Figure 6.

Tumors exposed to proinflammatory e-cigarette components are responsive to immune checkpoint blockade. (A) Mean fluorescence intensity of T cell immune checkpoints CTLA4, PD-1, TIM3 and LAG-3 on splenic CD8+ T cells from mice exposed to PG/VG with or without 36 ml/mg nicotine as described in Figure 4A . CTLA4 and TIM3 were significantly elevated only in the presence of nicotine (p=0.011, p=0.015 respectively). PD-1 was significantly elevated in the presence of PG/VG (p=0.024) with no nicotine effect (p=0.431). No significant differences were found for LAG-3 expression. Summarized results can be found in Table 1 . (B) The frequency of immune checkpoint co-expression (none (0), any given one (1), any combination of 2 (2), any combination of 3 (3) or all markers (4)) is shown in splenic CD8+ T cells as assessed by flow cytometry Boolean gating. (C) Percentage of quadruple-positive CTLA4+TIM3+LAG-3+PD-1+ splenic CD8+ T cells. Significantly elevated quadruple-positive cells were found in the presence PG/VG (p<0.001, Table 1 ) with a significant additive nicotine effect (p<0.001). (D) Experimental scheme to test the effect of immune checkpoint inhibition in e-cigarette preconditioned subcutaneous tumors. After preconditioning MC38 cells in the presence of 2.5 μM PG/VG with or without 36 mg/ml nicotine for 14 days, mice (n=5 per experiment) were implanted with tumors subcutaneously. Starting 3 days after implantation., mice were treated with intraperitoneal anti-CTLA4 (aCTLA4) or the relevant isotype control (IgG2k/a) on days 3, 6 and 9 after implantation. (E) Endpoint tumor volumes. Error bars represent standard error mean (SEM). Treatment with anti-CTLA4 showed a significant effect on tumor volume (p<0.001; Table 2 ) but not observed for PG/VG+nicotine (p=0.572). No significant interaction between anti-CTLA4 treatment and presence of PG/VG+nicotine was observed (p=0.934). (F) Metastasis rates: 6/15 (40%) animals with PG/VG-preconditioned MC38 tumors showed peritoneal metastases, which were absent (0/15) in both anti-CTLA4-treated groups and nearly absent (1/15) in control mice. There was no significant difference in metastases among these three groups (p=0.762 by Fisher Exact Test (p=0.762) and a significant increase in the PG/VG+nicotine mice compared to the others (p=0.005 by Fisher Exact Test).