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

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.