Light-sheet micro-aspiration microscopy for the isolation and analysis of rare tumor cells from intact clinical specimens

项目总结 此赠款申请响应计划公告编号PA-18-398，并概述了培训和 研究计划，这将使我为专注于光学发展的长期研究生涯做好准备 更好地了解、诊断和治疗人类前列腺癌的影像方法。该提案概述了 指导培训计划，将我置于NIH资助的一个研究团队的直接指导下 来自华盛顿大学(UW)和邻近的弗雷德·哈钦森癌症研究中心 (FHCRC)。我的培训计划包括三个免费的重点领域：(1)接受组织的进一步培训 清除和标记，(2)获得单细胞分离和分析方面的教育和实践培训，以及 (3)建立分子检测和前列腺癌的知识库，以促进有意义的未来 与这些领域的专家合作。为了实现这些目标，我将利用这笔资金将时间用于 参加研究生课程、研讨会、培训研讨会、大型讲座，最重要的是定期参加 在我的职业发展计划中，与我的整个职业奖励团队会面。研究项目 该提案的组成部分是因为已知稀有细胞群在 许多最具挑战性的癌症和下游分析问题(即基因组学、蛋白质组学和 转录学)，因此可以针对这些单个细胞进行更有效的治疗 通常隐藏的疾病的关键因素。尽管如此，单细胞分离(单个细胞 有针对性地收集以供进一步研究)仍然对当前的方法具有技术挑战性，特别是对于 罕见的肿瘤细胞。这尤其有问题，因为这些罕见的肿瘤细胞通常决定转移、治疗 耐药性和复发，所有这些最终导致患者死亡。虽然这一挑战存在于 几乎所有形式的癌症，这项提案将特别关注已经侵袭 前列腺癌的淋巴管空间，因为对这些稀有细胞的分离和分析可以使 开发新的诊断和治疗策略。为了实现这一目标，我建议在我的职位上- 博士研究，并经过上述必要的培训，开发出一种新的光片微抽吸 首次从完整的临床标本中分离出这些罕见的肿瘤细胞 时间到了。该项目由三个具体目标组成：(1)多分辨率开顶式光板的研制 用于识别罕见肿瘤细胞的显微镜系统；(2)光片显微镜与 用于分离罕见肿瘤细胞的显微抽吸；以及(3)该系统的概念验证临床验证 从有淋巴管侵袭的人前列腺组织中分离罕见侵袭性肿瘤细胞。前列腺 华盛顿大学和FHCRC的癌症研究环境是我培训和从事这项研究的理想场所 项目，并在我的指导团队的指导下，我将成功地完成这一职业发展 奖励并追求我成为一名独立调查员的长期职业目标。

PROJECT SUMMARY This grant application responds to Program Announcement Number PA-18-398 and outlines a training and research program which will prepare me for a long-term research career focused on the development of optical imaging methods to better understand, diagnose, and treat human prostate cancer. The proposal outlines a mentored-training program which places me under the direct guidance of a team of NIH funded researchers from the University of Washington (UW) and the neighboring Fred Hutchinson Cancer Research Center (FHCRC). My training plan involves three complimentary areas of focus: (1) receiving further training in tissue clearing and labeling, (2) obtaining educational and hands-on training in single-cell isolation and analysis, and (3) developing a knowledge-base in molecular assays and prostate cancer to facilitate meaningful future collaborations with experts in these fields. To achieve these goals, I will use the funding to dedicate time for attending graduate courses, workshops, training seminars, grand rounds lectures, and most importantly regular meetings with my entire career award team, as outlined in my career development plan. The research project component of the proposal is driven by the fact that rare cell populations are known to play an outsized role in many of the most challenging problems in cancer, and downstream profiling (i.e., genomics, proteomics, and transcriptomics) of these individual cells can therefore allow for more-effective therapies targeted against these often-hidden lynchpins of the disease. Despite this, single-cell isolation (the process by which individual cells are targeted and collected for further study) is still technically challenging with current methods, particularly for rare tumor cells. This is particularly problematic, as these rare tumor cells often dictate metastasis, treatment resistance, and recurrence, all of which ultimately result in patient mortality. While this challenge exists for nearly all forms of cancer, this proposal will focus specifically on metastatic tumor cells which have invaded the lympho-vascular space in prostate cancer, as the isolation and analysis of these rare cells can enable the development of new diagnostic and treatment strategies. To achieve this, I propose to build upon my post- doctoral research, and with the necessary training above, develop a novel light-sheet micro-aspiration microscope which will enable the isolation of these rare tumor cells from intact clinical specimens for the first time. The project is comprised of three specific aims: (1) development of a multi-resolution open-top light-sheet microscope system for identifying rare tumor cells; (2) combined demonstration of light-sheet microscopy with micro-aspiration for isolating rare tumor cells; and (3) proof-of-concept clinical validation of the system for isolating rare invading tumor cells from human prostate tissues with lympho-vascular invasion. The prostate cancer research environment at UW and the FHCRC is an ideal place for me to train and pursue this research project, and with the guidance of my mentorship team, I will successfully complete this career development award and pursue my long-term career goal of becoming an independent investigator.