thilti khawhnak a langhter.
Kan hlathlainak nih mizaw chung le mizaw karlak ah cell phun hna i a fekmi thlennak zong a langhter (Figure 2D le Figure S4A). A bik in, mizaw dang he tahchunh tikah, mizaw 70 nih cun a dangdang metabolic sining a langhter (Figure 2E le Figure S4B), cu nih cun mizaw karlak ah metabolic heterogeneity a um kho ti a langhter. Mizaw dang (1.2 in 2 liters; Table S1) he tahchunh tikah, mizaw 70 (80 ml) ah an lakmi ascites dihlak cu a tlawm deuh ti kha hngalh awk tlak a si. Principal component hlathlainak (tahchunhnak ah, a cheuchum redundancy hlathlainak hmannak) chungah mizaw karlak i aa khat lonak uknak nih cell phun karlak ah aa khatmi thlennak a langhter, cun cell phun le/asiloah microenvironment cu metabolite profile ningin fiang tein an i fonh (Figure 2F ). Metabolite pakhat lawng hlathlainak nih hi thil sining hna hi a langhter i cell phun le pawngkam hmete karlak ah a biapi mi thleidannak a langhter. A hlei in hmuhmi aa dannak cu MNA a si ti kha hngalh awk tlak a si, mah cu a tlangpi in CD45- cells le CD4+ le CD8+ cells ah a rummi a si i cu nih cun tumor a luhhnawh (Figure 3A). CD4 + cells caah cun, hi thil sining hi a fiang bik, cun CD8 + cells chung i MNA zong pawngkam nih fakpi in a hnorsuanmi a lo. Sihmanhsehlaw, mah cu a biapi lo, zeicahtiah mizaw paruk chungin pathum lawng kha tumor CD8+ mark caah zohfel khawh an si. MNA pin ah, ascites le tumor chung i cell phun dangdang ah, TIL ah a ttha lomi a dang metabolites zong aa dang tein an rum (Figures S3 le S4). Cucaah, hi data nih hin hlathlainak tuah chapnak caah ruahchannak a ummi immunomodulatory metabolites phu pakhat a langhter.
(A) Ascites le tumor in CD4+, CD8+ le CD45- cells ah MNA a tlawm deuhmi. Box plot nih a karlak (line), interquartile range (frame hinge) le data range, interquartile range (frame whisker) let 1.5 tiang a langhter. Mizaw thilri le lamthluan ah langhtermi bantukin, P man (*P<0.05 le **P<0.01) hngalh khawhnak ding caah mizaw limma man kha hmang. (B) MNA thlennak hmanthlak (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 (a sen): 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 of scRNA-seq of ascites (grey) le tumor (sen; n = 3 mizaw). (D) scRNA-seq hmangin hngalhmi cell phun dangdang ah NNMT langhternak. (E) SK-OV-3, minung hrinmi kal (HEK) 293T, T cells le MNA-thlopmi T cells ah NNMT le AOX1 langhternak. A tthenmi langhternak cu SK-OV-3 he pehtlai in langhter a si. SEM he langhternak pattern cu langhter a si (n = a ngandammi bawmchantu 6). Ct man 35 nak tam cu hmuh khawh lomi (UD) ah ruah a si. (F) 8mM MNA in thlopmi SK-OV-3, HEK293T, T cells le T cells ah SLC22A1 le SLC22A2 langhternak. A tthenmi langhternak cu SK-OV-3 he pehtlai in langhter a si. SEM he langhternak pattern cu langhter a si (n = a ngandammi bawmchantu 6). Ct man 35 nak tam cu hmuh khawh lomi (UD) ah ruah a si. (G) MNA he suimilam 72 chung chiah hnu ah a cawlcangmi a ngandammi bawmchantu T cells chung i cell MNA dat. SEM he langhternak pattern cu langhter a si (n = a ngandammi bawmchantu 4).
MNA cu nicotinamide N-methyltransferase (NNMT; Figure 3B) nih S-adenosyl-1-methionine (SAM) in nicotinamide (NA) ah methyl phu thlennak in a chuak. NNMT cu minung cancer phunphun ah a tam tuk i a karh, a luhhnawh i a karh (25-27). TME chung i T cells ah MNA a chuahkehnak hngalhthiam deuh awkah, HGSC mizaw pathum hna i an ascites le an tumor ah cell phun kip ah NNMT langhternak kha scRNA-seq hmangin kan hman (Table S5). Cell 6,500 hrawng hlathlainak nih a langhter mi cu ascites le tumor pawngkam ah, NNMT langhternak cu fibroblast le tumor cell population tiah ruahmi lawng ah a tlawm (Figure 3, C le D). PTPRC (CD45 +) a langhtermi mibu pakhat hmanh ah a fiangmi NNMT langhternak a um lo ti kha hngalh awk tlak a si (Figure 3D le Figure S5A), cucu metabolite spectrum ah hmuhmi MNA cu T cells chungah luhter a si cang ti a langhter. Aldehyde oxidase 1 (AOX1) langhternak nih MNA cu 1-methyl-2-pyridone-5-carboxamide (2-PYR) asiloah 1-methyl-4-pyridone-5-carboxamide (4- PYR) ah a thlen; Figure 3B) cu COL1A1 a langhtermi fibroblasts milu zong ah a tlawm (Figure S5A), cu hna nih cun T cells nih a hlanlio MNA metabolism thiamnak an ngei lo ti kha a langhter. Hi MNA he aa pehtlaimi genes pawl an langhternak pattern cu HGSC mizaw hna sinin ascites chungin a pahnihnak aa hngat lomi cell data set hmangin fehter a si (Figure S5B; n = 6) (16). Cun, MNA in thlopmi a ngandammi bawmchantu T cells hna i quantitative polymerase chain reaction (qPCR) hlathlainak nih control SK-OV-3 ovarian tumor cells he tahchunh tikah, NNMT asiloah AOX1 cu a lang lo (Figure 3E). Hi ruahlopi in hmuhmi nih hin MNA cu fibroblasts asiloah tumors in TME chung i a pawngkam T cells ah a chuak kho ti a langhter.
Candidate hna ah hin organic cation phurtu 1 in 3 tiang (OCT1, OCT2 le OCT3) chungkhar an i tel ko nain, a cawhmi phurtu 22 (SLC22) chungkhar (SLC22A1, SLC22A2 le SLC22A3) nih an code, MNA phurtu a si khomi hna cu an fiang rih lo (28) . A ngandammi bawmchantu T cells chungin mRNA QPCR nih SLC22A1 langhternak tlawmte lawng a langhter nain SLC22A2 hmuh khawh lomi a langhter, cu nih cun cauk chung ah a hlan ah an rak langhter cang ti kha a fehter (Figure 3F) (29). Cu he aa dang in, SK-OV-3 ovarian tumor cell line nih cun transporter pahnih a sangmi a langhter (Figure 3F).
T cells nih ramdang MNA lak khawhnak an ngeih khawhnak hneksaknak caah, a ngandammi bawmchantu T cells cu MNA aa dangmi a ummi ah suimilam 72 chung an cawm hna. Exogenous MNA a um lo ahcun, MNA chung i a ummi cell chung thilri cu hmuh khawh a si lo (Figure 3G). Sihmanhsehlaw, exogenous MNA in thlopmi a cawlcangmi T cells nih cells chung i MNA dat ah dose-dependent karhternak a langhter, 6 mM MNA tiang (Figure 3G). Hi hmuhmi nih hin transporter langhternak a niam ko nain le intracellular MNA metabolism caah rian a ttuanmi enzyme bik a um lo nain, TIL nih MNA cu a lak khawh rih ko ti a langhter.
Mizaw T cells chung i metabolites spectrum le in vitro MNA laknak hneksaknak nih cancer he aa pehtlaimi fibroblasts (CAF) nih MNA an chuahter khawhnak a karhter i tumor cells nih TIL phenotype le riantuannak kha a uk khawh. MNA nih T cells cung ah a chuahpimi thil hngalh khawhnak dingah, a ngandammi bawmtu T cells pawl cu MNA a um le um lo ah in vitro ah an cawlcangter hna, cun an karh ning le cytokine chuahternak kha an zohfel hna. Ni 7 chung MNA kha a sang bik dose in an chap hnu ah, milu let hnih in a karhmi zat cu a zor deuh, cu lioah thazaang cu dose vialte ah a hmun (Figure 4A). Cun, lenglei MNA thlopnak nih CD4 + le CD8 + T cells nih tumor necrosis factor-α (TNFα; Figure 4B) a langhtermi zat a karhter. Cu he aa dang in, IFN-γ chuahnak cu CD4 + T cells ah a zor ngaingai, asinain CD8 + T cells ah cun a zor lo, cun interleukin 2 ah thlennak a um lo (IL-2; Figure 4, C le D). Cucaah, hi MNA-thlopmi T cell cinnak chungin a chuakmi thil hna i enzyme-linked immunosorbent assay (ELISA) nih TNFα ah a karh ngaingai, IFN-γ ah a zor, cun IL-2 ah thlennak a um lo (Figure 4, E in G tiang). . IFN-γ a zornak nih MNA nih T cells hna i an anti-tumor cawlcanghnak khamnak ah rian a ttuan kho ti a langhter. T cell-mediated cytotoxicity cung i MNA nih a chuahpimi thil kha zohchunh awk ah, chimeric antigen receptor T (FRα-CAR-T) cells nih folate receptor α le CAR-T (GFP) kha green fluorescent protein (GFP) -CAR-T) cells nih a ukmi kha a dammi donor peripheral thisen mononuclear cells (PBMC) nih an chuah. CAR-T cells cu MNA a um lio ah suimilam 24 chung an cin hna, cun minung SK-OV-3 ovarian tumor cells he folate receptor α a langhtermi effector le target ratio 10:1 ah an cin hna. MNA thlopnak nih FRα-CAR-T cells thah khawhnak a zorter ngaingai, cucu adenosine in thlopmi FRα-CAR-T cells he aa lo (Figure 4H).
(A) Ni 7 ah nunphung in direct in a nung khomi cell relnak dihlak le milu zat let hnih karhnak (PD). Bar graph nih a ngandammi bawmchantu paruk hna i an tlangpi + SEM kha a langhter. A tlawmbik n = 3 aa hngat lomi hneksaknak chungin data a aiawh. (B in D) CD3/CD28 le IL-2 cu T cells pawl kha anmah le an MNA tlawmtam cio ah ni 7 chung riantuanternak caah hman an si. Hlathlainak tuah hlanah, cells pawl cu PMA/ionomycin le GolgiStop he suimilam 4 chung an forh hna. T cells ah TNFα (B) langhternak. A nungmi cells chung i TNFα langhternak tahchunhnak hmanthlak (orhlei) le tabular data (orhlei). T cells ah IFN-γ (C) le IL-2 (D) langhternak. Cytokines langhternak cu flow cytometry in tah a si. Bar graph nih a tlangpi (n = a ngandammi bawmchantu 6) + SEM a langhter. P man hngalh khawhnak dingah aa dannak le tahfung (*P<0.05 le **P<0.01) kha lam khatlei in hlathlainak hmang. A tlawmbik n = 3 aa hngat lomi hneksaknak chungin data a aiawh. (E in G) CD3/CD28 le IL-2 cu T cells pawl kha anmah le an MNA tlawmtam cio ah ni 7 chung riantuanternak caah hman an si. PMA/ionomycin thazaang peknak suimilam 4 hlan le hnu ah medium cu an lak. TNFα (E), IFN-γ (F) le IL-2 (G) an tlawmtam ning cu ELISA in tah a si. Bar graph nih a tlangpi (n = a ngandammi bawmchantu 5) + SEM a langhter. P man cu aa dannak le tahnak tuah tthannak (*P<0.05) in lam khatlei hlathlainak hmangin tuak a si. Dotted line nih cun hmuh khawhnak ri kha a langhter. (H) Cell tthencheunak hneksaknak. FRα-CAR-T asiloah GFP-CAR-T cells cu adenosine (250μM) asiloah MNA (10 mM) in suimilam 24 chung remh an si, asiloah thlopbul loin an chiah hna (Ctrl). SK-OV-3 cells thahnak zatuak kha tah a si. P man cu Welch t hneksaknak in tuakmi a si (*P<0.5 le **P<0.01).
MNA-a hngatmi TNFα langhternak uknak kong hngalhthiamnak hmuh khawhnak ding caah, MNA-thlopmi T cells TNFα mRNA thlennak kha an zohfel (Figure 5A). MNA in thlopmi a ngandammi bawmchantu T cells nih TNFα ttialmi tlukruannak ah a let hnih in a karh, cucu MNA cu TNFα ttialmi uknak cungah aa hngatmi a si ti a langhter. Hi a si khomi uknak lamthluan hlathlainak tuah awkah, TNFα a ukmi theihmi ttialmi thil pahnih, cu hna cu activated T cell nuclear factor (NFAT) le specific protein 1 (Sp1) an si, cu hna cu MNA nih a naihmi TNFα promoter ( 30) he an i pehtlaihnak ah an zohfel hna. TNFα promoter nih hin NFAT pehtlaihnak hmun 6 le Sp1 pehtlaihnak hmun 2 a ngei, hmun pakhat ah aa tluk [5'cap in -55 base pairs (bp)] (30). Chromatin immunoprecipitation (ChIP) nih MNA in thlop tikah, Sp1 le TNFα promoter pehtlaihnak cu a let thum in a karh ti a langhter. NFAT luhternak zong a karh i a biapitnak a naih (Figure 5B). Hi data nih a langhter mi cu MNA nih Sp1 ṭialnak in TNFα langhternak kha a uk, cun a tlawm deuh in NFAT langhternak kha a uk.
(A) MNA tel loin cinmi T cells he tahchunh tikah, MNA in thlopmi T cells ah TNFα langhternak a let in aa thleng. SEM he langhternak pattern cu langhter a si (n = a ngandammi bawmchantu 5). A tlawmbik n = 3 aa hngat lomi hneksaknak chungin data a aiawh. (B) NFAT le Sp1 hnu ah 8 mM MNA he asiloah um loin thlopmi T cells TNFα promoter cu (Ctrl) le PMA/ionomycin thazaang peknak he suimilam 4 chung an fonh hna. Immunoglobulin G (IgG) le H3 cu immunoprecipitation caah a ttha lomi le a ttha lomi uknak ah an hman hna. ChIP zat tuaknak nih a langhter mi cu MNA-thlopmi cells ah Sp1 le NFAT nih TNFα promoter he an i pehtlaihnak cu control he tahchunh tikah a let zeimawzat a karh. A tlawmbik n = 3 aa hngat lomi hneksaknak chungin data a aiawh. P man cu t-test tampi in tuakmi a si (*** P <0.01). (C) HGSC ascites he tahchunh tikah, T cells (cytotoxic a si lomi) nih tumor ah TNF langhternak a karhter. Ron nih mizaw phunphun a aiawh. Langhtermi cells pawl cu 300 tiang random in an lak hna i a hlei in suaimi (** Padj = 0.0076) kha tlawmter awkah an hninh hna. (D) Ovarian cancer caah MNA model ruahnak cheuhnak. MNA cu TME chung i tumor cells le fibroblasts ah a chuak i T cells nih a lak. MNA nih Sp1 le TNFα promoter pehtlaihnak a karhter, cu nih cun TNFα ṭialnak le TNFα cytokine chuahternak a karhter. MNA nih IFN-γ zong a zorter. T cell riantuannak khamnak nih thah khawhnak a zorter i hmawm ṭhanchonak a rangter.
Report nih a chim ning ahcun, TNFα nih hin hmailei le hnulei ah aa hngatmi hmawm le hmawm dohnak tthathnemnak a ngei, asinain nauinn cancer tthanchonak le karhternak ah theihhngalhmi rian a ngei (31-33). Report ning ahcun, ovarian cancer a ngeimi mizaw hna i ascites le tumor tissues ah TNFα a tlawmtam ning cu benign tissues nakin a tam deuh (34-36). Mechanism lei ah, TNFα nih thi rang cells hna an riantuannak, an riantuannak le an karhnak kha a uk khawh, cun cancer cells hna i an muisam kha a thlen khawh (37, 38). Hi hmuhmi he aa tlak in, aa dangmi gene langhternak hlathlainak nih TNF cu ascites he tahchunh tikah tumor tissues chung i T cells ah a tthangcho ngaingai ti a langhter (Figure 5C). TNF langhternak a karhmi cu cytotoxic a si lomi phenotype a ngeimi T cell milu lawng ah a lang (Figure S5A). A tawinak in chim ahcun, hi data nih hin MNA nih HGSC ah immunosuppressive le tumor a karhtertu thil pahnih a ngei timi ruahnak kha a dirpi.
Flow cytometry cung ah hram bunhmi ceunak hmelchunhnak cu TIL metabolism hlathlainak caah a biapi bikmi lam ah a cang. Hi hlathlainak hna nih a langhter mi cu, a pahnihnak lymphoid organs chungin peripheral thisen lymphocytes asiloah T cells he tahchunh ahcun, murine le human TIL nih hin glucose (4, 39) le mitochondrial riantuannak a tlau deuhdeuh (19, 40) an ngei deuh. Hi hlathlainak ah aa lo ngaimi phichuak kan hmuh ko nain, a biapi bikmi tthanchonak cu aa khatmi thlehmi tumor tissue chungin tumor cells le TIL hna i an thlennak kha tahchunh a si. Hi hlan i report cheukhat he aa tlak in, ascites le tumor chungin a chuakmi tumor (CD45-EpCAM +) cells nih CD8 + le CD4 + T cells nakin glucose laknak a sang deuh, cu nih cun tumor cells nih glucose a lakmi a sangmi cu T cells he tahchunh khawh a si ti kha a dirpi. T cell zuamnak ruahnak. TME. Sihmanhsehlaw, tumor cells hna i mitochondrial cawlcanghnak cu CD8 + T cells nakin a sang deuh, asinain mitochondrial cawlcanghnak cu CD4 + T cells he aa lo. Hi hmuhmi nih hin oxidative metabolism cu tumor cells caah a biapi timi a chuakmi tlangtar kha a fehter (41, 42). Cun CD8 + T cells cu CD4 + T cells nakin oxidative dysfunction ah an tuar deuh kho men, asiloah CD4 + T cells nih mitochondrial cawlcanghnak kilvennak caah glucose ti lo carbon sources dang an hman khawh tiah ruahnak an chuah (43, 44). Ascites ah CD4 + T effectors, T effector memory le T central memory cells karlak ah glucose laknak le mitochondrial cawlcanghnak ah thleidannak kan hmu lo ti kha hngalh a herh. Cu bantuk cun, CD8 + T cells hna i an i dannak dirhmun cu hma ah glucose laknak thlennak he pehtlaihnak a ngei lo, cu nih cun in vitro ah cinmi T cells le in vivo ah minung TIL karlak ah a biapi mi thleidannak a langhter (22). Hi hmuhmi hna cu thleidannak um loin mah tein cell milu tthencheunak hmannak zong nih a fehter, cu nih cun CD45 + / CD3- / CD4 + / CD45RO + cells cu tumor cells nakin glucose laknak le mitochondrial cawlcanghnak a sang deuhmi an tam deuh nain Metabolic active cell population an ngei ti kha a langhter chin. Hi milu zat nih hin scRNA-seq hlathlainak ah hmuhmi myeloid suppressor cells asiloah plasmacytoid dendritic cells hna i an ruahmi milu hmete kha a aiawh men lai. Hi pahnih hi minung ovarian tumor ah report an tuah ko nain [45], an herh rih.
Flow cytometry-based methods nih cell phun karlak ah glucose le oxidative metabolism ah a tlangpi in aa dannak kha a fianter khawh ko nain, TME ah mitochondrial metabolism caah glucose asiloah a dang carbon sources nih a chuahtermi a dikmi metabolites cu hngalh khawh a si rih lo. TIL subset pakhatkhat ah metabolites a um le um lo kha chiahnak nih cun thlehmi tissue chungin cell population kha thianh a hau. Cucaah, kan cell rumternak lam cu mass spectrometry he fonh in mizaw sample aa tlakmi ah T cells le tumor cell population ah aa dang tein rumtermi metabolites kong ah theihhngalhnak a kan pek khawh. Hi lam hi fluorescence-activated cell thleidannak nakin tthatnak a ngei ko nain, metabolite library cheukhat cu a hramthawk tein a fekmi le/asiloah a rangmi thlennak rate ruangah a hnorsuan khawh hna (22). Sihmanhsehlaw, kan phunglam nih theihhngalhmi immunosuppressive metabolites pahnih, adenosine le kynurenine kha a hmuh khawh, zeicahtiah annih cu sample phunkhat le pakhat karlak ah an i dang tuk.
Tumor le TIL phun hme tete kan metabolomic hlathlainak nih ovarian TME ah metabolites riantuannak kong ah theihhngalhnak tam deuh a kan pek. Pakhatnak ah, flow cytometry hmangin, tumor le CD4 + T cells karlak ah mitochondrial cawlcanghnak ah thleidannak a um lo ti kha kan hngalh. Sihmanhsehlaw, LC-MS/MS hlathlainak nih hi milu hna lakah metabolites tamnak ah thlennak nganpi a langhter, cu nih cun TIL metabolism le a dihlak in metabolic cawlcanghnak kong biachahnak tuah tikah ralring tein fianternak a herh ti a langhter. Pahnihnak ah, MNA cu ascites ah si loin CD45-cells le T cells karlak ah aa dannak nganbik a ngeimi metabolite a si. Cucaah, tthencheunak le hma umnak nih TIL thlennak ah thlennak a chuahpi kho, cu nih cun a ummi pawngkam hmete ah aa khat lomi a si khomi kha a langhter. Pathumnak ah, MNA-chuahtertu enzyme NNMT langhternak cu CAF ah a tlawm deuh, cucu tumor cells a si, asinain hmuh khawhmi MNA levels cu tumor-in a chuakmi T cells ah hmuh khawh a si. Ovarian CAF ah NNMT a tam tukmi nih cancer a chuahter khomi thil a ngei ti theih a si, a ruang cheukhat cu CAF metabolism, tumor luhhnawhnak le metastasis a chuahter caah a si (27). TIL a dihlak in a tlawm ko nain, CAF ah NNMT langhternak cu Cancer Genome Atlas (TCGA) mesenchymal subtype he aa naih ngai, cucu a ttha lomi hmailei kong he aa pehtlaimi a si (27, 46, 47). A donghnak ah, MNA rawhnak caah tuanvo a ngeimi enzyme AOX1 langhternak zong CAF milu ah a tlawm, cucu T cells nih MNA thlen khawhnak an ngei lo ti a langhter. Hi hmuhmi nih hin hi hmuhmi fehternak caah riantuan chap a herh ko nain, T cells ah MNA a sangmi nih hin immunosuppressive CAF microenvironment a um ti a langhter khawh timi ruahnak kha a dirpi.
MNA phurtu hna i an langhternak a niam tukmi le MNA thlennak ah aa telmi a biapimi protein pawl an hmuh khawh lomi hna ruangah, T cells ah MNA a um cu ruahlopi in a si. NNMT le AOX1 cu scRNA-seq hlathlainak le aa hngatmi phu hnih i target qPCR in hmuh khawh an si lo. Hi hmuhmi nih hin MNA cu T cells nih an sermi a si lo, asinain a pawngkam TME in an lakmi a si ti a langhter. In vitro hneksaknak nih T cells nih lenglei MNA an khon tawn ti a langhter.
Kan in vitro hlathlainak nih a langhter mi cu lenglei MNA nih T cells ah TNFα langhternak a chuahter i Sp1 le TNFα promoter pehtlaihnak a ṭhanchoter. TNFα nih cun hmawm dohnak le hmawm dohnak riantuannak a ngei ko nain, hnuk cancer ah, TNFα nih hnuk cancer tthanchonak a bawmh khawh (31-33). Ovarian tumor cell culture ah TNFα tlawmternak asiloah mouse model ah TNFα signal hlohnak nih TNFα-mediated inflammatory cytokine chuahternak a tthanchoter khawh i tumor tthanchonak a kham khawh (32, 35). Cucaah, hi kongah, TME-in chuahmi MNA cu autocrine loop hmangin TNFα-a hngatchanmi mechanism hmangin pro-inflammatory metabolite bantukin rian a ttuan khawh, cu nih cun ovarian cancer a chuahter i a karhter (31). Hi thil a cang khomi cungah hram bunh in, TNFα khamnak cu nauinn cancer caah a si khomi thlopnak sii pakhat in hlathlainak tuah a si (37, 48, 49). Cun, MNA nih CAR-T cells hna i ovarian tumor cells hna caah cytotoxicity a hrawh, MNA-mediated immune suppression caah tehte dang a pek. A dihlak in, hi hmuhmi nih hin tumor le CAF cells nih MNA kha extracellular TME ah an chuahternak model pakhat a chuahpi. (i) TNF-induced ovarian cancer tthanchonak thazaang peknak le (ii) MNA-induced T cell cytotoxic cawlcanghnak khamnak thawngin, hi nih hin tumor tthatnak pahnih a ngei kho (Figure 5D).
A donghnak ah, rang tukin cell rumternak, cell pakhat lawng zulhnak le metabolic profiling fonh in hmannak thawngin, hi hlathlainak nih HGSC mizaw hna ah tumor le ascites cells karlak ah immunometabolomic thleidannak nganpi a langhter. Hi a tlingmi hlathlainak nih hin T cells karlak ah glucose laknak le mitochondrial cawlcanghnak ah aa dannak a um ti a langhter, cun MNA cu non-cell autonomous immune regulatory metabolite tiah a langhter. Hi data nih hin minung cancer ah TME nih T cell metabolism zeitindah a thlen timi cungah huham a ngei. T cells le cancer cells karlak ah rawlchuannak caah direct in zuamnak a um tiah report an tuah ko nain, metabolites nih hin tumor karhternak le endogenous immune responses a kham khawhnak ding caah indirect regulators rian an ttuan khawh. Hi uknak metabolites hna riantuannak kong fianternak nih hin anti-tumor immune response thanchoternak caah lam dang a hun khawh.
Mizaw zohchunhnak le sii lei data cu Canadian Tissue Repository Network nih fehtermi BC cancer tumor tissue chiahnak in hmuh a si. BC Cancer Hlathlainak Zulhphung Committee le University of British Columbia (H07-00463) nih an cohlanmi phunglam ningin, mizaw vialte an zohchunhmi le sii lei data nih theihternak ca in hnatlaknak an hmuh asiloah phungning tein an hnatlaknak an hlawt. Sample pawl cu fehtermi BioBank (BRC-00290) ah chiah an si. Mizaw sining tling tein Table S1 le S5 ah langhter a si. Cryopreservation caah, scalpel hmangin mizaw i a hmawm sample kha hriamnam in hrawh a si i cu hnuah 100-micron filter chungin a hnuh i cell pakhat lawng a ummi hmuh khawh a si. Mizaw ascites cu 1500 rpm in 4°C ah minutes 10 chung centrifuge tuah a si i cells pawl kha pellet tuah i a cunglei ti kha chuah. Tumor le ascites in hmuhmi cells cu 50% lumnak in a cawlcang lomi minung AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) le 10% dimethyl sulfoxide ah an chiah hna. Hi kilvenmi cell pakhat lawng a ummi hna cu an hnim hna i a tanglei ah langhtermi metabolomics le metabolite hngalh khawhnak caah hman an si.
A tlingmi thilri cu 0.22 μm thletmi 50:50 chapmi RPMI 1640: AimV in aa tel. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) 10% heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) he chapmi, x1 Penicillin Streptomycin (PenStrep) cawhmi (Thermo Fisher Scientific) le 50 μMB-mercaptoethanol. AimV (Invitrogen) cu 20 mM Hepes (Thermo Fisher Scientific) le 2 mM l-glutamine (Thermo Fisher Scientific) he chap a si. Flow cytometer staining buffer cu 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) 3% heat-inactivated AB human serum (Sigma) he chapmi a si. Cell rumternak buffer cu 0.22μm filtered PBS in sermi a si i 0.5% lumnak in a cawlcang lomi minung AB serum (Sigma-Aldrich) he chap a si.
37°C a tlingmi thilri ah, cells pawl cu 10 nM MT DR le 100 μM 2-NBDG in minutes 30 chung an thuh hna. A hnu ah, cells pawl cu 4°C ah minutes 15 chung nun khawhnak rong eF506 in an thuh hna. FC Block (eBioscience) le Brilliant Stain Buffer (BD Biosciences) ah cell pawl kha hnim ṭhan hna, flow cytometer staining buffer ah cawh (sertu nih a chimmi ningin), cun khaan lum ah minutes 10 chung chiah. Cell pawl kha antibodies (Table S2) he flow cytometry staining buffer ah 4°C ah minutes 20 chung rawn hna. Hlathlainak tuah hlanah flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ah cells pawl kha hnim ṭhan hna. Cell relnak data hlathlainak caah SpectroFlo le FlowJo V10 hmang law, data sernak caah GraphPad Prism 8 hmang. 2-NBDG le MT DR i a tlangpi ceunak thazaang (MFI) cu log-normalized a si, cun aa tlakmi mizaw hna tuaktan awkah zatlang hlathlainak caah paired t test hman a si. Hlathlainak chungin thil cangmi 40 nak tlawm deuh a ngeimi milu vialte kha hloh dih hna; zatlang hlathlainak le data hmuhsaknak tuah hlanah a ttha lomi man paohpaoh caah MFI man 1 kha khumh.
A cunglei thil kalning panel i kut in tuahmi kharnak strategy kha chap awkah, FlowJo chung i a thimi cell pawl hloh hnu ah cell pawl kha milu ah amah tein chiah awkah a sining khamnak thingkung (FAUST) (21) nih a tlingmi fianternak kha kan hman. A dik lomi tthencheunak (PD1+ le PD1-tumor cells fonh) le chiahmi milu pawl fonh awkah chuahmi kha kut in kan tawlrel. Sample pakhat ah a tlangpi in 2% nak tam cells aa tel, a dihlak ah 11 population caah.
Ficoll gradient density centrifugation cu PBMC le leukocyte thleidannak thilri (STEMCELL Technologies) thleidannak caah hman a si. CD8 + T cells cu CD8 MicroBeads (Miltenyi) hmangin PBMC chungin an thleidan hna i sertu nih a chimmi ningin zarh 2 chung TransAct (Miltenyi) hmangin a tlingmi thilri ah an kauhter hna. Cell pawl cu IL-7 (10 ng/ml; PeproTech) aa telmi a tlingmi thilri ah ni 5 chung an dirter hna, cun TransAct in an thawhter ṭhan hna. Ni 7 ni ah, sertu nih a chimmi ningin, minung CD45 MicroBeads (Miltenyi) cu cells pawl kha a pehzulh in voi thum tthanchoternak caah hman an si. Cell pawl cu flow cytometry hlathlainak caah (a cunglei langhtermi bantuk in) tthen an si, cun LC-MS/MS hlathlainak caah cell million khat cu voi thum tthen an si. A tanglei langhtermi bantuk in sample pawl cu LC-MS/MS in an tuah hna. A tlaumi metabolite man cu ion nambar 1,000 he kan tuak. Sample pakhat cio cu a dihlak ion nambar (TIC) in a tlarimi a si, logarithm in thlen a si i hlathlainak tuah hlanah MetaboAnalystR ah amah tein a tlarimi a si.
Mizaw pakhat cio i an cell pakhat lawng a ummi cu a lum i 40 μm filter in a tlingmi medium ah a thlet (a cunglei langhtermi bantuk in). Sertu phunglam ningin, MicroBeads (Miltenyi) hmangin magnetic bead tthennak in a ttha mi thimnak voi thum pehtlai tein tuah a si i CD8+, CD4+ le CD45- cells (tikhor cung) caah sample pawl kha tthanter awkah hman a si. A tawinak in, cells pawl cu cell enrichment buffer (a cunglei langhtermi bantuk in) ah an hren hna i an rel hna. Cell pawl cu minung CD8 beads, minung CD4 beads asiloah minung CD45 beads (Miltenyi) he 4°C ah minutes 15 chung an chiah hna, cun cell enrichment buffer in an tawl hna. Sample cu LS column (Miltenyi) chungin an kalpi, cun a ttha le a chia tthencheu pawl kha an lak hna. Caan sauhternak le cell tthanchonak tthanchonak caah, CD8-fraction cu CD4+ tthanchonak a voihnihnak caah hman a si, cun CD4-fraction cu a hnu CD45-rumternak caah hman a si. Tthennak tuah chung vialte tikhor cungah a cawhmi kha chiah.
Metabolite hlathlainak caah sample pawl timhtuahnak caah, cells pawl cu tikhor-a kikmi chiti cawhmi in voikhat an tawl hna, cun sample pakhat ah 80% methanol 1 ml an cawh, cun vortex an tuah i ti nitrogen ah an hnim. Sample pawl cu a kik-a lumternak voithum tiang an tuah hna i 4°C ah minutes 15 chung 14,000 rpm ah an cawh hna. Metabolites aa telmi supernatant cu a ro tiang a hnim. Metabolite pawl cu 0.03% formic acid 50 μl ah an cawh tthan hna, cawh awkah an cawh hna, cun a rawkmi thilri pawl hloh awkah an cawh hna.
A cunglei langhtermi bantuk in metabolite pawl chuah. Metabolomics hlathlainak caah a cunglei ti kha a ttha ngaimi ti chromatography thawl ah thlen. Batch effects hrial khawhnak ding caah sample pakhat cio kha aa khatmi cell zat in thlopnak caah random thlopnak phunglam hmang. AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50) ah a hlan ah chuahmi vawleicung thilri hna i an sining zohfelnak kan tuah. Chromatographic hlathlainak le a sangbik hmunhma fonhnak cu MultiQuant version 2.1 software (Applied Biosystems SCIEX) hmangin tuah a si.
A tlaumi metabolite man tuaktannak caah ion relnak 1000 hman a si, cun sample pakhat cio i TIC cu hmuhmi metabolite pakhat cio i a tlarimi peak area tuaktannak caah hman a si i sample thlennak in hmanmi thilri hlathlainak nih a chuahpimi thlennak hna kha remh a si. TIC cu a tlarimi a si hnu ah, MetaboAnalystR(51) (a tlarimi parameter) cu logarithmic thlennak le amah tein a tlarimi tthencheunak caah hman a si. Sample phun karlak ah metabolome aa dannak hlathlainak tuah awkah vegan R package he PCA kan hman, cun mizaw hna hlathlainak caah a cheuchum tlawmtam hlathlainak kan hman. Sample karlak Euclidean hlatnak kha fonh awkah lumnak map dendrogram sernak caah Ward phunglam kha hmang. Cell phun dihlak le pawngkam hmete ah aa dangmi tamnak metabolite pawl hngalh khawhnak ding caah a tlarimi metabolite tamnak ah limma (52) kan hman. Fianternak a fawiter deuh nakhnga, model fianternak caah phu tlawmpal parameter kan hman, cun microenvironment chung i cell phun pawl kha phu pakhat cio (n = phu 6) in kan ruah hna; sullam hneksaknak caah, metabolite pakhat cio caah tahnak voithum kan tuah A hmaan lomi thlennak hrial khawhnak ding caah, mizaw cu limma suaisamnak ah dawnkhantu ah telh a si. Mizaw dangdang karlak ah metabolites aa dannak zoh khawhnak ding caah, mizaw hna telh chih in limma model kha a fekmi lam in kan remh. Padj <0.05 (Benjamini-Hochberg remhnak) i cell phun le pawngkam hme tete karlak ah a hlankan in langhtermi thleidannak a biapitnak kha kan thanh.
Miltenyi Dead Cell Removal Kit (>80% viability) hmangin thazaang tthanchoter hnu ah, 10x 5′gene langhternak protocol hmangin a nungmi a kikmi ascites le hmawm sample dihlak cung ah single-cell transcriptome sequencing tuah a si. Aa tlakmi hmawm le ascites a ngeimi case panga cu an hlathlai hna, asinain hmawm sample pakhat chungin a tlawmmi nun khawhnak nih a telh khawh lo. Mizaw thimnak tampi hmuh khawhnak ding caah, 10x chromium controller lamthluan ah mizaw pakhat cio i an sample pawl kha kan fonh hna i, ascites le tumor hmun pawl kha a dang tein kan hlathlai hna. Sequencing tuah hnu ah [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; tumor le ascites caah cell pakhat ah 73,488 le 41,378 relmi a tlangpi in]], CellSNP le Vireo (53) (CellSNP cung ah hram bunh in) kan hman. GRCh38 nih a pekmi minung SNP (VCF) cu bawmchantu sinak pek a si. SNPRelate kha kan hman i a nai bikmi sinak (IBS) kha mizaw phun (IBS), thleidan lomi cells le duplexes tiah hngalhmi cells le ascites le tumor samples karlak ah aa tlakmi donor pawl telh loin (54). (tumor le ascites in 2792 le 4183 cells, ciocio) hlathlainak caah Jaccard hlatnak in cluster cells cung ah hram bunh in igraph (57) Louvain clustering kha kan hman. cells cu CD8A le GZMA langhternak in fianter an si, ribosomal protein langhternak a tlawmmi subclusters telh loin. Izar et al. (16) nih an chuahmi data kha kan hmuh, an t-SNE luhternak telh chih in, immune cell hmelchunhnak le NNMT langhternak karlak i langhternak aa tluknak kha a uk khawh.
PBMC cu leukocyte tthennak thilri (STEMCELL Technologies) in Ficoll gradient density centrifugation in tthen an si. CD3 + cells cu CD3 beads (Miltenyi) hmangin PBMC chungin an thleidan hna. MNA a um le um lo ah, CD3+ cells cu plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) le IL-2 (300 U/ml; Proleukin) in an cawlcang. Kauh ni hmanung bik ah, nun khawhnak (Fixable Viability Dye eFluor450, eBioscience) le karhternak (123count eBeads, Thermo Fisher Scientific) cu flow cytometry in an tuak. Suimilam 4 chung GolgiStop he PMA (20 ng/ml) le ionomycin (1μg/ml) in cells thazaang peknak in effector riantuannak kha tuaktan, cun CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) le TNFα-fluoresce isothiansothianate (TCB1, MA1 BD). qPCR le ChIP cells pawl kha PMA (20 ng/ml) le ionomycin (1μg/ml) in suimilam 4 chung thazaang pe hna. ELISA supernatant cu PMA (20 ng/ml) le ionomycin (1 μg/ml) he suimilam 4 chung thazaang peknak hlan le hnu ah lak a si.
RNeasy Plus Mini Kit (QIAGEN) hmangin RNA thleidan khawhnak ding caah sertu phunglam kha zulh. QIAshredder (QIAGEN) hmangin sample kha aa khat tein ser. Complementary DNA (cDNA) sernak caah a sangmi RNA in cDNA thilri (Thermo Fisher Scientific) kha hmang. TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) kha a tanglei hlathlainak hna he gene langhternak (sertu phunglam ningin) tahnak caah hmang: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs0042755m15S, 2012 Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] le Hs01010726_m1 (SLC22A2). Sample pawl cu StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) ah MicroAmp fast optical 96-well reaction plate (Applied Biosystems) ah MicroAmp optical film he an kalpi hna. Ct man 35 a lonh ahcun hmuh khawhnak threshold cunglei ah ruah a si i hmuh khawh lomi tiah hmelchunh a si.
A hlan ah langhtermi (58) bantuk in ChIP tuah. A tawinak in, cells pawl cu formaldehyde (a donghnak 1.42%) in an thlop hna i khaan lum ah minutes 10 chung an chiah hna. Minute 10 chung tikhor cung ah a chapmi a linmi khamnak (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl le 0.1% NP-40) kha hmang law, a langhtermi bantukin immunoprecipitation buffer ah chiah ṭhan (58 ). Cu hnu ah zohchunhmi cu a tanglei tluknak hna he sonicated a si: tthen 10 (20 1-second pulses) le aa thleng lomi caan second 40. ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) le SP1 (Cell Signaling Technology; 3μl) antibodies pawl kha 4°CC ah zan khuadei hninh in sample he cawh. Protein A beads (Thermo Fisher Scientific) kha 4°C ah suimilam 1 chung nem tein hninh in sample he cawh, cun chelex beads (Bio-Rad) hmangin DNA kha rumter, cun proteinase K (Thermo Fisher) kha protein rawl ei khawhnak caah hman. TNFα promoter cu PCR in hmuh a si: hmailei, GGG TAT CCT TGA TGC TTG TGT; cu he aa ralchanh in, GTG CCA ACA ACT GCC TTT ATA TG (207-bp thilchuak). Hmanthlak pawl cu Image Lab (Bio-Rad) nih an chuah i ImageJ software hmangin an tah.
Cell cinnak supernatant cu a cunglei langhtermi bantuk in lak a si. Hlathlainak cu minung TNFα ELISA thilri (Invitrogen), minung IL-2 ELISA thilri (Invitrogen) le minung IFN-γ ELISA thilri (Abcam) sertu phunglam ningin tuah a si. Sertu phunglam ningin, a cunglei ti cu TNFα le IL-2 hmuh khawhnak ding caah 1:100 in cawh a si, cun IFN-γ hmuh khawhnak ding caah 1:3 in cawh a si. EnVision 2104 Multilabel Reader (PerkinElmer) hmangin 450 nm ah a laknak kha tah.
PBMC cu leukocyte tthennak thilri (STEMCELL Technologies) in Ficoll gradient density centrifugation in tthen an si. CD3 + cells cu CD3 beads (Miltenyi) hmangin PBMC chungin an thleidan hna. MNA a um le um lo ah, CD3+ cells cu plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) le IL-2 (300 U/ml; Proleukin) he ni 3 chung an cawlcangter hna. Ni 3 hnu ah, cells pawl cu an lak hna i 0.9% saline in an tawl hna, cun pellet cu a rannak in an lumter. Cell relnak cu 123count eBeads hmangin flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) in tuah a si.
A cunglei langhtermi bantuk in metabolite pawl chuah. A ro cangmi chuahmi cu 4000 cell tlukruannak/μl tluk in an cawh ṭhan. Sample cu reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) le CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters) hmangin hlathlainak tuah. Polar mass spectrometer (6470, Agilent), cu ah cun electrospray ionization nih a ttha lei in a ttuan. Mobile phase A cu 0.1% formic acid (H2O ah) a si, mobile phase B cu 90% acetonitrile, 0.1% formic acid a si. LC gradient cu 100% A caah 0 in 2 minutes, 99% B caah 2 in 7.1 minutes, le 99% B caah 7.1 in 8 minutes a si. Cun mobile phase A he column kha minutes 3 chung 0.6 ml/min a luannak in tlukruanter ṭhan. . A luan ning cu 0.4ml/min a si, cun column khaan cu 50°C tiang lumter a si. MNA's a thiangmi chemical standard (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) kha hmang law chiah caan (RT) le thlennak (RT = 0.882 minutes, thlennak 1 = 137→94.1, thlennak 2 = 137→92 , thlennak = 137→77) kha dirh. Thlennak pathum hna cu a dikmi tlaih caan ah an cang tikah, thlennak 1 cu a hleiin a si le si lo hngalh khawhnak ding caah zat tuaknak caah hman a si. MNA (Toronto Research Chemical Company) i a tlarimi tthencheunak cu 0.1, 1.0, 10 le 100 ng/ml le 1.0 le 10μg/ml tlukruannak hmuh khawhnak dingah stock cawhmi (1 mg/ml) kha voi ruk tthennak in sermi a si. Hmuh khawhnak ri cu 1 ng/ml a si, cun a tlarimi lehnak cu 10 ng/ml le 10μg/ml karlak a si. LC/MS hlathlainak caah sample le standard microliter pahnih thunnak pakhat cio hman a si, cun hlathlainak platform a fekmi si khawhnak ding caah thunnak pariat hnu ah cawhmi tthatnak zohchunhnak sample pakhat cio tuah a si. MNA-thlopmi cell sample vialte MNA lehnak cu hneksaknak i a tlarimi chung ah a um. Data hlathlainak cu MassHunter quantitative analysis software (v9.0, Agilent) hmangin tuah a si.
A pahnihnak chan αFR-CAR sermi cu Song et al. sin in lakmi a si. (59). A tawinak in, sermi nih a tanglei thilri pawl aa tel: CD8a hruaitu zulhphung, minung αFR-specific single-chain variable fragment, CD8a hinge le transmembrane region, CD27 intracellular domain le CD3z intracellular domain. CAR zulhphung tling cu GenScript nih a ser, cun thlennak tthatnak tuaktannak caah hmanmi GFP langhternak cassette cunglei ah a pahnihnak chan lentiviral langhternak vector ah a thlen.
Lentivirus cu HEK293T cells thlennak in a chuakmi a si [American Type Culture Collection (ATCC); Dulbecco's remhmi Eagle medium ah 10% fetal bovine serum (FBS) le 1% PenStrep aa telmi ah cin, cun CAR-GFP vector le The packaging plasmids (psPAX2 le pMD2.G, Addgene) nih lipofection amine (Sigma-Aldrich) an hman. Virus aa telmi supernatant cu transfection tuah hnu suimilam 48 le 72 ah an lak, an thlet, cun ultracentrifugation in an cawh. Transduction tiang -80°C ah a tlawmmi virus supernatant kha chiah.
PBMC cu a dammi donor leukocyte tthennak thilri (STEMCELL Technologies) in Ficoll gradient density centrifugation in tthen an si. PBMC chungin CD8+ cells thleidan awkah a ttha mi thimnak CD8 microbeads (Miltenyi) hmang. TransAct (Miltenyi) le TexMACS medium [Miltenyi; 3% lumnak in a cawlcang lomi minung thi, 1% PenStrep le IL-2 (300 U/ml) he chapmi]. Thazaang peknak hnu suimilam 24 ah, T cells cu lentivirus (106 cells ah 10 μl a tlawmmi virus supernatant) in an thlen hna. Cytek Aurora (FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+) ah thlen hnu ni 1 in ni 3 karlak ah, a tlawmbik 30% thlennak tthatnak langhter awkah cells GFP langhternak kha tuaktan.
CAR-T cells cu Immunocult (STEMCELL Technologies; 1% PenStrep he chapmi) ah suimilam 24 chung a tanglei thil sining hna tangah an cin hna: thlop lomi, 250 μM adenosine asiloah 10 mM MNA in thlopmi. Thlop hmasa hnu ah, CAR-T cells cu PBS in an tawl hna i SK-OV-3 cells 20,000 he an fonh hna [ATCC; McCoy 5A medium (Sigma-Aldrich) ah 10% FBS le 1% PenStrep he 10 ah chapmi: Immunocult medium chapmi ah effector le target ratio 1 cu voithum in a karhter. SK-OV-3 cells le SK-OV-3 cells cu digitalis saponin (0.5mg/ml; Sigma-Aldrich) he lysed an si i, negative le positive control ah an hman hna. Suimilam 24 chung hmunkhat ah cin hnu ah, a cunglei ti cu lak a si i lactate dehydrogenase (LDH) cu sertu nih a chimmi ningin tah a si (LDH Glo Cytotoxicity Assay Kit, Promega). LDH supernatant cu LDH buffer ah 1:50 in cawh a si. Thahnak zatuak cu a tanglei formula hmangin tah a si: thahnak zatuak = remhnak zatuak / thahnak tam bik x 100%, cuka ahcun remhnak zatuak = co-culture-T cells lawng, le thahnak zatuak = a ttha mi uknak-a chia mi uknak.
Cafang asiloah thilri le lam hna ah langhtermi bantukin, zatlang hlathlainak caah GraphPad Prism 8, Microsoft Excel asiloah R v3.6.0 hmang. Mizaw pakhat chungin (ascites le tumor tibantuk) sample tampi lak a si ahcun, t test pahnih kan hman asiloah aa tlak ning in linear asiloah generalized model ah mizaw cu random effect in kan telh. Metabolomics hlathlainak caah, a biapitnak hneksaknak cu a let thum in tuah a si.
Hi capar caah bawmtu thilri caah, http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1 zoh
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MNA nih T cells hna i an pumsa ralthatnak khamnak ah a bawmh i minung cancer thlopnak caah a si khomi immunotherapy target a aiawh.
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. Dephardini (R.R. Bellnis), Deraph. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA nih T cells hna i an pumsa ralthatnak khamnak ah a bawmh i minung cancer thlopnak caah a si khomi immunotherapy target a aiawh.
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