Synergistic Potential of CDK4/6 Inhibitors and Radiotherapy with Anti-PD-L1 Immunotherapy in Triple-Negative Breast Cancer

Purpose/Objective(s)

Triple-negative breast cancer (TNBC) represents the subtype with the poorest survival outcomes among all breast cancer molecular classifications. While immunotherapy has shown promising anti-tumor effects in TNBC treatment, its efficacy is not universal across all patients. CDK4/6 inhibitors have been recognized for their ability to radiosensitize and modulate the immune system. Additionally, high-dose radiotherapy (RT) has been observed to bolster the effects of immunotherapy. This study investigates the potential of enhancing immunotherapy effectiveness in TNBC by integrating RT with CDK4/6 inhibitors, focusing on modulating the tumor microenvironment.

Materials/Methods

We employed three human TNBC cell lines—MDA-MB-231, MDA-MB-453, and MDA-MB-468—along with the 4T1 mouse TNBC cell line to assess CDK4/6 inhibitor, abemaciclib, in radiosensitizing effects of TNBC using the clonogenic assays. We assessed the anti-tumor efficacy of RT, abemaciclib, and anti-PD-L1 antibody combination through the 4T1 cell line-derived immunocompetent mouse model. Interferon-γ (IFN-γ) levels in mouse blood were monitored before, during, and after treatment to gauge the immune response. Tumor-infiltrating lymphocytes (TILs) were analyzed in excised tumor samples through flow cytometry assays and immunohistochemical staining.

Results

Clonogenic assays showed that RT combined with abemaciclib pretreatment had synergistic effects across all tested TNBC cell lines. A single 8 Gy fraction of RT increased PD-L1 expression on the surface of tumor cells, while abemaciclib (at 100nm or 200nm concentrations) did not alter PD-L1 expression in all TNBC cell lines. The combination of abemaciclib, RT, and anti-PD-L1 in the 4T1 mouse model significantly decreased the tumor growth (P < 0.05) and elevated circulating IFN-γ levels during treatment (P < 0.001) when compared with control, RT alone, abemaciclib with anti-PD-L1, abemaciclib with RT, and anti-PD-L1 with RT. TILs assays showed the combination treatment of abemaciclib, RT, and anti PD-L1 increased CD4 and CD8 positive T cells proportion (P < 0.05 and P <0.01, respectively) as well as tumor associated macrophage (P < 0.001) when compared to the control group. Immunohistochemical staining revealed an increase in CD8 positive T cells and monocyte chemoattractant protein (MCP)-1 positive macrophages in the triple-combination treatment group compared to the other four groups.

Conclusion

Combined CDK4/6 inhibitors with RT enhance anti-tumor effects of anti-PD-L1 immunotherapy in TNBC through increasing secretion of IFN-γ and modulation of tumor microenvironments via recruiting CD4 and CD8 positive T-cells, as well as M1 type tumor associated macrophage.