An investigation into the extracellular matrix characteristics of osteosarcoma cells using Raman spectroscopy

Shepherd, Laura and Taylor, Adam and Kerns, Jemma (2020) An investigation into the extracellular matrix characteristics of osteosarcoma cells using Raman spectroscopy. Masters thesis, Lancaster University.

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Abstract

Osteosarcoma is an aggressive, rare, and highly heterogeneous malignancy that quickly metastasises and predominantly affects young adults in the pubertal growth phase. This sarcoma has multiple subtypes, with few unifying features. The production of tumour osteoid from the malignant mesenchymal cells is a hallmark feature and histological requirement for diagnosis, identified from an invasive biopsy. A non-invasive technique with the ability to analyse tumour osteoid, diagnose osteosarcoma and detect early changes, would be clinically useful. The aim of this investigation was to analyse the extracellular matrix characteristics of the MG63, SaOS-2, and Te85 osteosarcoma cell lines, using Raman spectroscopy and Alizarin Red staining. COVID-19 implications disrupted the completion of planned/prepared experiments, so the investigation refocused on the analysis of the MG63 cell line using Raman spectroscopy. MG63 cells were grown in osteogenic medium and fixed in formalin at 0, 7, 14, 21, 28, and 35 days. An inVia Raman microspectrometer (Renishaw plc) with a 785nm laser was used to obtain spectra at each time point. The data was pre-processed, and principal component and linear discriminant analyses were performed, revealing protein and lipid associated peaks predominantly contributed to the variance between time points. Early mineral species were also identified, contributing to the separation of day 35 spectra from the other time points. Differences in protein secondary structure were identified by the univariate analysis, indicating increased collagenous organisation in the later time points. This investigation demonstrated the ability of Raman spectroscopy to differentiate between MG63 cells at different time points, based on biomolecular differences, associated with osteoid production and proliferation related changes. This is clinically valuable for the development of screening/diagnostics tools in osteosarcoma, as Raman spectroscopy could be used to detect early molecular changes, before clinical signs and symptoms. This versatile technique has the potential to revolutionise oncological screening and diagnostics.