BIO-POD: PALLET MICRO-ARRAY FOR RARE CELL ANALYSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们已经开发出一种托盘阵列纳米技术，可以分离单个贴壁细胞。将这项纳米技术与基于抗体的检测方法、先进的光学成像和乳腺肿瘤的细针抽吸(FNA)样本相结合，我们正在试行一种方法，该方法1)克服了现有技术的限制，如激光显微解剖，2)患者在诊断时和肿瘤切除后都可以使用，3)可以枚举肿瘤内存在的各种细胞成分，这些元素可能为预后或预测疗效提供重要信息，4)高通量自动分析，以及4)有可能评估单个细胞分区的分子轮廓，这可能为个性化治疗的设计提供更多信息。 这些研究的假设是，托盘阵列纳米技术将允许从原发乳腺肿瘤中识别、计数和分离以下单个细胞成分：乳腺癌干细胞、内皮祖细胞、肌上皮细胞和炎性浸润物。这一假设将通过追求以下具体目标来检验： 1.建立稀有贴壁细胞的检测阈值。 我们将使用混合的细胞，以不同的比例表达细胞表面分子的独特组合，通过流式细胞仪确认，应用于托盘阵列。由于多个肿瘤细胞亚群的检测需要多色/多抗原检测，我们将进行多色荧光成像，以确定稀有细胞的检测阈值。此外，我们还将改进托盘阵列的设计。 2.将此方法应用于采用细针抽吸(FNA)采样的原代乳腺癌细胞。 将利用切除后立即进行的FNAs对乳腺肿瘤标本进行试点可行性研究，以测试将发现从细胞系推广到原发肿瘤组织的能力。这些研究将建立乳腺癌干细胞、内皮祖细胞、肌上皮细胞、肿瘤上皮细胞和炎性浸润物的分离、鉴定和检测方法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have developed a pallet array nanotechnology that permits isolation of individual adherent cells. Combining this nanotechnology with antibody-based detection methods, advanced optical imaging and fine needle aspirate (FNA) sampling of breast tumors, we are piloting a methodology that 1) overcomes limitations to existing technologies, such as laser microdissection, 2) could be available to patients at the time of diagnosis vs. after tumor resection, 3) permits enumeration of various cellular elements present within a tumor potentially yielding important information for prognosis or predictive of therapeutic efficacy, 4) is designed for high throughput automated analyses, and 4) has the potential to assess the molecular profile of individual cellular compartments, which could provide additional information for the design of tailored individualized therapy. The HYPOTHESIS for these studies is that the pallet array nanotechnology will permit the identification, enumeration, and isolation of the following individual cellular elements from primary breast tumors: breast cancer stem cells, endothelial progenitor cells, myoepithelial cells, and inflammatory infiltrate. This hypothesis will be tested by pursuing the following Specific Aims: 1. Establish the detection threshold for identifying rare adherent cells. We will employ mixtures of cells expressing unique combinations of cell surface molecules in varying proportions, confirmed by flow cytometry, applied to the pallet array. As detection of multiple tumor cellular subsets will require multicolor/multi-antigen detection, we will perform multi-color fluorescence imaging to establish the detection threshold for rare cells. Additionally, we will refine the design of the pallet array. 2. Apply this methodology to primary breast cancer cells using Fine Needle Aspirate (FNA) sampling. Pilot feasibility studies will be conducted on breast tumor specimens using FNAs performed immediately after resection, in order to test the ability to generalize the findings from cell lines to primary tumor tissues. These studies will establish isolation, identification, and detection methods for the enumeration of breast cancer stem cells, endothelial progenitor cells, myoepithelial cells, tumor epithelial cells, and inflammatory infiltrates.