Figure 5.

The PaSSS-based drug combinations induced an immune response of PBMC in SCC25 and Cal27 models in vitro and reduced tumor growth in vivo. (A) To examine IFN-γ secretion (as illustrated in the scheme on the left) SCC25 cells (middle panel) and Cal27 cells (right panel) were treated with either anti-EGFR monotherapy (Er) or the PaSSS-based combination. C stands for control. After 48h and 96h respectively the supernatants were collected for IFN-γ levels quantification (*P < 0.05, **P < 0.001). (B) SCC25 (middle panel) and Cal27 cells (right panel) were co-cultured (CC) with PBMCs (as illustrated in the scheme on the left) and treated for 48h and 96h respectively with either anti -EGFR monotherapy or the predicted combination (with or without the addition of 10μg/ml Keytruda, Ky). PBMCs were then collected and CD3 levels in CD8 positive cells were measured (*P < 0.02 for SCC25,*P < 0.007 for Cal27). (C) SCC25 (C, upper right panel) and Cal27 (C, lower right panel) were CC with non-activated /activated PBMCs (AC PBMC) and then the cells were treated with either anti-EGFR monotherapy or the predicted combination with or without the addition of 10 μg/ml Keytruda for 96h. Cell survival was measured via methylene blue. (D) SCC25 (D, left panel) or Cal27 (D, right panel) were injected subcutaneously into mice, and once tumors reached 50 mm³, treatments were initiated. In both cases, the PaSSS-based drug combinations (see black arrows) inhibited tumor growth and demonstrated an effect superior to monotherapy of erlotinib or to the drug combinations predicted to partially target the PaSSS (*P < 0.03 for SCC25) (*P < 0.03 for Cal27) (see Figures 3,4 for details regarding the altered signaling signatures and the PaSSS-based drug combination predictions). (E) Representative, treated and untreated SCC25 and Cal27 tumors, harvested after 25 days and 14 days respectively, are shown. Panels (A-C) were created using BioRender.com.