Harnessing label-free Raman spectroscopy for metastatic cancer diagnosis and biologic development

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Johns Hopkins University
Optical spectroscopy is unique amongst experimental techniques in that it can be performed in near-physiological conditions, achieve high molecular specificity, and explore dynamics on timescales ranging from nanoseconds to days. In particular, Raman spectroscopy has emerged in the last two decades as a uniquely versatile method to investigate the structures and properties of molecules in diverse environments through interpreting vibrational transitions. In this thesis, we present four interconnected biomedical and biopharmaceutical applications of Raman spectroscopy that exploit its exquisite molecular specificity, non-perturbative nature, and near real-time measurement capability. In the first presented study, we harness spontaneous Raman spectroscopy in conjunction with multivariate analysis to rapidly and quantitatively determine antibody-drug conjugate aggregation with the goal of eventual application as an in-line tool for monitoring protein particle formation. By exploring subtle, but consistent, differences in spectral vibrational modes of various monoclonal antibodies (mAb) aggregations, a support vector machine-based regression model is developed which is able to accurately predict a wide range of protein aggregation. In addition, the investigation of these spectral vibrational modes also offers new insights into mAb product-specific aggregation mechanisms. Second, leveraging surface-enhanced Raman scattering (SERS) and localized surface plasmon resonance (LSPR), we present a design of plasmonic nanostructures based on rationally structured metal-dielectric combinations, which we call composite scattering probes (CSP). Specifically, we design CSP configurations that have several prominent resonance peaks enabling higher tunability and sensitivity for self-referenced multiplexed analyte sensing. The CSP prototypes were used to demonstrate differentiation of subtle changes in refractive index (as low as 0.001) as well as acquire complementary untargeted plasmon-enhanced Raman measurements from the biospecimen’s compositional contributors. In the third study, we demonstrate that Raman spectroscopy offers vital biomolecular information for early diagnosis and precise localization of breast cancer-colonized bone alterations. We show that as early as two weeks after intracardiac injections of breast cancer cells in mouse models, Raman measurements in femur and spine uncover consistent changes in both bone matrix and mineral composition. This research effort opens the door for improved understanding of breast metastatic tumor-related bone remodeling and establishing a non-invasive tool for detection of early metastasis and prediction of fracture risk. In parallel with this effort, we also seek to identify the differences between organ-specific isogenic metastatic breast cancer cells. By interpreting the informative spectral bands, we are able to unambiguously identify these isogenic cell lines as unique biological entities. Our spectroscopic study and corresponding metabolic research indicate that tissue-specific adaptations generate biomolecular alterations on cancer cells.
Raman spectroscopy, Chemometrics, Biophotonics