DataSheet_2_Metabolic-related gene pairs signature analysis identifies ABCA1 expression levels on tumor-associated macrophages as a prognostic biomarker in primary IDHWT glioblastoma.docx

BackgroundAlthough isocitrate dehydrogenase (IDH) mutation serves as a prognostic signature for routine clinical management of glioma, nearly 90% of glioblastomas (GBM) patients have a wild-type IDH genotype (IDHWT) and lack reliable signatures to identify distinct entities.MethodsTo develop a robust prognostic signature for IDHWT GBM patients, we retrospectively analyzed 4 public datasets of 377 primary frozen tumor tissue transcriptome profiling and clinical follow-up data. Samples were divided into a training dataset (204 samples) and a validation (173 samples) dataset. A prognostic signature consisting of 21 metabolism-related gene pairs (MRGPs) was developed based on the relative ranking of single-sample gene expression levels. GSEA and immune subtype analyses were performed to reveal differences in biological processes between MRGP risk groups. The single-cell RNA-seq dataset was used to examine the expression distribution of each MRG constituting the signature in tumor tissue subsets. Finally, the association of MRGs with tumor progression was biologically validated in orthotopic GBM models.ResultsThe metabolic signature remained an independent prognostic factor (hazard ratio, 5.71 [3.542-9.218], P ConclusionsThe metabolic signature is expected to be used in the individualized management of primary IDHWT GBM patients.</p

968 blocks the activity of glutaminase and decreases GSH/GSSG ratio in HCC cells.

<p>a, The activity of glutaminase in LM3 cells was tested after cells were treated with 968 at 20 μM for 24 h. Means ± SD of triplicates are shown. Similar results were obtained in two independent experiments. ** P < 0.01 compared with untreated controls. b, LM3 cells were treated with 968 at 20 μM for 36 h, then GSH/GSSG ratio was measured. The results are shown as the means ± SD of two independent experiments. ** P < 0.01 compared with untreated controls.</p

968 and DHA cooperatively increase intracellular ROS leading to enhanced cytotoxicity in LM3 cells.

<p><b>a</b> & <b>b,</b> The intracellular ROS level was assessed after cells were treated with 968 and/or DHA for 12 or 24 h. The results are shown as the means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 compared with the intracellular ROS level in cells treated with 968 or DHA only. Peak values associated with treatment for 12 h (left) and 24 h (right) are representative of three independent experiments. <b>c,</b> Cells were pretreated with NAC (3 mM) for 2 h and then treated with 968 and DHA for 48 h. The results are shown as the means ± SD of three independent experiments. ** P < 0.01 compared with cells without NAC pretreatment (UT group).</p

The combination of 968 with DHA exerts synergistic cytotoxic effect in HCC cells.

<p>a-d, The combinatory cytotoxicity of 968 and DHA (a-HepG2, b-7402, c-LM3, d-ECV304) was assessed by MTT assay after cells were treated with 968 and DHA at a serial of doses as indicated for 48 h. The molar ratio of 968 to DHA was 1:1. Data are presented as means ± SD (n = 6). Similar results were obtained in two independent experiments. ^# not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001. e, The combinatory effect of 968 and DHA was assessed by trypan blue exclusion using a cell counter after LM3 cells were treated with 968 and DHA at 20 μM for 48 or 72 h. Data shown are means ± SD of triplicates. Similar results were obtained in three independent experiments. ** P < 0.01 and *** P < 0.001.f, LM3 cells were treated with 968 (20 μM) and/or DHA (20 μM) for 48 h, then cell viabilities were assessed by crystal violet staining assay. The representative plate of three independent experiments is shown.</p

Cytotoxic effect of 968 on HCC and human endothelial cells.

<p>a-d, The inhibitory effect on cell proliferation was detected by the MTT assay after cells were treated with 968 at depicted concentrations for 48 h. Four cell lines HepG2 (a), 7402 (b), LM3 (c), and ECV304 (d) were used. Means ± SD are shown (n = 6). ^# not significant, *P < 0.05, **P < 0.01, and ***P < 0.001. e, LM3 cells were treated with 20 μM 968 and the viability was measured by trypan blue exclusion on 1, 2, 3 and 4, respectively. Untreated cells were used as control. Data are presented as the means ± SD of triplicates. Similar results were obtained in two independent experiments. ^# not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.</p

Antitumor efficacy of DHA on HCC.

<p>The cytotoxicity of DHA was assessed by MTT assay after cells (<b>a</b>-HepG2, <b>b</b>-7402, <b>c</b>-LM3, <b>d</b>-ECV304) were treated with DHA at a serial doses as indicated for 48 h. Means ± SD (n = 6) are shown. Similar results were obtained in two independent experiments. ^# not significant, ** P < 0.01, *** P < 0.001.</p

GLS1 silencing enhances DHA-induced cytotoxicity in LM3 cells.

<p><b>a</b>, The expression of GLS1 protein was decreased after cells were transfected with GLS1-siRNA for 48 h. Blots are representative of three independent experiments. <b>b</b>, The combinatory cytotoxicity of GLS-siRNA and DHA was assessed by the MTT assay after LM3 cells were treated with GLS-siRNA and/or DHA (20 μM) for 48 h. Means ± SD are shown. Similar results were obtained in three independent experiments. * P < 0.05, *** P < 0.001.</p