Mass Spectrometry Detection of Drugs in Single Bladder Cancer Cells from Patients

 描述(由申请人提供)：从患者的单个膀胱癌细胞中进行药物的质谱学检测癌症越来越被理解为一个在单细胞水平上定义和传播的过程。癌症干细胞生物学、化疗耐药发展和转移性疾病的研究都集中在单个癌细胞的作用上。为了在单个癌细胞水平上进行癌症研究和治疗，需要新的生物分析能力和技术。在个性化药物和单细胞分析的界面上，一个尚未得到满足的主要生物分析需求将是在单个癌细胞水平上监测患者给药化疗的剂量和有效性的能力。目前，还没有临床生物分析方法能够确定患者单个癌细胞内化疗药物的浓度，这种方法将是建立理想的剂量方案的有力工具，从而提供有效的化疗浓度和最低的毒性。此外，单细胞分析方法也可以评估接受化疗的患者对单个癌细胞的健康状况的有效性(例如，细胞凋亡水平)，将提供有关治疗效果的实时相关信息。我们开发了一种新的设备--单探针--可以与质谱仪(MS)联用进行生物分析。这种单探针的采样尖端小于真核细胞(&lt；10μm)，可以插入到个体生命中 对癌细胞内的化合物进行采样，以便立即进行MS分析。在我们发表的结果中，我们已经证明，这项技术可以用于检测体外培养和剂量的单个癌细胞中抗癌化合物及其代谢物的存在。我们的长期研究目标是充分发展单探针MS技术作为一种生物分析方法，以提高患者的化疗效果。这项应用的目的是建立使用单探针MS技术对患者分离的癌细胞中的化疗药物进行定量单细胞MS(QSCMS)的方案。本研究的目标将通过完成以下两个具体目标来完成：目的1：利用单探针技术，建立包括标准护理(SOC)药物在内的几种抗癌化合物在膀胱癌细胞内的QSCMS实验方案。目的：我们将应用AIM 1中开发的体外QSCMS方法来定量检测从接受治疗的斯蒂芬森癌症中心患者的尿液中分离的膀胱癌细胞中化疗药物的细胞内水平。所提出的研究具有新颖性和重要意义。这将是首次定量测量进入患者癌细胞的抗癌药物。该项目的成功将允许对单细胞和肿瘤生物学的新理解，这是目前不可能的。

 DESCRIPTION (provided by applicant): Mass Spectrometry Detection of Drugs in Single Bladder Cancer Cells from Patients Cancer is increasingly understood as a process defined and propagated at the single cell level. Research in cancer stem cell biology, chemotherapeutic resistance development, and metastatic disease all focus on the action of individual cancer cells. To approach cancer research and therapy on the single cancer cell level, new bioanalytical capabilities and technologies are needed. A major unmet bioanalytical need at the interface of personalized medicine and single cell analysis will be the capability to monitor the dosing and effectiveness of patient-administered chemotherapeutic therapies on the single cancer cell level. Currently, there are no clinical bioanalytical methods capable of determining the concentration of chemotherapeutic agents inside of a patient's individual cancer cells, and such a method would be a powerful tool in establishing ideal dosing regiments that deliver effective chemotherapeutic concentrations with minimal toxicities. Further, a single cell analysis method that could also assess the effectiveness of the patient administered chemotherapeutic on the health of the individual cancer cells (e.g. apoptosis level) would give real-time relevant information about the therapeutic efficacy. We have developed a novel device-the Single-probe-that can be coupled with mass spectrometry (MS) for bioanalysis. The Single-probe, with a sampling tip smaller than eukaryotic cells (<10 μm), can be inserted into individual living cancer cells to sample the intracellular compounds for immediate MS analysis. In our published results, we have proven that this technique can be used to detect the presence of anti-cancer compounds and their metabolites inside of single cancer cells cultured and dosed in vitro. Our long term research goal is to fully develop the Single-probe MS technique as a bioanalytical method to improve the effectiveness of chemotherapy in patients. The objective of this application is to establish protocols to use the Single-probe MS technique for quantitative single cell MS (qSCMS) of chemotherapeutic agents in patient isolated cancer cells. This research objective will be accomplished through completing the following two Specific Aims: Aim 1: Using the Single-probe technology, we will establish an experimental protocol for the qSCMS of several anti-cancer compounds, including standard of care (SOC) drugs, inside bladder cancer cells. Aim 2: We will apply the in vitro qSCMS protocol developed in Aim 1 to quantitate the intracellular levels of chemotherapy drugs in bladder cancer cells isolated from the urine of treated Stephenson Cancer Center patients. The proposed research is novel and significant. It will be the first time to quantitatively measure anti-cancer drugs delivered into patient's cancer cells. The success of this project will allow for new understandings in single cell and tumor biology that are not currently possible.