Fig 2: Forest plot of hazard ratios for overall survival assessed by PD-L2 expression and clinicopathological factors. PD-L2 expression was an independent prognostic factor after adjusting age, grade, IDH status and 1p19q status in TCGA and CGGA datasets.

Fig 3: Synergistic effect of eFT-508 and adriamycin on the in vitro chemosensitivity of BC cell lines. A BC cell lines are chemoresistant to adriamycin with high values of IC50. Exponentially growing cultures of BC cell lines, MDA-MB-231, MDA-MB-468, MCF7, T47D, and SUM149, were treated with increasing concentrations of adriamycin for 24 h. Cell viability was evaluated using MTT assay, and percent viability after 24 h were plotted. No significant difference was observed between the cell lines for any of the concentrations of adriamycin tested. The selection criteria of drug dilution were scalar dilution factor 3 and then factor 2. B Differential sensitivity of BC cell lines to Tomivosertib (eFT-508). Same as A but was treated with increasing concentrations of eFT-508. All cell lines tested had more chemosensitivity to eFT-508 compared to the same concentration of adriamycin. MDA-MB-468 and MDA-MB-231 were more chemosensitive compared to the other three cell lines. The selection criteria of drug dilution involved scalar dilution factor 3 and then factor 2. #P < 0.05; ##P < 0.01; **P = 0.001; ***P < 0.001. C Adriamycin, in combination with eFT-508, showed a higher in vitro therapeutic index. Same as A but instead treated with both adriamycin and eFT-508. Combination therapy resulted in significantly less IC50 in all cell lines compared to monotherapy. The IC50 in the MDA-MB-468 cells was significantly lower compared to the other four cell lines. The selection criteria of drug dilution included scalar dilution factor 3 and then factor 2. Data in A–C are represented as staggered plots, with each point representing an experimental replicate (n = 3). D Western blot was used to detect the expression of PD-L1 in BC cell lines. E Western blot was used to detect the expression of PD-L2 in BC cell lines. F Treatment with eFT-508 resulted in the inhibition of all five cell lines, and Western blot was used to detect the expression of PD-L1 and PD-L2 in cell lines. G Treatment with eFT-508 resulted in the suppression of MNK1/2-mediated phosphorylation of eIF4E in all five cell lines. Immunoblot analysis of phosphorylated eIF4E (S209) and total eIF4E in the cell lines treated with eFT-508. Shown are representative blots from three biological replicates. GAPDH was used as a loading control. H Treatment with eFT-508 resulted in the inhibition of global translation. Representative polysome profile obtained from the post-nuclear extract of vehicle or eFT-508 treated MDA-MB-231 cells. Treatment with eFT-508 resulted in the lower 80S and polysome peaks. Similar results were obtained in the other four cell lines as well (data not shown). I Results in E were additionally verified using the SUrface SEnsing of Translation (SUnSET) technique to assess changes in global translation. Untreated or eFT-508-treated MCF7 and MDA-MB-231 cells were labeled with puromycin for 15 min. Shown are immunoblot analyses of lysates made from these cells using an anti-puromycin antibody (top). The membrane was subsequently stained with Coomassie Blue to ensure equal protein loading across lanes. Shown are representative blots from three experiments. J Treatment with adriamycin had no noticeable effect on global translation. Untreated or adriamycin-treated MCF7 and MDA-MB-231 cells were labeled with puromycin for 15 min. Shown are immunoblot analyses of lysates made from these cells using an anti-puromycin antibody (top). The membrane was subsequently stained with Coomassie Blue to ensure equal protein loading across lanes. Shown are representative blots from three experiments

Fig 4: Immunostaining of lung cancer. (A) A squamous cell carcinoma with a high density of TIL and (B) positive expression of PD-L1 on TCs. (C) A squamous cell carcinoma with a high density of TIL and (D) positive expression of PD-L1 on ICs. (E) An adenocarcinoma with a high density of TIL and (F) positive expression of PD-L2 on TCs. (G) A squamous cell carcinoma with a high density of M2 TAM and (H) positive expression of PD-L2 on ICs. Scale bar=100 µm. TIL, tumor-infiltrating lymphocyte; PD-L, programmed death-ligand; TCs, tumor cells; ICs, tumor-infiltrating immune cells; TAM, tumor-associated macrophage.

Fig 5: Comparison of PD-L2 expression level in CGGA and TCGA cohorts with different TCGA molecular subtypes (a, c), PD-L2 was significantly increased in mesenchymal subtype (p < 0.0001). ROC curves exhibited highly sensitivity in predicting mesenchymal subtype (b, d). Receiver operating characteristic (ROC) curve for mesenchymal subtype prediction in CGGA and TCGA datasets. ROC curve analysis showed that PD-L2 had highly sensitivity and specificity to predict mesenchymal subtype in CGGA and TCGA database. Area under curve(AUC) was 0.903 and 0.816, respectively.