Capturing cancer cells with peptide bead technology

本提案的目的是开发一种基于肽的技术来捕获和扩大体液中的癌细胞，特别是胸腔积液。胸腔积液是恶性肿瘤尤其是肺癌的常见并发症。常规细胞学方法在检测胸腔积液中的癌细胞时，灵敏度一般较低。体液中癌细胞的检测通常受到少数恶性细胞的限制，这些细胞被间皮细胞、正常血细胞、成纤维细胞、微生物和细胞碎片的存在所掩盖。在我们的实验室里，我们利用联合研究者Dr. Kit Lam发明的“一面包一肽”组合文库技术，鉴定出了对恶性细胞具有高亲和力的肽配体。我们的实验室还开发了一种敏感和特异性的细胞生长检测方法，用于筛选数百万个肽珠，以寻找特定的肽基序，以便上皮癌细胞可以附着和生长。利用这种方法，我们已经确定了人类恶性细胞系（如非小细胞肺癌）细胞表面受体特异性的肽特异性基序。到目前为止，我们已经确定了两种对肺癌细胞具有高亲和力的特异性六肽。利用与这些肽结合的微珠，初步研究已经证明了从胸膜液中分离上皮细胞的可行性，胸膜液中含有大量红细胞和细胞碎片中已证实的恶性细胞。我们假设，从胸膜液中提取的恶性细胞可以通过细胞生长试验分离、富集和保存，该试验使用的珠子与我们迄今已确定的两种特定的六肽配体结合。本建议的具体目标如下：1。大规模合成含有这两种特定六肽基序之一的微球；2. 利用这些肽珠从胸膜液中捕获和扩增上皮细胞，优化细胞生长的条件；3. 建立保存和鉴定从细胞头部生长试验中富集的恶性细胞的方法；4. 目的：建立高效、高通量磁肽珠分离富集胸腔积液恶性细胞的技术。这项技术的发展将大大提高我们对恶性胸腔积液的诊断能力，并将为捕获和扩大其他体液中脱落的恶性细胞提供一个模型。

The purpose of this proposal is to develop a peptide-based technology to capture and expand cancer cells in body fluids, specifically pleural effusion. Pleural effusion is a common complication associated with malignancies especially lung cancer. The sensitivity is generally low in detecting cancer cells in pleural fluid by conventional cytologic methods. Detection of cancer cells in body fluids is commonly limited by the small number of malignant cells which are obscured by the presence of mesothelial cells, normal blood cells, fibroblasts, microorganisms and cellular debris. In our laboratory, we have identified peptide ligands with high affinity for malignant cells using the technology of 'one-bread one-peptide' combinatorial library invented by the co-investigator, Dr. Kit Lam. A sensitive and specific cell-growth-on-bead assay has also been developed in our laboratory for screening millions of peptide beads for specific peptide motifs to use epithelial cancer cells can attach and grow. Using this method, we have identified peptide specific motifs specific for cell- surface receptors of human malignant cell lines such as that from non- small cell lung cancers. To date, we have identified two specific hexapeptides with high affinity for lung cancer cells. Using beads conjugated with these peptides, pilot studies have demonstrated the feasibility of isolating epithelial cells from pleural fluids with proven malignant cells amid a large number of red blood cells and cell debris. We hypothesize that malignant cells from pleural fluid can be isolated, enriched and preserved by the cell-growth-on-bead assay using beads conjugated with the two specific hexapeptide ligands that we have identified to date. The specific aims of this proposal are formulated as cells: 1. To synthesize, in large scale, beads carrying one of these two specific hexapeptide motifs; 2. To optimize conditions of cell-growth- on-bead assay using these peptide beads to capture and expand epithelial cells from pleural fluid; 3. To develop methods to preserve and identify malignant cells enriched from the cell-growth-on-bead assay; 4. To develop a high-yield and high-throughput technology of magnetic peptide-beads for isolating and enriching malignant cells from pleural fluid. The development of this technology will greatly enhance our ability of diagnosing malignant pleural effusion, and it will provide a model for capturing and expanding exfoliated malignant cells in other body fluids.