Transport of Large Molecules and Viral Vectors in Tumors: Effects of Cytotoxic an

描述（申请人提供）：溶瘤病毒疗法是一种很有前途的方法，因为有效的转导和癌细胞特异性病毒复制可以提高治疗效果。但是，溶瘤病毒的大尺寸阻碍了它们的跨血管和间质运动，从而显著限制了它们向癌细胞的递送和抗肿瘤功效。我们在当前资助期的研究结果表明，癌细胞凋亡产生空隙和通道样结构，这增强了肿瘤内注射后溶瘤单纯疱疹病毒（HSV）的初始递送、病毒传播和治疗效果。但是为了提高靶向原发性和转移性肿瘤的溶瘤病毒疗法的全身功效，鉴定药剂（例如，细胞毒性剂）或将改善静脉内注射的病毒颗粒的血管外渗和肿瘤内分布的策略。在所提出的研究中，我们将测试靶向肿瘤血管将改善病毒载体的递送和溶瘤HSV的抗肿瘤功效的假设。肿瘤血管系统将用内皮细胞靶向剂（抗整联蛋白14或15阻断抗体）或靶向内皮细胞和癌细胞的细胞毒素（环磷酰胺、多西他赛）靶向。已知所选择的药剂可诱导内皮细胞凋亡并重塑肿瘤血管。我们将使用多光子激光扫描显微镜来测量大颗粒的血管孔径截止尺寸、血管重塑（例如血管密度、直径、灌注）以及植入免疫缺陷小鼠乳腺脂肪垫窗口的人乳腺癌细胞中的病毒外渗和扩散。由于病毒在血液中的半衰期较短（HSV ~ 3小时），我们将确定每种药物的大颗粒外渗峰值时间。根据颗粒外渗的峰值时间，将全身注射溶瘤HSV，并确定与感染细胞相关的血管分数。将在肿瘤切片中评估内皮细胞凋亡和颗粒外渗之间的相关性。我们将测试在静脉注射溶瘤病毒之前给予内皮靶向剂或细胞毒素是否会在乳腺脂肪垫肿瘤中产生更长的肿瘤生长延迟并减少肺中的自发转移形成。我们还将确定细胞毒素或内皮靶向剂与溶瘤HSV的重复给药（循环）是否会改善大颗粒的瘤内分布以及溶瘤HSV的扩散和治疗效果。我们希望这些转化研究的结果将确定单一药物和/或方法，这将增强血管外渗和肿瘤内传播的病毒，并针对肿瘤的溶瘤病毒疗法的疗效。公共卫生相关性：溶瘤病毒疗法的临床疗效受到病毒颗粒大尺寸的限制，这阻止了它们穿过肿瘤血管的内皮壁和癌细胞之间的运动。我们的研究结果表明，诱导癌细胞凋亡提高了肿瘤渗透和肿瘤内注射溶瘤病毒的治疗效果。在拟定的研究中，我们将确定用临床可用的细胞毒素或内皮靶向剂靶向肿瘤血管是否会改善静脉注射溶瘤病毒的瘤内分散和疗效。

DESCRIPTION (provided by applicant): Oncolytic virotherapy is a promising approach because the efficient transduction and cancer cell-specific viral replication can boost therapeutic efficacy. However, the large size of oncolytic viruses hinders their transvascular and interstitial movement, thus significantly limiting their delivery to cancer cells and anti- tumor efficacy. Our findings from the current funding period showed that cancer cell apoptosis produced void spaces and channel-like structures, which enhanced the initial delivery, viral spread and therapeutic efficacy of oncolytic herpes-simplex-virus (HSV) after intratumor injection. But in order to improve the systemic efficacy of oncolytic virotherapy targeted both at primary and metastatic tumors, it is essential to identify agents (e.g., cytotoxic agents) or strategies that will improve the vascular extravasation and intratumoral distribution of viral particles injected intravenously. In the proposed studies we will test the hypothesis that targeting tumor blood vessels will improve the delivery of viral vectors and the anti-tumor efficacy of oncolytic HSV. The tumor vasculature will be targeted with endothelial cell targeting agents (anti-integrin 14 or 15 blocking antibodies) or cytotoxics (cyclophosphamide, docetaxel) that target both endothelial and cancer cells. The agents selected are known to induce endothelial cell apoptosis and remodel the tumor vasculature. We will use multiphoton laser scanning microscopy to measure the vascular pore cutoff size of large particles, vascular remodeling (e.g. vessel density, diameter, perfusion), and virus extravasation and spread in human mammary carcinoma cells implanted in mammary fat pad windows in immunodeficient mice. Because of the short-half life of virus in blood (HSV ~ 3 hours), we will determine for each agent the peak-time of large particle extravasation. Based on the peak-time of particle extravasation, oncolytic HSV will be injected systemically and the fraction of vessels associated with infected cells will be determined. The correlation between endothelial apoptosis and particle extravasation will be assessed in tumor sections. We will test if the administration of endothelial targeting agents or cytotoxics before the intravenous injection of oncolytic virus will produce a longer tumor growth delay in mammary fat pad tumors and reduce spontaneous metastasis formation in the lung. We will also determine if the repeated administration (cycling) of cytotoxics or endothelial targeting agents with oncolytic HSV will improve the intratumoral distribution of large particles and the spread and therapeutic efficacy of oncolytic HSV. We expect that the results of these translational studies will identify single agents and / or approaches that will enhance the vascular extravasation and intratumoral spread of virus, and the efficacy of oncolytic virotherapy targeted at tumors. PUBLIC HEALTH RELEVANCE: The clinical efficacy of oncolytic virotherapy is limited by the large size of virus particles, which prevents their movement across the endothelial wall of tumor vessels and between cancer cells. Our results demonstrate that the induction of cancer cell apoptosis improves the tumor penetration and therapeutic efficacy of oncolytic virus injected intratumorally. In the proposed studies we will determine if the targeting of tumor blood vessels with clinically available cytotoxics or endothelial targeting agents will improve the intratumoral dispersion and efficacy of oncolytic virus injected intravenously.