Dual-modal Microscopy of Metabolic Reprogramming in Cancer

 描述(申请人提供)：代谢重编程，从氧化代谢到糖酵解代谢的转变，越来越被认为是癌症的核心标志。我们最近的体外研究表明，代谢重编程可能是致癌RAS诱导的线粒体功能障碍推动肿瘤发生和发展的潜在机制。检验这一创新和重要的假说将极有希望促进我们对癌症代谢信号的理解，并启发早期癌症检测和靶向治疗的新策略。然而，由于缺乏能够在体内微观水平上动态成像代谢变化的技术，这一研究一直受到阻碍。本研究旨在通过研制反射式双模显微镜平台来满足这一苛刻的要求。该平台以一种全新的方式集成了两种尖端技术-光声显微镜(PAM)和多光子荧光寿命成像显微镜(MPFLIM)-将能够同时成像两个金标准代谢指标：氧代谢率(MRO2)和光学氧化还原比。这两个指数的共同进化将定量和动态地描绘癌症发展过程中的代谢重新编程。为此，我们围绕三个具体目标组织了这个项目。首先，我们将通过集成高频超声换能器和反射显微镜物镜来开发一种光声物镜。这种新颖的设计将在反射模式下实现光激发(用于PAM和MPFLIM)和声学检测(仅用于PAM)的自然集成。然后，我们将在现有MPFLIM系统的基础上实现双模式显微镜平台。通过适当设计的双模式扫描和数据采集同时检测光吸收的血液血红蛋白和自身荧光代谢辅酶(即nadh和fad)，将首次能够在活体内进行高分辨率成像。 MRO2和光学氧化还原比在相同的时空尺度上。最终，在小鼠肿瘤模型中验证该平台的性能将为后续RAS诱导的线粒体功能障碍在代谢重编程和肿瘤发生中的作用的机制研究做好准备。成功完成这一发展拨款将导致代谢成像领域的技术突破，并使我们能够解决重要问题，包括线粒体分裂的遗传抑制是否可以逆转新陈代谢重新编程，以及哪些遗传因素对癌症的代谢表型起作用。这些研究不仅将使我们更清楚地了解癌症代谢信号，还可能为RAS驱动的恶性肿瘤的治疗确定新的治疗靶点。双模式显微镜的潜在影响可能非常广泛，因为代谢重新编程有助于癌症以外的许多疾病的发展，包括但不限于阿尔茨海默病、肥胖症和糖尿病。这种新型光学-声学物镜的强健性能和易于实施，将为在整个研究界传播这项有前途的技术铺平道路。

 DESCRIPTION (provided by applicant): Metabolic reprogramming, the shift from oxidative to glycolytic metabolism, has been increasingly considered as a core hallmark of cancer. Our recent in vitro study suggests that metabolic reprogramming may be the underlying mechanism through which oncogenic Ras-induced mitochondrial dysfunction drives tumor initiation and progression. Examining this innovative and important hypothesis holds great promise to advance our understanding of the metabolic signaling in cancer and inspire new strategies for early cancer detection and targeted therapy. However, it has been impeded by the lack of a technology capable of dynamically imaging the metabolic shift at the microscopic level in vivo. The proposed research aims to meet this stringent demand by developing a reflection-mode dual-modal microscopy platform. Integrating two cutting-edge techniques- photoacoustic microscopy (PAM) and multiphoton fluorescence lifetime imaging microscopy (MPFLIM)-in a radically new way, this platform will enable concurrent imaging of two gold-standard metabolic indices: the metabolic rate of oxygen (MRO2) and optical redox ratio. Co-evolution of the two indices will quantitatively and dynamically delineate metabolic reprogramming during cancer development. To this end, we have organized this project around three specific aims. First, we will develop an optical-acoustic objective by integrating a high- frequency ultrasonic transducer and a reflective microscope objective. This novel design will enable the natural integration of optical excitation (for both PAM and MPFLIM) and acoustic detection (for PAM only) in reflection mode. Then, we will implement the dual-modal microscopy platform based on our existing MPFLIM system. Concurrent detection of optically-absorbing blood hemoglobin and autofluorescent metabolic coenzymes (i.e., NADH and FAD) through properly designed dual-modal scan and data acquisition will enable, for the first time, in vivo high-resolution imaging of MRO2 and optical redox ratio at the same spatiotemporal scale. Ultimately, validating the performance of the platform in a mouse tumor model will prepare us for follow-up mechanistic studies of the role of Ras-induced mitochondrial dysfunction in metabolic reprogramming and tumorigenesis. Successful completion of this developmental grant will lead to a technical breakthrough in the field of metabolic imaging and allow us to tackle important questions, including whether genetic inhibition of mitochondrial fission can reverse metabolism reprogramming and which genetic factors contribute to the metabolic phenotypes in cancer. These studies not only will give us a clearer understanding of cancer metabolic signaling but also may identify new therapeutic targets for the treatment of Ras-driven malignancies. The potential impact of the dual- modal microscopy could be very broad, because metabolic reprogramming contributes to the development of numerous diseases besides cancers, including but not limited to Alzheimer disease, obesity, and diabetes. The robust performance and ease of implementation, enabled by the novel optical-acoustic objective, will pave the way for disseminating this promising technique throughout the research community.