Label-Free, Longitudinal, Multi-Metric Viability Imaging of 3D Tissue Spheroid Array

摘要 在选择癌症治疗方法时，医生通常从一线治疗方案开始，并监测患者 在观察和等待的基础上取得进展，遵循一套基于大型患者临床试验的指南 人口。但这种传统的方法受到了质疑，因为它是否为个别患者提供了 最佳治疗方案。为了更好地单独找到匹配的治疗方法，一种名为精准癌症医学的概念 或者个性化的癌症药物已经被研究过了。在其他方法中，功能精准医学 直接测试从患者身上活检的肿瘤细胞的化疗方案，以找到最佳匹配的治疗方案 针对特定的病人。 然而，这种有希望的方法并没有被临床医生广泛采用，因为肿瘤 实验室内的微环境与患者体内的微环境不同，导致药物不一致 样本和患者之间的反应，活组织细胞的数量通常不足以 可供测试的选项数量可靠。第一个问题正在通过以下三个方面的最新进展得到解决： 三维(3D)细胞培养技术，更好地模拟实验室中的人体微环境。但是 第二个问题是可检测选择的数量有限，这主要是由于目前检测方法的局限性。 在3D培养中评估化疗敏感性的技术。在目前的大多数检测方法中，样品只能进行检测。 一次，具有不同作用机制的多种药物不能同时进行一次测试 化验。这些限制加在一起，会以指数级的方式减少涉及到 多个评估时间点以设计序贯疗法或增加要测试的药物数量 一种综合疗法。 在这里，我们将开发一种新的技术来评估3D培养中的化学敏感性，通过最大化 一种名为光学相干层析成像(OCT)的无标记3D显微镜技术的潜力。大多数人 之前的OCT研究仅测量了一种或两种类型的信号，并显示了对应于 每项研究中只有一种类型的细胞活力中断过程。但这种做法引发了人们的担忧 关于特异性(即，研究中测试的其他类型的过程可以产生类似的OCT 信号)。这种低特异性，加上不清楚的生存评估机制，阻碍了OCT的发展。 方法从功能精准医学这一前景看好的概念出发。 因此，我们将开发至少18种不同类型的OCT信号，并建立它们的灵敏度和 对四种主要类型的生存能力破坏过程的特异性。这种方法的可行性已经得到了 我们对6,000多个3D培养细胞进行成像和分析的试点研究提供了强有力的支持 椭球体。这个R01项目将对多达100,000个球体进行成像和分析，以获得前所未有的系统 研究OCT信号类型的综合范围。

SUMMARY When selecting cancer therapy, physicians generally begin with first-line treatment options and monitor patient progress on a watch-and-wait basis, following a set of guidelines based on clinical trials from a large patient population. But this traditional method has been questioned on whether it provides individual patients with the optimal treatment. To better find a matching treatment individually, a concept called precision cancer medicine or personalized cancer medicine has been studied. Among other approaches, functional precision medicine directly tests chemotherapy options on tumor cells biopsied from a patient to find the best matching treatment for the specific patient. This promising approach, however, has not been widely adopted by clinicians because the tumor microenvironment in a lab differed from the one within the patient’s body, leading to inconsistent drug responses between the sample and patient, and the quantity of biopsied cells is generally insufficient for a reliable number of options to be tested. The first problem is being addressed by recent advances in three- dimensional (3D) cell culture techniques, which better mimic the body’s microenvironment in a lab. But the second problem, the limited number of testable options, is mainly due to limitations in the current assay techniques that assess chemosensitivity in 3D culture. With most current assays, a sample can only be tested once, and multiple drugs with different mechanisms of action cannot be simultaneously tested by a single assay. Combined, these limitations exponentially reduce the number of testable options when involving multiple assessment time points to design a sequential therapy or when increasing the number of drugs to test a combination therapy. Here, we will develop a new technique for the assessment of chemosensitivity in 3D culture, by maximizing the potential of a label-free 3D microscopy technology, called optical coherence tomography (OCT). The majority of prior OCT research measured only one or two types of signals and showed the signals corresponding to only a single type of cell viability disruption process in each study. But this approach has led to a concern about specificity (i.e., other types of processes than the one tested in the study can generate similar OCT signals). This low specificity, along with unclear mechanisms of viability assessment, have prevented OCT methods from being adopted for the promising concept of functional precision medicine. Therefore, we will develop at least 18 different types of OCT signals and establish their sensitivity and specificity to four major types of viability disruption processes. The feasibility of this approach has been strongly supported by a pilot study where we imaged and analyzed more than 6,000 3D-cultured cell spheroids. This R01 project will image and analyze up to 100,000+ spheroids for an unprecedentedly systemic investigation of the comprehensive range of OCT signal types.