Single Cell to Spheres in High-Throughput Microfluidics for Cancer Drug Screening

用于癌症药物筛选的高通量微流体中的单细胞到球体

• 批准号: 8492473
• 负责人: Euisik Yoon
• 金额: $ 17.87万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8492473
• 关键词: Accounting; Address; Adherent Culture; Adhesions; Antineoplastic Agents; Architecture; Behavior; Biological Assay; Breast Cancer Cell; Cancer Model; Cancer cell line; Cell Count; Cell Culture Techniques; Cell Line; Cell model; Cells; Characteristics; Chemical Agents; Chemicals; Clinical; Clinical Trials; Collection; Complex; Data; Development; Devices; Dose; Ensure; FDA approved; Force of Gravity; Frequencies; Grant; Growth; Harvest; Heterogeneity; Human; Implant; In Vitro; Individual; Knowledge; Label; Libraries; MCF7 cell; MDA MB 231; Malignant Neoplasms; Measures; Methods; Microfluidics; Morbidity - disease rate; NOD/SCID mouse; Patients; Pattern; Performance; Pharmaceutical Preparations; Population; Population Heterogeneity; Preclinical Drug Evaluation; Procedures; Recording of previous events; Resistance; Resolution; Robotics; Scheme; Screening for cancer; Seeds; Small Molecule Chemical Library; Sorting - Cell Movement; Surface; Suspension Culture; Suspension substance; Suspensions; System; Therapeutic; Time; United States National Institutes of Health; Xenograft procedure; cancer cell; cancer stem cell; cancer therapy; cancer type; clinically relevant; drug use screening; high throughput screening; improved; in vivo; in vivo Model; innovation; malignant breast neoplasm; mortality; mouse model; neoplastic cell; novel; operation; prostate cancer cell; public health relevance; research study; response; screening; single cell analysis; small molecule; small molecule libraries; stem; stemness; success; tool; traditional therapy; tumor; user-friendly

DESCRIPTION (provided by applicant): A controversial but well founded cancer cell model proposes that a small subset of tumor initiating cells (TIC) or cancer "stem-like" cells (CSC) are necessary to initiate and sustain cancer growth. They are resistant to traditional therapy and capable of division and differentiation to give rise to a heterogeneous population of tumor cells. Therapeutics which target TIC/CSC have the potential to drastically improve patient survival. However, there are several obstacles to the study of TIC/CSC, First, they are very rare, representing typically <5% of cells in cell lines and <1% of cells in tumors. Additionally, there i considerable evidence that several subpopulations of tumor initiating cells may exist within one tumor and their identification would require the use of many cell markers in combination with other identifying characteristics. Traditional screening methods typically focus on reduction of overall tumor cell number and will therefore miss these rare, transient cells. There is a clear need to provide tools to expedite the characterization of these rare but critical subtypes to aid i the development of more effective targeted therapies. This grant will focus on the development of high throughput single cell microfluidic platforms for the marker-free enrichment and study of cancer stem cells (CSC) or tumor initiating cells (TIC) using single-cell derived cancer spheroids. This platform will provide high efficiency single-cell capture (>90% capture) and long-term suspension and adherent culture from single cells. Suspended sphere culture of single cancer cells provides the ability to not only screen cancer heterogeneity at high throughput, but also provides the capability for label-free CSC/TIC drug screening. Preliminary Data: We have demonstrated a user-friendly microfluidic approach capable of automated capture (>80% rate) of single cells into high throughput arrays, using no external systems. With this platform we have successfully tracked captured single prostate cancer cells, grown clonal colonies, and analyzed their heterogeneous drug response. Additionally, we have investigated the capabilities of topographically patterned PDMS for non-adherent culture of cancer cells. These surfaces were integrated into our single cell microfluidic platform for the formation of single-cell derived spheres. Sphere forming efficiencies were measured for multiple breast cancer cell lines including SUM159, MCF7, and MDA-MB231. Specific Aims: In Specific Aim 1, we will characterize our novel patterned PDMS surface for non-adherent culture use, integrate them into an optimized single cell capture platform, and modify the device architecture to interface high throughput chemical screening libraries. In Specific Aim 2, we will use our integrated system to characterize the sphere forming efficiency of multiple breast cancer lines, screen the CSC/TIC targeting efficiency of the NIH Clinical Collection chemical library, and perform a secondary dose response assay on those selected in the drug screen. Finally in Specific Aim 3, we will develop a method for harvesting spheres from our device and validate the top chemical agents from Aim 2 using a NOD/SCID mouse model.

描述（由申请人提供）：一个有争议但有充分依据的癌细胞模型提出，一小部分肿瘤起始细胞（TIC）或癌症“干细胞样”细胞（CSC）是启动和维持癌症生长所必需的。它们对传统疗法具有抗性，并且能够分裂和分化以产生异质性肿瘤细胞群体。靶向TIC/CSC的治疗药物有可能大大改善患者的生存率。然而，TIC/CSC的研究存在几个障碍，首先，它们非常罕见，通常代表细胞系中<5%的细胞和肿瘤中<1%的细胞。此外，有相当多的证据表明，在一个肿瘤中可能存在几个肿瘤起始细胞亚群，并且它们的鉴定需要使用许多细胞标志物与其他鉴定特征的组合。传统的筛选方法通常侧重于减少总体肿瘤细胞数量，因此会错过这些罕见的瞬时细胞。显然需要提供工具来加速这些罕见但关键的亚型的表征，以帮助开发更有效的靶向疗法。该资助将专注于开发高通量单细胞微流体平台，用于使用单细胞衍生的癌症球体进行癌症干细胞（CSC）或肿瘤起始细胞（TIC）的无标记富集和研究。该平台将提供高效的单细胞捕获（>90%捕获）和来自单细胞的长期悬浮和贴壁培养。单个癌细胞的悬浮球培养不仅提供了以高通量筛选癌症异质性的能力，而且还提供了无标记CSC/TIC药物筛选的能力。初步数据：我们已经证明了一种用户友好的微流体方法，能够自动捕获（>80%率）的单细胞进入高通量阵列，不使用外部系统。通过这个平台，我们成功地追踪了捕获的单个前列腺癌细胞，生长了克隆集落，并分析了它们的异质性药物反应。此外，我们还研究了形貌图案化PDMS用于癌细胞非贴壁培养的能力。将这些表面整合到我们的单细胞微流体平台中，用于形成单细胞衍生的微流体。 球体。测量了包括SUM 159、MCF 7和MDA-MB 231在内的多种乳腺癌细胞系的球体形成效率。具体目标：在具体目标1中，我们将表征用于非贴壁培养的新型图案化PDMS表面，将其整合到优化的单细胞捕获平台中，并修改设备架构以连接高通量化学筛选库。在具体目标2中，我们将使用我们的集成系统来表征多个乳腺癌细胞系的球体形成效率，筛选NIH临床收集化学库的CSC/TIC靶向效率，并对药物筛选中选择的那些进行二次剂量反应测定。最后，在具体目标3中，我们将开发一种从我们的设备中收获球体的方法，并使用NOD/SCID小鼠模型验证目标2中的顶级化学试剂。