Journal: Journal for Immunotherapy of Cancer
Article Title: Afatinib boosts CAR-T cell antitumor therapeutic efficacy via metabolism and fate reprogramming
doi: 10.1136/jitc-2024-009949
Figure Lengend Snippet: AFA boosted the survival and enhanced the antitumor capacity of CAR-T cells. ( A ) Schematic of the experimental procedure. CAR-T cells were pretreated with AFA, after which CAR expression, differentiation, exhaustion and RNA sequencing were performed. The cytotoxicity of Nalm-6-luc cells and AFA-CAR-T cells was assessed in media without IL-2 and AFA. ( B ) The ratio of CAR expression in T and CAR-T cells after AFA pretreatment compared with that in the NC group. The data are reported as the means±SEMs (n=3). ( C–D ) Representative western blot of TCR ( C ) and PI3K-AKT-mTOR ( D ) in AFA-pretreatment CAR-T cells. ( E ) The relative fluorescence of OCR ( L ) and ECAR ( M ) in T cells treated with AFA. The data are reported as the means±SEMs (n=3). ( F ) Relative CD62L and CD45RO levels in AFA-pretreatment CAR-T cells. The data are reported as the means±SEMs (n=3). ( G ) The percentages and counts of living CAR-T cells. The data are reported as means±SEMs (n=3). ( H ) Cytotoxicity of AFA-pretreatment CAR-T cells in media containing E:T at ratio of 1:4, 1:8, or 1:16 for 48 hours, 72 hours, and 96 hours. The data are reported as the means±SEMs (n=3). ( I ) The expression levels of cytotoxicity-related factors of AFA-pretreatment CAR-T cells were co-cultured with Nalm-6-luc cells. The data are reported as the means±SEMs (n=3). ( J ) AFA-pretreatment CAR-T cells were categorized into Glu+ and Glu− groups based on the presence or absence of glucose in the culture medium and cultured for 2 days. Cytotoxicity of CAR-T cells in media containing E:T at ratio of 1:4, 1:8, or 1:16 for 72 hours. The data are reported as the means±SEMs (n=3). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant according to unpaired t-tests ( B , and E–J ). AFA, afatinib; AKT, protein kinase B; CAR, chimeric antigen receptor; ECAR, extracellular acidification rate; E:T, effector-to-target; FCM, flow cytometry; Glut1, glucose transporter 1; INF, interferon; IL, interleukin; LAT, linker for activation of T cells; LCK, lymphocyte-specific protein tyrosine kinase; mTOR, mechanistic target of rapamycin; NC, negative-control; OCR, oxygen consumption rate; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; RPS6, ribosomal protein S6; Tcm, central memory T; Tef, terminal effector T cells; Tem, effector memory T cells; Tn, naïve T; TNF, tumor necrosis factor; ZAP 70, zeta-chain-associated protein kinase 70.
Article Snippet: After treated with AFA for 2 days, CAR-T cells were resuspended using 1,640 medium and extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) detection working solution (Elabscience), maintained a cell concentration of 5×10 6 cells/mL.
Techniques: Expressing, RNA Sequencing, Western Blot, Fluorescence, Cell Culture, Flow Cytometry, Activation Assay, Negative Control
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