Bionanoconjugates for Detection of Circulating Tumor Cells in Lung Cancer

描述(由申请人提供)：循环肿瘤细胞(CTC)的测量有可能填补肺癌患者临床护理和管理方面的主要空白。CTC作为乳腺癌患者预后标志物的确认开创了CTC测量的重要先例；然而，组织化学CTC计数现在用于乳腺癌和前列腺癌的临床相关性已被证明不够可靠，不足以应用于肺癌。这在很大程度上是由于与其他实体肿瘤相比，肺肿瘤的表型异质性和较高的抗原变异性。肿瘤蛋白转录本的PCR扩增是一种概念上有吸引力的替代CTC测量方法，它为多种抗原的测量提供了灵敏度和无限的能力。虽然文献表明，CTC的聚合酶链式反应检测与肺癌分期、疾病复发和生存率有关，但该方法仍处于研究阶段。由于信使核糖核酸转录物被用来替代肿瘤蛋白的表达，聚合酶链式反应因缺乏特异性而受到批评。我们设计了一种结合了免疫组织化学的特异性和聚合酶链式反应的敏感性的方法。这项申请详细介绍了一种新的基于纳米颗粒的系统的测试和优化，以克服肺癌CTC测量中遇到的技术和生物学障碍。具体地说，构建的生物纳米结合物携带一个抗原特异性单抗和一个结合物特异性双链DNA标签序列，用于纳米特异性鉴定。当偶联物与细胞表面的抗原结合时，通过短暂加热复杂的混合物，可以将单链DNA标签释放到悬浮液中；通过聚合酶链式反应扩增释放的DNA链，提供了高度敏感和可量化的细胞结合和相应抗原表达的测量方法。抗原结合的生物纳米结合物的信号放大允许检测外周血液中低浓度CTC的低频蛋白质，该技术固有的多路复用功能提供了更高的灵敏度而不会降低特异性，使其特别适合于肺癌CTC的测量。该方法的敏感性将通过外周血以不同浓度添加具有已知抗原表达的培养的肿瘤细胞来确定。通过抗体介导从外周血单核细胞中分离CTC进行CTC浓缩，有望进一步提高检测限，同时也降低了假阳性结果的可能性。我们期望能够达到无与伦比的检测极限，对肺癌CTC具有高度的特异性。本申请详细说明了这一前景看好的倡议的初步开发和测试的比较和系统阶段。

DESCRIPTION (provided by applicant): Measurement of circulating tumor cells (CTC) has potential to fill major gaps in the clinical care and management of lung cancer patients. Validation of CTC as a prognostic marker in breast cancer patients set an important precedent for CTC measurement; however, histochemical CTC enumeration now used as a clinical correlate in breast and prostate cancer has proven to be neither robust nor reliable enough for application to lung cancer. This is due in large part to the phenotypic heterogeneity and high antigen variability associated with lung tumors as compared to other solid tumors. PCR amplification of cancer protein transcripts is a conceptually appealing alternative approach to CTC measurement that offers sensitivity and unlimited capabilities for multiple antigen measurements. Although literature indicates that PCR detection of CTC correlates with lung cancer stage, disease recurrence and survival, this approach remains investigational. Since mRNA transcript is used as a surrogate for tumor protein expression, PCR is criticized for lack of specificity. We have devised an approach that combines the specificity of immuno-histochemistry with the sensitivity of PCR. This application details testing and optimization of a novel nanoparticle-based system to overcome technical and biological hurdles encountered in lung cancer CTC measurement. Specifically, bio-nanoconjugates are constructed to carry one antigen specific monoclonal antibody and a conjugate-specific double stranded DNA tag sequence for nano-specific identification. When conjugates bind antigen on a cell surface, a single strand of the DNA tag can be released into suspension by briefly heating the complex mixture; liberated DNA strands amplified by PCR provide a highly sensitive and quantifiable measure of cellular binding and corresponding antigen expression. Signal amplification of the antigen-bound bio-nanoconjugate allows detection of low frequency proteins on low concentration CTC in peripheral blood, and multiplexing capabilities inherent to the technology offer increasing sensitivity without reduced specificity, making this particularly well suited for the measurement of lung cancer CTC. Sensitivity of the approach will be determined with peripheral blood spiked at various concentrations with cultured tumor cells with known antigen expression. CTC enrichment using antibody-mediated separation of CTC from peripheral blood mononuclear cells is expected to further increase the limits of detection, while also reducing the potential for false positive results. We anticipate being able to achieve unrivaled detection limits with high degrees of specificity for lung cancer CTC. This application details the comparative and systematic phases of initial development and testing of this promising initiative.