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的主要空白。CTC作为乳腺癌患者预后指标的验证为CTC的测量开创了重要先例；然而，组织化学CTC计数现在被用作乳腺癌和前列腺癌的临床相关性，已被证明既不稳健也不够可靠，不能应用于肺癌。这在很大程度上是由于与其他实体肿瘤相比，与肺肿瘤相关的表型异质性和高抗原变异性。PCR扩增癌症蛋白转录本是CTC测量的一种概念上吸引人的替代方法，为多种抗原测量提供了灵敏度和无限能力。虽然文献表明PCR检测CTC与肺癌分期、疾病复发和生存相关，但这种方法仍处于研究阶段。由于mRNA转录物被用作肿瘤蛋白表达的替代品，PCR被批评缺乏特异性。我们设计了一种结合免疫组织化学特异性和PCR敏感性的方法。本应用程序详细测试和优化了一种新型纳米颗粒为基础的系统，以克服在肺癌CTC测量中遇到的技术和生物学障碍。具体来说，生物纳米偶联物被构建为携带一个抗原特异性单克隆抗体和一个偶联物特异性双链DNA标记序列，用于纳米特异性鉴定。当偶联物与细胞表面的抗原结合时，通过短暂加热复合混合物，可以将单链DNA标签释放到悬浮中；通过PCR扩增的释放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。抗原结合生物纳米偶联物的信号放大允许检测外周血中低浓度CTC上的低频蛋白，该技术固有的多路复用能力提供了更高的灵敏度而不降低特异性，使其特别适合于肺癌CTC的测量。该方法的敏感性将通过在不同浓度的外周血中加入已知抗原表达的培养肿瘤细胞来确定。利用抗体介导的从外周血单个核细胞中分离CTC来富集CTC，预计将进一步提高检测限，同时也减少假阳性结果的可能性。我们期望能够达到无与伦比的检测限和肺癌CTC的高度特异性。此应用程序详细说明了这个有希望的计划的初始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.