Stimulated Brillouin Flow Cytometry for biomechanical assessment of metastatic potential

摘要 虽然我们发现和治疗原发肿瘤的能力在过去十年中显著提高，但它仍然 很难诊断转移的来源，转移仍然是近90%与癌症相关的原因 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是 公认为肿瘤进展、恶性肿瘤转化和转移，但仍未得到充分利用 由于缺乏合适的测量工具。最近，许多微流控变形性方法 出现了，但它们只给出了细胞整体机械性能的平均值，而它将是 分离细胞核和细胞骨架很重要，它们需要机械刺激才能探测 属性，这是有害的，因为细胞对机械刺激有强烈的反应。在过去的几年里，我们 我一直在开发一种名为布里渊显微镜的全光学方法来应对这一挑战，并强烈地 在肿瘤生物学中建立了它，以表征转移级联过程中的细胞力学。但是，当前 技术本身速度有限(~50ms/点)，因为它依赖自发的布里渊相互作用，并且 因此不适合肿瘤细胞的快速筛选/分选。在这里，我们将发展受激布里渊源 细胞术，1)速度提高约100倍，2)提供额外的对比机制，如 粘度和质量密度具有微米级的分辨率。在这一突破的基础上，我们将发展和 使用无标记的弹性模数、粘度和密度验证流式细胞仪/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是否 微流控技术用于评估细胞的迁移和增殖以及在小鼠模型中用于评估细胞的转移能力 体内实验(目标2)。严格的技术开发、验证、基准和现场测试过程 将产生一个具有前所未有的能力的平台，可以根据细胞的机械特性对细胞进行表征和分类 仪器内的特性与传统的流式细胞仪兼容，因此准备广泛采用 癌症生物界。该提案的特点是光学技术专家之间的合作 (UMD)和癌症转移先驱在体外(JHU)和体内(UMB)环境中都建立了 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.