PHOTOACOUSTIC MICROSCOPY OF NEOVASCULARIZATION IN RENAL CANCER GROWTH AND THERAPY

描述（由申请人提供）：靶向肿瘤新生血管的承诺尚未实现。因此，有必要对微血管功能如何影响肿瘤生物学以及肿瘤或内皮信号通路如何调节新生血管形成有新的认识。我们开发了一种新的无创成像技术，光声显微镜（PAM）。PAM利用激光激发血红蛋白（Hb），在毛细血管水平分辨率下测定每个肿瘤微血管的新生血管结构、血红蛋白浓度（红细胞压积）、氧饱和度（SO2）和血流，无需外源性造影剂或组织窗口构建。这些数据独特地实现了肿瘤耗氧量代谢率（MRO2）的微区域测定。我们将结合PAM与生物学、药理学和遗传学操作来验证肿瘤新生血管结构和功能调节的假设，并受肿瘤或内皮细胞中VEGF和PI3K信号的调节。我们将研究肾癌，因为它是由于缺氧诱导因子(HIF)-2和-1的过度表达而导致的血管增生，这些因子上调VEGF和其他血管生成因子。我们将在免疫缺陷小鼠中使用人类786-O （VHL和PTEN阴性）异种移植物来询问所有肿瘤中由相同动静脉对提供的相同血管网络。我们将用这些具体目标来检验我们的假设：1.0。开发一种集成的无标签光声显微镜，纵向成像血管横截面，红细胞压积，SO2，血流和MRO2。目前，我们使用两种PAM仪器分别成像血细胞比容(CHb)/SO2和血管横截面/血管流量。两种量化MRO2的系统由于重定位和异步性容易产生误差。2.0. 确定786-O型肾癌异种移植生长期间的新生血管功能、肿瘤代谢和细胞生物学。我们将使用纵向PAM成像来阐明微血管功能、肿瘤MRO2、肿瘤和内皮细胞增殖、生存、血管生成和PI3K信号通路在肿瘤生长过程中是如何交错的。3.0. 抑制VEGF信号传导并决定新生血管和肾癌细胞的功能反应。我们将使用一种针对人类和小鼠VEGF的抗VEGF抗体，并测试每个PAM参数的正常化，内皮细胞和肿瘤细胞增殖和存活的减少，以及逃避血管生成信号的上调。4.1. 药理学测定mTORC1或mTORC1和-2在肾癌细胞和肿瘤相关内皮中的功能。我们将使用拉帕罗格（依维莫司）或双重mTORC1/2抑制剂（PP242），并测试差异新生血管功能和癌细胞生物学敏感性的机制。4.2. 检测TORC2在肾癌内皮细胞中的功能。我们将有条件地删除成人受体内皮中必要的mTORC2成分Rictor，以检测新血管功能、MRO2和肿瘤细胞存活和增殖信号的正常化。这项研究的影响将是提高肾癌和其他实体恶性肿瘤患者的生存率。

DESCRIPTION (provided by applicant): The promise of targeting tumor neovascularization remains unrealized. Therefore new insights into how microvessel function impacts tumor biology and how tumor or endothelial signaling pathways regulate neovascularization are necessary. We developed a novel noninvasive imaging technique, photoacoustic microscopy (PAM). PAM uses laser excitation of hemoglobin (Hb) to determine neovascular architecture, Hb concentration (hematocrit), oxygen saturation (SO2), and flow in each tumor microvessel at capillary level resolution without exogenous contrast or tisue window construction. These data uniquely enable microregional determination of tumor metabolic rate of oxygen consumption (MRO2). We will combine PAM with biological, pharmacological, and genetic manipulations to test the hypothesis that tumor neovascular architecture and function regulate, and are regulated by, VEGF and PI3K signaling in tumor or in endothelial cells. We will study renal cancer because it is hypervascular due to overexpression of hypoxia- inducible factors (HIF)-2 and -1 that upregulate VEGF and other angiogenic factors. We will use human 786-O (VHL and PTEN negative) xenografts in immunodeficient mice to interrogate the same vascular network supplied by the same arteriovenous pair in all tumors. We will test our hypothesis with these Specific Aims: 1.0. Develop an integrated label-free photoacoustic microscope that longitudinally images vessel cross- section, hematocrit, SO2, blood flow, and MRO2. Currently we use two PAM instruments to image separately hematocrit (CHb)/SO2 and vessel cross section/flow vessel-by-vessel. Two systems quantifying MRO2 are prone to eror due to repositioning and asynchronicity. 2.0. Determine neovascular function, tumor metabolism, and cell biology during 786-O renal cancer xenograft growth. We will use longitudinal PAM imaging to elucidate how microvessel function, tumor MRO2, tumor and endothelial proliferative, survival, angiogenic, and PI3K signaling pathways are interlaced during tumor growth. 3.0. Inhibit VEGF signaling and determine the functional response of the neovasculature and renal cancer cells. We will use an anti- VEGF antibody, targeting human and mouse VEGF, and test for normalization of each PAM parameter, diminutions in endothelial and tumor cell proliferation and survival, and evasive angiogenic signaling upregulation. 4.1. Pharmacologically determine mTORC1 or both mTORC1 and -2 function in renal carcinoma cels and tumor-associated endothelium. We will use a rapalog (everolimus) or a dual mTORC1/2 inhibitor (PP242) and test for mechanisms of differential neovascular functional and cancer cell biological sensitivity. 4.2. Determine TORC2 function in the endothelial cells of renal carcinomas. We will conditionally delete the necesary mTORC2 component, Rictor, in adult recipient endothelium, testing for normalization of neovascular function, MRO2 and tumor cell survival and proliferative signaling. The impact of this proposed study will be to improve survival of patients with renal cancer and other solid malignancies. PUBLIC HEALTH RELEVANCE: The blood supply to tumors is important for their growth and spread throughout the body. The vasculature is an emerging target for anti-tumor therapy. The function of tumor vessels and their interrelationship to tumor molecular signaling pathways regulating these vessels are incompletely understood. This project will use a novel technique to determine tumor neovessel function and tumor cell signaling during progressive growth and under the stress of vascular- or cancer cell targeted therapies. This project will change our understanding of how tumors grow and become therapy resistant.