SUMMARY This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 21-083. The interplay between radiation therapy (RT) in cancer treatment and the host immune system is complex. RT can have local therapeutic effects through direct damage to cancer cells, or can stimulate a systemic anti-tumor immune response. The benefits of combining RT and immunotherapy have been reported in several pre-clinical models and case reports. However, despite the sustained and deep responses observed in some patients, many cancer patients do not respond to these agents emphasizing the need for further improvements in treatment strategies. Interestingly, the effects of radiation on the TME vary with dose and fractionation schedules. In addition to its role in immune activation, RT can also cause chronic inflammation, release of cytokines, and increased infiltration of immunosuppressive cells in the TME potentially rendering decreased tumor responses in a paradoxical manner. However, knowledge is lacking on the immunologic impact of different RT regimens, especially in comparison with changes to RT in human tumor tissues. This proposal aims to investigate the effects of different doses and fractionation of RT on innate and adaptive immune cells using both pre-clinical syngeneic murine melanoma and non-small cell lung cancer (NSCLC) flank models. We will correlate and compare these changes to those within formalin-fixed paraffin-embedded (FFPE) tissue surgical samples from human patients with brain metastases from melanoma and NSCLC recently treated with RT or non-irradiated patient samples. Comparing with non-irradiated control samples, we will determine the effects of RT on tumor immunogenicity (PD-L1, MHC Class I levels) and on immune cell absolute numbers in the tumor microenvironment (TME). Using a multispectral imaging technique, we will measure RT induced CD4+ T cell, CD8+ effector T cells, regulatory T cells, myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs), and dendritic cell influx into the TME and secondary lymphoid tissues in animal models. Serum cytokine levels following RT will also be measured using cytokine array multiplex panels. Similarly, a multispectral imaging technique will measure RT induced changes to immune cell populations in human tumor surgical specimens. Since RT can induce changes in the gene and protein expression profiles of both tumor and immune cells, and transcriptomic landscapes can be associated therapeutic outcomes, the cell-type specific transcriptional profile in the TME of FFPE human tumor samples will be determined using single cell transcriptomic assays. Our multi-disciplinary team plans to systematically categorize the effects of different RT regimens on immune cells and define a radiation response index (i-RRI) on a dose scale (in Gy). We aim to define immune cells as being either “radiation sensitive” or “radiation resistant” at particular dose...