2933 - Radiotherapy Plus Aurora A Kinase Inhibitor Reshape the Immune Microenvironment and Potentiate Anti-Tumor Responses

X. Zhang¹, W. Wen¹, and D. Chen²; ¹Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ²Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China

Purpose/Objective(s): Aurora A kinase is a serine/threonine protein kinase that plays a crucial role in the regulation of cell cycle and is overexpressed in multiple cancers, associated with poor prognosis of patients. In clinical trials, Aurora A kinase inhibitors have not benefited patients well by exerting anti-tumor effects through targeting Aurora A kinase. Our aim is to explore the immune mechanism of radiotherapy combined with Aurora A kinase inhibitor against tumors, and to apply better combined treatment methods to clinical practice, serving patients. Materials/

Methods: Through WB and cell counting kit experiments, we selected 4T1 and CMT167, two cell lines with high Aurora A expression and sensitivity to Aurora A kinase inhibitors. Then, we subcutaneously implanted respectively these two cell lines into BALB/c and C57BL/6J mice, followed by different treatments after grouping, to evaluate the tumor progression, survival, and prognosis of the mice. We performed flow cytometry analysis on the tumor and spleen immune microenvironment of mice, and observed the changes. We performed RNA-seq to identify differentially expressed genes, validate them and explore the mechanisms. And using immunohistochemistry to assess the association between Aurora A kinase expression and prognosis in clinical radiotherapy cases. Finally, we combined immune checkpoint inhibitor, and evaluated the anti-tumor effect of the triple therapy on the mice.
Results: Radiotherapy combined with Aurora A kinase inhibitor effectively inhibited tumor growth and improved the survival of tumor-bearing mice. The safety of the combined treatment was confirmed by the monitoring of mouse body weight and the HE staining of vital organs. Flow cytometry results showed that radiotherapy combined with Aurora A kinase inhibitor could reshape the tumor immune microenvironment, increase the number and cytotoxicity of CD8⁺ T cells, and improve the immune status of the spleen, increasing the number of total T cells, CD8⁺ T cells, effector CD4⁺ T cells and effector CD8⁺ T cells. Additionally, the depletion of CD8⁺ T cells experiment also confirmed that the combined treatment no longer had a significant tumor-suppressing effect after the removal of CD8⁺ T cells. RNA sequencing results showed that the expression of chemokines recruiting T cells, such as CXCL9 and CXCL11, increased in the combined treatment group. In vitro flow cytometry experiments confirmed that the combined treatment increased the expression of PD-L1 on the tumor surface, providing a theoretical basis for the combined application of immune checkpoint inhibitors.
Conclusion: Our results confirm that radiotherapy combined with Aurora A kinase inhibitor not only reshaped the tumor immune microenvironment and improve the immune status of the spleen, but also enhance the therapeutic effect of combined immune checkpoint inhibitors.