Microfluidic sorting of lung cancer cells from leukapheresis product as an alternative to metastatic tumor biopsy

随着精确肿瘤学的到来，包括靶向特定基因驱动因素的小分子抑制剂 癌症和对癌症新抗原的免疫反应的调节器，迫切需要先进的诊断方法来 指导和监测治疗干预措施。目前，这种实时监控是通过重复进行的，或者 耐药，活组织检查；然而，这些手术活检是侵入性的，可能会有严重的并发症，包括 没有足够的组织进行预期的分析，并且只采样了一个特定的肿瘤部位，这可能不具有代表性 患者体内的所有肿瘤细胞群。液体活检有望为癌症治疗带来革命性的变化， 实现对肿瘤衍生材料的频繁基于血液的监测，因为癌症因治疗而演变 干预措施。从常规血液样本中分离出足够数量的稀有CTC的技术障碍是 是阻碍临床部署基于CTC的诊断机会的唯一主要限制。启动者 这里提出的技术，我们从处理常规的10毫升血管(占全血容量的0.2%)转移到 利用临床白细胞分离来采集接近全血体积(40%-100%)的样本可解决这一挑战。这个 这项技术的基本基础是高效地去除抗体标记的血细胞 未经处理的CTCs(与“阳性选择”相反的“阴性耗尽”)，从而使肿瘤抗原- 独立浓缩不受干扰的、可行的四氯化碳。这一策略适用于从几乎所有 实体肿瘤类型，而不对肿瘤细胞的物理或生物学特性做出任何先验假设。我们的 假设使用白细胞分离法对大量CTC进行液体活检与侵入性活检是相同的。 转移性肿瘤病变，目前是在肺癌疾病进展的同时进行的。 为了解决我们的假设，我们制定了三个目标。在目标1中，我们将开发一种大容量CTC隔离技术 基于微流控阴性选择。在目标2中，我们将把目标1中设计的微流控部件集成到 用于从白斑中分选CTC的单片芯片。在目标3中，我们将通过直接比较 肿瘤活检和液体活检，彼此在几周内进行，评估它们的成功率和 诊断准确率。我们相信，我们团队现有的资源和多学科专业知识的融合 将带来变革性的生物工程技术，伴随着高度临床相关的临床挑战， 为临床前肺癌治疗提供了新的工具。这项初步研究的积极结果将为 测试更复杂的临床应用，范围从测量药物治疗的癌细胞信号效应 (“非侵入性药代动力学”)和用于癌症免疫治疗的细胞表面蛋白靶点的定量(蛋白质- 基于预测标志物)，并且最终甚至能够检测和组织定位高危早期浸润性癌症 个人，如家族性乳腺癌、有环境风险的人(例如吸烟者的肺结节、#年的肝癌 肝硬变)、不明意义的异常X线表现或无法取材活检。

With the advent of precision oncology, including both small molecule inhibitors targeting specific genetic drivers of cancer and modulators of the immune response to cancer neoantigens, there is a pressing need for advanced diagnostics to direct and monitor therapeutic interventions. Currently, such real time monitoring is performed through repeat, or resistance, biopsies; however, these surgical biopsies are invasive, may have significant complications including insufficient tissue for the intended analyses, and only sample one specific site of tumor, which may not be representative of the entire tumor cell population within a patient. Liquid biopsies are poised to revolutionize cancer therapies, by enabling frequent blood-based monitoring of tumor-derived materials, as cancers evolve in response to therapeutic interventions. The technological hurdle in isolating sufficient numbers of rare CTCs from routine blood specimens has been the single major limitation preventing the clinical deployment of CTC-based diagnostic opportunities. Enabled by the technology proposed here, our shift from processing routine 10 mL blood tubes (0.2% of whole blood volume) to making use of clinical leukapheresis to sample near-whole blood volumes (40-100%) addresses this challenge. The fundamental basis of the technology is the highly efficient depletion of antibody-tagged blood cells away from unmanipulated CTCs (“negative depletion” as opposed to “positive selection”), thereby enabling tumor antigen– independent enrichment of unperturbed, viable CTCs. This strategy is applicable to cells disseminated from virtually any solid tumor type without making any a priori assumption on the physical or biological properties of tumor cells. Our hypothesis is that liquid biopsy of large number of CTCs using leukopheresis is equivalent to the invasive biopsies of metastatic tumor lesions, currently performed at the time of on-treatment disease progression in lung cancer. To address our hypothesis, we formulated 3 Aims. In Aim 1, we will develop a large-volume CTC isolation technology based on microfluidic negative selection. In Aim 2, we will integrate microfluidic components designed in Aim 1 into a monolithic chip for sorting of CTCs from leukopaks. In Aim 3, we will test our hypothesis by a direct comparison of tumor biopsy and liquid biopsy, performed within a few weeks of each other, assessing both their success rate and diagnostic accuracy. We believe that the convergence of resources and multidisciplinary expertise available in our team will lead to a transformative bioengineering technology, paired with a highly clinically-relevant clinical challenge, providing a new tool for preclinical lung cancer therapeutics. A positive outcome in this pilot study would set the stage for testing more complex clinical applications, ranging from measuring cancer cell signaling effects of drug therapy (“noninvasive pharmacokinetics”) and quantitation of cell surface protein targets for cancer immunotherapy (protein- based predictive markers), and ultimately even enable detection and tissue localization of early invasive cancers in at-risk individuals such as familial breast cancer, people with environmental risks (e.g., lung nodules in smokers, liver cancer in cirrhosis), abnormal radiographic findings of unknown significance or inaccessible biopsy.