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

每年有近50%的癌症患者接受单独或联合化疗的放射治疗。在放射治疗的情况下，这是该项目的重点，缺氧（非常低的氧气水平）是辐射抵抗或治疗失败的重要原因。最近的证据表明，经历短暂缺氧的肿瘤也可能含有耐辐射的癌细胞。特别感兴趣的是位于肿瘤中接近氧扩散极限的癌细胞，该区域由于血管系统发育不良而经历氧可用性的波动。目前尚不清楚这些癌细胞对氧气输送波动的代谢适应（减少能量消耗的适应）是否有助于促进辐射抗性。该项目的研究目标是开发一个多模态成像平台，以研究微血管氧合与细胞代谢之间的关系，以及这种关系如何有助于促进治疗抵抗。了解这些与放射治疗反应的关系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机会，获得宝贵的研究经验，在设计和开发的光学成像技术和癌症生物学的基础科学研究。该提案的教育和推广目标是开发基于光和光学的全年学习包，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配备积极的教学模块，以更好地向学生传达光学和基于光的概念。首席研究员的长期职业目标是开发光学成像技术，可以可视化和回答有关肿瘤微环境的基本生物学问题，这将最佳地导致开发新的生物标志物，可以刺激成本的发展。有效的工具，以帮助促进公共卫生。 为了实现这一目标，该项目将建立和验证一个无标记的多模式成像平台（与高光谱暗场显微镜（TP-HDMI）集成的双光子显微镜），以定量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辐射抗性。 将使用高光谱显微镜根据血红蛋白的光吸收以及氧合（HbO 2）和脱氧（dHb）血红蛋白吸收曲线的差异评估氧合水平。 用于空间比较的氧合水平指标为[HbO 2]/（[HbO 2]+[dHb]）。双光子显微镜将用于评估细胞代谢的基础上，该系统的能力，图像自然荧光NADH和FAD，分子是至关重要的能源生产过程。 用于空间比较的细胞代谢度量将是光学氧化还原比FAD/（FAD + NADH）。该项目的变革性在于能够研究体内肿瘤氧合和细胞代谢之间时空关系的动态变化，以响应放射治疗，以及这些变化如何在放射抵抗性和敏感性肿瘤中不同。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耐辐射表型。研究计划是根据两个目标组织的。 第一个目标是建立多模式系统，并验证其能力，调查血管氧合和细胞代谢之间的时空关系。一旦组装，将在无胸腺裸鼠中建立的窗室模型内的正常组织中验证集成成像平台。 将使用图像分析算法分离血管和非血管组织。 当小鼠经受不同程度的缺氧时，将对氧气供应变化的动态代谢反应进行成像。 将确定作为与最近血管的距离的函数的光学氧化还原比。 第二个目的是研究放射抵抗和敏感肿瘤对放射治疗反应的肿瘤生物能量学的时空变化。 集成成像平台将用于研究植入小鼠窗口室中的已知辐射抗性和敏感性的人类头颈部肿瘤中细胞代谢响应于不同剂量的辐射治疗的动态时空变化。 所有成像相关终点将使用免疫组织化学测定法进行进一步验证。 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以及放射治疗后时间的函数。 所获得的结果预计将显着扩大我们的理解如何空间位置的细胞相对于氧气扩散限制影响其抵抗treatment.This奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

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.