The impact of chronic stress on radiation induced cell death and the anti-tumor immune response - Radiation Therapy (RT) is a common form of cancer treatment that can be effective in treating numerous malignancies. Two key components of an effective RT regimen are a dose of irradiation that is sufficient to cause tumor cell death, and an innate immune response, driven by dendritic cells and fueled by the debris from dying tumor cells, that goes on to activate anti-tumor adaptive immunity. Collectively, this process has come to be known as the in situ vaccine effect of radiation. Unfortunately for many patients, a deficiency in one of these two key components can occur from the onset of treatment, or develop over time, and result in resistance to RT. For example, if an insufficient amount of tumor cell death occurs from a given dose of radiation, not only will more live cancer cells remain within the tumor, but this lack of cell death will also ultimately limit the activation and recruitment of adaptive immune cells. Without adaptive immune activation, the remaining live cells within the tumor, and potential metastases that could be present throughout the body, can survive and proliferate. We have determined that chronic stress mediated by β-adrenergic signaling is capable of inducing tumor cell resistance to irradiation induced cell death in vitro, and we have also determined that this same stress results in a subdued anti-tumor immune response generated from RT in vivo. The goal of this proposal is to resolve the mechanism through which adrenergic stress induces tumor cell radioresistance, and to determine whether this change in cell death is driving the immunologic changes observed in vivo, in addition to the direct effects of stress on immune cells. To address these goals, we will use pharmacologic and genetic approaches to induce or inhibit signaling cascades downstream of the β1, β2, and β3-ARs, and determine which receptor, and which signaling pathways, are responsible for the observed increase in tumor cell survival after irradiation. We will define how this signaling drives survival by evaluating cell death pathways including apoptosis, necrosis, and necroptosis, and determine whether inhibiting this signaling also leads to a potentially more immune stimulating tumor microenvironment. To do so, we will assess cGAS/STING signaling and damage associated molecular pattern (DAMP) production (including ATP, HMGB1, and Calreticulin) in vitro. Using a series of co-culture experiments where dendritic cells (DCs) are cultured with irradiated tumor cells experiencing varying levels of β-AR signaling, we will evaluate whether changes in the radiation induced cell death processes described above affect DC maturation and function. In vivo, we will utilize various β-AR deficient mouse strains to evaluate whether increased β-AR signaling in tumor cells alone is sufficient to drive resistance to therapy and impaired anti-tumor immunity. Changes in DAMP production in vivo will also be evaluated. Taken together, this project has the potential to produce paradigm shifting discoveries which outline a new and important mechanism of radiation resistance that is driven by the human physiologic response to chronic stress and anxiety, β-adrenergic signaling. Ultimately, these discoveries could enhance the efficacy RT, improve patient outcomes, and increase patient quality of life.
