Improving the Efficacy of Checkpoint Blockade Inhibitors in Breast Cancer by Combining with Radiation Therapy

Abstract

Large cohorts of recent clinical studies have established that increased levels of tumor-infiltrating lymphocytes (TILs) in patients with the triple-negative breast cancer (TNBC) subtype predicted better clinical outcomes compared to other subtypes. These observations led to the hypothesis that women with TNBC may respond to checkpoint blockade therapy. However, early results from these trials using checkpoint inhibitors alone or with chemotherapy has shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, we hypothesized that radiation therapy can be utilized to increase the efficacy of check point blockade inhibitors of the stress-induced heat shock protein 70 (HSP70) in order to reverse immunosuppression and induce neo-antigens to improve the outcomes of TNBC. To test the effectiveness of a combination of checkpoint blockade inhibitors and radiation therapy, this study utilized 6-week old female BALB/c 4T1 tumor-bearing mice as a model for TNBC. Combination treatment of HSP70 inhibitors and targeted irradiation was performed prior to tumor resection and data collection. Specifically, Radiation Therapy doses were planned and delivered using a Small Animal Radiation Platform (SARRP) equipped with on-board cone-beam CT. A single fraction of 17 Gy (Two fractions of 11.5 Gy) doses were delivered based on EQD2 calculation (alpha Beta ratio = 4). To achieve highly localized dose delivery, an x-ray collimator was used to focus the broad beam from a 225 kVp x-ray tube. Our preliminary data provide evidence that targeting HSP70 in combination with radiation therapy has dual activity on the tumor and the immunosuppressive microenvironment. Furthermore, reversal of HSP70 blockade significantly reduces the immunosuppressive MDSC infiltration in lungs by more than 50% compared to lungs from control mice. This also led to increased tumor infiltration of effector T cells from 1-2% in controls to 12% in lungs from mice treated with HSP70 inhibitor in combination with radiation. Interestingly, T cell infiltration is further increased to 20% when check point blockade inhibitor is combined with HSP70 inhibitor and radiation. Therefore, we showed that HSP70 regulates three fundamental processes; i) protecting tumor cells from cytotoxic cell death, ii) serving as a powerful tumor antigen attracting TILs, and iii) modulating immunosuppressive MDSCs. In summary, our studies elucidate the molecular mechanism by which HSP70 cytoprotects tumor cells from cytotoxic agents while regulating the immunosuppressive MDSCs which limits the anti-tumor activity of TILs. They also provide evidence that blocking HSP70 potentiates the efficacy of the checkpoint blockade particularly when combined with radiation in syngeneic mouse models representing TNBC subtype.