Integration of Flexible Micro Spring Array and High Throughput Microfluidics for

描述（由申请人提供） 摘要：转移性癌症释放循环肿瘤细胞（CTC）的能力，这些细胞穿过血液并侵入身体的不同部位，占癌症相关死亡的90%以上。迫切需要用于改进诊断和治疗策略的新技术来改善晚期转移性癌症患者的患者结局。一种这样的技术涉及通过从患者血液样品中分离来分析这些CTC。由于CTC的分子谱可能与原发性肿瘤的分子谱完全不同，而与转移性肿瘤的分子谱更相似，因此CTC更适合于转移性癌症的预后和诊断。目前的CTC技术由于灵敏度和选择性差、成本高和处理时间长而在临床应用中存在严重的困难和限制。CellSearchTM是目前FDA唯一批准的CTC分析系统，仅用于检测CTC，不能保存活细胞。从血液样本中获得CTC的根本挑战是它们非常罕见，在数十亿个血细胞中只有少数肿瘤细胞。由于肿瘤细胞几乎总是显著大于正常血细胞，基于尺寸的分离已被证明是CTC捕获的有效方法。我们已经采取了一种新的方法来建立微过滤技术，通过实施一系列的灵活的微弹簧和使用一个可调节的低压流系统，以尽量减少过滤过程中细胞所经历的机械应力。这是一种高效且具有成本效益的系统，能够在仅10分钟内从临床相关的7.5 mL血液中富集活性CTCs。尽管实现了针对白细胞的大于104富集，但富集细胞的纯度不足以获得 临床相关的遗传信息。我们提出了一个高通量的微流控系统，将物理分区这些细胞进行分析，在一个单一的细胞level.By利用微技术的这种固有的优势，大量的平行反应可以用来克服纯度的问题。基因表达谱和突变检测可用于改善诊断，并导致开发针对每个患者优化的高度个性化的治疗计划。此外，使用微流体进行CTC的多孔分配将用于尝试建立用于CTC培养的有利条件，即使在初始低接种数下。CTC的成功原代培养将允许药物功效测试，其可用于离体测定潜在的药物，而不会使患者暴露于化疗的不必要的成本或毒性作用。这些新的方法是基于对可行的CTC的分析，代表了一种尚未得到证实的不同方法。然而，由于这种技术平台适用于几乎所有类型的癌症，它可以相当快地彻底改变转移性癌症患者的治疗方法。 公共卫生相关性：最致命的癌症形式可以释放出通过血液循环并扩散到全身的侵略性细胞。已经开发了微制造技术，以有效地从患者血液样品中分离这些细胞，从而允许微创“液体活检”，其可以经常用于监测肿瘤进展。该项目探索了高通量分析方法的整合，这将使人们能够在这些细胞上测试各种抗癌药物，而不会对患者造成风险，并获得遗传信息，这对制定高度个性化的治疗计划至关重要。

DESCRIPTION (Provided by the applicant) Abstract: The ability of metastatic cancer to release circulating tumor cells (CTCs) that travel through the blood and invade different parts of the body accounts for over 90% of cancer related deaths. New techniques for improved diagnosis and therapeutic strategies are desperately needed to improve patient outcomes in late stage metastatic cancer patients. One such technique involves the analysis of these CTCs through isolation from patient blood samples. Since molecular profiles of CTCs can be quite different from those of the primary tumor and more similar to the metastatic tumors, CTCs are better suited for metastatic cancer prognosis and diagnosis. Current CTC technologies have serious difficulties and limitations for clinic applications due to poor sensitivity and selectivity, high cost, and long processing times. CellSearchTM, the only current FDA approved system for CTC analysis, is only used for detection of CTCs and is not capable of preserving viable cells. The fundamental challenge with obtaining CTCs from blood samples is the fact that they are so rare, with only a few tumors cells occurring among billions of blood cells. Since tumor cells are almost always significantly larger than normal blood cells, size based separation has been demonstrated as an effective method for CTC capture. We have taken a novel approach to established microfiltration technology by implementing an array of flexible microsprings and using a regulated low pressure flow system to minimize the mechanical stresses experienced by cells during the filtration process. This is an efficient and cost effective system that is capable of the enrichment of viable CTCs from a clinically relevant blood volume of 7.5 mL in only 10 minutes. Despite achieving greater than 104 enrichment against leukocytes, the purity of the enriched cells is not sufficient for obtaining clinically relevant genetic information. We propose the incorporation of a high throughput microfluidic system that will physically partition these cells for analysis on a single cell level.By exploiting this inherent advantage of microtechnology, a large volume of parallel reactions can be used to overcome the issues with purity. Genetic expression profiles and mutation detections may be used for improved diagnosis, and lead to the development of highly personalized therapy plans that are optimized for each patient. Furthermore, the use of microfluidics for multiwell partitioning of CTCs will be used to attempt the establishment of favorable conditions for the culture of CTCs, even at an initially low seeding number. Successful primary culture of CTCs will allow drug efficacy tests that may be used to assay potential drugs ex vivo without exposing a patient to the unnecessary cost or toxic effects of chemotherapy. These new approaches based on the analysis of viable CTCs represent a different approach that has not been proven. However, since this technological platform is applicable to almost every type of cancer, it could fairly quickly revolutionize the way that therapies are derived for metastatic cancer patients. Public Health Relevance: The most deadly forms of cancer can release aggressive cells that circulate through the bloodstream and spread throughout the body. Microfabrication technology has been developed to effectively isolate these cells from a patient blood sample, allowing a minimally invasive "liquid biopsy" that may be performed often for monitoring tumor progression. This project explores the integration of a high throughput approach to analysis that will make it possible to test various anticancer drugs on these cells at no risk to the patient, and obtain genetic information that will be crucial to developing a highly personalized treatment plan.