Ading color to cancer, adenovirus and flow cytometry to identify and capture CTCs

描述(由申请人提供)：我们提出了一种利用工程化腺病毒和复杂的流式细胞术来识别和捕获循环肿瘤细胞(CTCs)的新技术。目前检测CTCs的技术包括逆转录聚合酶链式反应(RT-PCR)、流式细胞术、荧光原位杂交，以及最近的微流控技术。不幸的是，RT-PCR不能区分存活的转移性CTC和源自原发肿瘤的核酸或细胞碎片。好了！基于抗体的技术不能用于检测所有癌症，但只能检测那些表达最常见和特征最好的标志物的癌症。因此，迫切需要开发新的诊断试剂和工具，不仅要检测和捕获CTC，而且要量化它们的恶性潜力，并预先确定在切除单个患者的肿瘤方面最有效的治疗方法。尽管癌症在基因组水平上的复杂性和多变性，但一个统一的主题是它们的生长去调控表型，即所谓的癌症特征，它是由相对较少的关键途径的突变所赋予的。与其专注于检测肿瘤之间众多且高度可变的单个遗传损伤，我们建议创建诊断病毒，在其基因组中整合多个转录和分子模块，以感染和检测患者的肿瘤，报告其分子“特征”及其对不同治疗方法的反应。使用这些试剂，任何特定肿瘤的分子损伤和恶性特征都将被快速识别(在24小时内)，并通过标准化的自动化平台进行评分。此外，这些试剂还可以用作记者，快速而直接地确定患者的肿瘤是否可能对特定的治疗产生反应。我们的目标是开发一个标准化的自动化平台，提供护理点诊断，以达到任何其他检测系统、生物标记物或相关基因表达签名无法实现的分子复杂性和预后能力水平上的临床决策。为了实现这一目标，我们将结合在Salk、UCSD和NanoSort开发的变革性新技术平台，这些平台基于肿瘤细胞获取的指示恶性进展和治疗反应的分子病变以不同的颜色标记肿瘤细胞，促进使用集成的“芯片实验室”流式细胞仪对其进行检测、量化和分离。 公共卫生相关性：为了帮助诊断和治疗癌症，我们将特别设计腺病毒，这种病毒会优先感染循环中的肿瘤细胞(CTCs，即离开肿瘤并进入血液的细胞)，将这些罕见的细胞与常规血样中发现的数十亿个血细胞明显区分开来。这项技术还可以检测癌症的状态，为医生提供关于癌症的侵袭性或可治疗性的信息。我们将这种腺病毒系统与一种先进的新型芯片实验室细胞分选设备相结合，该设备将病毒感染的细胞沿着微流体通道传递，以从血液中进行测量和分类，并检查癌症的特征特征。

DESCRIPTION (provided by applicant): We propose a novel technique to identify and capture circulating tumor cells (CTCs) using engineered adenoviruses and sophisticated flow cytometry. Current techniques for detection of CTCs include reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, fluorescence in situ hybridization, and, more recently, microfluidics. Unfortunately, RT-PCR does not distinguish between viable metastatic CTC versus nucleic acids or cellular fragments originating from the primary tumor. ! Antibody-based techniques cannot be used for detection of all cancers, but only those cancers that express the most common and well- characterized markers. As such, there is a desperate need to develop new diagnostic agents and tools that not only detect and capture CTCs but also quantify their malignant potential and identify 'up-front' the therapies that are most effective in ablating an individual patient's tumor. Despite the complexity and variability of cancers at a genome scale, a unifying theme is their growth deregulation phenotypes, the so-called hallmarks of cancer, which are conferred by mutations in a relatively small number of key pathways. Rather than focus on detecting individual genetic lesions that are numerous and highly variable between tumors, we propose to create diagnostic viruses that incorporate multiple transcriptional and molecular modules in their genomes to infect and detect a patient's tumor, report its molecular 'hallmarks' and its response to different therapies 'up- front'. Using these agents, the molecular lesions and malignant characteristics of any given tumor will be rapidly discerned (within 24 hours) and scored via a standardized automated platform. Furthermore, these agents could also be used as reporters to determine rapidly and directly if a patient's tumor is likely to respond to a particular therapy. Our goal is to develop a standardized automated platform that provides point-of-care diagnostics to inform clinical decisions at a level of molecular sophistication and prognostic power that is not possible with any other detection system, biomarkers or correlative gene expression signatures. To achieve this, we will combine transformative new technological platforms developed at the Salk, UCSD, and NanoSort that label tumor cells in different colors based on their acquisition of molecular lesions that dictate malignant progression and response to therapy, facilitating their detection, quantification and isolation using an integrated 'lab-on a chip' flow cytometer. PUBLIC HEALTH RELEVANCE: To aid in the diagnosis and treatment of cancer, we will specially design adenoviruses that preferentially infect circulating tumor cells (CTCs, cells that have left a tumor and entered the bloodstream), drastically distinguishing these rare cells from the billions of blood cells found in a routine blood sample. This technology also detects the status of the cancer, giving doctors information about how aggressive or treatable a cancer might be. We combine this adenoviral system with a sophisticated new lab-on-a-chip cell-sorting device that passes the virally infected cells along microfluidic channels to be measured and sorted from the blood, and examined for characteristic hallmarks of cancer.