31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Thioridazine Sensitizes Apoptotic Effect of TRAIL in Human Lung Cancer PC9 Cells Through ER Stress Mediated Up-regulation of DR5

Background and objective Tumor necrosis factor-related apoptosis-inducting ligand (TRAIL) can induce apoptosis of tumor cells, however, various of tumor cells may survive because of resistance to TRAIL-mediated apoptosis. This study is to observe the proliferation inhibition effect of TRAIL sensitized by thioridazine on PC9 cells through endoplasmic reticulum (ER) stress mediated up-regulation of death receptor 5 (DR5) and investigate its mechanism. Methods PC9 cells were treated with different concentrations of thioridazine and TRAIL alone or in combination. Cell proliferation was measured by MTT assay, and cell apoptosis and cell-surface DR5 were detected by flow cytometry. Western blotting was utilized to measure the expressions of ER stress-related proteins glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), p-PKR-like ER kinase (PERK), p-eukaryotic initiation factor-2α (eIF2α), activating transcription factor 4 (ATF4) and apoptosis-related proteins caspase-3, caspase-9, caspase-8, PARP, DR5. Results Thioridazine inhibited the proliferation of PC9 cells in a dose-dependent manner (P<0.05). Thioridazine increased the inhibition and apoptosis of PC9 cells and up-regulated the expression of cell-surface DR5 induced by TRAIL. Flow cytometry showed that compared with TRAIL group, combination group of TRAIL and thioridazine increased cell apoptotic rates significantly (P<0.05). Western blotting indicated that compared with TRAIL group, expressions of Cleaved-caspase-8, Cleaved-PARP and DR5 increased significantly in combination group of TRAIL and thioridazine. The induction of DR5 and pro-apoptotic effect were mediated through activation of ER stress accompanying by increased synthesis of GRP78 and CHOP, which can be blocked by adding of ER stress inhibitor 4-PBA. Conclusion Thioridazine enhanced proliferation inhibition effect of TRAIL in PC9 cells may be facilitated through ER stress mediated upregulation of DR5

Mechanism of Killing Effect of Thioridazine on Human Lung Cancer PC9 Cells

Background and objective Recent research shows thioridazine which is a kind of phenothiazine antipsychotic drugs can inhibit the proliferation of various tumor cells in vitro, but the role of thioridazine on lung cancer has not been reported. So we choose PC9 cell lines as the research object, the aim is to oberve the killing effect of thioridazine on PC9 cells and investigate its possible mechanism. Methods After being treated with different concentrations of thioridazine, the proliferation of PC9 cells was determined by methyl thiazolyltetrazolium (MTT) assay. Flow cytometry was used to measure the cell cycle distribution and apoptosis. The expressions of cell cycle-associated protein CyclinD1 and apoptosis-related proteins Caspase-3, Caspase-8, Caspase-9, Bcl-2, Bax and Bcl-xl were detected by Western blot. Results The proliferation of PC9 cells was significantly inhibited by thioridazine in a dose- and time-dependent manner. Flow cytometry showed that cell cycle was arrested in G0/G1 phase and the apoptotic rates were significantly increased with the increasing concentration of thioridazine. Compared with the control group, the differences were statistically significant (P<0.05). Western blot analysis showed that, compared with the control group, thioridazine reduced the expressions of CyclinD1, Bcl-2 and Bcl-xl (P<0.01) and increased the expression of Bax (P<0.01). In the mean time, thioridazine promoted the activities of Caspase-3, Caspase-8 and Caspase-9 (P<0.01). Conclusion The mechanism of thioridazine inhibiting the proliferation of PC9 cells may be related to stimulation of Caspase apoptotic pathway, down-regulation of CyclinD1, Bcl-2, Bcl-xl and up-regulation of Bax