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

描述(申请人提供)：一个有争议但有充分依据的癌细胞模型提出，肿瘤启动细胞(TIC)或癌症“干细胞样”(CSC)的一小部分是启动和维持癌症生长所必需的。它们对传统疗法具有抵抗力，并能够分裂和分化，从而产生不同种类的肿瘤细胞。以TIC/CSC为靶点的治疗技术有可能显著提高患者的存活率。然而，TIC/CSC的研究有几个障碍：第一，它们非常罕见，通常占细胞系细胞的5%和肿瘤细胞的1%。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，它们的识别需要结合其他识别特征使用许多细胞标志物。传统的筛查方法通常侧重于减少总的肿瘤细胞数量，因此会错过这些罕见的瞬时细胞。显然需要提供工具来加快这些罕见但关键的亚型的特征，以帮助开发更有效的靶向治疗。这笔赠款将集中于开发高通量单细胞微流控平台，用于使用单细胞来源的癌症球体进行癌症干细胞(CSC)或肿瘤起始细胞(TIC)的无标记浓缩和研究。该平台将提供高效的单细胞捕获(90%捕获)和单细胞的长期悬浮和贴壁培养。单个癌细胞的悬浮球体培养不仅提供了高通量筛选肿瘤异质性的能力，而且提供了无标记CSC/TIC药物筛选的能力。初步数据：我们已经展示了一种用户友好的微流控方法，能够在不使用外部系统的情况下将单个细胞自动捕获(80%比率)到高通量阵列中。利用这一平台，我们成功地追踪了捕获的单个前列腺癌细胞，培养了克隆克隆，并分析了它们的异质性药物反应。此外，我们还研究了用于非贴壁培养癌细胞的局部图案化PDMS的能力。这些表面被整合到我们的单细胞微流控平台中，形成单细胞衍生 球体。对包括SUM159、MCF7和MDA-MB231在内的多种乳腺癌细胞株的球体形成效率进行了测量。具体目标：在特定目标1中，我们将表征我们用于非贴壁培养的新型图案化PDMS表面，将其集成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文库。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株的球体形成效率，筛选NIH临床收集化学文库的CSC/TIC靶向效率，并对在药物筛选中选择的那些进行二次剂量反应分析。最后，在特定的目标3中，我们将开发一种从我们的设备中获取球体的方法，并使用NOD/SCID小鼠模型验证来自目标2的顶级化学试剂。

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.