MOLECULAR ANALYSIS OF CANCER PROGRESSION WITH LABEL-FREE RAMAN SPECTROSCOPY

Due to its ability to probe water-containing samples using visible and near-infrared frequencies with high chemical specificity, Raman spectroscopy is an attractive tool for label-free investigation of biological samples. While Raman spectroscopy has been leveraged for exploratory studies in clinical cancer diagnostics, only limited studies have used it to understand the molecular mechanisms driving key characteristics of cancer progression. In this thesis, we present three progressively complex applications of Raman spectroscopy that take advantage of its specificity and synergistic combination with plasmonic nanoparticles and multivariate data analysis for molecular study of cancer. First, we used Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) to investigate the roles of microcalcification status and the composition of tumor microenvironment in breast tissue for identification of a range of breast pathologies. We developed a partial least squares-discriminant analysis-based classifier to correlate the spectra with their pathology to obtain high prediction accuracy. A parallel investigation of the genetic drivers of microcalcification formation in breast cancer cells revealed that stable silencing of the Osteopontin gene decreased the formation of hydroxyapatite in breast cancer cells and reduced their migration. Next, we demonstrated the ability to detect premetastatic changes in the lungs of mice bearing breast tumors, in advance of tumor cell seeding, using Raman spectroscopy and multivariate data analysis. Our measurements showed reliable differences in the collagen and proteoglycan features of the premetastatic lungs which uniquely identify the metastatic potential of the primary tumor. Consistent with histological assessment, our results hint at a continuous premetastatic niche formation model dependent on the metastatic potential of primary tumor. Finally, we exploited Raman mapping to elucidate radiation therapy-induced biomolecular changes in murine tumors and uncovered latent microenvironmental differences between treatment-resistant and -sensitive tumors. We used multivariate curve resolution-alternating least squares (MCR-ALS) and support vector machine (SVM) to quantify biomolecular differences in the tumor microenvironment and constructed classification models to predict therapy outcome and resistance. We found significant differences in lipid and collagen content between unirradiated and irradiated tumors. Taken together, these studies pave the way for applications of Raman spectroscopy beyond clinical diagnostics such as metastatic risk assessment and treatment monitoring

Discerning the differential molecular pathology of proliferative middle ear lesions using Raman spectroscopy

Despite its widespread prevalence, middle ear pathology, especially the development of proliferative lesions, remains largely unexplored and poorly understood. Diagnostic evaluation is still predicated upon a high index of clinical suspicion on otoscopic examination of gross morphologic features. We report the first technique that has the potential to non-invasively identify two key lesions, namely cholesteatoma and myringosclerosis, by providing real-time information of differentially expressed molecules. In addition to revealing signatures consistent with the known pathobiology of these lesions, our observations provide the first evidence of the presence of carbonate- and silicate-substitutions in the calcium phosphate plaques found in myringosclerosis. Collectively, these results demonstrate the potential of Raman spectroscopy to not only provide new understanding of the etiology of these conditions by defining objective molecular markers but also aid in margin assessment to improve surgical outcome.National Institute for Biomedical Imaging and Bioengineering (U.S.) (9P41EB015871-26A1)Connecticut Institute for Clinical and Translational ScienceJHU Whiting School of Engineering (Startup Funds

Primary breast tumor induced extracellular matrix remodeling in premetastatic lungs

Abstract The premetastatic niche hypothesis proposes an active priming of the metastatic site by factors secreted from the primary tumor prior to the arrival of the first cancer cells. We investigated several extracellular matrix (ECM) structural proteins, ECM degrading enzymes, and ECM processing proteins involved in the ECM remodeling of the premetastatic niche. Our in vitro model consisted of lung fibroblasts, which were exposed to factors secreted by nonmalignant breast epithelial cells, nonmetastatic breast cancer cells, or metastatic breast cancer cells. We assessed ECM remodeling in vivo in premetastatic lungs of female mice growing orthotopic primary breast tumor xenografts, as compared to lungs of control mice without tumors. Premetastatic lungs contained significantly upregulated Collagen (Col) Col4A5, matrix metalloproteinases (MMPs) MMP9 and MMP14, and decreased levels of MMP13 and lysyl oxidase (LOX) as compared to control lungs. These in vivo findings were consistent with several of our in vitro cell culture findings, which showed elevated Col14A1, Col4A5, glypican-1 (GPC1) and decreased Col5A1 and Col15A1 for ECM structural proteins, increased MMP2, MMP3, and MMP14 for ECM degrading enzymes, and decreased LOX, LOXL2, and prolyl 4-hydroxylase alpha-1 (P4HA1) for ECM processing proteins in lung fibroblasts conditioned with metastatic breast cancer cell media as compared to control. Taken together, our data show that premetastatic priming of lungs by primary breast tumors resulted in significant ECM remodeling which could facilitate metastasis by increasing interstitial fibrillar collagens and ECM stiffness (Col14A1), disruptions of basement membranes (Col4A5), and formation of leaky blood vessels (MMP2, MMP3, MMP9, and MMP14) to promote metastasis

Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy

The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo

Rapid Identification of Biotherapeutics with Label-Free Raman Spectroscopy

Product identification is a critical and required analysis for biotheraputics. In addition to regulatory requirements for identity testing on final drug products, in-process identity testing is implemented to reduce business risks associated with fill operations and can also be used as a tool against counterfeiting. Biotherapeutics, in particular monoclonal antibodies, represent a challenging cohort for identity determination because of their similarity in chemical structure. Traditional methods used for product identification can be time and labor intensive, creating a need for quick, inexpensive and reliable methods of drug identification. Here, driven by its molecular-specific and nonperturbative nature, we present Raman spectroscopy as an alternate analytical tool for identity testing. By exploiting subtle differences in vibrational modes of the biologics, we have developed partial least-squares-discriminant analysis derived decision algorithms that offer excellent differentiation capability using spontaneous Raman spectra as well as label-free plasmon-enhanced Raman spectra. Coupled with the robustness to spurious correlations due to its high information content, our results highlight the potential of Raman spectroscopy as a powerful method for rapid, on-site biotherapeutic product identification

Rapid Identification of Biotherapeutics with Label-Free Raman Spectroscopy

Product identification is a critical and required analysis for biotheraputics. In addition to regulatory requirements for identity testing on final drug products, in-process identity testing is implemented to reduce business risks associated with fill operations and can also be used as a tool against counterfeiting. Biotherapeutics, in particular monoclonal antibodies, represent a challenging cohort for identity determination because of their similarity in chemical structure. Traditional methods used for product identification can be time and labor intensive, creating a need for quick, inexpensive and reliable methods of drug identification. Here, driven by its molecular-specific and nonperturbative nature, we present Raman spectroscopy as an alternate analytical tool for identity testing. By exploiting subtle differences in vibrational modes of the biologics, we have developed partial least-squares-discriminant analysis derived decision algorithms that offer excellent differentiation capability using spontaneous Raman spectra as well as label-free plasmon-enhanced Raman spectra. Coupled with the robustness to spurious correlations due to its high information content, our results highlight the potential of Raman spectroscopy as a powerful method for rapid, on-site biotherapeutic product identification

Lithium in breast milk transiently affects the renal electrolytic balance of infants

Background: The use of lithium during breast-feeding has not been comprehensively investigated in humans due to concerns about lithium toxicity. Procedure: We analyzed lithium in the kidneys of nursed pups of lithium medicated mothers, using analytical spectroscopy in a novel rat model. The mothers were healthy rats administered lithium via gavage (1000 mg/day Li2CO3 per 50 kg body weight). Results: Lithium was detected in the breast milk, and in the blood of pups (0.08 mM), of lithium-exposed dams at post-natal day 18 (P18), during breast-feeding. No lithium was detected after breast-feeding, at P25 (4 days after cessation of nursing). The lithium pups blood had elevated urea nitrogen at P18 and reduced total T4 at P18 and P25, indicating a longer-term effect on the kidneys and the thyroid gland. Multivariate machine-learning analysis of spectroscopy data collected from the excised kidneys of pups showed elevated potassium in lithium-exposed animals both during- and after breast-feeding. The elevated renal potassium was associated with low nephrin expression in the kidneys measured immunohistochemically during breast-feeding. After lithium exposure is stopped, the filtration of lithium from the kidneys reverses these effects. Our study showed that breastfeeding during lithium use has an effect on the kidneys of the offspring in rats