Isolation and Identification of Heterogeneous Circulating Tumor Cells Using a Microchip with Hyperuniform Patterns

癌症转移是癌症相关死亡的主要原因，涉及恶性肿瘤细胞从原发肿瘤扩散到其他器官中形成肿瘤。 计数血液中的循环肿瘤细胞（CTC）可以测量癌症进展并评估癌症治疗的有效性。 然而，CTC是高度多样化的，并且只有特定的CTC亚型负责癌症转移，而目前的CTC计数技术不能区分亚群。在这个项目中，CTC亚群将被分离，并使用含有新结构（不同间距和直径的柱子排列）的微芯片进行识别，该微芯片可以控制局部流动模式。 不同的流动模式使得能够在不同的芯片位置捕获亚型。该芯片将允许在捕获CTC的地方对其进行分析，以确保分析反映这些细胞的真实特征。从这项工作中获得的变革性知识将指导CTC技术在辅助癌症诊断、预测肿瘤进展和监测治疗效果方面的应用。如果成功的话，这种方法可以适用于类似地分离免疫细胞或干细胞的亚群，用于治疗癌症或其他疾病。拟议的教育和推广计划将加强TTU的生物医学工程课程和计划，并向本科生和研究生介绍现代生物工程技术。外联活动将与现有的TTU初中和高中女生方案协调，并激发她们对科学和工程的兴趣。此外，由于TTU是一个西班牙裔服务机构，将努力将更多的代表性不足的少数民族纳入该项目。该项目的目标是通过无标记分离和分析循环肿瘤细胞（CTC）亚群来了解CTC的转移潜力。具有超均匀结构的微芯片将用于分离、原位鉴定和选择性分离CTC。超均匀性（HU）是一个新兴的概念，包装模式，包含局部异质性或随机性和全球的规则性或同质性。超均匀性的概念将被集成到基于亲和力的微流体装置中用于CTC分离。 由于超均匀结构引起的局部流动模式的受控差异，预期微芯片上不同位置的细胞停滞需要不同的粘合强度。此外，预期该粘合强度与捕获的CTC上的表面标记物的类型和密度相关，因此与其转移特性相关。为了使捕获效率最大化，微芯片涂层将用肿瘤特异性抗体修饰。 单个CTC组的分离将通过捕获的细胞和微柱表面之间的可生物降解的纳米膜的选择性降解来实现。为了将CTC异质性特征与转移状态相关联，将进行下游表征和初步体内验证。研究计划有三个目标。 本论文的第五个目的是设计和表征用于CTC捕获的HU结构微芯片，并分析装置中的流动模式和粘附力。将通过实验和建模来研究局部流体模式，以确定癌细胞积聚的潜在“口袋”，并将给定口袋内细胞的存在与特征性表面粘附相关联。 第二个目标是使用HU微芯片捕获和鉴定具有可变表面标志物表达的单细胞和混合癌细胞系的亚群。 原型微芯片将用于研究具有不同大小、培养条件和细胞周期的细胞的PC 3（人前列腺癌细胞系）中的亚群，并用于区分混合前列腺癌细胞系（PC 3和LNCaP（人前列腺癌细胞系））中的亚群。第三个目标是开发从HU微芯片选择性释放癌细胞亚群，并进行下游表征和初步体内验证。 将通过降解细胞和微柱表面之间的纳米膜和受控的局部机械搅拌来进行HU微芯片上捕获的癌细胞混合物（PC 3和LNCaP）的选择性释放。为了研究转移的进展，将原代人前列腺癌细胞系（CP3TX）移植到小鼠的股骨中，并在不同时间点收集血液样品，并通过HU微芯片运行以捕获可用作转移指标的CTC。 该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

Cancer metastasis, the leading cause of cancer-related death, involves the spreading of malignant tumor cells from the primary tumor to form tumors in other organs. Counting circulating tumor cells (CTCs) in blood can measure cancer progression and evaluate the effectiveness of a cancer treatment. However, CTCs are highly diversified and only specific CTC subtypes are responsible for cancer metastasis, while current CTC-counting technologies do not distinguish between subpopulations. In this project, CTC subpopulations will be isolated and identified using a microchip containing novel structures (arrangements of posts with different spacings and diameters) that can control local flow patterns. The different flow patterns enable the capture of subtypes in different chip locations. This chip will permit CTCs to be analyzed where they are captured to ensure that the analyses reflect the true characteristics of these cells. Transformative knowledge obtained from this work will guide application of CTC technologies in assisting cancer diagnosis, predicting tumor progression, and monitoring therapeutic efficacy. If successful, the approach can be adapted to similarly separate subpopulations of immune cells or stem cells for curing cancers or other diseases. The proposed education and outreach plan will enhance the Biomedical Engineering curriculum and program at TTU and introduce undergraduate and graduate students to modern bioengineering techniques. Outreach activities will coordinate with existing TTU programs for junior-high and high school girls and are armed at sparking their interest in science and engineering. In addition, since TTU is a Hispanic Serving Institution, efforts will be made to include more underrepresented minorities into the project.The goal of this project is to understand the metastatic potential of circulating tumor cells (CTCs) through label-free fractionation and profiling of CTC subpopulations. A microchip with hyperuniform structure will be used to isolate, in-situ identify, and selectively separate CTCs. Hyperuniformity (HU) is an emerging concept of a packing pattern that contains local heterogeneity or randomness and global regularity or homogeneity. The concept of hyperuniformity will be integrated into affinity-based microfluidic devices for CTC isolation. Due to the controlled differences in local flow patterns induced by the hyperuniform structure, cell arrest in different locations on the microchip are expected to require different adhesive strengths. Further, this adhesive strength is anticipated to be related to the types and densities of surface markers on the captured CTCs and therefore, their metastatic character. To maximize capture efficiency, microchip coatings will be modified with tumor specific antibodies. Separation of individual CTC groups will be achieved by selective degradation of biodegradable nanofilm between the captured cells and the surface of the microposts. In order to correlate of CTC heterogeneity profile with metastatic status, downstream characterization and preliminary in vivo validation will be performed. The Research Plan is organized under three objectives. The FiRST Objective is to design and characterize HU structured microchip for CTC capture and analyze flow pattern and adhesion force in the device. The local fluidic pattern will be studied through experiments and modeling to determine the potential "pockets" where cancer cells accumulate and to correlate the presence of a cell within a given pocket with a characteristic surface adhesion. The SECOND Objective is to capture and identify subpopulations of single cell and mixed cancer cell lines with variable expression of surface markers using a HU microchip. A prototype microchip will be used for studying subpopulations in PC3 (human prostate cancer line) with cells with different sizes, culture conditions and cell cycles and for distinguishing subpopulations in mixed prostate cancer cell lines (PC3, and LNCaP (human prostate adenocarcinoma line)). The THIRD Objective is to develop selective release of subpopulations of cancer cells from a HU microchip and perform downstream characterization and preliminary in vivo validation. Selective release of a captured cancer cell mixture (PC3 and LNCaP) on a HU microchip will be conducted by degrading the nanoflm between cells and micropost surfaces and controlled local mechanical agitation. To study the progression of metastasis, a primary human prostrate cancel cell line (CP3TX), will be transplanted into the femurs of mice and blood samples will be collected at different time points and run through HU microchips to capture CTCs that can be used as metastasis indicators. Indicator profiles are expected to correlate with prostate cancer metastasis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.