Advancing Vibrational Spectroscopy for Cellular and Sub Cellular Analysis: Raman Spectroscopy as an in Vitro Chemotherapeutic Screeening and Assessment Protocol

Vibrational spectroscopy, including Raman micro-spectroscopy, has attracted considerable attention over the last few years, as a powerful, non-invasive tool for clinical applications, especially in cancer diagnosis, in vivo and ex vivo. As a molecular fingerprinting technique with optical resolution, Raman micro-spectroscopy is able to monitor biochemical processes, drug uptake, efficacy and mode of action and mechanisms of interaction of chemotherapeutic drugs at a subcellular level. In vitro applications may be more strategically achievable, and can help guide drug design and discovery, and eventually evaluate To this end, different lung cell lines were used and Raman micro-spectroscopy was coupled to valuable other techniques such as Confocal Laser Scanning Fluorescence Microscopy, Flow Cytometry and Atomic Force Microscopy, in order to explore itssignatures of drug resistance, towards potential applications in personalised therapy and as a companion diagnostic tool. However, to evaluate accurately the potential of Raman micro-spectroscopy for such applications, it is essential to optimise measurement and data processing protocols associated with subcellular analysis in order to extract all valuable spectroscopic information and to demonstrate the ability of this technique to distinguish not only between normal and cancer cells but also between cancer cell lines before exploring its potential as a chemotherapeutic screening and assessment protocol using commercially available chemotherapeutic agents. To be considered as an in vitro companion diagnostics technique to screen for personalised therapies, Raman micro-spectroscopy should be able to monitor subcellular interaction with chemotherapeutic drugs and to characterise cellular resistance. To this end, different lung cell lines were used and Raman micro-spectroscopy was coupled to valuable other techniques such as Confocal Laser Scanning Fluorescence Microscopy, Flow Cytometry and Atomic Force Microscopy, in order to explore its potential to elucidate drug pathways, chemical binding signature, mechanisms of action and efficacy and physiological cellular responses to the drug exposure. As chemotherapeutic agents, Doxorubicin and Actinomycin D, both anthracyclines widely used in clinics especially for lung cancer were employed as pilot molecules. Multivariate data analysis, consisting of Principal Component Analysis, Linear discriminant and Partial Least Square Regression analysis were employed to deeply investigate the spectral features related to drug effects and cellular responses. Investigations demonstrate the ability of Raman micro-spectroscopy not only to track the subcellular accumulation of the drug as function of time but also to identify its mechanism of action, the subsequent cellular response and to differentiate cellular resistance. Moreover, despite the fact that different cell lines show different chemotherapeutic resistance, the chemical binding signature appears to be identical from anti-cancer drugs which belongs to same chemotherapeutic group with implications of different mechanisms of action function of time and dose. In human lung cancer cell lines which show different cytotoxic sensitivities to the drugs, different spectroscoic response profiles to the drugs are observed, which can be potentially linked to cellular defence mechanisms, such as the expression of anti-apoptotic proteins, and DNA repair