BCAT2 Puab Ib Cov Kabmob Tsis Muaj Kabmob Microenvironment Thiab Induces Resistance To Anti-PD-1/PD-L1 Immunotherapy Los Ntawm Kev Tswj Xyuas Cov Tshuaj Proinflammatory Chemokines Thiab Anticancer Immunity

Txhawm rau txhim kho cov lus teb ntawm monotherapy ntawm kev tiv thaiv kab mob tiv thaiv kab mob (ICB), nws yog ib qho tsim nyog yuav tsum nrhiav lub hom phiaj tshwm sim hauv kev kho ua ke. Los ntawm kev txheeb xyuas cov qog microenvironment (TME) cov ntsuas ntsig txog, nws tau lees paub tias BCAT2 ua rau TME tsis muaj mob qog noj ntshav hauv lub zais zis. Cov txiaj ntsig ntawm multiomics qhia tias BCAT2 muaj kev cuam tshuam rau kev ua haujlwm ntawm cytotoxic lymphocyte los ntawm kev txwv cov kev ua ub no ntawm proinflammatory cytokine / chemokine-txog txoj hauv kev thiab T-cell-chemotaxis txoj hauv kev. Immunoassays qhia tias kev tso tawm ntawm CD8+T-cell-txog chemokines ua rau muaj kev cuam tshuam tsis zoo nrog BCAT2, ua rau txo qis ntawm CD8+T hlwb nyob ib ncig ntawm BCAT2+ cov qog hlwb los ntawm deb mus rau nyob ze. Kev kho mob ntawm BCAT2 tsis muaj peev xwm thiab tiv thaiv PD-1 cov tshuaj tiv thaiv kab mob muaj cov nyhuv synergistic hauv vivo, implying lub peev xwm ntawm BCAT2 hauv kev kho ua ke. Tsis tas li ntawd, tus nqi ntawm BCAT2 hauv kev kwv yees qhov ua tau zoo ntawm kev tiv thaiv kab mob tiv thaiv kab mob tau raug lees paub nyob rau hauv ntau pawg tshuaj tiv thaiv kab mob. Ua ke, raws li ib qho tseem ceeb molecule nyob rau hauv TME, BCAT2 yog ib tug tshiab lub hom phiaj nyob rau hauv ua ke nrog nrog ICB thiab ib tug biomarker ntawm kev taw qhia precision kho.

Cov txiaj ntsig ntawm cistanche tubulosa-Antitumor

1. Taw qhia

Bladder cancer (BLCA) yog ib qho ntawm cov kab mob uas tshwm sim ntau tshaj plaws hauv cov zis.[1] Nws kwv yees tias ntau dua 430,000 cov neeg mob tau kuaj pom thoob ntiaj teb txhua xyoo.[2] Txawm hais tias kev kho mob phais radical, yuav luag ib nrab ntawm cov neeg mob uas mob qog nqaij hlav zais zis tshwm sim metastasis.[3] Yog li ntawd, txoj kev kho mob ua lub luag haujlwm tseem ceeb hauv kev mob qog noj ntshav siab. Nrog rau kev txhim kho kev tiv thaiv kab mob, tshwj xeeb tshaj yog kev tiv thaiv kab mob tiv thaiv kab mob (ICB), cov ntaub ntawv pov thawj qhia tau tias ICB muaj kev ua tau zoo heev hauv kev tshem tawm cov qog nqaij hlav. Txawm li cas los xij, tseem muaj coob tus neeg mob uas tsis teb rau monotherapy ntawm ICB los ntawm qhov laj thawj ntawm thawj qhov kev tawm tsam lossis tau txais kev tawm tsam.[5] Rau kev daws qhov tsis xav tau kev kho mob, kev sib xyaw ua ke ntawm lwm cov kev kho mob muaj txiaj ntsig nrog ICB muab kev pom tshiab rau kev kho mob monotherapy. Cov qog microenvironment (TME) yog qhov nyuaj thiab muaj kev cuam tshuam loj heev rau kev ua tau zoo ntawm kev tiv thaiv kab mob.[6] Nrog rau theem infiltration tsis txaus ntawm cytotoxic T lymphocytes (CTLs), noninflamed TME tau suav tias yog ib qho tseem ceeb hauv kev ua tsis tau kom muaj zog tiv thaiv kab mob tiv thaiv kab mob thaum lub sij hawm kho mob. Yog li ntawd, nws yog ib qho tseem ceeb kom nrhiav tau ib qho tseem ceeb molecule uas tuaj yeem hloov kho TME uas tsis yog mob rau hauv TME thiab muaj peev xwm los ua ib lub hom phiaj kho mob. Branched-chain aminotransferase 2 (BCAT2) yog ib qho tseem ceeb enzyme nyob rau hauv cov txheej txheem ntawm sulfur amino acid metabolism.[8] Li et al. pom tias BCAT2 yog qhov tseem ceeb rau kev txhim kho mob qog noj ntshav ntawm lub txiav los ntawm kev sib kho cov ceg ntoo-chain amino acids (BCAAs) catabolism.[9] Lee et al. pom tau tias BCAT2 deficiency inhibited qog loj hlob ntawm pancreatic ductal adenocarcinoma (PDAC) los ntawm kev tswj cov lipid metabolism.[10] Txawm hais tias muaj ntau qhov kev tshawb fawb tau sau tseg tias BCAT2 cuam tshuam ncaj qha rau cov txheej txheem lom neeg ntawm cov qog los ntawm kev tswj cov txheej txheem metabolic, nws lub luag haujlwm hauv kev tswj hwm TME tiv thaiv kab mob tsis tau tshawb pom. Hauv peb txoj kev tshawb fawb, los ntawm kev txheeb xyuas tag nrho ntawm TME cov ntsuas ntsuas hauv Xiangya BLCA pawg thiab ntau pawg BLCA pej xeem, peb tau tshuaj xyuas tias BCAT2 tej zaum yuav ua rau muaj kev tiv thaiv TME hauv BLCA. Rau kev tshawb nrhiav cov txheej txheem molecular, peb tau ua ntxiv ib leeg-cell RNA sequencing (siRNA seq) thiab bulk-RNA seq, qhia tias BCAT2 plays ib qho kev nyuaj siab hauv kev nrhiav neeg ua haujlwm ntawm CTLs rau hauv TME, los ntawm inhibiting kev ua ub no ntawm cytokine / chemokine-koom nrog cov teeb liab txoj hauv kev thiab Tcell-chemotaxis teeb liab txoj kev. Hauv vitro, multi-immunoassays tau pom tias kev tso tawm ntawm CTL-txog chemokines muaj kev sib raug zoo tsis zoo nrog kev qhia ntawm BCAT2. Raws li kev txheeb xyuas panoramic ntawm cov ntaub so ntswg microarray (TMA), peb pom muaj kev sib raug zoo ntawm CTLs thiab BCAT2+ qog hlwb hauv spatial faib. Hauv vivo, qhov kev sib koom tes tau tshwm sim hauv kev kho mob ua ke ntawm BCAT2 poob thiab ICB. Qhov tseem ceeb tshaj, nws lub luag haujlwm hauv kev kwv yees cov txiaj ntsig ntawm kev tiv thaiv kab mob tau raug lees paub hauv Xiangya BLCA immunotherapy cohort.

cistanche tubulosa- txhim kho lub cev tiv thaiv kab mob

2. Cov txiaj ntsig

2.1. BCAT2 tsis zoo cuam tshuam nrog Anticancer Immunity ntawm TME hauv BLCA

Hauv ntau hom mob qog noj ntshav, qhov kev qhia ntawm BCAT2 yog siab dua hauv cov ntaub so ntswg ntau dua li cov ntaub so ntswg ib txwm muaj (Daim duab S1A, Cov Ntaub Ntawv Txhawb Nqa) thiab nws cov qauv qhia hauv BLCA tau lees paub hauv Xiangya BLCA Cohort (Daim duab S1B, Cov Ntaub Ntawv Txhawb Nqa). Tsis tas li ntawd, BCAT2 tau nthuav dav dav hauv ntau cov kab mob qog noj ntshav (Daim duab S1C, Cov Ntaub Ntawv Txhawb Nqa). Tsis tas li ntawd, cov txiaj ntsig ntawm kev txheeb xyuas kab mob qog noj ntshav ntawm cov kab mob chemokine, MHCs, immunostimulators thiab TIICs tau qhia tias BCAT2 muaj cov tshuaj tiv thaiv kab mob tseem ceeb hauv ob peb hom mob qog noj ntshav, suav nrog mob qog noj ntshav (BLCA), mob qog noj ntshav mis (BRCA), raum raum papillary cell. carcinoma (KIRP), pancreatic adenocarcinoma (PAAD), sarcoma (SARC) thiab thyroid carcinoma (THCA) (Daim duab S2A, B, Cov ntaub ntawv txhawb nqa). Tsis tas li ntawd, kev sib raug zoo tsis zoo ntawm BCAT2 thiab cov chaw tiv thaiv kab mob sib kis (PD- 1, PD-L1, CTLA-4, thiab LAG-3) kuj tau pom (Daim duab S2C–F, Cov Ntaub Ntawv Txhawb Nqa ). Los ntawm kev txheeb xyuas kab mob qog noj ntshav, peb pom tias BCAT2 muaj cov tshuaj tiv thaiv kab mob zoo tshaj plaws hauv BLCA. Yog li, peb tau tshawb nrhiav ntxiv nws lub luag haujlwm tiv thaiv kab mob hauv BLCA. Raws li lub hauv paus ntawm tus nqi nruab nrab ntawm BCAT2, peb faib cov tib neeg rau hauv pawg siab-BCAT2 thiab qis-BCAT2 pawg hauv pawg TCGA-BLCA. Obviously, ib tug series ntawm chemokines, chemokine receptors, MHC-txog cov molecules, thiab immunostimulators tau down-expressed nyob rau hauv lub high-BCAT2 pab pawg neeg thiab tshaj-qhia nyob rau hauv qis-BCAT2 pawg (Daim duab 1A). Ib qho txiaj ntsig zoo sib xws tau pom nyob rau hauv lub voj voog tiv thaiv kab mob qog noj ntshav, uas suav nrog ntau cov kauj ruam tseem ceeb hauv kev nrhiav thiab nkag mus rau lub cev tiv thaiv kab mob (Daim duab 1B). Ntxiv mus, peb tau pom muaj kev sib txuas tsis zoo ntawm cov qib infiltration ntawm lub cev tiv thaiv kab mob (CD8+T cell, CD4+T cell, xwm killer (NK) cell, thiab dendritic (DC) cell) thiab BCAT2 sib txawv. algorithms (Daim duab 1C). Ib yam li ntawd, txo qis ntawm kev tiv thaiv kab mob ntawm cov kab mob hauv lub cev (CD8+T cell, NK cell, Macrophage, T helper 1 (Th1) cell, thiab DC cell) tau pom nyob rau hauv pawg high-BCAT2 piv rau cov qis-BCAT2. pab pawg (Daim duab 1D). Qhov tseem ceeb tshaj, thaum sib txheeb BCAT2 nrog TIS cov qhab nia (Daim duab S3, Cov Ntaub Ntawv Txhawb Nqa) thiab cov tshuaj tiv thaiv tiv thaiv kab mob (Daim duab 1E), pom tseeb kev sib raug zoo tsis zoo tau tshwm sim ntawm lawv. Yog li, peb tau kwv yees tias BCAT2 tuaj yeem tswj hwm TME tiv thaiv kab mob tsis zoo thiab cuam tshuam rau kev ua tau zoo ntawm kev tiv thaiv kab mob siab heev. Tsis tas li ntawd, cov txiaj ntsig saum toj no tau raug lees paub zoo hauv cuaj pawg neeg ywj pheej BLCA (GSE31684, GSE32894, GSE48075, GSE48276, GSE69795, GSE83586, GSE86411, GSE87304, thiab GSE412870, Kev Txhawb Nqa Cov Ntaub Ntawv (S. Thaum kawg, peb tau txheeb xyuas lub koom haum ntawm BCAT2 thiab TME dua hauv Xiangya BLCA Cohort. Raws li qhov tshwm sim, qhov kev qhia ntawm BCAT2 muaj kev sib raug zoo tsis zoo rau kev ua haujlwm ntawm kev tiv thaiv kab mob qog noj ntshav, qib infiltration ntawm TIICs, TIS cov qhab nia, thiab kev qhia theem ntawm kev tiv thaiv kab mob (Daim duab 2A-C). Sib sau ua ke, peb tau nthuav tawm tias qhov kev qhia siab ntawm BCAT2 ua rau tsis muaj mob TME hauv BLCA thiab kev qhia qis ntawm BCAT2 ua rau TME mob hauv BLCA.

2.2. Tshawb xyuas cov txheej txheem ntawm BCAT2 hauv Shaping TME los ntawm Bulk RNA-seq thiab siRNA-seq

Hauv pawg TCGA-BLCA, DEGs ntawm cov pab pawg siab / qis BCAT2, pawg neeg siab / tsis muaj zog tiv thaiv kab mob, thiab pawg qhab nia siab / qis stromal tau txheeb xyuas thiab coj kev sib tshuam (Daim duab S13A, Cov ntaub ntawv txhawb nqa). Interestingly, BCAT2 zoo-txog DEGs tsis muaj kev sib tshuam nrog kev tiv thaiv kab mob thiab stromal qhab nia zoo-txog DEGs (Daim duab 2D). Lub caij no, tib lub phenomenon tshwm sim nyob rau hauv qhov tsis zoo EGs ntawm lawv (Daim duab S13B, Cov ntaub ntawv txhawb nqa). Cov kev tshawb pom no tau qhia tias BCAT2 tej zaum yuav ua rau tsis zoo rau kev tiv thaiv kab mob tiv thaiv kab mob. Qhov xwm txheej, cov txiaj ntsig ntawm GO thiab KEGG kev tshuaj ntsuam tau qhia tias BCAT2- cuam tshuam DEGs tau ua kom muaj txiaj ntsig zoo tshaj plaws hauv txoj hauv kev ntawm leukocyte tsiv teb tsaws, T cell ua kom, thiab cytokine--cytokine receptor kev sib cuam tshuam (Daim duab 2E, F). Yog li ntawd, peb tau kwv yees tias BCAT2 ua rau TME tsis muaj mob hauv BLCA los ntawm kev cuam tshuam cov kev ua ub no ntawm cytokine / chemokine-txog txoj hauv kev. Txawm li cas los xij, tus yam ntxwv ntawm ntau NA sequencing txiav txim siab nws cov kev txwv, uas nws qhia theem ntawm cov noob yog xam los ntawm tus nqi nruab nrab ntawm tag nrho cov hlwb hauv cov ntaub so ntswg. Txhawm rau kov yeej qhov kev txwv, siRNA tau ua ntawm peb BLCA cov qauv. Ntau tshaj 19 000 hlwb tau txheeb xyuas thiab muab faib ua rau pawg: epithelial cell, fibroblast cell, T/NK cell, endothelial cell, B cell, thiab myeloid cell (Daim duab 3A), xa mus rau cov biomarkers paub (EPCAM, LYZ, CD3D, COL1A1, CD79A, CD19, PECAM1, and VWF).[11] Strikingly, BCAT2 feem ntau yog qhia nyob rau hauv epithelial hlwb (EPCAM +), es tsis yog endothelial hlwb thiab lub cev tiv thaiv kab mob. Raws li lub hauv paus ntawm CNV tsub zuj zuj, EPCAM + epithelial hlwb raug suav hais tias yog malignant urothelial hlwb. Txhawm rau txheeb xyuas tus qauv qhia ntawm BCAT2, ob pawg sab nraud siRNA tau suav nrog hauv txoj kev tshawb no. Hauv GSE135337 scRNA cohort, ntau dua 36, ​​000 cov hlwb tau ua tiav thiab muab faib ua tsib pawg, Feem ntau cov cell subtype ntawm BCAT2 tseem yog lub zais zis urothelial cell (Daim duab 3B). Ib qho piv txwv zoo sib xws tau tshwm sim hauv GSE145137 siRNA cohort (Daim duab 3C). Yog li ntawd, cov qauv ntawm feem ntau cov lus qhia ntawm malignant hlwb inferred tias BCAT2 ua raws li ib tug immunoregulator ib feem ntawm TME feem ntau yog los ntawm kev sib txawv ntawm cov qog nqaij hlav cancer. Raws li kev qhia theem ntawm BCAT2, malignant urothelial hlwb tau muab faib ua pawg siab-BCAT2 thiab pawg qis-BCAT2. GSEA tsom xam ntawm GO cov ntsiab lus nyob rau hauv GSE135337 thiab GSE145137 siRNA cohorts qhia tau hais tias ib pawg ntawm cytokine / chemokine-txog txoj hauv kev tau raug txo qis hauv cov pab pawg siab-BCAT2, suav nrog kev tsim cov tshuaj chemokine, chemokine secretion, kev tswj ntawm chemokine mediated rau chemokine cov lus teb, Chemokine receptor binding, cytokine kev ua haujlwm, cytokine khi thiab cytokine receptor binding (Daim duab 3D-G; Daim duab S14B, Cov ntaub ntawv txhawb nqa). Lub caij no, leukocyte chemotaxis, lymphocyte chemotaxis, T cell chemotaxis, thiab lawv txoj kev tswj hwm tau muaj kev cuam tshuam tsis zoo rau BCAT2 (Daim duab 3E-I; Daim duab S14A, Cov ntaub ntawv txhawb nqa). GSEA tsom xam ntawm KEGG cov ntsiab lus tau qhia tias txoj hauv kev ntawm cytokine-cytokine receptor kev sib cuam tshuam thiab kev ua cov tshuaj antigen thiab kev nthuav qhia tau pom tseeb hauv cov pab pawg siab-BCAT2 (Daim duab 3F). Ua ke, kev qhia siab ntawm BCAT2 txwv tsis pub cov kev ua ub no ntawm proinflammatory cytokines thiab chemokines-txog txoj hauv kev, ua rau txo qis infiltration theem ntawm TIICs, uas ua rau TME tsis muaj tshuaj lom thaum kawg.

Daim duab 1. BCAT2 tsis zoo cuam tshuam nrog kev tiv thaiv kab mob qog noj ntshav hauv TME ntawm BLCA. A) Kev nthuav qhia tus qauv ntawm chemokines, chemokine receptors, MHC molecules, thiab immunostimulators hauv siab thiab qis BCAT2 pawg. B) Kev ua haujlwm ntawm kev tiv thaiv kab mob qog noj ntshav hauv cov pab pawg siab thiab qis BCAT2. Xya xim sawv cev rau xya kauj ruam hauv lub voj voog. *p < 0 05; **p < 0.01; ***p <0.001. C) Kev sib raug zoo ntawm BCAT2 thiab ntau hom kev tiv thaiv kab mob (CD8+T cell, CD4+T cell, DC cell, thiab NK cell) nyob rau hauv 6 txoj kev ywj pheej algorithms. D) Cov qauv kev nthuav qhia ntawm ntau yam subtypes ntawm lub cev tiv thaiv kab mob (CD8+T cell, NK cell, Macrophage, Th1 cell, thiab DC cell) ntsig txog cov noob caj noob ces hauv siab thiab qis BCAT2 pawg. E) Kev sib raug zoo ntawm BCAT2 thiab ICB ntsig txog cov noob caj noob ces. Number nyob rau hauv lub voj voog txhais tau hais tias correlation coefficient.

Daim duab 2. Kev lees paub txoj haujlwm ntawm BCAT2 los ntawm Xiangya BLCA pawg. A) Kev sib raug zoo ntawm BCAT2 / kab mob qog noj ntshav (sab laug) thiab BCAT2 / TIICs (txoj cai). Cov kab thiab cov dotted txhais tau hais tias kev sib raug zoo thiab tsis zoo, thickness ntawm cov kab txhais tau tias coefficient ntawm kev sib raug zoo, kab nrog cov xim sib txawv sawv cev rau p-tus nqi ntawm kev sib raug zoo. B) Kev sib raug zoo ntawm BCAT2 thiab ICB ntsig txog cov noob caj noob ces. C) Kev sib raug zoo ntawm BCAT2 thiab TIS cov qhab nia cuam tshuam txog cov noob caj noob ces. Tus lej hauv lub voj voog sawv cev rau kev sib raug zoo coefficient. D) Kev sib tshuam ntawm BCAT2, cov qhab nia tiv thaiv kab mob thiab stromal qhab nia zoo txog cov noob caj noob ces. E,F) Sab saum toj 20 enrichment signaling pathways of GO and KEGG analys in the BCAT{10}}related genes.

Daim duab 3. Tshawb xyuas cov txheej txheem ntawm BCAT2 hauv kev tsim TME los ntawm ntau RNA-seq thiab scRNA-seq. A, B) tSNE cov phiaj xwm ntawm cov qauv qhia ib leeg ntawm BCAT2 hauv Xiangya siRNA cohort thiab GSE135337 pawg. C) Violin zaj duab xis ntawm ib leeg-cell qhia qauv ntawm BCAT2 hauv GSE145137 pawg. D, E) GSEA tsom xam ntawm GO lub sij hawm qhia txog kev ua ub no ntawm cytokine/chemokine-txog txoj hauv kev thiab lub cev tiv thaiv kab mob chemotaxis-txog txoj hauv kev ntawm ntau pawg BCAT2 hauv pawg GSE135337. F) GSEA tsom xam ntawm KEGG lub sij hawm qhia txog kev ua ub no ntawm antigen kev nthuav qhia thiab kev sib cuam tshuam ntawm cytokine thiab cytokine receptor txoj kev ntawm ntau pawg BCAT2 hauv pawg GSE145137. G-I) GSEA tsom xam ntawm GO lub sij hawm qhia txog kev ua ub no ntawm cytokine / chemokine-txog txoj hauv kev, cov kab mob tiv thaiv kab mob chemotaxis-txog txoj hauv kev, thiab cov kab mob tiv thaiv kab mob kev tsiv teb tsaws ntawm cov pab pawg BCAT2 sib txawv hauv GSE145137 cohort. J, K) GO thiab KEGG tsom xam ntawm DEGs ntawm BCAT2 OE thiab BCAT2 KD cell kab.

2.3. BCAT2 Varies nthuav qhia cov qauv ntawm CD8+T-Cell-Related Chemokines thiab Inhibits Cytotoxic Capacity ntawm CTLs

Txhawm rau txheeb xyuas txoj hauv kev txhawb nqa tau hais los saum no, BCAT2 overexpression (BCAT2 OE) thiab BCAT2 knockdown (BCAT2 KD) tib neeg (T24) / murine (MB49) zais zis kab mob qog noj ntshav tau tsim ua tiav (Daim duab S15A-D, Cov Ntaub Ntawv Txhawb Nqa) thiab tau sim nrog high-throughput RNA sequencing. Kev cia siab, GO tsom xam ntawm T24 cov kab ntawm tes tau qhia tias DEGs tau ua kom muaj txiaj ntsig zoo hauv txoj hauv kev ntawm chemokine-mediated signaling, teb rau chemokine, leukocyte migration, thiab leukocyte migration (Daim duab 3J). KEGG txoj kev tsom xam ntawm T24 kab xov tooj ntawm tes tau pom tias txoj hauv kev chemokine signaling, cytokine--cytokine receptor kev sib cuam tshuam txoj hauv kev, thiab txoj kev mob qog nqaij hlav zais zis tau ua kom muaj txiaj ntsig zoo (Daim duab 3K). Ib yam li ntawd, ob peb txoj hauv kev tseem ceeb cytokine / chemokine-txog txoj hauv kev tau pom nyob rau hauv GO thiab KEGG kev tshuaj xyuas ntawm MB49 kab ntawm tes (Daim duab S16A, B, Cov ntaub ntawv txhawb nqa). Nws tus kheej pom tseeb, ntau yam cytokines thiab chemokines ua lub luag haujlwm tseem ceeb hauv kev tswj TME. Yog li, nws yog ib qho tsim nyog los tshuaj xyuas cytokines / chemokines uas tau tswj hwm los ntawm BCAT2. Li no, ProcartaPlex ntau yam immunoassays tau siv los txheeb xyuas qhov txawv txav ntawm cytokines / chemokines hauv tib neeg thiab nas zais zis kab mob qog noj ntshav. Kuj ceeb tias, qib qis ntawm CCL3, CCL4, CCL5, thiab CXCL10 upregulated nyob rau hauv tib neeg thiab murine BCAT2-KD cell kab thiab downregulated nyob rau hauv tib neeg thiab murine BCAT2-OE cell kab (Daim duab 4A, B; daim duab S16C , D, Cov ntaub ntawv txhawb nqa). Hauv cov kev tshawb fawb yav dhau los, CCL3, CCL4, CCL5, CXCL9, thiab CXCL10 tau suav tias yog cov tshuaj tua kab mob tseem ceeb rau kev nrhiav CD8+T hlwb rau hauv TME.[12] Yog li ntawd, rau kev soj ntsuam zoo dua ntawm CD8+T-cell-txog chemokines, tag nrho cov ntawm lawv tau suav nrog rau kev siv tau ntxiv. Strikingly, RNA qhia theem ntawm CCL3, CCL4, CCL5, CXCL9, thiab CXCL10 tau txo qis hauv BCAT2 OE hlwb thiab ua rau muaj zog hauv BCAT2 KD hlwb (Daim duab 4C). Ib yam li ntawd, cov txiaj ntsig ntawm ELISA tau pom tias cov qib protein secreted kuj muaj kev cuam tshuam tsis zoo nrog kev qhia ntawm BCAT2 (Daim duab 4D). Cov kev tshawb pom no tau qhia tias kev nthuav tawm ntau dhau ntawm BCAT2 inhibits transcriptome thiab protein ntau ntawm CD8+T-cell-related chemokines, suav nrog CCL3, CCL4, CCL5, CXCL9, thiab CXCL10. Qhov tseem ceeb tshaj, qhov kev ntsuam xyuas chemotaxis tau qhia tias BCAT2 OE kab xov tooj ntawm tes tau cuam tshuam qhov chemotaxis peev xwm ntawm CD8+T hlwb (Daim duab 4E, sab laug). Hloov pauv, cell supernatants ntawm BCAT2 KD cell kab muaj peev xwm nyiam ntau CD8+T hlwb dua li nws cov kev tswj tsis zoo (Daim duab 4E, txoj cai). Cov kev tshawb pom no tau qhia tias BCAT2 tuaj yeem cuam tshuam rau chemotaxis muaj peev xwm ntawm CD8+T hlwb los ntawm kev tswj hwm mRNA thiab cov protein ntau ntawm cov tshuaj chemokines. Tsis tas li ntawd, T-cell-mediated cancer cell-tilling assay tau siv los tshawb txog cytotoxicity variation ntawm T hlwb tom qab kev sib cuag ncaj qha nrog cov qog hlwb qhia txog qib sib txawv ntawm BCAT2. Raws li pom nyob rau hauv daim duab 4F thiab daim duab S17A, B (Cov ntaub ntawv txhawb nqa), overexpression ntawm BCAT2 ntawm cov qog hlwb ncaj qha suppressed lub cytotoxic muaj nuj nqi ntawm T hlwb thiab knockdown ntawm BCAT2 ntawm cov qog hlwb ho rov qab lub siab nyiam. Flow cytometry tsom xam ntawm cov T cells tau pom tias feem ntau ntawm CD8+TNF-𝛼+ T hlwb thiab CD8+IFN-𝛾+ T hlwb muaj qhov sib txawv tseem ceeb hauv cov kab ke sib txawv, uas txhais tau tias muaj kev hloov pauv loj hauv cov kev ua ntawm CD8+T cells (Daim duab 4G; Daim duab S17C, D, Cov ntaub ntawv txhawb nqa). Sib sau ua ke, BCAT2 muaj peev xwm tiv thaiv kev muaj peev xwm nrhiav neeg ua haujlwm ntawm CD8+T hlwb los ntawm kev tswj hwm cov tshuaj chemokines nrog rau kev txwv cov haujlwm ntawm CTLs los ntawm kev sib cuag ncaj qha. Tsis tas li ntawd, qhov tshwm sim ntawm T-cell-mediated cancer cell tua assay (tsis muaj T cells pawg) tuaj yeem ua pov thawj tias BCAT2 ua lub luag haujlwm oncogenic hauv zais zis. Yog li ntawd, kev ntsuam xyuas phaj colony thiab transwell migration/invasion assay tau siv los xyuas qhov kev xav no. Raws li kev cia siab, overexpression ntawm BCAT2 tau txhim kho qhov kev loj hlob, kev tsiv teb tsaws, thiab kev cuam tshuam ntawm cov qog hlwb, thiab knockdown ntawm BCAT2 tau txwv tsis pub cov cwj pwm no (Daim duab S18A–F, Cov Ntaub Ntawv Txhawb Nqa).

2.4. Validation ntawm Exclusive Spatial Relationship ntawm BCAT2+ Tumor Cell thiab CD8+T Cell los ntawm TMA ntawm Xiangya BLCA Cohort

Raws li cov txiaj ntsig ntawm ntau RNA-seq, scRNA-seq, thiab kev sim vitro, peb tau pom tias BCAT2 ua lub luag haujlwm tiv thaiv kab mob hauv BLCA los ntawm kev txwv kev nrhiav neeg ua haujlwm thiab cytotoxicity ntawm CD8+T hlwb. Txawm li cas los xij, kev sib cuam tshuam ntawm BCAT2+ qog hlwb thiab CD8+T hlwb tseem tsis paub txog ntawm tib neeg cov ntaub so ntswg. Hauv Xiangya BLCA pawg, cov duab sawv cev thiab tag nrho cov qhab nia ntawm IHC tau qhia qhov tsis zoo ntawm BCAT2 thiab CD8 (R=−0.38, p=0.0 038) (Daim duab 5A, B). Multicolor IF staining ntawm BCAT2+ qog hlwb (BCAT2+CK{16}}) thiab BCAT2+CD8+T hlwb tau ua nyob rau hauv TMA thiab semiautomatic soj ntsuam siv cov TissueFAXS panoramic quantification platform. Raws li pom nyob rau hauv daim duab 5C, qhov kev nthuav qhia ntawm BCAT2 thiab CK19 tau sib tshooj ntau heev. Hloov pauv, qhov kev nthuav qhia ntawm BCAT2 thiab CD8 yog qhov sib txawv thiab sib cais. Cov ncauj lus kom ntxaws coexpression proportions ntawm BCAT2+CK19+ hlwb thiab BCAT2+CD8+T hlwb yog 87.29% thiab 5.09% (Daim duab 5D, E), uas tau ua raws li cov qhia qauv ntawm BCAT2 hauv scRNA-seq. Hauv tag nrho TMA, tseem muaj qhov sib txawv tseem ceeb hauv kev sib koom ua ke ntawm lawv (Daim duab S19A, Cov ntaub ntawv txhawb nqa). Qhov tseem ceeb tshaj, nyob rau hauv ntau qhov kev ncua deb gradient tsom xam (0–25, 25–50, 50–100, thiab 100–150 μm) nyob ib ncig ntawm BCAT2+ qog hlwb, suav ntawm CD8+T hlwb tau maj mam nce. los ze rau qhov deb (Daim duab 5F; Daim duab S19B, Cov Ntaub Ntawv Txhawb Nqa). Ua ke, peb tau ua pov thawj tias ntawm tib neeg cov ntaub so ntswg, BCAT2 kuj tseem qhia hauv cov qog hlwb, thiab BCAT2+ qog cell muaj kev sib raug zoo nrog CD8+T cell.

Cov txiaj ntsig ntawm cistanche tubulosa-Antitumor

2.5. Poob BCAT2 Txhim Kho Kev Ua Haujlwm ntawm Anti-PD-1 Kev Kho

Nrog rau qhov cuam tshuam tsis zoo rau TME thiab qhov cuam tshuam tsis zoo rau inhibitory checkpoint blockade, nws ua rau muaj kev txaus siab los tshawb txog cov txiaj ntsig zoo ntawm BCAT2 poob thiab tiv thaiv PD- 1 kho. Ua ntej ntawm kev tiv thaiv kab mob, tus qauv qog nqaij hlav qog nqaij hlav hauv plab tau tsim los ntawm BCAT2 KD thiab tswj murine cell kab. Tom qab ntawd, cov tshuaj tiv thaiv PD-1 kev kho thiab tswj kev kho mob tau siv rau cov nas uas muaj kabmob (Daim duab 6A). Raws li nws lub luag haujlwm oncogenic thiab kev tswj hwm ntawm cov tshuaj chemokines hauv vitro, BCAT2 deficiency inhibited qog kev loj hlob thiab ntev lub sij hawm ciaj sia nyob rau hauv vivo, los ntawm upregulating CD8+T muaj feem xyuam rau chemokines (Daim duab 6B–E; Daim duab S20A, Cov ntaub ntawv txhawb nqa). Nyob rau hauv nam ntawm immunotherapy kev ua tau zoo, txawm hais tias anti-PD-1 monotherapy tuaj yeem ua rau txo cov qog nqaij hlav, kev kho mob ntawm BCAT2 KD thiab anti-PD-1 monoclonal antibody (mab) qhia tau hais tias muaj kev tiv thaiv qog nqaij hlav zoo dua thiab tau txais ntau dua. kev muaj sia nyob. Nyob rau hnub teem tseg, cov qog tau sau thiab npaj rau kev tshuaj xyuas ntxiv. Ib feem ntawm lawv tau digested rau hauv ib leeg-cell ncua kev kawm ntawv thiab ua ndlwg cytometry tsom xam. Nrog cov tib neeg zoo sib xws ntawm leukocytes thiab T hlwb (Daim duab S20B-E, Cov Ntaub Ntawv Txhawb Nqa) hauv cov qog, feem ntau ntawm CD8+T hlwb tau pom nyob rau hauv pawg BCAT2 deficiency dua li hauv pawg tswj tsis zoo. Ib yam li ntawd, pab pawg kho mob ua ke tau muaj kev nkag mus ntawm CTLs ntau dua li pawg kws kho mob monotherapy (Daim duab 6F-H). Qhov tseem ceeb tshaj, cov ntsuas cytotoxicity (GZMB, IFN-𝛾, TNF-𝛼, thiab Perforin) ntawm CTLs kuj tau ntxiv dag zog rau cov qog murine nrog BCAT2 poob thiab kev kho mob ua ke piv rau kev tswj hwm (Daim duab 6G, I–L). Lwm qhov chaw ntawm cov qog tau ua rau hauv cov seem khov thiab siv IF staining. Raws li qhov xav tau, qhov ceev ntawm CD8+T hlwb hauv thaj tsam ntawm kev txaus siab (ROI) tau pom zoo ib yam nrog kev ntsuas cytometry (Daim duab 6M, N). Cov kev tshawb pom no tau qhia tias BCAT2 tsis muaj peev xwm ntawm cov qog nqaij hlav tuaj yeem ua rau mob TME thiab muaj txiaj ntsig zoo nrog kev kho cov tshuaj tiv thaiv kab mob. Txhawm rau txheeb xyuas lub luag haujlwm ntawm CD8+T hlwb hauv kev sib koom ua ke ntawm kev kho mob, CD8𝛼 mab tau siv los tiv thaiv lawv hauv cov nas uas muaj peev xwm tiv thaiv kab mob (Daim duab S21A, Cov ntaub ntawv txhawb nqa) thiab tau lees paub nws cov txiaj ntsig depletion los ntawm flow cytometry thiab IF (Daim duab S21B, C, Cov ntaub ntawv txhawb nqa). Obviously, tom qab depletion, qog lub nra thiab lub sij hawm ciaj sia tau hloov pauv ntau heev (Daim duab S21D-G, Cov ntaub ntawv txhawb nqa). Cov kev tshawb pom no tau qhia tias CD8+T hlwb ua si ib qho tseem ceeb hauv kev sib koom ua ke ntawm kev kho ua ke.

Daim duab 4. BCAT2 txawv cov qauv qhia ntawm CD8+T-cell-txog chemokines thiab inhibits cytotoxic peev ntawm CTLs. A,B) Heatmaps ntawm ProcartaPlex ntau yam immunoassays tso tawm qhov sib txawv ntawm cov cytokines thiab chemokines hauv BCAT2 OE, BCAT2 KD thiab pawg tswj tsis zoo (n=3 ib pawg). C, D) Histograms qhia cov qib mRNA normalized thiab protein secretion concentrations ntawm CCL3, CCL4, CCL5, CXCL9, thiab CXCL10 hauv BCAT2 OE (sab laug), BCAT2 KD (txoj cai) thiab pawg tswj tsis zoo (n {{ 15}} ib pawg). *p < 0 {20}}5; **p < 0.01; ***p <0.001. E) Chemotaxis assay qhia tias muaj peev xwm sib txawv chemotaxis ntawm CTLs hauv BCAT2 OE, BCAT2 KD, thiab pawg tswj tsis zoo (n=3 ib pawg). p <0.05; ** p <0.01. F) T-cell-mediated cancer cell killing assay qhia tias muaj peev xwm tua tau sib txawv ntawm T cell cocultured nrog BCAT2 OE, BCAT2 KD, thiab cov kab tswj tsis zoo ntawm tes (n=3 ib pawg). G) Flow cytometry tsom xam qhia cov kev ua ub no sib txawv ntawm CD8+T hlwb hauv ntau pawg coculture (n=3 ib pawg).

Daim duab 5. Kev lees paub tshwj xeeb ntawm kev sib raug zoo ntawm BCAT2+ qog hlwb thiab CD8+T hlwb los ntawm TMA ntawm Xiangya BLCA pawg. A) IHC duab ntawm BCAT2 thiab CD8 nyob rau hauv cov kab mob thiab tsis mob ntawm TME. Scale bar: 5 0 μm. B) Kev sib raug zoo ntawm BCAT2 thiab CD8 raws li IHC cov qhab nia ntawm lawv hauv TMA tag nrho. C) Multicolor IF daim duab ntawm BCAT2, CK19, CD8, thiab kev sib xyaw ua ke hauv cov kab mob thiab tsis mob ntawm TME. BCAT2+ cell (liab), CK19+ cell (cyan), CD8+T cell (txiv kab ntxwv), thiab cell nucleus (xiav). Scale bar: 50 μm. D,E) Cov ncauj lus kom ntxaws coexpression tus nqi ntawm BCAT2+CK{16}} thiab BCAT{17}}CD8+ hlwb hauv cov qauv raug. F) Distance gradient tsom xam (0–25, 25–50, 50–100, thiab 100–150 μm) ntawm CD8+T hlwb nyob ib ncig ntawm BCAT2+CK19+ hlwb nyob rau hauv ib txwm qauv.

Daim duab 6. Kev poob ntawm BCAT2 txhim kho qhov ua tau zoo ntawm kev tiv thaiv PD-1 kho. A) Flow daim duab ntawm txoj kev npaj kho mob. B) Cov qog nqaij hlav ntawm cov kev kho mob sib txawv (n=5 ib pab pawg). C–E) Kev ntsuas qhov ntim ntawm cov qog, lub cev qhov hnyav, thiab lub sijhawm muaj sia nyob hauv cov kev kho mob sib txawv (n=5 ib pab pawg). ns: tsis muaj qhov tseem ceeb; *p< 0.05; **p < 0.01; ***p < 0.001. F) Contour plots indicate the proportion of CD8+T cells; G) proportions of GZMB+CD8+T cells, IFN-𝛾+CD8+T cells, TNF- 𝛼+CD8+T cells, and Perforin+CD8+T cells in different therapy regimens. H) Quantified scatter plots exhibit proportion of CD8+T cells; I–L) proportions of GZMB+ CD8+T cells, IFN-𝛾+ CD8+T cells, TNF-𝛼+ CD8+T cells, and Perforin+ CD8+T cells in different therapy regimens (n = 5 per group). ns: no significance; *p<0.05; **p < 0.01; ***p < 0.001. M, N) IF image and quantified histogram show densities of CD8+T cells in different therapy regimens (n = 3 per group). Scale bar: 20 μm. CD8+T cell (green) and cell nucleus (blue). ns: no significance, *p<0.05; **p < 0.01; ***p < 0.001.

Tsis tas li ntawd, nrog rau cov qog hlwb, ib feem me me ntawm BCAT2 kuj tau qhia hauv CD8+T hlwb. Yog li, peb tau tshawb nrhiav ntxiv seb qhov sib txawv ntawm BCAT2 ntawm CD8+T hlwb puas tuaj yeem cuam tshuam rau lawv cov dej num. Tom qab staining ib leeg-cell raug tshem tawm ntawm murine spleen nrog BCAT2 thiab T cell-txog cov tshuaj tiv thaiv fluorescent, peb piv cov feem ntawm CD8+ TNF-𝛼+T thiab CD8+ IFN-𝛾+T hlwb ntawm BCAT 2+CD8+ thiab BCAT2-CD8+ pawg. Interestingly, peb pom tias cov proportions zoo sib xws ntawm ob pawg, qhia tias cov kev ua ntawm CD8+T cell yog ywj siab ntawm nws cov theem qhia ntawm BCAT2 (Daim duab S22A–C, Cov ntaub ntawv txhawb nqa).

2.6. BCAT2 Predicts Teb rau Immunotherapy nyob rau hauv ntau Real-World Immunotherapy Cohorts

Cov kev tshawb pom saum toj no tau pom tias muaj lub luag haujlwm tiv thaiv kab mob ntawm BCAT2 hauv TME thiab cov txiaj ntsig sib xyaw ua ke ntawm kev sib txuas BCAT2 poob nrog kev tiv thaiv PD-1 kho. Txawm li cas los xij, nws qhov kev kwv yees muaj peev xwm ntawm kev siv tshuaj tiv thaiv kab mob tsis paub meej. Yog li ntawd, nyob rau hauv TCGA-BLCA pawg, cov qhab nia ntxiv ntawm kev tiv thaiv kab mob tiv thaiv kab mob tau muab piv rau cov pab pawg siab thiab qis BCAT2. Raws li pom nyob rau hauv daim duab S23A (Cov ntaub ntawv txhawb nqa), pawg qis-BCAT2 muaj ntau cov noob caj noob ces nyob rau hauv txoj kev tiv thaiv kab mob tiv thaiv kab mob, uas tuaj yeem pom tias qhov qis qis ntawm BCAT2 sawv cev rau lub xeev rhiab heev rau kev tiv thaiv kab mob. Rau kev siv tau ntxiv, 58 tus neeg mob qog nqaij hlav qog nqaij hlav qog nqaij hlav (MIBC) uas tau txais kev kho neoadjuvant anti-PD-1 hauv peb lub tsev kho mob tau suav nrog hauv Xiangya BLCA immunotherapy cohort. Tus neeg sawv cev IHC, IF, thiab CT cov duab ntawm tus neeg teb thiab cov neeg tsis teb tau hais tias qhov kev qhia ntawm BCAT2 muaj kev sib raug zoo rau kev siv tshuaj tiv thaiv kab mob-ib tus neeg uas muaj BCAT2 qhia qib qis dua yuav teb rau kev siv tshuaj tiv thaiv kab mob ntau dua li tus neeg uas muaj tus kab mob. siab BCAT2 qhia theem (Daim duab 7A–C). Ntxiv mus, IHC cov qhab nia ntawm Xiangya BLCA immunotherapy cohort thiab mRNA qhia matrix ntawm IMvigor210 pawg qhia tau hais tias muaj kev sib raug zoo tsis zoo ntawm BCAT2 thiab PD-L1 (R=−{{30 }}}.4, p=0.002; R=−0.41, p < 0.001) (Daim duab S23B, C, Cov ntaub ntawv txhawb nqa). Yog li ntawd, peb ncaj qha piv cov kev ua tau zoo ntawm kev tiv thaiv kab mob ntawm cov pab pawg siab thiab qis BCAT2 hauv Xiangya BLCA immunotherapy cohort thiab pom tias cov pab pawg BCAT2 qis muaj feem ntau ntawm cov neeg teb ntau dua li cov pab pawg BCAT2 siab (p=0.001) (Daim duab 7D). Ntxiv mus, peb tau soj ntsuam qhov kwv yees qhov tseem ceeb ntawm BCAT2, PD-L1, thiab kev sib xyaw ua ke Performance index (BCAT2+PD-L1) ntawm pathological teb rau immunotherapy thiab pom qhov ua tau zoo ntawm kev sib xyaw ua ke Performance index (BCAT2+PD- L1) ntawm kev kwv yees qhov tseeb (Daim duab 7E). Kev tshoov siab, ntawm qhov tshwm sim ntawm kev muaj sia nyob, pawg BCAT2 qis tau muaj kab mob tsis muaj sia nyob ntev (DFS) dua li pawg BCAT2 siab (p=0.032) (Daim duab 7F), qhia txog qhov ntsuas tus nqi ntawm BCAT2 hauv BLCA tshuaj tiv thaiv kab mob. Hauv kev kho mob, cov tib neeg uas muaj suab puam TME feem ntau yuav muaj kev tiv thaiv kab mob tsawg dua li cov tib neeg uas muaj TME, ntxiv qhov tseem ceeb ntawm qhov ntsuas ntsuas. Yog li ntawd, peb tau xaiv txhua tus neeg uas muaj suab puam TME hauv IMvigor210 pawg thiab ua qhov kev ntsuam xyuas dav. Hauv kev sib piv ncaj qha ntawm theem qhia ntawm BCAT2 ntawm cov pab pawg lus teb sib txawv, pawg CR (tus neeg teb) muaj qhov qhia qis qis ntawm BCAT2 dua li SD thiab PD pawg (tsis teb) (Daim duab 7G). Qhov tseem ceeb tshaj, qhov sib xyaw ua ke Performance index (BCAT2+PD-L1) kuj muaj qhov ua tau zoo heev ntawm kev kwv yees qhov tseeb (Daim duab 7H). Txawm hais tias tsis muaj qhov sib txawv tseem ceeb hauv kev ciaj sia tag nrho (OS) ntawm cov pab pawg siab thiab qis BCAT2 nyob rau hauv cov suab puam hom IMvigor210 cohort, prognosis tendency tseem zoo ib yam li cov txiaj ntsig ntawm Xiangya BLCA immunotherapy cohort (Daim duab 7I). Sib nrug los ntawm PD-L1, microsatellite instability (MSI) yog lwm qhov tseem ceeb kwv yees ntawm kev ua tau zoo ntawm kev tiv thaiv kab mob. Cov xwm txheej ntawm kev kho qhov tsis sib haum xeeb (MMR) - kev paub zoo MMR (pMMR) thiab qhov tsis zoo MMR (dMMR) ua kom muaj kev sib raug zoo nrog tsawg zaus MSI (MSI-L) thiab siab-frequency MSI (MSI-H).[13] Yog li, peb tau ua qhov kev ntsuam xyuas dav dav ntawm txhua tus neeg hauv Xiangya BLCA immunotherapy cohort raws li cov txiaj ntsig ntawm plaub MMR cim cov noob (MLH1, MSH2, MSH6, thiab PMS2). Cov txiaj ntsig tau nthuav tawm ntau dua ntawm dMMR (MSI-H) hauv pawg BCAT2 qis piv rau pawg BCAT2 siab (p=0.02) (Daim duab S23D, E, Cov ntaub ntawv txhawb nqa). Tsis tas li ntawd, peb tau soj ntsuam qhov kev kwv yees qhov tseem ceeb ntawm MMR, BCAT2, thiab kev ntsuas kev sib xyaw ua ke (MMR + BCAT2) ntawm kev siv tshuaj tiv thaiv kab mob thiab pom tias qhov sib xyaw ua ke (MMR + BCAT2) muaj qhov tsim nyog tau txais txiaj ntsig ntawm kev kwv yees qhov tseeb (Daim duab S23F, Cov ntaub ntawv txhawb nqa). Ua ke, cov kev tshawb pom no tau pom tias BCAT2 tsim nyog los ua qhov kev kwv yees ntxiv ntawm kev ua tau zoo ntawm BLCA immunotherapy. Thaum kawg, qhov kwv yees tus nqi ntawm BCAT2 hauv kev teb rau kev tiv thaiv kab mob tau tshawb pom hauv ntau hom mob qog noj ntshav. Peb pom tias cov tib neeg uas muaj kev qhia siab ntawm BCAT2 tau pom cov lus teb tsis zoo (p=0.04) hauv GSE35640 cohort (melanoma) (Daim duab 7J). Txawm hais tias tsis muaj qhov sib txawv tseem ceeb hauv cov lus teb ntawm qhov siab thiab qis BCAT2 pawg hauv GSE173839 (mob qog noj ntshav), GSE135222 (NSCLC), thiab Gide 2019 cohort (melanoma), cov neeg mob uas tsis tshua muaj kev qhia ntawm BCAT2 tseem yuav muaj cov lus teb zoo. rau immunotherapy (Daim duab 7K–M).

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2.7. Tus nqi ntawm BCAT2 nyob rau hauv Forecasting Molecular Subtype thiab Guiding Precision Therapy

Nrog zoo heterogeneity uas twb muaj lawm nyob rau hauv ib txwm histology kev faib ua feem, nce pov thawj pom tau hais tias molecular subtype muaj peev xwm muab tau ntau dua kev faib ua feem raws li transcriptome profile nyob rau hauv BLCA.[14] Tsis tas li ntawd, nrog cov yam ntxwv zoo sib xws ntawm cov tshuaj tiv thaiv kab mob hauv tib lub subtype, kev faib tawm molecular muaj peev xwm los kwv yees TME thiab qhia kev kho qhov tseeb hauv kev kho mob.[15] Los ntawm kev tsom xam ntawm cov kab ke sib txawv ntawm cov kab ke sib txawv hauv TCGA-BLCA pawg, peb pom tias cov tib neeg uas tsis tshua muaj kev qhia ntawm BCAT2 feem ntau yuav muab faib ua ib hom kab mob basal, nrog rau txoj hauv kev ua haujlwm ntawm basal sib txawv, EMT sib txawv, kev tiv thaiv kab mob sib txawv, interferon teb, thiab lwm yam. Cov tib neeg uas muaj kev qhia siab ntawm BCAT2 feem ntau yog muab faib ua luminal subtypes, nrog rau txoj hauv kev ua haujlwm ntawm luminal sib txawv, urothelial sib txawv, thiab Ta (Daim duab S24A, Cov ntaub ntawv txhawb nqa). Raws li kev pom zoo ntawm molecular subtype, basal subtype muaj ntau qhov kev nkag mus ntawm cov nyhuv lymphocytes thiab muaj zog IFN-𝛾 signaling txoj kev ntau dua li cov luminal subtype, [16] uas txhais tau hais tias muaj txiaj ntsig zoo ntawm kev tiv thaiv kab mob. Tom qab ntawd, AUC tau siv los ntsuas qhov tseeb ntawm kev twv ua ntej. Amazingly, tshwj tsis yog Baylor subtype system (AUC=0.76), feem ntau ntawm AUC tau dhau ntawm 0.90 hauv lwm lub tshuab (Daim duab S24B, Cov Ntaub Ntawv Txhawb Nqa), uas txhais tau hais tias qhov kev twv ua tau zoo ntawm BCAT2 hauv lub molecular subtype. Txhawm rau tshawb nrhiav nws lub luag haujlwm hauv kev kwv yees qhov kev ua tau zoo ntawm lwm cov kev kho mob, peb muab piv rau kev ua haujlwm ntawm epidermal growth factor receptor (EGFR) lub hom phiaj kev kho mob thiab kev siv hluav taws xob cuam tshuam ntawm cov pab pawg siab thiab qis BCAT2. Nrog rau kev ua kom pom tseeb txog kev tswj hwm, cov neeg mob hauv pawg tsawg-BCAT2 feem ntau yuav muaj cov lus teb zoo rau EGFR-target therapy thiab radiotherapy (Daim duab S24C, Cov ntaub ntawv txhawb nqa). Rau kev ua kom ua ntej ua ntej kev ntseeg siab rau ntawm molecular subtystivity, yim blucta cohorts (saib Gse52329, Gse52674, gse52329, thiab e-}} vigor210) yog thov rau sab nraud validation. Nrog rau qhov tseeb ntawm kev twv ua ntej, cov kev tshawb pom zoo sib xws tau pom nyob rau hauv cov pab pawg no (Figures S25–S27, Cov Ntaub Ntawv Txhawb). Tus kab mob Hyperprogressive (HPD) yog ib qho kev tsis txaus ntseeg heev rau kev tiv thaiv kab mob. Rau kev tshawb nrhiav kev cuam tshuam ntawm BCAT2 ntawm HPD, HPD-txuas biomarkers tau sau los ntawm cov kev tshawb fawb yav dhau los [17] thiab CNV ntawm biomarkers tau muab piv rau cov pab pawg qis thiab siab BCAT2. Tshwj tsis yog rau EGFR, CNV amplification tus nqi ntawm lwm cov HPD-positive txuam biomarkers (liab cim) yog siab dua nyob rau hauv high-BCAT2 pawg, tshwj xeeb tshaj yog MDM2 (p=0.0116). CNV tshem tawm cov nqi ntawm HPD-tsis zoo txuam nrog biomarkers (ntsuab cim) tau qis dua hauv pawg BCAT2 siab (Daim duab S24D, Cov ntaub ntawv txhawb nqa). Cov txiaj ntsig tau qhia tias cov neeg mob uas muaj kev qhia siab ntawm BCAT2 muaj peev xwm pheej hmoo ntawm HPD thaum immunotherapy. Neoadjuvant chemotherapy (NAC) yog ib qho kev kho tseem ceeb hauv BLCA. Cov cim kev kwv yees cov lus teb rau NAC hauv BLCA tau sau los ntawm kev tshuaj xyuas ntawm Buttigliero li al.[18] thiab lawv qhov kev hloov pauv tau muab piv rau ntawm ob pawg. Raws li tag nrho cov kev hloov pauv ntau dua ntawm biomarkers thiab kev hloov pauv ntau zaus ntawm RB1 hauv pawg qis-BCAT2, peb xav tias tus neeg uas tsis tshua muaj kev qhia ntawm BCAT2 muaj qhov ua tau zoo ntawm NAC hauv kev kho mob (Daim duab S24E, Cov ntaub ntawv txhawb nqa). Yog li ntawd, cov ntaub ntawv Drugbank tau siv los sib piv cov txiaj ntsig ntawm ntau cov tshuaj khomob sib xyaw ua ke ntawm ob pawg thiab pom tias cov tib neeg uas tsis tshua muaj kev qhia ntawm BCAT2 feem ntau yuav teb rau cov kws khomob. Tsis tas li ntawd, cov pab pawg tsawg-BCAT2 kuj tau pom tias muaj txiaj ntsig zoo dua hauv kev siv tshuaj tiv thaiv kab mob thiab kev kho ERBB. Kev npaj txhij txog, cov neeg mob uas muaj kev qhia siab ntawm BCAT2 tej zaum yuav tau txais txiaj ntsig zoo hauv kev kho mob antiangiogenic (Daim duab S24F, Cov Ntaub Ntawv Txhawb Nqa). Sib sau ua ke, cov tib neeg uas tsis tshua muaj kev qhia ntawm BCAT2 muaj rau cov kab mob molecular subtype, basal subtype, uas txhais tau hais tias cov lus teb zoo dua rau kev tiv thaiv kab mob, tshuaj tua kab mob, xov tooj cua, EGFR-kev kho lub hom phiaj, thiab ERBB txoj kev kho. Hmoov tsis zoo, cov neeg mob uas muaj kev qhia siab ntawm BCAT2 muaj cov lus teb tsis zoo rau cov kev kho mob no. Txawm li cas los xij, txoj kev kho antiangiogenic tuaj yeem yog lub teeb ci rau lawv.

Daim duab 7. BCAT2 kwv yees cov lus teb rau immunotherapy nyob rau hauv ntau lub ntiaj teb no immunotherapy cohorts. A) IHC duab ntawm BCAT2 thiab PD-L1 ntawm cov neeg teb thiab cov neeg tsis teb nyob hauv Xiangya BLCA immunotherapy cohort. Scale bar: 5 0 μm. B) Multicolor IF duab ntawm BCAT2 thiab PD-L1 ntawm cov neeg teb thiab cov neeg tsis teb nyob hauv Xiangya BLCA immunotherapy cohort. Scale bar: 50 μm. BCAT2+cell (ntsuab), PD-L1+cell (daj), thiab cell nucleus (xiav). C) CT duab ntawm qhov sib txawv ntawm kev siv tshuaj tiv thaiv kab mob ntawm cov tib neeg uas muaj qib siab thiab qis ntawm BCAT2. Lub xub liab taw qhia rau thaj chaw qog. D) Tag nrho qhov sib txawv ntawm cov lus teb ntawm cov siab thiab qis BCAT2 pawg hauv Xiangya BLCA immunotherapy cohort. E) Kev kwv yees qhov tseeb ntawm BCAT2, PD-L1, thiab kev sib xyaw ua ke (BCAT2+PD-L1) ntawm cov lus teb rau kev tiv thaiv kab mob hauv Xiangya BLCA immunotherapy cohort. F) Qhov sib txawv ntawm cov kab mob tsis muaj sia nyob (DFS) ntawm cov pab pawg siab thiab qis BCAT2 hauv Xiangya BLCA immunotherapy cohort. G) Qhov sib txawv ntawm kev qhia theem ntawm BCAT2 ntawm CR, PR, SD, thiab PD pawg hauv cov suab puam hom IMvigor210 pawg. p <0.05; ns: tsis muaj qhov tseem ceeb. H) Kev kwv yees qhov tseeb ntawm BCAT2, PD-L1, thiab kev sib xyaw ua ke (BCAT2+PD-L1) ntawm cov lus teb rau kev tiv thaiv kab mob hauv hom suab puam ntawm IMvigor210 cohort. I) Qhov sib txawv ntawm tag nrho cov ciaj sia taus (OS) ntawm siab thiab qis BCAT2 pawg nyob rau hauv ib hom suab puam ntawm IMvigor210 cohort. J–M) Qhov sib txawv ntawm cov lus teb rau kev tiv thaiv kab mob ntawm cov pab pawg siab thiab qis BCAT2 hauv GSE35640, GSE173839, GSE135222, thiab Gide2019 pawg.

3. Kev sib tham

Raws li cov enzyme tseem ceeb hauv cov txheej txheem catabolism ntawm BCAAs, kev tshawb fawb yav dhau los tau tsom mus rau lub luag haujlwm metabolic ntsig txog BCAT2 hauv cov kab mob, xws li rog rog, ntshav qab zib, thiab arrhythmia. Ma et al. pom tau hais tias khob tawm BCAT2 nyob rau hauv cov ntaub so ntswg adipose tuaj yeem ua kom adipose browning thiab thermogenesis, uas ua rau txo cov rog rog hauv nas.[19] Los ntawm kev kuaj ntshav ntawm ntau dua 2000 tus neeg, Gerszten li al. tau piav qhia txog cov kab mob ntshav qab zib mellitus thiab pom tias BCAT2- hloov BCAAs ua lub luag haujlwm tseem ceeb hauv kev txhim kho cov kab mob.[20] Portero et al. qhia tias kev hloov pauv tsis tseem ceeb ntawm BCAT2p.Q300*/p.Q300* ua rau nce qib ntawm BCAAs thiab induce arrhythmias hauv nas.[21] Tsis ntev los no, ob peb txoj kev tshawb fawb pom tias BCAT2 muaj kev sib raug zoo nrog mob qog noj ntshav. Lei et al. qhia tau tias acetylation-mediated degradation ntawm BCAT2 tuaj yeem cuam tshuam txoj kev loj hlob ntawm pancreatic ductal adenocarcinoma (PDAC) los ntawm kev txo qis cov metabolism hauv BCAAs.[22] Wang et al. suav hais tias txwv tsis pub hloov pauv qib ntawm BCAT2 ua rau txo qis hauv qib glutamate, uas txhawb nqa ferroptosis ntawm hepatoma hlwb.[23] Txawm li cas los xij, tag nrho cov kev tshawb fawb uas twb muaj lawm tau hais txog kev cuam tshuam ntawm BCAT2- kho BCAA catabolism ntawm cov txheej txheem mob qog noj ntshav es tsis yog tshawb nrhiav lub tshuab tshiab. Hauv txoj kev tshawb no, peb tau nthuav tawm lub luag haujlwm tiv thaiv kab mob ntawm BCAT2 hauv TME thawj zaug. Daim ntawv thov ntawm immunotherapy rede- fines cancer kho, nrog zoo heev nyob zoo thiab ntev ciaj sia taus lub sij hawm. Txawm li cas los xij, nyob rau hauv tus account ntawm heterogeneity ntawm cov qog tiv thaiv kab mob ecological, ib feem ntawm cov tib neeg tau txais txiaj ntsig zoo. TME yog cov txheej txheem nyuaj, muaj cov qog hlwb, lub cev tiv thaiv kab mob, cov hlwb stromal, thiab cov hlab ntsha. Raws li qib infiltration ntawm CTLs, TME tuaj yeem muab faib ua hom kab mob thiab tsis muaj kab mob feem ntau.[25] Cov pov thawj loj hlob tau qhia tias mob TME ua lub luag haujlwm zoo hauv kev tshem tawm cov tshuaj tiv thaiv kab mob tiv thaiv kab mob thaum lub sijhawm kho.[26] Yog li, peb tau txheeb xyuas ntau yam kev ntsuas TME ntsig txog thiab pom tias kev qhia siab ntawm BCAT2 ua rau TME tsis muaj mob, nrog rau kev tiv thaiv kab mob qog noj ntshav, kev tawm tsam ntawm chemokine profile, MHC molecules, thiab tsis txaus infiltration theem ntawm TIICs. Tsis tas li ntawd, BCAT2 muaj kev cuam tshuam tsis zoo rau TIS thiab cov noob caj noob ces ntawm ICB, uas txhais tau hais tias cov neeg uas muaj kev qhia siab ntawm BCAT2 feem ntau yuav tsis teb rau cov tshuaj tiv thaiv kab mob. Txhawm rau kom ntseeg tau peb qhov kev xav, peb muab piv cov lus teb ntawm cov pab pawg siab thiab qis BCAT2 hauv Xiangya BLCA immunotherapy cohort thiab lwm cov kab mob tiv thaiv kab mob. Kuj ceeb tias, muaj qhov sib txawv tseem ceeb hauv ntau pawg, uas qhia tau hais tias BCAT2 kuj muaj peev xwm ua si qhov kev kwv yees ntawm kev siv tshuaj tiv thaiv kab mob, tshwj xeeb tshaj yog hauv BLCA. Tsis tas li ntawd, scRNA-seq tau siv los tshawb nrhiav cov txheej txheem tswj hwm ntawm BCAT2 hauv TME thiab qhia tias kev ua haujlwm ntawm cytokine / chemokine ntsig txog kev taw qhia txoj hauv kev, T cell chemotaxis signaling pathway, thiab T cell migration signaling pathway muaj qhov cuam tshuam tsis zoo nrog BCAT2. Raws li txoj cai tseem ceeb hauv lub network, cytokines thiab chemokines yog qhov tseem ceeb rau kev lag luam ntau lub cev tiv thaiv kab mob rau hauv TME. Cov kab ke chemokine muaj plaub lub tsev neeg loj: C, CC, CXC, thiab CX3C, nrog rau kev rov ua dua tshiab hauv kev ua khub ntawm ligands thiab receptors.[27] Cytokine tuaj yeem muab faib ua Th1, Th2, thiab Th17 subfamilies raws li lawv txoj haujlwm lom neeg. Los ntawm kev tso tawm ntawm ntau theem ntawm lawv, kev sib tw ntawm cov qog hlwb thiab lub cev tiv thaiv kab mob yog txaus los rov tsim cov yam ntxwv ntawm TME.[29] Txawm li cas los xij, ntawm feem ntau ntawm cytokine thiab chemokine, nws yog qhov tsim nyog los tshuaj xyuas cov pawg tseem ceeb tshaj plaws. Siv lub vaj huam sib luag 34-cytokine thiab chemokine immunoassay, peb tau qhia tias cov tshuaj chemokines xws li CCL3, CCL4, CCL5, CXCL9, thiab CXCL10, muaj kev cuam tshuam tsis zoo nrog kev qhia ntawm BCAT2 hauv tib neeg thiab murine zais zis cell. Nws paub zoo tias CXCL9 thiab CXCL10 yog lub luag haujlwm rau kev nrhiav CD8+T hlwb rau hauv TME.[30] Txhawm rau kom paub meej txog kev ua haujlwm ntawm CCL3, CCL4, thiab CCL5, peb tau saib cov kev tshawb fawb yav dhau los. Harlin et al. tau siv cov protein arrays thiab qPCR los qhia tias qhov upregulation ntawm CCL3, CCL4, thiab CCL5 muaj peev xwm txhawb kev tsiv teb tsaws ntawm CD8+T hlwb rau hauv melanoma.[31] Noman et al. pom tias muaj coob tus neeg zais cia ntawm CCL5 pab tsim kom muaj ib tug proinflammatory TME los ntawm kev lag luam CTLs rau hauv cov nqaij mos mob qog noj ntshav.[32] Los ntawm IHC thiab RNA-seq ntawm chemokine profile hauv ntau pawg, Romero li al. pom tau tias CCL4 thiab CCL5 muaj kev sib koom ua ke zoo nrog qib infiltration ntawm CD8+T hlwb hauv pancreatic cancer.[33] Li no, peb tau txiav txim siab tias CD8+T-cell-related chemokines yog cov tshuaj chemokines loj tswj los ntawm BCAT2 hauv zais zis. Txawm hais tias peb tau ua kom pom qhov tsis zoo ntawm kev sib raug zoo ntawm BCAT2 thiab CD8+T cell-related chemokines, cov txheej txheem tswj hwm ntawm lawv tseem yuav tsum tau tshawb nrhiav ntxiv. Kev kawm ntawm Peterson et al. pom tau hais tias kev ua kom ntawm MAP kinase (MAPK) kev taw qhia txoj hauv kev tuaj yeem ua rau kev tsim cov CX3CL1.[34] Xu et al. qhia tias JAK-STAT txoj kev taw qhia txoj hauv kev tswj kev tso tawm ntawm Th1-txog chemokines, ua rau txo qis qhov nkag mus ntawm TILs.[35] Tsis ntev los no, Peng et al. pom tias demethylase JMJD3 tuaj yeem cuam tshuam qhov kev qhia ntawm CD4+Tcell-txog chemokines thiab txo cov qib infiltration ntawm cytotoxicity T hlwb hauv TME, sawv cev rau lub luag haujlwm tseem ceeb ntawm kev hloov pauv epigenetic hauv kev tswj cov chemokine qhia.[36] Mayo et al. kuj tau nthuav tawm tias tus kab mob epigenetic inhibitor, histone deacetylase 1, muaj lub peev xwm loj los cuam tshuam qhov kev qhia ntawm CXCL8 los ntawm kev ua kom muaj qhov cuam tshuam ntawm nuclear-𝜅B (NF-𝜅B) signaling pathway.[37] Ntxiv mus, raws li cov cim ntawm qog cell metabolism, aerobic glycolysis thiab reactive oxygen hom (ROS), qhia tau hais tias ua tau zoo ntawm inducing qhia ntawm CXCL8 thiab CXCL14 los ntawm elevating cov dej num ntawm signaling pathways ntawm NF-𝜅B thiab transcription factor activator protein 1 (AP{{93). }})[38] ib. Qhov zoo siab, hauv peb txoj kev tshawb fawb, kev taw qhia txoj hauv kev ntawm NF-𝜅B, STAT, thiab MAPK tau ua kom muaj txiaj ntsig zoo ntawm cov kab hluav taws xob siab thiab qis BCAT2 (Daim duab 3K; Daim duab S16A, B, Cov ntaub ntawv txhawb nqa). Yog li ntawd, ua ke nrog cov kev tshawb fawb saum toj no, peb yuav tshawb nrhiav cov molecule tseem ceeb hauv cov txheej txheem tswj hwm ntawm BCAT2 thiab CD8+T-txog chemokines. Qhov txwv lub hom phiaj teb tus nqi ntawm kev kho mob monotherapy nrog ICB ua rau muaj kev tshwm sim ntawm kev kho mob ua ke. Rau kev hloov cov nonimmunologic TME rau immunologic TME, Wolchok li al. siv cotreatment ntawm nivolumab (anti-PD-1 kho) thiab ipilimumab (anti-CTLA{105}} kho) hauv cov neeg mob uas muaj melanoma thiab pom muaj kev txhawb nqa curative nyhuv, ascribing rau synchronous enhanced priming, ua kom thiab tua ntawm CTLs. [39] Ntau tshaj li kev sib txuas nrog lwm hom ICB, chemotherapy plus immunotherapy yog lwm txoj kev kho mob. Raws li thawj kab kev kho mob rau cov kab mob urothelial siab heev, ob peb txoj kev tshawb fawb pom tias cisplatin-based chemotherapy muaj peev xwm ua kom muaj kev ua kom muaj zog TME los ntawm kev nce qib ntawm MHC I chav kawm ntawm cov hlwb dendritic thiab ua rau MDSCs thiab Tregs puas tsuaj.[40] Coincidentally, Homma et al. qhia tias lwm yam tshuaj khomob rau cov qog nqaij hlav zais zis - gemcitabine - tuaj yeem txhim kho qhov nkag mus rau CD{110}}T hlwb thiab ua rau lub cev tsis muaj zog tiv thaiv kab mob hauv TME.[41] Hauv peb qhov kev sim ua tsiaj preclinical, kev kho mob ntawm BCAT2 poob thiab tiv thaiv PD-1 mab tau nthuav tawm cov txiaj ntsig antitumor ntau dua piv rau monotherapy ntawm anti-PD-1 mab hauv cov nas tiv thaiv kab mob, uas muab kev kho tshiab. lub tswv yim rau cov neeg mob tiv thaiv ICB. Lub caij no, los ntawm kev ntsuam xyuas cytometry thiab IF, peb tau pom tias kev poob qis ntawm BCAT2 tsis tsuas yog nrhiav neeg coob coob ntawm CTLs rau hauv TME tab sis kuj txhim kho kev tua ntawm CTLs. Ib yam li ntawd, Peng et al. pom tias overexpression ntawm LGALS2 txo qhov ntau ntawm infiltrating CTLs nrog rau cytotoxic biomarkers nyob rau hauv lub mis mob cancer-cov nas nas.[42] Yang et al. piav qhia tias CXCL13 muaj peev xwm ua kom cov lus teb rau cov tshuaj tiv thaiv kab mob hauv cov qog nqaij hlav qog nqaij hlav zes qe menyuam los ntawm kev nce qib infiltration ntawm CD8+T hlwb thiab qib secretion ntawm GZMB, IFN-𝛾, thiab IL-2.[43] Ua ke nrog cov lus teb muaj zog tiv thaiv kab mob, kev sib xyaw ua ke ntxiv kev ntxhov siab rau lub cev tiv thaiv kab mob uas twb muaj lawm, uas tej zaum yuav ua rau muaj kev phiv loj heev. Raws li cov qauv qhia ntawm BCAT2 ntawm qib siRNA, tshwj xeeb tshaj yog nyob rau hauv cov qog hlwb es tsis yog nyob rau hauv lub cev tiv thaiv kab mob thiab endothelial hlwb, peb muaj peev xwm ua ntej infer hais tias BCAT2 poob los yog inhibitor ntawm BCAT2 yog tsis tshua muaj yuav ua rau muaj kev phiv tsis zoo. Txawm li cas los xij, qhov kev siv tshuaj zoo tshaj plaws thiab lub sijhawm luv luv ntawm BCAT2 inhibitor thiab anti-PD-1 mab hauv kev sib xyaw ua ke tseem yuav tsum tau tshawb nrhiav.

Rau kev taw qhia txog kev kho mob rau cov tib neeg, peb tau txheeb xyuas BCAT2 rau cov kab mob molecular ntawm BLCA thiab biomarkers tseem ceeb ntawm ntau yam kev kho mob. Raws li kev kwv yees muaj txiaj ntsig hauv ntau pawg, peb pom tias kev tiv thaiv kab mob tiv thaiv kab mob, tshuaj tua kab mob, tshuaj tua hluav taws xob, thiab EFGR-kev kho lub hom phiaj yog tsim nyog rau cov neeg mob uas tsis tshua muaj kev qhia ntawm BCAT2. Kev kho antiangiogenic raug pom zoo rau cov neeg mob uas muaj kev qhia siab ntawm BCAT2. Sib txawv los ntawm cov txheej txheem tshawb nrhiav nyuaj ntawm molecular subtypes, BCAT2 yog ib qho kev kwv yees portable rau kev kho kom raug. Inevitably, muaj qee qhov kev txwv hauv peb cov kev tshawb fawb. Ua ntej, cov qauv loj thiab lub sijhawm ua raws ntawm Xiangya BLCA cohort thiab Xiangya BLCA immunotherapy cohort raug txwv. Peb yuav tsum tau ua kom cov qauv loj ntxiv thiab txuas ntxiv ua raws li cov qauv. Thib ob, raws li kev sib koom ua ke tiag tiag, Xiangya BLCA immunotherapy cohort muaj cov kev phais sib txawv uas tej zaum yuav ua rau muaj kev tsis ncaj ncees. Peb yuav ua kom peb pawg neeg loj ntxiv thiab ua cov kev soj ntsuam pab pawg raws li kev xaiv kev phais tib yam. Thib peb, kev sib koom ua ke ntawm kev sib koom ua ke ntawm kev kho mob hauv vivo yuav tsum tau ua kom muaj txiaj ntsig ntxiv nrog kev siv cov txheej txheem ntawm BCAT2 inhibitor. Hauv cov ntsiab lus, raws li lub luag haujlwm tiv thaiv kab mob hauv TME ntawm BLCA, BCAT2 yog lub hom phiaj tshwm sim hauv kev sib xyaw ua ke ntawm ICB thiab qhov tseeb biomarker ntawm kev kho kom raug.

Cov txiaj ntsig ntawm cistanche tubulosa- ua kom muaj zog tiv thaiv kab mob

4. Ntu kev sim

cohort: As illustrated in a previous study,[44] 56 eligible BLCA patients accepted surgical treatment (TURBT (transurethral resection of bladder tumor) or radical cystectomy) in Xiangya Hospital. All the samples were conducted through high throughput RNA sequencing (RNA-seq) to acquire transcriptome information. Then, data on RNA-seq, clinicopathologic features, and follow-up information of these patients were utilized to construct the Xiangya BLCA cohort (Table S1, Supporting Information). Xiangya BLCA immunotherapy cohort: 58 MIBC patients were included in the Xiangya BLCA immunotherapy cohort. All the samples were obtained by diagnostic TURBT before the implementation of neoadjuvant anti-PD-1 therapy. After at least two cycles standard neoadjuvant immunotherapy (NAI), TURBT, or radical cystectomy (RC) were conducted based on treatment response and patients' willingness. 17 individuals with complete response (CR) and 16 individuals with partial response (PR) were classified into responders, 17 individuals with stable disease (SD), and 8 individuals with progressive disease (PD) were classified into nonresponders. The detailed clinicopathological characteristics are listed in Table S2 (Supporting Information). Xiangya scRNA cohort: Three samples of muscle-invasive bladder cancer were implemented scRNA-seq, constituting the Xiangya scRNA cohort. Detailed preparation of single-cell suspension, data transformation, and cell quality control have been elaborated in previous articles.[45] In simple terms, three tumor samples were loaded on a Chromium Single Cell Controller instrument (10×Genomics, USA) to produce single-cell gel beads in suspension. Seurat R package was applied to transform the count matrix into Seurat format. Four standards were set up for excluding low-quality cells in the matrix: unique molecular identifier (UMI) amount < 1000, gene quantity < 200, log10GenesPerUMI < 0.70, and mitochondrial-originated UMI counts > 20%. After integrating the samples based on the top 3000 variable traits, principal component analysis (PCA) and the Findclusters algorithm were employed to identify main cell clusters. Based on the level of copy number variation (CNV) in epithelial cells, malignant bladder carcinoma cells were selected from clusters. The study plan was approved by the ethics committee of Xiangya Hospital, Central South University (Item number: 2021101175). All the specific men were collected complying with informed consent rights. The Cancer Genome Atlas (TCGA) Database: RNA expression matrix, survival outcome, and CNV of 33 types of carcinomas were acquired from the UCSC Xena database. Log2 transformation was employed to normalize RNA-seq data and the GISTIC algorithm was applied to process CNV data. Gene Expression Omnibus (GEO) Database: Transcriptome data of 1740 samples from nine BLCA cohorts: GSE31684 (93 samples), GSE48075 (142 samples), GSE69795(61 samples), GSE32894 (308 samples), GSE48276 (116 samples), GSE83586 (307 samples), GSE86411 (132 samples), GSE52329 (20 samples), GSE87304 (305 samples), and GSE128702 (256 samples) were obtained from GEO database. RNA-seq data of three immunotherapy cohorts: GSE35640 (14 samples, melanoma, and MAGE-A3 therapy), GSE173839 (105 samples, breast cancer, and anti-PD-L1 therapy), and GSE135222 (27 samples, nonsmall cell lung carcinoma (NSCLC), and anti-PD-1/PD-L1 therapy) were downloaded from GEO database. Single-cell transcriptomic characterization of two BLCA scRNA cohorts: GSE135337 (8 samples) and GSE145137 (3 samples) was obtained from the GEO database. Data processing was similar to the Xiangya siRNA cohort. Other Databases: RNA expression matrix and clinicopathologic characteristics of immunotherapy cohorts: IMvigor210 (348 samples, bladder cancer, and anti-PD-1 therapy) and Gide2019 (58 samples, melanoma, anti-PD-1, or anti-CTLA-4 therapy) were collected from http://researchpub.gene.com/IMvigor210CoreBiologies and TIDE database (http://tide. dfci.harvard.edu/). Data of RNA-seq and clinical characteristics of a BLCA cohort—E-MTAB-1803 (85 samples)—was downloaded from ArrayExpress (https://www.ebi.ac.uk/arrayexpress/). BCAT2 expression levels in various types of cancer cell lines were downloaded from the Cancer Cell Line Encyclopedia (CCLE) database. Specific clinicopathologic and follow-up information from public databases were listed in our previous studies.[44,46] Assessment of Immunological Identity of TME in BLCA: As depicted in our previous study,[44] a comprehensive evaluation of the immunological trait of TME in BLCA was summarized. The tracking tumor immunophenotype (TIP) website (http://biocc.hrbmu.edu.cn/TIP/) was used to assess the activities of cancer immunity cycles, which reflected the effectiveness of antitumor immunity. It is separated into seven concrete steps: release and presentation of tumor antigen (steps 1 and 2), startup and activation of effector T cells (step 3), trafficking and assisting diverse immune cells into TME (steps 4 and 5), recognition and killing cancer cells (steps 6 and 7).[47] Six independent algorithms (TIMER, CIBERSORT, CIBERSORT-ABS, MCP-COUNTER, TISIDB, and XCELL) were employed to calculate the infiltration levels of tumor-infiltrating immune cells (TIICs).[48] Furthermore, effector genes of immune cells, ligands and receptors of chemokines, major histocompatibility complex (MHC) molecules, and immunostimulators were collected for evaluating immunological characteristics of TME multi-dimensionally. T cell-inflamed score (TIS) and markers of ICB were utilized to assess the host sensitivity state to immunotherapy.[49] Enrichment Pathways Analysis: Limma R package with empirical Bayesian function was employed to screen differentially expressed genes (DEGs) between high and low expression of BCAT2 in the RNA expression matrix.[50] Screening thresholds were |log (fold change) (log FC) |>1.5 thiab kho p-tus nqi < 0 05. Gene Ontology (GO) thiab Kyoto Encyclopedia of Genes thiab Genomes (KEGG) kev soj ntsuam tau ua los ntawm DEGs txheeb xyuas los ntawm ClusterProfiler R pob.[51] Seurat R pob nrog Findmarker algorithm tau siv los xam cov theem qhia ntawm BCAT2 ntawm cov hlwb epithelial hauv scRNA-seq. Raws li qhov sib txawv ntawm cov noob qhia qeb los ntawm kev hloov pauv (FC) tus nqi, gene set enrichment analysis (GSEA) tau siv los txiav txim qhov zoo thiab qhov tsis zoo. Kev txheeb xyuas ntawm Immune-Related DEGs: ESTIMATE R pob tau ua haujlwm los suav cov qhab nia tiv thaiv kab mob thiab stromal hauv TCGA-BLCA pawg. Raws li tus nqi nruab nrab ntawm ob qhov qhab nia thiab qib qhia ntawm BCAT2, Limma R pob tau siv los txheeb xyuas qhov zoo / tsis zoo ntawm lub cev tiv thaiv kab mob thiab stromal-txog DEGs. Venn daim duab R pob tau siv los ua kev sib tshuam ntawm ntau pab pawg thiab ua kom paub tseeb txog DEGs. Kev faib tawm ntawm Cov Neeg Siv Molecular Subtypes ntawm BLCA: Xya molecular subtypes ntawm BLCA (UNC, Baylor, TCGA, MDA, Lund, CIT, thiab Kev Pom Zoo) tau siv dav hauv kev kho mob rau kev ntsuas tus yam ntxwv ntawm TME thiab kev ua tau zoo ntawm ntau yam kev kho mob.[52] Hauv kev txiav txim siab ntawm kev sib cuam tshuam ntawm ntau yam subtypes, peb muab faib ua ob pawg tseem ceeb - basal thiab luminal subtypes.[16] R pob ntawm Consensus MIBC thiab BLCAsubtyping tau ua haujlwm los faib cov tib neeg rau hauv cov subtypes tshwj xeeb. Tom qab normalization, thaj tsam hauv qab ROC nkhaus (AUC) tau siv los ntsuas qhov kev ntseeg tau ntawm BCAT2 hauv kev kwv yees kev faib tawm. Precision Therapy Assessment of Patients: Raws li tau piav qhia hauv peb txoj kev tshawb fawb yav dhau los, [53] muaj cov npe tseem ceeb ntawm cov noob caj noob ces, uas tuaj yeem kwv yees qhov ua tau zoo ntawm kev kho mob, xov tooj cua, kev kho mob, thiab kev tiv thaiv kab mob, tau sau los ntawm cov kev tshawb fawb sib txawv thiab cov ntaub ntawv pov thawj.[16, 54] ssGSEA algorithm tau siv los txheeb xyuas cov qhab nia ntxiv ntawm cov npe kos npe rau noob.[55] Cells Kab: Tib neeg lub zais zis qog hlwb (T24) thiab murine zais zis hlwb (MB49) tau yuav los ntawm Procell Life Science & Technology (Wuhan, Tuam Tshoj) thiab Meisen CTCC (Jinhua, Tuam Tshoj), feem. Lawv tau khaws cia hauv DMEM nruab nrab (BasalMedia, Tuam Tshoj) nrog 10% fetal bovine serum (FBS) (BI, Ixayees), 1% penicillin thiab streptomycin (NCM Biotech, Tuam Tshoj), thiab kab lis kev cai nyob rau hauv incubator ntawm 37 degree kub muaj 5% CO2. Kev tsim kho thiab RNA-seq ntawm Stable Transfection Cells: Lentiviral vector plenti-BCAT2-flag-puromycin (GV341) tau tsim thiab tsim los ntawm GeneChem (Shanghai, Tuam Tshoj) kom ruaj khov BCAT2-overexpression (BCAT2 OE) kab ntawm tes thiab nws cov kev tswj tsis zoo (oe-vector). Tsis tas li ntawd, BCAT2- luv hairpin RNA (shRNA) tau cloned rau hauv GV112-lentiviral vector los ntawm GeneChem (Shanghai, Tuam Tshoj) rau ib tug ruaj khov BCAT2-knock down (BCAT2 KD) cell kab . Target sequences of shRNA tau teev nyob rau hauv Table S3 (Cov ntaub ntawv txhawb nqa). Tom qab ntawd, recombinant BCAT2 lentiviruses tau kis mus rau hauv lub hom phiaj hlwb raws li cov chaw tsim khoom cov lus qhia. 2 ug mL-1 puromycin (Amersco, USA) tau ua hauj lwm los lim cov cell ruaj khov rau peb hnub. Western blot thiab quantitative reverse-transcription PCR (qRT-PCR) tau siv los ntsuas qhov ua tau zoo ntawm kev hloov pauv. Thaum kawg, lub cell transfection ruaj khov nrog qhov ua tau zoo tshaj plaws tau xaiv rau RNA-seq thiab kev sim ntxiv. Peb qhov piv txwv ntawm txhua kab ntawm tes raug xa mus rau BGI RNA-seq platform (Shenzhen, Suav). Cov txheej txheem rau kev xaiv DEGs thiab kev txheeb xyuas ntawm txoj hauv kev txhawb nqa tau zoo ib yam li tau piav qhia hauv 2.3 (kev txheeb xyuas txoj hauv kev zoo dua). ProcartaPlex Multiple Immunoassays rau Kev Tshawb Fawb Qib Qib ntawm Chemokines thiab Cytokines ntawm Cov Kab Mob Cancer Cell: Tib Neeg ProcartaPlex immunoassay vaj huam sib luag (Cat: EPX340-12167-901) thiab nas ProcartaPlex immunoassay vaj huam sib luag (Cat: EPX{{5}{}) 364} tau yuav los ntawm ThermoFisher Scientific (Massachusetts, USA) txhawm rau kuaj pom cov protein qhia ntau dua 30 cov tshuaj chemokines tseem ceeb thiab cytokines hauv cov kab mob qog noj ntshav ruaj khov. Nyob rau hauv luv luv, cell kab lis kev cai supernatants tau sau los ntawm ib tug 24- zoo phaj thiab centrifugation kom tshem tawm cov hlwb thiab cov khib nyiab ntawm tes. Tom qab ntawd, clarifying supernatants tau incubated nrog hlaws dai nyob rau hauv lub vaj huam sib luag raws li cov chaw tsim tshuaj paus cov lus qhia. Luminex nrhiav tau platform (ThermoFisher Scientific, USA) tau siv los ua kom muaj nuj nqis ntawm txhua tus qauv. Cov ntsuas ntsuas tsis tau raug cais tawm ntawm qhov kev tshuaj ntsuam. qRT-PCR: Tag Nrho RNA raug cais tawm ntawm cov hlwb nrog Cell Total RNA Isolation Kit thiab Tsiaj Tag Nrho RNA Isolation Kit (Foregene, Tuam Tshoj) raws li cov chaw tsim khoom raws tu qauv. cDNA tau tsim los siv UeIris II RTPCR System rau First-Strand cDNA Synthesis (US Everbright, Tuam Tshoj). qRTPCR tau ua tiav siv SYBR Green qPCR Master Mix (US Everbright, Tuam Tshoj) ntawm CFX Txuas System (Bio-Rad, Tebchaws Asmeskas). GAPDH tau siv los ua tus txheej txheem tswj hwm sab hauv. Cov primers tau tsim thiab tsim los ntawm Sangon Biotech (Shanghai, Tuam Tshoj) thiab cov ncauj lus kom ntxaws primer sequences tau teev nyob rau hauv Table S4 (Cov ntaub ntawv txhawb nqa). ELISA: Concentrations ntawm CCL3, CCL4, CCL5, CXCL9 (MIG), thiab CXCL10 (IP10) nyob rau hauv tib neeg lub zais zis mob cancer cell kab lis kev cai supernatants tau txiav txim los ntawm tib neeg ELISA cov khoom siv raws li cov chaw tsim tshuaj paus raws tu qauv (Proteintech, USA). Tus nyeem ntawv biotech microplate (ThermoFisher Scientific, USA) tau ua haujlwm los ntsuas qhov ntsuas qhov muag (OD) qhov tseem ceeb. Nyob rau hauv kev saib ntawm ntau haiv neeg ntawm secretion theem ntawm txawv cytokines thiab chemokines, peb ua cov ntaub ntawv standardization (log 2 transformation) ua ntej tsom xam. Western Blot: RIPA buffer (NCM biotech, Tuam Tshoj) ntxiv nrog 1% Protease Inhibitor Cocktail (NCM biotech, Tuam Tshoj) tau siv los ua cov hlwb. BCA Protein Assay Kit (NCM biotech, Tuam Tshoj) tau ua haujlwm los txiav txim siab cov protein ntau. Tom qab raug xa mus rau PVDF daim nyias nyias, cell proteins tau incubated nrog thawj cov tshuaj tiv thaiv thiab tshwj xeeb HRP-conjugated secondary antibodies successively. Cov teeb liab tau raug ntes los ntawm XRS qhov kev pom zoo (Bio-Rad, Tebchaws Asmeskas). Cov tshuaj tiv thaiv tseem ceeb suav nrog cov tshuaj tiv thaiv BCAT2 (Cat: ab95976, Abcam, USA) thiab cov tshuaj tiv thaiv GAPDH (Cat: ab8245, Abcam, USA). Cov tshuaj tiv thaiv thib ob suav nrog HRP tshis tiv thaiv luav IgG (Cat: 7074, Cell Signaling Technology, USA) thiab HRP tshis tiv thaiv nas IgG (Cat: 7076, Cell Signaling Technology, USA). T Lymphocyte-Mediated Cancer Cell-Killing Assay thiab Coculture Assay: Cov ntshav tau sau los ntawm 10 tus neeg pub noj qab haus huv hauv peb lub tsev kho mob. Raws li cov chaw tsim khoom cov lus qhia, gradient centrifugation yog ua hauj lwm los extract peripheral ntshav mononuclear hlwb (PBMCs) los ntawm Lymphoprep (Cat: 07851, StemCell Technologies, USA). Tsis tas li ntawd, Red Blood Cell Lysis Buffer (Solarbio, Tuam Tshoj) tau siv los tshem tawm cov hlwb liab sib xyaw nrog PBMCs. Txhawm rau qhib T hlwb, PBMCs tau coj mus rau hauv DMEM nruab nrab (Gibco, USA), ntxiv rau Recombinant Human IL- 2 (10 ng mL−1, Cat: 202-1L-050, R&D , USA), thiab ImmunoCult Human CD3/CD28/CD2 T cell activator (25 μL mL−1, Cat: 10970; STEMCELL Technologies, USA) rau 1 lub lis piam. Ntawm qhov piv ntawm 1: 5, tib neeg lub zais zis qog nqaij hlav cancer (BCAT2-OE, BCAT2-KD, thiab lawv cov kev tswj tsis zoo) tau cocultured nrog activated T hlwb hauv DMEM nruab nrab nrog anti-CD3 antibody (100 ng mL−1, Thermo Scientific, USA) thiab IL-2 (10 ng mL−1) los ntawm ib tus neeg pub dawb hauv 12- lub phaj zoo rau 72 teev. Tom qab ntawd, T cells tau sau rau kev ntsuas cytometry. Cov ncauj lus kom ntxaws qhov rooj zoo ntawm kev soj ntsuam ntws tau qhia hauv daim duab S28 (Cov ntaub ntawv txhawb nqa). Cov qog nqaij hlav cancer ntxiv tau stained nrog crystal violet thiab ntsuas OD tus nqi ntawm 570 nm los ntawm tus nyeem ntawv microplate. Antibodies rau flow cytometry tsom xam suav nrog Zombie Aqua Fixable Viability Kit (Cat: 423101, Biolegend, USA), APC / Cy7 anti- human CD45 (Cat: 368516, Biolegend, USA), Pacific Blue anti- human CD3 Antibody (Cat: 300329, Biolegend, USA), PerCP/Cy5.5 anti- human CD4 Antibody (Cat: 317427, Biolegend, USA), FITC anti-tib neeg CD8a Antibody (Cat: 301006, Biolegend, USA), PE/Dazzle 594 anti-human TNF- 𝛼 Antibody (Cat: 502946, Biolegend, USA), thiab Brilliant Violet 711 anti-human IFN- 𝛾 Antibody (Cat: 502540, Biolegend, USA). Chemotaxis Assay: Chemotaxis assay tau ua tiav nyob rau hauv 24-zoo transwell phaj nrog 3 μm core inch (Corning, USA). Tib neeg CD8+T Cell Isolation Kit (Cat: 480012, Biolegend, USA) tau siv los rho CD8+T cells los ntawm PBMC. 1 × 105 cov hlwb raug rho tawm hauv 200 μL ntim tau ntxiv rau hauv lub chamber sab saud thiab 600 μL supernatants ntawm cov kab sib txawv ruaj khov ntawm tes tau muab ntxiv rau hauv lub chamber qis. Tom qab incubation rau 6 h ntawm 37 degree, hlwb tsiv mus rau hauv lub qis chamber thiab raug sau thiab suav los ntawm cytometry ntws. Cell Proliferation, Migration, and Invasion Assays: Ib qho kev ntsuam xyuas ntawm lub phaj colony tau siv los ntsuam xyuas lub peev xwm ntawm cell proliferation. BCAT2-OE, BCAT2-KD, thiab kev tswj tsis zoo ntawm tib neeg lub zais zis qog nqaij hlav qog noj ntshav tau raug coj mus rau hauv 6-zoo daim hlau rau ib qhov dej. Tom qab incubating nyob rau hauv ib tug 37 degree humidified huab cua rau 2 lub lis piam, colonies tau tsau thiab dyed nrog paraformaldehyde thiab siv lead ua violet, feem. Transwell chambers nrog 8.0 μm pore polycarbonate membrane inserts (Corning, USA) tau ua hauj lwm los soj ntsuam lub peev xwm ntawm cell migration thiab ntxeem tau hauv vitro. 1 × 105 cov hlwb hloov pauv ruaj khov tau raug tshem tawm hauv qhov nruab nrab tsis muaj ntshav qab zib thiab cov noob mus rau hauv lub chamber sab saum toj nrog lossis tsis muaj Matrigel (Corning, USA), rau kev cuam tshuam thiab kev tsiv teb tsaws chaw sib cais. Tom qab incubating rau 24 h (migration assay) thiab 48 h (invasion assay), hlwb adhering rau hauv qab ntawm daim nyias nyias tau tsau thiab stained. Cells uas tseem nyob rau hauv lub sab saum toj chamber raug tshem tawm los ntawm swabs. Tsib random teb raug xaiv nyob rau hauv ib lub tshuab tsom xam los xam cov xov tooj ntawm tes. Immunofluorescence (IF) thiab Immunohistochemistry (IHC): Multicolor IF ntawm TMAs ntawm Xiangya BLCA Cohort thiab Xiangya BLCA immunotherapy Cohort tau siv los ntawm ntau yam khoom siv fluorescent immunohistochemical staining (Absin, Suav). Nyob rau hauv luv luv, tom qab dewaxing, rehydrating, thiab antigen renovating, TMA tau incubated nrog thawj cov tshuaj tiv thaiv thiab lwm yam tshuaj tiv thaiv kab mob uas prompts lub binding ntawm txawv luciferins los ntawm tyramide teeb liab amplification (TSA). Tom qab so tawm cov tshuaj tiv thaiv tsis sib xws nrog citrate tsis, cov txheej txheem ntawm incubation tau rov ua dua ob zaug. DAPI tau ua haujlwm los tiv thaiv cell nuclei thiab cov duab tau raug tshuaj xyuas los ntawm Pannoramic MIDI platform (3DHISTECH, Hungary). Thawj cov tshuaj tiv thaiv ntawm TMA ntawm Xiangya BLCA pawg muaj xws li anti-BCAT2 (Cat: ab95976, Abcam, USA), anti-Cytokeratin 19 (CK19) (Cat: ab52625, Abcam, USA), anti-CD8 (Cat: ab237709, Abcam, USA ) thiab DAPI (Invitrogen, USA). Secondary antibodies thiab fluorochrome on TMA of Xiangya BLCA cohort muaj xws li HRP Tshis Anti-Labbit/Mouse IgG, Absin 520 TSA Plus, Absin 570 TSA Plus, Absin 650 TSA Plus (abs50012, Absin). Thawj cov tshuaj tiv thaiv ntawm TMA ntawm Xiangya BLCA immunotherapy cohort suav nrog tshuaj tiv thaiv BCAT2 (Cat: ab95976, Abcam, USA), anti-PD-L1 (Cat: ab213524, Abcam, USA), thiab DAPI (Invitrogen, USA). Secondary antibodies thiab fluorochrome ntawm TMA ntawm Xiangya BLCA immunotherapy cohort suav nrog HRP Tshis Anti-Labbit / Nas IgG, Absin 520 TSA Plus, thiab Absin 650 TSA Plus (abs50012, Absin). IF ntawm murine qog cov ntaub so ntswg tau incubated nrog thawj antibody-anti-CD8 (Cat: 372902, BioLegend, USA)-thiab theem nrab antibody-anti-nas Alexa Fluor 488 dye conjugate (Invitrogen, USA) sequentially. DAPI tau siv rau qhov pom ntawm lub cell nucleus. Tsib random teb raug xaiv nyob rau hauv ib lub tshuab tsom xam los xam qhov zoo staining xov tooj ntawm tes. IHC ntawm TMAs ntawm Xiangya BLCA Cohort thiab Xiangya BLCA immunotherapy Cohort tau ua los ntawm SP-9000 Cov Khoom Siv (ZSGB-BIO, Suav). Tom qab antigen retrieval thiab thaiv, thawj cov tshuaj tiv thaiv, thiab cov tshuaj tiv thaiv theem nrab tau incubated nrog qog nqaij hlav. Tom qab ntawd, diaminobenzidine (DAB) tau siv rau dye phiaj antigens. Brown teeb liab tau suav tias yog qhov zoo staining. Cov qhab nias ntawm staining siv (tsis tuaj yeem=0, tsis muaj zog=1, nruab nrab=2, thiab muaj zog=3) muab cov qhab nia ntawm qhov zoo feem pua ​​(tsis muaj staining=0, staining hlwb tsawg dua 25%=1, 25%–50% staining cells=2, 50%–75% staining cells=3, thiab staining cells ntau dua 75%=4) yog Qhov kawg IHC cov qhab nia ntawm cov qauv. Cov qhab nia ntawm BCAT2 / CD8 / PD-L1 tsawg dua rau lub ntsiab lus tau muab cais raws li kev qhia qis, cov qhab nia ntawm lawv ntau dua lossis sib npaug rau rau lub ntsiab lus tau muab cais ua cov lus qhia siab. Rau kev txiav txim ib tus neeg cov xwm txheej ntawm MMR, tag nrho cov qauv los ntawm Xiangya BLCA immunotherapy cohort raug soj ntsuam raws li cov txiaj ntsig ntawm plaub MMR cim cov noob (MLH1, MSH2, MSH6, thiab PMS2). Raws li tau piav qhia hauv cov kev tshawb fawb yav dhau los, [56] thaum tag nrho plaub lub cim cim cim tau zoo, tus neeg raug cim tias yog MMR. Thaum ib qho ntawm plaub tus cim cim muaj qhov tsis zoo, tus neeg raug cim tias yog dMMR. Ob tus kws kho mob ywj pheej tau raug caw los ua qhov kev ntsuam xyuas. Antibodies muaj xws li: anti-BCAT2 (Cat: ab95976, Abcam, USA), anti-CD8 (Cat: ab4055, Abcam, USA), anti-PD-L1 (Cat: ab213524, Abcam, USA), anti-MLH1 (Cat: A4858, Abcolonal, Tuam Tshoj), anti-MSH2 (Cat: A22177, Abcolonal, Tuam Tshoj), anti-MSH6 (Cat: A16381, Abcolonal, Tuam Tshoj), anti-PMS2 (Cat: A4577, Abcolonal, Tuam Tshoj), thiab HRP tshis Anti -Lavbit/Mouse IgG (ZSGB-BIO, Suav). TissueFAXS Panoramic Analyzes of Spatial Interaction hauv TME: Rau kev soj ntsuam spatial kev sib cuam tshuam ntawm BCAT2+ malignant cells thiab effector T cells, TissueFAXS panoramic platform (Tissue Gnostics, Austria) tau siv los luam theej duab thiab semiautomatically txheeb xyuas lub hom phiaj ntawm lub hlwb los ntawm ntau xim IF. TMA ntawm Xiangya BLCA Cohort. Lub TMA tau tsim los ntawm 1.5 hli core biopsies los ntawm paraffin-embedded specimens ntawm qog nqaij hlav. Hauv kev nthuav dav, BCAT2+ hlwb, cov kab mob malignant, thiab CD8+T cells tau stained los ntawm cov tshuaj tiv thaiv tshwj xeeb thiab cov tshuaj tiv thaiv thib ob. DAPI (Invitrogen, Tebchaws Asmeskas) tau siv los ua kom cov cell nucleus. Cov kauj ruam ntxaws ntxaws tau piav qhia hauv kev kawm ntawm Makarevic li al.[57] Hauv luv luv, raws li kev siv fluorescence ntawm cov ntsuas sib txawv thiab lub cev lub cev ntawm cov hlwb, cov hlwb zoo tau raug cim thiab ntsuas. Qhov tseem ceeb tshaj, qhov chaw faib tawm ntawm CD8+T hlwb nyob ib puag ncig BCAT2+CK19+ cov hlwb tau piav qhia los ntawm cov phiaj xwm tawg vim qhov chaw nyob deb (0–25, 25–50, 50– 100 thiab 100-150 μm). Ob tus kws paub txog kab mob tau raug caw tuaj xyuas qhov kev txiav txim siab ntawm TissueFAXS panoramic platform. Kev sim tsiaj: Poj niam C57BL / 6 nas (6-7 lub lis piam) tau txais los ntawm Department of Laboratory Tsiaj, Central South University. Tag nrho cov haujlwm ntawm cov nas tau raug kuaj xyuas thiab pom zoo los ntawm Pawg Saib Xyuas Tsiaj thiab Siv Tsiaj ntawm Xiangya Tsev Kho Mob, Central South University (tus lej khoom: 2021101175). Qhov tseem ceeb tshaj plaws, txhawm rau tshawb xyuas lub luag haujlwm ntawm BCAT2 ntawm cov qog loj hlob hauv vivo, BCAT2 KD MB49 hlwb (5 × 105) thiab nws cov hlwb tswj hauv 100 μL nruab nrab ntim tau txhaj subcutaneously rau hauv txoj cai flank ntawm nas. Tsis tas li ntawd, tom qab tsim cov qauv qog ua tiav tiav (cov qog ntim txog li 100 mm3), 100 ug InVivomAb anti-nas PD-1 (Cat: BE0146, Bioxcell, USA) thiab IgG2a isotype tswj (Cat: BE0089, Bioxcell, Teb Chaws Asmeskas) tau txhaj tshuaj intraperitoneally rau hauv ib tus nas, rau kev soj ntsuam cov txiaj ntsig zoo ntawm BCAT2 poob thiab tiv thaiv PD-1 kho. Anti-PD-1 kev kho mob tau siv rau cov nas txhua 3 hnub thiab kav mus txog tsib qhov kev kawm. Tsis tas li ntawd, rau kev txiav txim seb qhov kev sib koom tes ntawm kev kho mob puas yog nyob ntawm CD8+T hlwb. Nrog kev sib xyaw ua ke, 100 ug InVivoPlus anti-nas CD8𝛼 (Cat: BP0117, Bioxcell, USA) thiab IgG2b isotype control (Cat: BP0090, Bioxcell, USA) tau ua hauj lwm kom deplete CD8+T hlwb hauv nas. Lub cev hnyav ntawm nas raug kaw txhua peb hnub. Nyob rau hnub teem sijhawm, cov qog tau sau, ntsuas, thiab npaj rau kev ntsuas cytometry, IF staining, thiab qRT-PCR. Rau kev tshawb nrhiav kev sib cuam tshuam ntawm theem qhia ntawm BCAT2 ntawm CD8+T hlwb thiab kev ua haujlwm ntawm CD8+T cells, spleens ntawm poj niam C57BL/6 nas (6- 7 lub lis piam) yam tsis muaj qog nqaij hlav. dissociated thiab npaj rau ib leeg-cell ncua kev soj ntsuam ntxiv. Flow Cytometry Analysis: Murine hlav tau hauv av thiab zom rau hauv ib leeg-cell ncua kev kawm los ntawm Collagenase IV (Cat: C5138, Sigma, USA), hyaluronidase (Cat: H3506, Sigma, USA), thiab DNase I (Cat: DN25, Sigma, USA). ). 70 μm cell strainers (BIOFIL, Tuam Tshoj) tau siv los lim tawm impurities thiab lub txee ntawm tes (Countstar, Tuam Tshoj) tau ua haujlwm kom nce (3-5) × 106 hlwb hauv txhua tus qauv. Tom qab txheeb xyuas cov hlwb nyob siv Zombie Aqua Fixable Viability Kit (Cat: 423101, Biolegend, USA) thiab thaiv Fc receptor nrog anti-nas CD16/32 antibody (Cat: 156603, Biolegend, USA), Cell membrane antigens tau stained los ntawm APC-Cy7 Anti-nas CD45 (Cat: 103116, Biolegend, USA), BV421 anti-nas CD3 (Cat: 100341, Biolegend, USA), BV605 anti-nas CD8a (Cat: 100744, Biolegend, USA) thiab PerCPCy5.5 anti-nas CD4 (Cat: 100540, Biolegend, USA). Tom qab ntawd, bioscience Foxp3 / Transcription Factor Staining Buffer Set (Cat: 2400632, Invitrogen, USA) tau siv los kho thiab permeabilizing lub cell thiab nucleus membrane. Cytotoxic nyhuv ntsig txog antigens raug dyed los ntawm PE anti-tib neeg / nas Granzyme B (Cat: 372208, Biolegend, USA), PE-Cy7 anti-nas TNF-𝛼 (Cat: 506324, Biolegend, USA), PE-Dazzle 594 anti- nas IFN-𝛾 (Cat: 505846, Biolegend, USA), APC anti-nas Perforin (Cat: 154304, Biolegend, USA). Lub gating lub tswv yim ntawm flow cytometry tsom xam muaj nyob rau hauv daim duab S29 (Cov ntaub ntawv txhawb nqa). Anti-tib neeg/nas BCAT2 (Cat: A7426, Abclonal, Suav teb) tau incubated nrog Flexible 488 antibody labeling cov khoom (Cat: KFA001, Proteintech, USA) rau synthesize tus kheej BCAT2 fluorescent antibody rau flow cytometry. Tom qab ntawd, BCAT2 cov tshuaj tiv thaiv kab mob thiab T-cell-txog cov tshuaj tiv thaiv kab mob tau muab sib xyaw thiab ntxiv rau hauv ib lub xov tooj ntawm kev ncua ntawm murine spleen rau kev tshawb nrhiav kev sib cuam tshuam ntawm qib qhia ntawm BCAT2 ntawm CD8+T cell thiab kev ua ntawm CD{{357} }T cell. Lub gating lub tswv yim ntawm flow cytometry tsom xam nyob rau hauv daim duab S30 (Cov ntaub ntawv txhawb nqa). Cov qauv stained tau kuaj pom ntawm Cytek DxpAthena Flow cytometry (Cytek Biosciences, USA) thiab cov ntaub ntawv raug tshuaj xyuas los ntawm Flowjo version software (BD Biosciences, USA). Kev Tshawb Fawb Txog Kev Tshawb Fawb: Cov ntaub ntawv tau nthuav tawm raws li txhais tau tias ± SD. t-test nrog lossis tsis muaj Welch kho tau siv los sib piv qhov sib txawv tsis tu ncua ntawm ob pawg. Ib txoj kev ANOVA tsom xam nrog lossis tsis muaj Brown-Forsythe thiab Welch cov kev xeem tau siv los sib piv cov hloov pauv tsis tu ncua ntawm ntau pawg. Chi-squared test los yog Fisher qhov tseeb xeem tau siv los sib piv dichotomous variables. Pearson lossis Spearman correlation coefficients test tau ua haujlwm los kwv yees qhov kev sib raug zoo ntawm qhov sib txawv ntawm qhov sib txawv. Kaplan-Meier ciaj sia taus nkhaus tau ua hauj lwm los qhia kev soj ntsuam kev soj ntsuam ntawm dichotomous variables, thiab cov cav-qib xeem tau siv los txiav txim qhov sib txawv ntawm kev txheeb cais. Ob sab p <0.05 tau txhais tias yog qhov tseem ceeb ntawm qhov pib. R software (version 4.0) thiab GraphPad Prism8 tau siv los ua cov ntaub ntawv hauv txoj kev tshawb no.

Kev siv

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