The impact of chronic stress on radiation induced cell death and the anti-tumor immune response

放射治疗(RT)是一种常见的癌症治疗形式，可以有效地治疗许多 恶性肿瘤。有效的RT方案的两个关键组成部分是足以引起 肿瘤细胞死亡和先天免疫反应，由树突状细胞驱动，由死亡碎片提供燃料 肿瘤细胞，进而激活抗肿瘤适应性免疫。总体而言，这一进程已经成为 被称为原位疫苗的辐射效应。不幸的是，对于许多患者来说，这两种缺陷中的一种 关键成分可以从治疗开始就发生，或者随着时间的推移而发展，并导致对RT的耐药性。为 例如，如果给定剂量的辐射导致的肿瘤细胞死亡数量不足，不仅会导致更多 活的癌细胞仍然留在肿瘤内，但这种细胞死亡的缺乏最终也会限制激活和 适应性免疫细胞的招募。如果没有适应性免疫激活，体内剩余的活细胞 肿瘤和可能存在于全身的潜在转移可以存活和增殖。我们有 确定β-肾上腺素能信号介导的慢性应激能够诱导肿瘤细胞耐药 在体外辐射诱导的细胞死亡，我们还确定了同样的应激导致驯服的 RT在体内产生抗肿瘤免疫反应。这项提议的目标是解决该机制 通过这种途径，肾上腺素能应激诱导肿瘤细胞辐射抵抗，并确定这种变化是否在细胞 除了压力对免疫的直接影响外，死亡也是体内观察到的免疫学变化的驱动因素 细胞。为了解决这些目标，我们将使用药理学和遗传方法来诱导或抑制信号转导 级联在β1、β2和β3-AR下游，并确定哪个受体和哪些信号通路， 是照射后观察到的肿瘤细胞存活率增加的原因。我们将定义该信号如何 通过评估包括细胞凋亡、坏死和坏死性下垂在内的细胞死亡途径来提高存活率，并确定 抑制这一信号是否也会导致潜在的更多免疫刺激肿瘤微环境。至 为此，我们将评估cGAS/STING信号和损伤相关分子模式(DAMP)的产生 (包括三磷酸腺苷、HMGB1和钙网蛋白)。利用一系列共培养实验，树突状细胞 (DC)与经历不同水平β-AR信号的照射的肿瘤细胞培养，我们将评估 上述辐射诱导的细胞死亡过程中的变化是否影响DC的成熟和 功能。在体内，我们将利用各种β-AR缺陷小鼠品系来评估β-AR增加 仅肿瘤细胞中的信号就足以导致对治疗的抵抗和抗肿瘤免疫的削弱。 还将评估体内湿气产生的变化。总而言之，这个项目有可能 产生范式转变的发现，勾勒出一种新的重要的辐射抵抗机制， 是由人类对慢性压力和焦虑的生理反应驱动的，β-肾上腺素能信号。最终， 这些发现可以提高RT的疗效，改善患者的预后，提高患者的生活质量。

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.