Stimulated Brillouin Flow Cytometry for biomechanical assessment of metastatic potential

摘要 虽然我们检测和治疗原发性肿瘤的能力在过去十年中显著提高，但仍然存在一些问题。 难以诊断转移的起源，而转移仍然是近90%的癌症相关疾病的原因。 死亡在这方面，最近，肿瘤和肿瘤细胞的机械状态的关键作用已经被发现。 它被认为是肿瘤进展、恶性转化和转移的关键因素，但仍然很少被利用。 因为缺乏合适的测量工具。许多微流体变形性方法最近 出现，但他们只给出了整体细胞机械性能的平均值，而这将是 重要的是分离细胞核与细胞骨架的贡献，他们需要机械刺激来探测 这是有害的，因为细胞对机械刺激有强烈反应。在过去的几年里，我们 一直在开发一种全光学方法来应对这一挑战，称为布里渊显微镜，并强烈 在肿瘤生物学中建立了它来表征转移级联过程中的细胞力学。但目前的 技术本身在速度上受到限制（~ 50 ms/点），因为它依赖于自发的布里渊相互作用， 因此不适合于肿瘤细胞的快速筛选/分选。在这里，我们将开发受激布里渊 流式细胞术，其1）将速度提高约100倍，以及2）提供额外的对比机制， 粘度和质量密度与微米级分辨率。基于这一突破，我们将开发和 使用弹性模量、粘度和密度验证流式细胞术/细胞分选平台， 对比机制（目标1）。然后，我们将验证我们对转移细胞的机械评估， 微流控测定以评估迁移和增殖，并在小鼠模型中评估细胞的转移能力 体内（目标2）。严格的技术开发、验证、基准测试和现场测试流程 将产生一个具有前所未有的能力的平台，根据细胞的机械特性对其进行表征和分类。 仪器内的特性与传统流式细胞术兼容，因此准备被广泛采用。 癌症生物学界。该提案的特点是光学技术专家之间的合作 (UMD)在体外（JHU）和体内（UMB）环境中， 卓有成效的合作记录。

ABSTRACT While our ability to detect and treat primary tumors has significantly increased in the past decade, it remains difficult to diagnose the origination of metastasis which remains responsible for nearly 90% of cancer-related deaths. In this respect, recently, the critical role of the mechanical state of tumors and tumor cells has been recognized for tumor progression, malignancy transformation and metastasis but it remains poorly exploited due to the lack of suitable measurement tools. Many microfluidic deformability approaches have recently emerged, but they only give an average value of the overall cell mechanical properties, while it would be important to separate nucleus vs cytoskeleton contributions, and they need mechanical stimulation to probe properties, which is deleterious since cells strongly react to mechanical stimuli. In the past few years, we have been developing an all-optical approach to this challenge, named Brillouin microscopy, and strongly established it in tumor biology to characterize cell mechanics during metastatic cascade. However, current technology is inherently limited in speed (~50ms/point), as it relies on spontaneous Brillouin interaction, and thus is not suitable for rapid screening/sorting of tumor cells. Here, we will develop stimulated Brillouin cytometry which 1) increases speed by ~100-fold and 2) provides additional contrast mechanisms such as viscosity and mass density with micron-scale resolution. Based on this breakthrough, we will develop and validate a flow cytometry/cell sorting platform using elastic modulus, viscosity, and density as label-free contrast mechanisms (Aim 1). We will then validate our mechanical assessment of metastatic cells against microfluidic assays to assess migration and proliferation and in mice models to assess cell’s metastatic ability in vivo (Aim 2). The rigorous process of technology development, validation, benchmarking and field-testing will yield a platform with unprecedented capabilities to characterize and sort cells based on their mechanical properties within an instrument compatible with traditional flow cytometry, thus ready to be widely adopted by the cancer biology community. The proposal features the collaboration between optical technology experts (UMD) and cancer metastasis pioneers in both in vitro (JHU) and in vivo (UMB) settings with an established track record of fruitful collaboration.