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

随着精确肿瘤学的出现，包括靶向特定遗传驱动因子的小分子抑制剂， 癌症和对癌症新抗原的免疫应答的调节剂，迫切需要先进的诊断， 指导和监测治疗干预。目前，这种真实的时间监测是通过重复执行的，或者 抵抗，活检;然而，这些手术活检是侵入性的，可能有严重的并发症，包括 用于预期分析的组织不足，仅对一个特定肿瘤部位进行采样，这可能不具有代表性 整个肿瘤细胞群的数量。液体活检有望彻底改变癌症疗法， 随着癌症响应于治疗药物的发展， 干预措施。从常规血液标本中分离足够数量的罕见CTC的技术障碍已经 是阻止基于CTC的诊断机会的临床部署的唯一主要限制。启用 本文提出的技术，我们从处理常规10 mL采血管（全血体积的0.2%）转变为 利用临床白细胞分离术来采集接近全血体积（40-100%）的样本解决了这一挑战。的 该技术的基本基础是高效地消耗抗体标记的血细胞， 未操作的CTC（“负消除”，与“正选择”相对），从而使肿瘤抗原- 独立富集未扰动的、有活力的CTC。这种策略适用于几乎任何来源的细胞 实体瘤类型，而不对肿瘤细胞的物理或生物学性质进行任何先验假设。我们 假设是使用白细胞去除法对大量CTC进行液体活检等同于侵入性活检 转移性肿瘤病变，目前在肺癌治疗中疾病进展时进行。 为了解决我们的假设，我们制定了三个目标。在目标1中，我们将开发大容量CTC分离技术 基于微流体负选择。在目标2中，我们将把目标1中设计的微流控组件集成到一个 用于从leukopaks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，在几周内进行对方，评估他们的成功率， 诊断准确性。我们相信，我们团队中的资源和多学科专业知识的融合 将导致一种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。这项试点研究的积极成果将为以下方面奠定基础： 测试更复杂的临床应用，从测量药物治疗的癌细胞信号作用， （“非侵入性药代动力学”）和定量用于癌症免疫治疗的细胞表面蛋白质靶（蛋白质- 基于预测标志物），最终甚至能够检测和组织定位处于风险中的早期浸润性癌症。 个体如家族性乳腺癌，具有环境风险的人（例如，吸烟者的肺结节， 肝硬化）、意义不明的异常放射学检查结果或无法进行活检。

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.