Harnessing label-free, second harmonic generation microscopy for research and diagnosis of osteosarcoma

Abstract

Osteosarcoma (OS), the most common primary cancer of bone, predominantly affects paediatric patients and is associated with poor prognoses. OS is characterised by the production of an immature bone matrix, resulting in aberrant extracellular matrix (ECM) components, including collagen. In recent years, label-free optical techniques have emerged as promising tools for non-invasive tumour identification, by detecting structural alterations in the ECM. This thesis explores the potential of label-free, second harmonic generation (SHG) microscopy as a tool for research and diagnosis of OS, aiming to enhance our understanding of this malignancy and provide valuable insights for OS diagnosis and treatment. The central hypothesis of this study is that label-free SHG microscopy can identify disease-specific matrix signatures in OS.
In the first results chapter, initial optimisation of SHG imaging and analysis protocols are described, enabling extraction of collagen fibre parameters from healthy and malignant bone biopsies. The optimised protocol highlights the importance of imaging parameters, including field of view size, for accurate quantification of collagen fibre parameters within human bone biopsies. Quantified collagen parameters included: fibre length, width, straightness, and orientation. The second results chapter presents the diagnostic capability of SHG microscopy and quantitative analyses of collagen in distinguishing OS biopsies from healthy bone and other skeletal malignancies. Here, SHG microscopy revealed a significant reduction in collagen fibre lengths in OS compared to healthy bone (average fibre length – bone: 27.88 µm  2.18, OS: 23.77 µm  1.00, p < 0.001) – a phenomenon which was exacerbated in late-stage OS biopsies. These findings provide increased awareness of the role of collagen alterations in OS stage and disease progression, highlighting the diagnostic and prognostic potential of aberrant collagen identified via SHG imaging. In the third results chapter, combined use of SHG microscopy and an established preclinical model of OS – c-fos transgenic mice – enabled temporal characterisation of OS-associated collagen phenotypes, enquiring whether collagen aberrations in this mouse model reflect human manifestations of the disease.
This research set out to advance our understanding of OS pathology and introduce the examination of aberrant collagen using label-free, SHG microscopy as a powerful diagnostic tool. It underscores the critical role of precise imaging parameters and offers a bridge between animal models and human disease, laying the foundation for possible innovative advancements in OS research and patient care.

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Identifiers

ORCID for Isabel Creith: orcid.org/0009-0006-4937-7345

ORCID for Sumeet Mahajan: orcid.org/0000-0001-8923-6666

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

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