FIGURE 4 from Dual Blockade of EP2 and EP4 Signaling is Required for Optimal Immune Activation and Antitumor Activity Against Prostaglandin-Expressing Tumors

TPST-1495 confers therapeutic efficacy in mice bearing PGE2-producing tumors. A, PGE2 detected by mass spectrometry in cell culture fluids of tumor cells lines after 24 hours in culture. Results depict six technical replicates. B, PGEM detected by mass spectrometry in urine of tumor-bearing mice from untreated animals (shown in C) approximately 17 days after tumor implantation. Results are representative of one experiment with five biological replicates. Tumor outgrowth (C) and percent TGI [TGI = 100*(Control Volume − Treatment Volume)/Control Volume] (D) measured in mice bearing flank implanted tumors of tumor types shown in the figure. A total of 1 × 106 cells were implanted on day 0 and treatment in mice randomized to assigned groups was initiated when average tumor volume within a tumor line reached approximately 80–100 mm3. Mice were treated with TPST-1495 (100 mg/kg twice a day, orally), E7046 (150 mg/kg every day, orally), PF04418948 (100 mg/kg every day, orally), or Celecoxib (60 mg/kg every day, orally) and followed for outgrowth until vehicle mice reached humane endpoints. Results are representative of two experiments with N = 5 mice per group. Mean and SD are depicted in all graphs and the Student two-sided t test was used to assess significance between individual groups at the last timepoint before mice were sacrificed. **, P P E) or IHC (F) from CT26 tumors after 14 days of treatment. A total of 1 × 106 CT26 cells were implanted in the flanks of animals and followed until the average was 80–100 mm3. Mice were then treated for 14 days at which time tumors were resected and stained. Results depict one experiment with 8 mice, representative of more than three similar experiments. G and H, Flow cytometry from CT26 tumors resected after 17 days of treatment. Tumors were implanted as in E, treated for 17 days with TPST-1495, and then time tumors were resected and stained for DC1+ dendritic cells (G) or macrophages (H). Results shown in G represent one experiment with 10 mice, representative of multiple similar experiments with an average of 2-fold increase over vehicle, but data depicted are the only replicate that met P t test was used to assess significance between individual groups. ****, P P P < 0.05.