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

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。它们对传统疗法有抵抗力，并且能够分裂和分化，从而产生异质的肿瘤细胞群。针对TIC/CSC的治疗方法有可能大大提高患者的生存率。然而，TIC/CSC的研究存在一些障碍，首先，它们非常罕见，通常在细胞系中占<5%的细胞，在肿瘤中占<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，它们的识别需要使用许多细胞标记，并结合其他识别特征。传统的筛选方法通常侧重于减少整体肿瘤细胞数量，因此将错过这些罕见的，短暂的细胞。显然需要提供工具来加快这些罕见但关键亚型的表征，以帮助开发更有效的靶向治疗。该资助将侧重于开发高通量单细胞微流控平台，用于使用单细胞衍生的癌球体对癌症干细胞（CSC）或肿瘤起始细胞（TIC）进行无标记富集和研究。该平台将提供高效率的单细胞捕获（>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培养不仅提供了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：我们已经展示了一种用户友好的微流体方法，能够在不使用外部系统的情况下，将单个细胞自动捕获（bbb80 %的速率）成高通量阵列。利用这个平台，我们已经成功地跟踪捕获的单个前列腺癌细胞，培养克隆菌落，并分析它们的异质药物反应。此外，我们还研究了地形图型PDMS用于癌细胞非贴壁培养的能力。这些表面被整合到我们的单细胞微流控平台，形成单细胞衍生

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.