Figure 4.

Inhibition of the Klf5/Cox2 axis increases the number and functionality of antineoplastic T lymphocytes in tumors. (A-B) Mice inoculated with control or Klf5-overexpression 67NR cells were treated every day with COX2 inhibitor (CEL) (total 6 times) when tumors were approximately 20 mm² in mean area. (C-E) Tumor size and weight distributions at Day 11 are shown. (F) Pge2 levels were evaluated in Klf5-overexpression versus control 67NR tumors receiving daily CEL (30 mg/kg) treatment for 6 days (n = 3). (G-J) The Klf5, Cox2 and Cd8 levels were quantified by ImageJ after staining with specific antibodies in paraffin-embedded tissues obtained from Klf5- overexpression versus control 67NR tumors. Representative images of Klf5, Cox2 and Cd8 (G). Klf5 expression was quantified in (H). Cox2 expression was quantified in (I). The level of Cd8⁺cells was quantified in (J). (K) Schematic of the experimental setup. Cytofluorometric analysis of tumor-infiltrating lymphocytes (TIL): Cd4⁺Foxp3⁺Cd25⁺ regulatory T cells (Treg) (L), Cd3⁺Cd8⁺ cytotoxic T lymphocytes (M), Cd8⁺Cxcr6⁺ T lymphocytes (N), and quantification of Icos, Ifnγ and Pd1 expression among both Cd4⁺ and Cd8⁺ T cells (O-T). Scale bar equals 50 μm. The data are represented as means ± SD. n ≥ 3 for mice in each group. (*p < 0.05; **, p <0.01; ***, p <0.001 significant vs. control; one-way or two-way ANOVA).