The researchers of the following article argued that \"metformin may play an imp
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Question
The researchers of the following article argued that "metformin may play an important role in modulating CD19-CAR T cell biological functions in an AMPK-dependent and mTOR/HIF1?-independent manner.” From the information provided below please find, list, and describe two panels from any figure that are evidence in supporting this claim.
Results
Metformin inhibits CD19-CAR T cell proliferation
The lentiviral expression vectors encoding the CD19-targeted CARs include CD19 scFv, human CD8a molecule transmembrane region, and intracellular signaling domains of CD28, 4-1BB, and CD3zeta and enhanced green fluorescent protein (eGFP) using the “self-cleaving” F2A peptide (Figure 1A). By using flow cytometry, we detected that the anti-CD19- CAR can be expressed efficiently on the T-cell surface. The transduction efficiency was about 63.1%±8.2% (Figure 1B). Based on these results, the CD19-CAR T cells can be used for the following experiments. To evaluate the effect of metformin on CD19-CAR T cells, the CCK-8 assay was used to detect CD19-CAR T-cell proliferation. As shown in Figure 1C, the proliferative ability of CD19-CAR T cells was significantly suppressed by metformin (1, 10, and 20 mM) in a dose-dependent manner. To assess the changes in the actual cell numbers, the effect of metformin on CD19-CAR T-cell proliferation was detected by direct cell counting. As shown in Figure 1D, we found that 1 mM metformin restrained CD19-CAR T-cell proliferation and 10 and 20 mM metformin had greater inhibitive effects. These results indicated that metformin inhibited CD19-CAR T-cell proliferation.
Metformin induces CD19-CAR T-cell apoptosis
To determine whether metformin has a direct effect on CD19-CAR T-cell apoptosis, the cells were incubated with metformin (1, 10, and 20 mM) for 24 hours. As shown in flow cytometry results, metformin (1, 10, and 20 mM) effectively induced CD19-CAR T-cell apoptosis (Figure 2A and B). These data demonstrated the pro-apoptotic role of metformin on CD19-CAR T cells.
Cytotoxicity of CD19-CAR T cells was inhibited by metformin
To verify the effect of metformin on CD19-CAR T-cell cytotoxicity, CD19-CAR T cells were cocultured with CD19+ Raji cells or CD19? K562 cells for 24 hours. The effector to target cell ratio (E:T) was from 1:1 to 40:1. As shown in Figure 3A, compared to CD19? K562 cells, the cytotoxic effect of CD19-CAR T cells was significantly activated when cocultured with CD19+ Raji cells at various E:T. Then, metformin was introduced in the cocultured CD19-CAR T cells and CD19+ Raji cells. As shown in Figure 3B, we found that metformin significantly inhibited the cytotoxicity of CD19- CAR T cells at various E:T. Furthermore, the IL-2 and INF-? release were detected by using ELISA assay. Our data showed that the secretion of IL-2 and INF-? at various E:T was significantly reduced when treated with metformin (Figure 3C and D). Collectively, these data indicated that metformin inhibited the cytotoxic activity of CD19-CAR T cells.
Metformin promotes AMPK phosphorylation and suppresses mTOR and HIF1? expression in CD19-CAR T cells
It is well documented that AMPK/mTOR/HIF1? signal pathway play a critical role in metformin-modulated lymphocytes energetic metabolism.20,24 To further explore the latent mechanism about metformin on CD19-CAR T cells, the AMPK, mTOR, and HIF1? signaling pathway was examined. As shown in Figure 4A, the AMPK phosphorylation was upregulated when treated with 10 mM metformin, whereas mTOR phosphorylation was downregulated. Meanwhile, metformin also downregulated the expression of HIF1?. The regulation was statistically significant, as quantified by densitometry (Figure 4B). These results indicated that AMPK/mTOR/HIF1? signal pathway may take part in the metformin-modulated glucose metabolism of CD19-CAR T cells.
AMPK pathway is involved in metforminregulated CD19-CAR T-cell apoptosis and cytotoxicity
To further delineate whether the role of metformin on CD19-CAR T cells was AMPK-dependent, compound C, a potent AMPK inhibitor, was applied to block AMPK phosphorylation (Figure 5A). As shown in Figure 5B–D, inhibition of AMPK phosphorylation with compound C reversed the metformin-modulated cytotoxicity and apoptosis in CD19-CAR T cells, whereas the metforminsuppressed mTOR and HIF1? expression was not affected by compound C (Figure 5E and F). Taken together, these results strengthen the opinion that AMPK pathway is involved in metformin-modulated biological functions of CD19-CAR T cells.
Metformin suppresses the cytotoxicity effect of CD19-CAR T cells in vivo
Having shown the effect of metformin on CD19-CAR T cells in vitro, next we were interested in determining whether metformin had the same role on suppressing the cytotoxicity of CD19-CAR T cells in vivo. NSG mice were engrafted with Raji-ffluc cells intravenously 3 days prior to CD19-CAR T-cell injection (Figure 6A). The data show that metformin treatment exhibits negative effect on the cytotoxicity of CD19-CAR T cells. Three of 5 mice in metformin treatment group showed tumor progression leading to their sacrifice at days 10, 15, and 17, respectively. The other two mice had large clusters of tumor cells but not sacrificed. However, tumor cells in the control group mice were mostly eliminated, and all mice survived at day 18 (Figure 6B). The survival rate of mice was reduced by the treatment of metformin (Figure 6C). Strikingly, the data demonstrated that metformin plays suppressant role on CD19-CAR T-cell cytotoxicity in vivo.
Figure 1 Metformin inhibits CD19-CAR T-cell proliferation. (A) Schematic of construct of CD19-targeted CAR. (B) Transfection efficiency of CD19-CAR T cells. The expression of CD19-targeted CAR was detected by flow cytometry. (C) CD19-CAR T cells were treated with metformin (0, 1, 10, and 20 mM) for 24 hours. The proliferation of CD19-CAR T cells was detected by the CCK-8 assay. (D) Growth curves of CD19-CAR T cells. CD19-CAR T cells treated with metformin (0, 1, 10, and 20 mM) were counted by using a hemocytometer over 12 days of incubation *P,0.05, **P,0.01 versus the control group. The results are represent as mean ± SD from three independent experiments. Abbreviations: CAR, chimeric antigen receptor; Met, metformin; LTR, long terminal repeat; CTS, Cis-acting termination zone; RRE, rev-response element; VH, variable region of heavy chain; VL, variable region of light chain; TM, transmembrane; OD, optical density.
Figure 2 Metformin induces CD19-CAR T-cell apoptosis. (A) CD19-CAR T cells were treated with metformin (0, 1, 10, and 20 mM) for 24 hours. The apoptotic rate of CD19-CAR T cells was detected by flow cytometry. (B) Statistical analysis of the flow cytometry results. The results are represented as mean ± SD from three independent experiments. *P,0.05, **P,0.01 versus the control group.
Figure 3 Cytotoxicity of CD19-CAR T cells was inhibited by metformin. (A) CD19-CAR T cells were cocultured with Raji and K562 cell lines at various E:T (1:1, 5:1, 10:1, 20:1, and 40:1) for 24 hours. The apoptotic rate was determined by flow cytometry. (B) CD19-CAR T cells were cocultured with Raji cell lines with 0, 1, and 10 mM metformin at various E:T (1:1, 5:1, 10:1, 20:1, and 40:1) for 24 hours. The apoptotic rate was determined by flow cytometry. (C) CD19-CAR T cells were cocultured with Raji cell lines with metformin (0, 1, 10, and 20 mM) at various E:T (1:1, 5:1, 10:1, 20:1, and 40:1) for 24 hours, and the supernatant was obtained from the cocultured system. The IL-2 secretion was detected by ELISA. (D) CD19-CAR T cells were cocultured with Raji cell lines with metformin (0, 1, 10, and 20 mM) at various E:T (1:1, 5:1, 10:1, 20:1, and 40:1) for 24 hours, and the supernatant was obtained from the cocultured system. INF-? secretion were detected by ELISA. The bar graph represents the results of three independent experiments, *P,0.05, **P,0.01 versus the control group.
Figure 4 Metformin increases the expression of p-AMPK and reduces the expression of p-mTOR and HIF1? inCD19-CAR T cells. (A) The CD19-CAR T cells were treated by 10 mM metformin for different times (0, 5, 10, 30, and 60 minutes), and the expression of p-AMPK, p-mTOR, and HIF1? was detected by Western blotting analysis. (B) Statistical analysis of relative p-AMPK, p-mTOR, and HIF1? protein expression results. *P,0.05, **P,0.01 versus the control group. Data are represented as mean ± SD from three independent experiments.
Figure 5 The AMPK inhibitor reverses the metformin-regulated apoptosis and cytotoxicity of CD19-CAR T cells but does not affect mTOR and HIF1? expression. (A) CD19-CAR T cells were treated with 10 mM metformin and/or compound C for 24 hours. The expression of p-AMPK was analyzed by Western blotting (up) and the statistical analysis of relative p-AMPK protein expression results is shown (down). (B) CD19-CAR T cells were cocultured with Raji cells with 10 mM metformin and/or compound C at various effector to target cell ratios (1:1, 5:1, 10:1, 20:1, and 40:1) for 24 hours, and the apoptotic rate was determined by flow cytometry. (C) CD19-CAR T cells were treated with 10 mM metformin and/or compound C for 24 hours, and the apoptotic rate of CD19-CAR T cells was detected by flow cytometry. (D) Statistical analysis of the flow cytometry results. (E) CD19-CAR T cells were treated with 10 mM metformin and/or compound C at different times (0, 5, 10, 30, and 60 minutes), and the expression of p-AMPK, p-mTOR, and HIF1? was analyzed by Western blotting analysis. (F) Statistical analysis of the Western blot results. *P,0.05, **P,0.01 versus the control group. Data are represented as mean ± SD from three independent experiments.
Figure Method/Brief Explanation Description of results/how they relate to the claim CTS 5' LTR RRE CD8 V VTMCD284-1BB CD3 3' LTR -Control -CD 19-CAR 1 5 100 Control Met (1 mM) Met (10 mM) Met (20 mM) 63.1% 8.2% c 80 1 0 C 60 40 5 0.5 ? 20 0.0 0 101 10% 10% 10% 10% CD19-CAR 0 2 46 8 10 12 Days post isolationExplanation / Answer
Ans- in figure 4 it is stated that Metformin increases the expression of p-AMPK and reduces the expression of p-mTOR and HIF1.
In figure 5 it is stated that
The AMPK inhibitor reverses the metformin-regulated apoptosis and cytotoxicity of CD19-CAR T cells but does not affect mTOR and HIF1
Above both statement are related to the fact as expression of one substance is not related to expression of other substance but indirectly they are related to each other.