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

 描述（由申请人提供）：来自患者的单个膀胱癌细胞中药物的质谱检测癌症越来越被理解为在单细胞水平上定义和传播的过程。癌症干细胞生物学、化疗耐药性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。在个体化医疗和单细胞分析的界面上，一个主要的未满足的生物分析需求将是在单个癌细胞水平上监测患者施用的化疗疗法的剂量和有效性的能力。目前，没有能够确定患者个体癌细胞内化疗剂浓度的临床生物分析方法，并且这种方法将是建立以最小毒性递送有效化疗剂浓度的理想给药方案的有力工具。此外，还可以评估施用化疗剂的患者对个体癌细胞的健康（例如凋亡水平）的有效性的单细胞分析方法将给出关于治疗功效的实时相关信息。 我们开发了一种新型的装置-单探针-可以与质谱（MS）耦合用于生物分析。单探针的取样尖端小于真核细胞（<10 μm），可插入个体活体细胞中， 癌细胞，以采样细胞内化合物用于立即MS分析。在我们发表的结果中，我们已经证明，这种技术可用于检测体外培养和给药的单个癌细胞内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质谱技术作为一种生物分析方法，以提高患者化疗的有效性。本申请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剂进行定量单细胞MS（qSCMS）的方案。本研究将通过完成以下两个具体目标来实现：目标1：使用单探针技术，我们将建立一个实验方案，用于几种抗癌化合物，包括标准治疗（SOC）药物，在膀胱癌细胞内的qSCMS。目标二：我们将应用目标1中开发的体外qSCMS方案定量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。本研究具有新颖性和重要意义。这将是第一次定量测量输送到患者癌细胞中的抗癌药物。该项目的成功将使人们对单细胞和肿瘤生物学有新的理解，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.