Figure 7. Aurora kinase A inhibition in combination with CTLA-4 blockade promotes antitumor immunity and cancer cell apoptosis. (A–B) Groups of C57BL/6 mice with mEER tumors were treated sequentially with alisertib (10 mg/kg) and anti-CTLA-4 antibody (aCTLA-4; 100 µg per dose) individually or in combination (combo). All mice were euthanized after two treatment cycles on day 23, and tumors were weighed (A, B). (C) Tumor-infiltrating lymphocytes and circulating lymphocytes from the tumor and spleen were isolated and analyzed by polychromatic flow cytometry to assess the frequencies of different immune cell populations. Changes in their expression are represented as a heatmap. The negative immune regulator LAG3 is highlighted in red. (D) Frequencies of circulating myeloid-derived suppressor cell levels for the tumor and spleen. The significance of differences in immune cell subsets among the treatment groups were assessed using one-way analysis of variance. *p<0.05, **p<0.005, ***p<0.0001, ****p<0.00005. (E) Immunoblotting was performed on tumor tissues from four mice per treatment group at day 23. The expression of cleaved caspase-3 (cl caspase-3), full-length PARP (Fl PARP), and cleaved PARP (cl PARP) were analyzed to assess apoptosis. The treatment groups are as follows: V – vehicle, I – anti-CTLA-4, A – alisertib, C – combination. CTLA-4, cytotoxic T-lymphocyte associated protein 4, IO-Immunotherapy; PARP, poly(ADP-ribose) polymerase .