CAREER: A multimodal imaging platform to investigate spatiotemporal changes in tumor bioenergetics that drive treatment resistance.

每年有近50%的癌症患者接受放射治疗，要么单独治疗，要么与化疗联合治疗。就本项目的重点放射治疗而言，缺氧（非常低的氧气水平）是导致放射抵抗或治疗失败的重要原因。最近的证据表明，经历短暂缺氧发作的肿瘤也可能含有抗辐射的癌细胞。我们特别感兴趣的是位于肿瘤中氧气扩散极限附近的癌细胞，这一区域由于脉管系统发育不良而经历氧气可用性波动。目前尚不清楚这些癌细胞对氧输送波动的代谢适应（减少能量消耗的适应）是否有助于提高抗辐射能力。本项目的研究目标是建立一个多模态成像平台，研究微血管氧合与细胞代谢之间的关系，以及这种关系如何促进治疗抵抗。对放射治疗反应的这些关系的了解可以导致靶向治疗的发展，以逆转耐药性。这些研究将为学生提供在光学成像技术的设计和开发以及癌症生物学基础科学研究方面获得宝贵的研究经验的机会。该提案的教育和推广目标是开发基于光和光学的一年学习工具包，这是中学课程的一部分。这些工具包的目的是为农村中学教师提供积极的教学模块，以便更好地向学生传达光学和光的概念。首席研究员的长期职业目标是开发光学成像技术，可以可视化并回答关于肿瘤微环境的基本生物学问题，这将最佳地导致新的生物标志物的开发，从而刺激成本效益工具的开发，以帮助促进公共卫生。为了实现这一目标，本项目将建立并验证一个无标签的多模态成像平台（双光子显微镜与高光谱暗场显微镜（TP-HDMI）集成），以定量地可视化微血管氧合和细胞代谢之间的关系，并确定这两个标志之间的关系如何促进抗辐射。高光谱显微镜将用于根据血红蛋白的光吸收以及含氧（HbO2）和脱氧（dHb）血红蛋白吸收谱的差异来评估氧合水平。用于空间比较的氧合水平度量为[HbO2]/([HbO2]+[dHb])。双光子显微镜将用于评估基于该系统成像天然荧光NADH和FAD的细胞代谢能力，这些分子对能量产生过程至关重要。用于空间比较的细胞代谢指标将是光学氧化还原比FAD/(FAD + NADH)。该项目的变革性质在于能够研究体内肿瘤氧合和细胞代谢在放射治疗下的时空关系的动态变化，以及这些变化在耐辐射和敏感肿瘤中的区别。这项工作的中心假设是，在这些间歇性缺氧条件下的代谢重编程可以导致细胞产生抗辐射表型。研究计划有两个目标。第一个目标是建立多模态系统，并验证其研究血管氧合和细胞代谢之间时空关系的能力。一旦组装完成，集成成像平台将在胸腺裸鼠建立的窗室模型的正常组织中进行验证。图像分析算法将用于分离血管和非血管组织。当小鼠受到不同程度的缺氧时，将对氧气供应变化的动态代谢反应进行成像。光学氧化还原比作为离最近血管距离的函数将被确定。第二个目的是研究放射耐药和敏感肿瘤对放射治疗的时空变化。该综合成像平台将用于研究植入小鼠窗室的已知辐射抗性和敏感性的人类头颈部肿瘤在不同剂量放射治疗下细胞代谢的动态时空变化。所有成像相关终点将使用免疫组织化学分析进一步验证。这些研究将建立癌细胞内代谢变化的模型，作为微血管氧合、与最近微血管的距离和放射治疗后时间的函数。所获得的结果有望显著扩展我们对细胞空间位置相对于氧扩散极限如何影响其对治疗的抵抗力的理解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Nearly 50% of all cancer patients each year are treated with radiation therapy, either alone or in combination with chemotherapy. In the case of radiation therapy, which is the focus of this project, hypoxia (very low levels of oxygen) is an important cause of radiation resistance or treatment failure. Recent evidence suggests that tumors experiencing brief episodes of hypoxia can also harbor radiation-resistant cancer cells. Of specific interest are cancer cells that are situated close to the oxygen diffusion limit in tumors, a region that experiences fluctuations in oxygen availability due to poorly developed vasculature. It is not known if metabolic adaptations (adaptations to decrease energy expenditure) of these cancer cells to fluctuations in oxygen delivery can help promote radiation resistance. The research goal of this project is to develop a multimodal imaging platform to investigate the relationship between microvascular oxygenation and cellular metabolism, and how this relationship helps promote treatment resistance. Knowledge of these relationships in response to radiation therapy can lead to the development of targeted therapeutics to reverse resistance. These studies will provide opportunities for students to gain valuable research experience in design and development of optical imaging technologies and basic science investigations of cancer biology. The educational and outreach goal of this proposal is to develop year-long learning kits based on light and optics, which is part of the middle school curriculum. The purpose of these kits is to equip rural middle school teachers with active teaching modules to better communicate optics and light-based concepts to students.The principal investigator's long-term career goal is to develop optical imaging technologies that can visualize and answer basic biological questions about the tumor micro-environment that would optimally lead to the development of new biomarkers that can spur the development of cost-effective tools to help advance public health. Towards this goal, this project will build and validate a label-free multimodal imaging platform (a two-photon microscope integrated with a hyperspectral darkfield microscope (TP-HDMI)) to quantitatively visualize the relationship between microvascular oxygenation and cellular metabolism and determine how the relationship between these two hallmarks promotes radiation resistance. Hyperspectral microscopy will be used to assess oxygenation levels based on light absorption by hemoglobin and the differences in absorption profiles of oxygenated (HbO2) and deoxygenated (dHb) hemoglobin. The oxygenation level metric for spatial comparison will be [HbO2]/([HbO2]+[dHb]). Two-photon microscopy will be used to assess cellular metabolism based on the system's ability to image natural fluorescing NADH and FAD, molecules that are critical for the energy producing processes. The cellular metabolism metric for spatial comparison will be the optical redox ratio FAD/(FAD + NADH). The transformative nature of the project lies in the ability to investigate dynamic changes in the spatiotemporal relationship between tumor oxygenation and cellular metabolism in vivo in response to radiation therapy, and how these changes might be distinct in radiation-resistant and sensitive tumors. The central hypothesis of this work is that metabolic reprogramming under these intermittently hypoxic conditions can cause cells to develop a radiation-resistant phenotype. The Research Plan is organized under two objectives. The FIRST OBJECTIVE is to build the multimodal system and to validate its ability to investigate spatiotemporal relationships between vascular oxygenation and cellular metabolism. Once assembled, the integrated imaging platform will be validated in normal tissue within a window chamber model established in athymic nude mice. Image analysis algorithms will be used to separate vascular and non-vascular tissue. Dynamic metabolic responses to changes in oxygen supply will be imaged while the mice are subjected to varying levels of hypoxia. The optical redox ratio as a function of distance from the nearest blood vessel will be determined. The SECOND OBJECTIVE is to investigate the spatiotemporal changes in tumor bioenergetics in radiation-resistant and sensitive tumors in response to radiation therapy. The integrated imaging platform will be used to investigate dynamic spatiotemporal changes in cellular metabolism in response to different doses of radiation therapy in human head and neck tumors of known radiation resistance and sensitivity implanted in the mouse window chambers. All of the imaging-related endpoints will be further validated using immunohistochemical assays. The studies will establish a model for metabolic changes within cancer cells as a function of microvascular oxygenation, distance from the nearest microvessel, and time after radiation therapy. Results obtained are expected to significantly expand our understanding of how the spatial location of cells with respect to the oxygen diffusion limit influences their resistance to treatment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.