PHOTOACOUSTIC MICROSCOPY OF NEOVASCULARIZATION IN RENAL CANCER GROWTH AND THERAPY

抽象的。靶向肿瘤新血管形成的承诺仍然没有实现。因此，新的见解 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 新血管形成是必要的。我们开发了一种新的非侵入性成像技术，光声 显微镜（PAM）。PAM使用血红蛋白（Hb）的激光激发来确定新生血管结构，Hb 浓度（红细胞压积）、氧饱和度（SO2）和毛细血管水平下每个肿瘤微血管中的流量 分辨率没有外来的对比度或组织窗口结构。这些数据使 肿瘤耗氧代谢率（MRO 2）的微区测定。我们将联合收割机PAM 通过生物学、药理学和遗传学操作来检验肿瘤新生血管 结构和功能调节肿瘤中或肿瘤中的VEGF和PI 3 K信号传导，并受其调节。 内皮细胞我们将研究肾癌，因为它是由于过度缺氧而导致的血管过多- 诱导因子（HIF）-2和-1上调VEGF和其它血管生成因子。我们将使用人类786-O (VHL和PTEN阴性）异种移植物中以询问相同的血管网络 在所有肿瘤中由相同的动静脉对供血。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记光声显微镜，纵向成像血管交叉， 切片、红细胞压积、SO2、血流量和MRO 2。目前，我们使用两台PAM仪器进行成像 分别为血细胞比容（CHb）/SO2和血管横截面/逐血管流动。两个系统量化 MRO 2由于重新定位和重复性而易于出错。2.0.确定新生血管功能， 786-O肾癌异种移植物生长期间的肿瘤代谢和细胞生物学。我们将使用纵向 PAM成像，以阐明微血管功能，肿瘤MRO 2，肿瘤和内皮细胞增殖，存活， 血管生成和PI 3 K信号通路在肿瘤生长期间交错。3.0.抑制VEGF信号传导， 确定新血管和肾癌细胞的功能反应。我们将使用反- VEGF抗体，靶向人和小鼠VEGF，并测试每个PAM参数的标准化， 内皮细胞和肿瘤细胞增殖和存活的减少，以及逃避性血管生成信号传导 上调。4.1.药理学确定mTORC 1或mTORC 1和-2在肾脏中的功能 癌细胞和肿瘤相关内皮细胞。我们将使用雷帕霉素（依维莫司）或双 mTORC 1/2抑制剂（PP 242）和用于差异新生血管功能和癌细胞的机制的测试 生物敏感性。4.2.确定肾癌内皮细胞中TORC 2的功能。我们将 有条件地删除成人受体内皮中的mTORC 2组分Rictor，测试 正常化的新生血管功能，MRO 2和肿瘤细胞存活和增殖信号。的影响 这项拟议的研究将提高肾癌和其他实体恶性肿瘤患者的生存率。

Abstract. 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.