Figure 4.

(see previous page). In vivo treatment with axitinib increases the number of tumor-infiltrating immune cells, especially CD11b⁺ cells, in subcutaneous and intracranial tumor models. Determination of tumor-infiltrating immune cells in subcutaneous and intracranial MO4-bearing mice on the 7th day of treatment with axitinib at 25 mg/kg bid or vehicle by oral gavage. After sacrifice, single cell suspensions of the subcutaneous or intracranial tumors were made and subsequently analyzed by flow cytometry. A-E. Subcutaneous tumor. The total number of CD45⁺ cells was increased in axitinib-treated mice as compared to vehicle-treated mice (A.). This increase was due to an increase of CD11b⁺Ly6C^highLy6G^- cells (E.). A. Percentage of CD45⁺ cells within the tumor. (B.) Percentage of CD45⁺CD3⁺ T cells within the CD45⁺ cell population. Within this population, the percentage of CD4⁺ and CD8⁺ T cells was determined. (C). Regulatory T cell population (T_reg) within the CD4⁺ T cell population determined as CD4⁺CD25⁺ cells. (D). Percentage of CD11c⁺ cells (DCs) within the CD45⁺ cells and expression of CD80 and CD86 maturation markers on their surface. (E). Percentage of CD11b⁺ cells within the CD45⁺ cells. Within this population further characterization of the MDSCs: Ly6C^highLy6G^- (moMDSC) and Ly6C^intLy6G⁺ (grMDSC). Three independent experiments were performed (3 mice per group) and results are presented as mean ± SEM. F-J. Intracranial tumor. Similar to the subcutaneous tumor, the total number of CD45⁺ cells was increased (F.) in axitinib-treated mice as compared to vehicle-treated mice. That was also due to an increase of CD11b⁺ Ly6C^highLy6G^- (J.) F. Percentage of CD45⁺ cells within the intracranial tumor. (G.) Percentage of CD3⁺CD45⁺ T cells within the CD45⁺ cell population. CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were determined within the CD3⁺ T cell population. (H.) Percentage of CD4⁺CD25⁺ cells (T_reg) within the CD45⁺CD4⁺ T cell population. (I.) Percentage of CD11c⁺ population (DCs) within the CD45⁺ cell population and expression of the maturation markers CD80 and CD80 within this CD11c⁺ population. (J.) Percentage of CD11b⁺ cells (myeloid cells) within the CD45⁺ population. Further determination of the different MDSC subpopualtions through expression of Ly6C⁺ and Ly6G⁺: Ly6C^highLy6G^- (moMDSC) and Ly6C^intLy6G⁺ (grMDSC). Three independent experiments were performed (3 mice per group). Intracranial tumors were pooled from each group and results are presented as mean ± SEM.