ANTICANCER DRUG EXPULSION IN SHED VESICLES

描述（由申请人提供）：在癌症化疗过程中，最初的缓解可能随后是耐药复发。耐药细胞群可能来自化疗的选择性压力，再加上赋予耐药肿瘤细胞选择性生长优势的突变或表观遗传变化。最近，人们发现癌细胞在体内和体外都会脱落大量纳米大小的细胞表面衍生颗粒。我们假设这些颗粒代表了一种抗癌药物外排机制，参与了耐药性。如果得到证实，了解这一机制将有助于设计更有效的抗癌药物，并有助于开发新的阻断耐药性的药物。为了支持这一假设，我的实验室获得的初步证据表明，囊泡脱落相关基因的表达与多药耐药有关。对来自不同组织的人类衍生癌细胞系的化学敏感性基因表达谱的统计分析表明，囊泡脱落相关基因可以预测耐药谱。用阿霉素和其他小荧光分子进行的脉冲追踪实验证实，从癌细胞表面脱落的囊泡可以介导药物排出。在这里，我们建议对囊泡介导的药物排出对总抗癌药物外排和耐药性的贡献进行初步研究。本研究的第一个目的是使用几种不同的放射性标记药物作为示踪剂，测量脱落囊泡中药物的排出，并将药物排出与各种乳腺肿瘤来源细胞系的多药耐药联系起来。第二个目的是检测乳腺癌患者乳头抽吸液和导管灌洗液中脱落囊泡或脱落囊泡相关抗原的存在。乳房切除术前获得的双侧乳头抽吸和导管灌洗液样本将使用显微镜、生化和生物物理技术分析是否存在脱落囊泡或脱落囊泡相关抗原。确定脱落囊泡特异性存在于癌性乳腺液中，将有助于未来研究囊泡脱落作为耐药性和癌症进展的预后标志物的作用，并将为脱落机制的生理学相关性提供证据。

DESCRIPTION (provided by applicant): In the course of cancer chemotherapy, an initial remission may be followed by drug-resistant relapse. Drug resistant cell populations can arise from selective pressure of chemotherapy, combined with mutations or epigenetic changes that confer a selective growth advantage to drug-resistant tumor cells. Recently, it has been found that cancer cells shed large numbers of nanometer-sized, cell surface-derived particles both in vivo and in vitro. We hypothesize that these particles represent an anticancer drug efflux mechanism, involved in drug resistance. If confirmed, understanding this mechanism would help design anticancer drugs with increased efficacy, and would facilitate development of novel pharmacological agents for blocking drug resistance. In support of this hypothesis, preliminary evidence obtained in my laboratory indicates that expression of vesicle shedding-associated genes correlates with multidrug resistance. Statistical analyses of gene expression profiles in relation to chemosensitivity profiles spanning human derived cancer cell lines from different tissues indicate that vesicle shedding associated genes predict drug resistance profiles. Pulse-chase experiments with doxorubicin and other small, fluorescent molecules confirm that vesicles shed from the surface of cancer cells can mediate drug expulsion. Here, we propose to perform a pilot study of the contribution of vesicle-mediated drug expulsion to total anticancer drug efflux and resistance. The first aim of this study will be to measure expulsion of drug in shed vesicles, using several different radiolabeled drugs as tracers, and relate drug expulsion to multidrug resistance across various breast tumor-derived cell lines. A second objective is to detect the presence of shed vesicles -or shed vesicle-associated antigens- in nipple aspirates and ductal lavage fluid obtained from breast cancer patients. Bilateral nipple aspirates and ductal lavage samples obtained prior to mastectomy will be analyzed for the presence of shed vesicles or shed vesicle-associated antigens using microscopic, biochemical and biophysical techniques. Establishing the presence of shed vesicles specifically in cancerous breast fluids would enable future studies of the role of vesicle shedding as a prognostic marker of drug resistance and cancer progression, and would constitute evidence for the physiological relevance of the shedding mechanism.