Cell experiment:

Bone marrow (BM) cells are flushed from femurs of BALB/c mice using sterile CM. Freshly harvested (BM) cells (0.5×106) are differentiated in the presence of 20 ng/mL recombinant mouse GM-CSF, ±PGE2 (10 nM), at 37℃, for 8 d. CM C fresh GM-CSF ± PGE2 is changed on days 3 and 6. After in vitro differentiation, cells are analyzed by flow cytometry. For certain experiments, CT26, 4T1 cell supernatants, and/or EP1 (SC-57089), EP2 (ER-880696), EP3 (L-798106), or EP4 (E7046) antagonists at 1 mM, are added to the BM cells. To assess the effect of differentiated BM cells on T cell proliferation, mouse BM cells differentiated are co-cultured for 72 hours with anti-CD3/CD28 Dynabeadsstimulated and CFSE (1 mM)-stained T cells. T cell proliferation is assessed by CFSE dilution using flow cytometry.

Animal experiment:

For the tumor isograft efficacy studies, 6-week old female BALB/c mice are implanted with cancer cells: 1×105 CT26 or 4T1 cells or 8×105 H22 cells per mouse s.c., or 1×105 EMT6 cells in the mammary fat pad. C57BL/6 mice are implanted s.c. with 1×106 Pan02 cells per mouse, and A/J mice are implanted s.c. with 2-3 mm3 SAI/N tumor fragments. To investigate the role of T cells in the anti-tumor response, 6 week old female nude mice (which lack T cells) are injected s.c. with 1×105 CT26 cells. When tumors reach approximately 50-100 mm3, tumor-bearing mice are randomly assigned to vehicle or treatment groups, and treatment regimens begin. E7046 is administered per oral (p.o.) as a 100 or 150 mg/kg suspension in 0.5% MC, daily for 21 d (QDx21). For the combination studies, E7777 is administered intravenously (i.v.) at 2.5 mg/mouse in saline, as 2 to 3 doses injected one week apart (Q7Dx2-3). Tumor volumes and body weights are recorded 2-3 times a week. For comparison with current immunotherapies, in addition to vehicle control and E7046 C E7777 groups, mice are assigned to anti-PD1 antibodies or anti-mouse PD-1 C antimouse CTLA4 antibodies treatment groups. Anti-PD-1 and anti-CTLA-4 antibodies (1 mg/mL), are administered i.p. in 100 mL, 3 times 4 d apart (Q4Dx3) for a total of 300 mg each. Isotype controls are administered i.p. at 1 mg/mL to the control group. For the CD4CT and CD8CT lymphocyte depletion, antimouse CD4 or anti-mouse CD8 antibodies or their isotype controls are administered in 100 mL, i.p. at 2.5 mg/mL every 4 days, for a total of 4 injections per mouse (1 mg).

E7046 is a potent and selective antagonist that potently competes with PGE2 binding to the type 4 prostaglandin E2 (PGE2) receptor EP4 [1,2].

Prostaglandin E2 receptor 4 (EP4) is a prostaglandin receptor for prostaglandin E2 (PGE2) implicated in various physiological and pathological responses in animal models and humans [3].

In vivo: In APCMin/+ mice, daily oral administration of E7046 slowed down the growth of established subcutaneous tumors and significantly delayed the recurrence of tumors after surgical resection. E7046 significantly reduced the combined colon polyp area and the size of individual polyps without influencing the total polyp number. E7046 exhibited great activity against colon and small intestine tumors [1]. E7046 delayed the growth of multiple syngeneic murine tumor types. E7046 showed no effect on tumor cell viability in vitro [2]. In the CT26 model, treatment with E7046 and anti-PD1 inhibited tumor growth. In the 4T1 model, E7046 in combination with anti-CTLA4 resulted in a nearly complete tumor growth inhibition [4].

References:
[1] Albu D I, Wu J, Huang K, et al.  Abstract B198: Pharmacological profile of the PGE2 EP4 receptor antagonist E7046[J]. 2015.
[2] Albu D I, Huang K C, Wu J, et al.  Abstract B034: Preclinical immune antitumor activity of myeloid-targeting E7046 and Treg depleting E7777[J]. 2016.
[3] Negishi M, Sugimoto Y, Ichikawa A.  Prostaglandin E receptors[J]. Journal of lipid mediators and cell signalling, 1995, 12(2-3): 379-391.
[4] Bao X, Albu D, Huang K C, et al.  Combination of EP4 antagonist and checkpoint inhibitors promotes anti-tumor effector T cells in preclinical tumor models[J]. Journal for immunotherapy of cancer, 2015, 3(2): P350.