Stimulated Raman scattering microscopy of three-dimensional models of disease.

了解细胞环境中的小分子相互作用对于新疗法的设计和生物学评估至关重要。在啮齿动物和哺乳动物中进行新化学实体的临床前测试，以评估药物的特性。拉曼光谱已广泛应用于研究药物与细胞的相互作用，包括可视化细胞环境中的吸收、保留和代谢。自从首次应用炔标记拉曼成像以来，已经检测到细胞内药物定位和富集。使用拉曼成像技术已经在细胞环境中检测到多种酪氨酸激酶抑制剂。例如，受激拉曼散射（SRS）显微镜已经能够检测多种弱碱性药物，这些药物已被证明在溶酶体隔室中显著富集。然而，这些研究中的大多数都是在单层细胞培养中进行的，忽略了组织和肿瘤微环境的复杂的3D性质。由于活体标本的组织异质性和3D性质，药物在动物模型中的可视化是一个重大挑战，同时需要减少临床前筛选中对活体动物模型的依赖。该项目旨在开发拉曼成像内的三维矩阵使用多细胞肿瘤球体（MTS）作为相关模型。它们展示了实体瘤的生理特征，包括结构复杂性和细胞-基质相互作用，可以在体外培养。小分子的渗透尚未在MTS中使用基于拉曼的方法进行研究。这个博士项目将开发MTS的无标记表征使用高光谱SRS与化学计量学图像分析。初步研究SRS成像用于MTS无标记表征的潜力。增殖外层的检测将使用指示细胞内容物的拉曼位移来可视化。除此之外，还将评估药物摄取的检测和球体内药物浓度的定量。Futibatinib是通过丙烯酰胺弹头的成纤维细胞生长因子受体（FGFR）蛋白家族的不可逆抑制剂。它还含有一个炔间隔基，以靶向疏水口袋，从而对表皮生长因子受体蛋白家族产生选择性。因此，炔基将用作标记物，以在整个MTS体积中可视化福替巴替尼。实现这一目标将显示在3D矩阵环境中药物成像的第一个这样的例子，并为研究MTS中的药物定位提供机会。MTS含有复杂的微环境，在球体的体积上具有pH梯度和氧化还原环境。然后，该项目将研究使用炔传感器使用高光谱SRS成像来成像MTS的pH平衡。pH和氧化还原活性的多重检测将通过生物正交拉曼窗口的SRS成像进行评估。最后，将在依赖于高酸性环境以释放活性药物分子的前药活化策略中研究这些作用。使用高光谱SRS成像的化学计量学分析，将实时评估前药活化的动力学，这是目前难以实现的，使用的技术是破坏性的，缺乏分辨率。

Understanding small molecule interactions in the cellular environment is crucial in the design and biological assessment of novel therapeutics. Preclinical testing of new chemical entities is conducted in rodents and mammals to assess the properties of the drug. Raman spectroscopy has been widely applied to studying drug-cell interactions, including visualising the uptake, retention and metabolism in the cellular environment. Since the first application of alkyne-tag Raman imaging, intracellular drug localisation and enrichment has been detected. A wide variety of tyrosine kinase inhibitors have been detected in the cellular environment using Raman imaging techniques. For example, stimulated Raman scattering (SRS) microscopy has enabled the detection of multiple weakly basic drugs that have been shown to significantly enrich within lysosomal compartments. However, the majority of these studies have been conducted in monolayer cell culture, neglecting the complex, 3D nature of tissue and tumour microenvironments. The visualisation of drugs in animal models represents a significant challenge because of tissue heterogeneity and the 3D nature of living specimens, whilst there is a need to reduce the reliance on live animal models in preclinical screening. This project aims to develop Raman imaging within 3D matrix using multicellular tumour spheroids (MTS) as a relevant model. They display a physiological profile of solid tumours, including the structural complexity and cell-matrix interactions, that can be cultured in vitro. The penetration of small molecules has yet to be investigated in MTS using a Raman-based approach. This PhD project will develop label-free characterisation of MTS using hyperspectral SRS with chemometric image analysis. The potential of SRS imaging for label-free characterisation of MTS will be investigated initially. The detection of the proliferating outer layer will be visualised using Raman shifts indicative of cellular contents. Beyond this first aim, the detection of drug uptake and quantification of intraspheroid drug concentration will be assessed. Futibatinib is an irreversible inhibitor of the fibroblast growth factor receptor (FGFR) family of proteins via an acrylamide warhead. It also contains an alkyne spacer to target a hydrophobic pocket giving selectivity over the epidermal growth factor receptor protein family. As such, the alkyne group will be used as a marker to visualise futibatinib throughout the MTS volume. Achieving this would show the first such example of drug imaging within a 3D matrix environment and open opportunities for investigating drug localisation in MTS. MTS contain a complex microenviorment with a gradient of pH and redox environment across the volume of the spheroid. The project will then investigate the use of alkyne sensors for imaging pH balance across MTS using hyperspectral SRS imaging. The multiplex detection of pH and redox activity will be assessed SRS imaging across the bio-orthogonal Raman window. Lastly, these effects will be investigated in prodrug activation strategies that are reliant upon a highly acidic environment to liberate the active drug molecule. Using chemometric analysis of hyperspectral SRS imaging, the kinetics of prodrug activation will be assessed in real-time, which is currently difficult to achieve, using techniques which are destructive and lacking in resolution.