A new modality for cholesterol impact tracking in colon cancer development - Raman imaging, fluorescence and AFM studies combined with chemometric analysis.

Colorectal cancer (CRC) is the third most common cancer worldwide. Obesity, alcohol consumption, smoking, high consumption of red or processed meat and a diet with low fibre, fruit, and vegetable intake increase CRC risk. Despite advances in surgery (the basic treatment for recovery), chemotherapy, and radiotherapy, CRC remains the second leading cause of cancer-related deaths in the world. Therefore the social importance of this problem stimulates research aimed at developing new tools for rapid CRC diagnosis and analysis of CRC risk factors. Considering the association between the cholesterol level and CRC, we hypothesize that cholesterol spectroscopic and AFM (atomic force microscopy) studies combined with chemometric analysis can be new, powerful tools used to visualize the cholesterol distribution, estimate cholesterol content and determine its influence on the biochemical and nanomechanical properties of colon cells. Our paper presents the analysis of human colon tissues: normal and cancer and human colon single cells normal CCD18-Co and cancer CaCo-2 in the physiological state and CaCo-2 upon mevastatin supplementation. Based on vibrational features we have shown that Raman spectroscopy and imaging allow cholesterol content in human colon tissues and human colon single cells of both types to be tracked and allow the effectiveness of mevastatin in the mevalonate pathway modulation and disruption of the cholesterol level to be proven. All observations have been confirmed by chemometric analysis including principal component analysis (PCA) and partial least squares discriminant analysis (PLSDA). The positive impact of statins on cholesterol content has also been studied by using fluorescence microscopy and atomic force microscopy (AFM). A significant increase in Young modulus as a mechanomarker for CaCo-2 human cancer colon cells upon mevastatin supplementation compared to CCD18-Co human normal colon cells was observed. This paper is one of the first reports about the use of Raman spectroscopic techniques in cholesterol investigations and the first one about cholesterol investigation using Raman spectroscopy (RS) on human cells ex vivo in the context of colon cancer development.