Figure 5.

EZM2302 inhibits, but JQ1 promotes, 4T1.2 lung metastasis and their differential effects on the regulation of CD8⁺ T cells. (A) A schematic of the workflow using vehicle, JQ1, EZM2302, or both for treatment of 4T1.2 orthotopic tumors. (B) 4T1.2 tumor growth curve under indicated treatment conditions (vehicle, JQ1, EZM2302, or both). Data are mean ± s.e.m. n = 9 (vehicle, JQ1, EZM2302 groups) and n = 8 (JQ1 + EZM2302 group). *P< 0.05; NS: not significant. (C) Drug-induced tumor volume changes normalized to the vehicle-treated group at 8 days or 28 days. Data are mean ± s.d. **P< 0.01; *P< 0.05. (D) Bioluminescence imaging of 4T1.2-luciferase tumors in mammary fat pads and lung after treatment with vehicle, JQ1, EZM2302, or both for 28 days. (E) Luciferase signal intensities of 4T1.2 tumor cells metastasize to lung in the indicated treatment groups. Data are mean ± s.d. *P< 0.05. (F) Flow cytometry analyses of CD4 and CD8 expressing cells from lungs of 4T1.2 syngeneic mouse models under indicated treatments. (G) Quantification of CD8⁺ T cells from CD45⁺ immune cells from lungs of 4T1.2 syngeneic model under indicated treatments. **P< 0.01; *P< 0.05; NS: not significant. (H) A schematic of antibody and drug treatment investigating the role of CD8⁺ T cells in 4T1.2 tumor growth. 4T1.2 tumors were treated with vehicle, EZM2302, anti-CD8 antibody or both, starting from the day of 4T1.2-luciferase injection to mammary fat pad. (I) Kaplan–Meier survival curves of 4T1.2 tumor bearing mice in indicated treatment groups. Two doses (25 and 100 μg) of anti-CD8 antibody were used. *P< 0.05. (J) Growth curve of primary 4T1.2 tumors under indicated treatment conditions. **P< 0.01. (K) Lung metastases detected by bioluminescence imaging of four treatment groups. *P< 0.05. (L) Quantification of CD8⁺/CD45⁺ cells from peripheral blood of control, anti-CD8, EZM2302 or anti-CD8 plus EZM2302 treatment groups after 10, 20 and 30 days. **P< 0.01; *P< 0.05; NS: not significant.