Altered Cholesterol Metabolism In Human Cancers Unraveled By Label-Free Spectroscopic Imaging

Despite tremendous scientific achievements, cancer remains the second leading cause of death in the United States. Metabolic reprogramming has been increasingly recognized as a core hallmark of cancer. My dissertation work identified novel diagnostic markers and therapeutic targets for human cancers through the study of cholesterol in cancer cells. Enabled by label-free Raman spectromicroscopy, we performed the first quantitative analysis of lipogenesis at single cell level in human patient cancerous tissues. Our imaging data revealed an unexpected, aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases, but not in normal prostate, benign prostatic hyperplasia, or prostatitis. Biochemical and molecular biological studies showed that such cholesteryl ester accumulation was a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway in prostate cancer cells. Furthermore, we found that such accumulation arose from significantly enhanced uptake of exogenous lipoproteins and required cholesterol esterification. Depletion of cholesteryl ester storage using pharmacological inhibitors or RNA interference significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth in mouse xenograft models with negligible toxicity. These findings open new opportunities for diagnosing and treating late-stage prostate cancer by targeting the altered cholesterol metabolism. My thesis work also found that cholesterol-rich domains on plasma membranes can be used as a marker for the loss of basoapical polarity, one of the earliest changes observed in breast neoplasia. Raman microspectroscopy revealed that in polarized acini lipids were more ordered at the apical membranes compared to basal membranes, and that an inverse situation occurred in acini that lost apical polarity upon treatment with Ca2+-chelator EGTA. This method allowed us to detect the disruption of apical polarity by dietary breast cancer risk factor, ω6 fatty acid, even when the effect was too moderate to permit a conclusive assessment by traditional immunostaining method. Collectively, label-free Raman analysis of cholesterol-rich membrane domains in mammary acini provides an effective screening platform to identify risk factors that initiate breast cancer

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

Image-guided surgery using invisible near-infrared fluorescent light : from pre-clinical studies to clinical validation

This thesis focuses on the use of near-infrared (NIR) fluorescence imaging during (cancer) surgery, using specialized intraoperative imaging systems. This technique is validated in preclinical studies on intraoperative tumor identification and image-guided resection, and and then translated to clinical studies using clinically available agents. Using experimental, tumor-specific NIR fluorescent agents (probes), tumors could successfully be identified during surgery and subsequently resected under image guidance, in several tumor models. Also, the clinically available indocyanine green (ICG) could be used to identify colorectal liver metastases during surgery, in an experimental model. Clinically, NIR fluorescence imaging could be used to identify sentinel lymph nodes (SLN, the lymph nodes that drain directly from a tumor and are most likely to contain tumor cells if metastasis has occurred). SLNs could be identified after ICG injection in breast cancer, vulvar cancer and cervical cancer. SLNs could also be identified ex vivo in colorectal cancer, after HSA800, an optimized experimental probe that was injected in freshly resected specimens. After intravenous ICG injection, NIR angiography could be performed in patients undergoing breast reconstructive surgery, bile ducts could be identified intraoperatively in patients undergoing pancreaticoduodenectomy, and colorectal liver metastases could be identified in patients undergoing liver surgery.Center for Translational Molecular Medicine (CTMM, DeCoDe and MUSIS projects), the Dutch Cancer Society, Foundation “De Drie Lichten” and the Leiden University Fund (LUF).LUMC / Geneeskund

Investigating Metabolic Activities and Phenotypes in Biological Systems with Vibrational Probes and Raman Techniques

In this dissertation, the emerging stimulated Raman scattering (SRS) microscopy in combination with various vibrational tags was extensively used to explore various aspects of biological systems. New techniques as well as new Raman active materials were also developed to facilitate the applications of SRS in biology. Chapter one introduces and comprehensively reviews vibrational tags that have been developed to date in combination with imaging techniques and their applications in biological sciences to investigate metabolism in living organisms. Chapter two studies lipotoxicity, a phenomenon that is well known but poorly understood. The study found phase separation can form on ER membrane in cells treated with long chain fatty acids due to the high transition temptation of their metabolites. It was also found that the phase separation severely disturbs normal distribution of ER membrane proteins because of hydrophobic mismatching. As the result, ER normal structure is disrupted, luminal space is collapsed, and interconnectivity of ER that ensures normal ER functions is lost. Additionally, ER stress sensor IRE1α was found to be activated directly by the formation of phase separation, which triggers apoptosis and ultimately leads to cell death. Chapter three describes the development of a new method termed as metabolic activity phenotyping (MAP) that acquires quantitative measurements of metabolic activities of individual cells, which is essential to understanding questions in diverse fields in biology. To achieve the goal, an automatic system was designed and built that improves the acquisition speed by more than 100 times compared to commercially available instruments. A set of vibrational probes with deuterium labeling was also carefully selected to enable accurate measurement of metabolic flux. Combining the merits of high throughput measurements and vibrational tags, MAP was applied to investigate the metabolic activity differences among various cancer cells, to study the heterogeneity of drug efficacy, and to facilitate breast cancer subtyping. Chapter four describes the development and application of a new class of Raman active nanoparticles, or Rdots. These Rdots were generated by non-covalently incorporating small molecule Raman probe into polymeric nanoparticles. The resulted Rdots are of compact size (~20 nm) and preserve all Raman spectral features of the small molecule probes used. Rdots were compared to other existing Raman active materials including SERS nanoparticles, and Rdots surpass all the other materials in terms of brightness. In addition, Rdots also possess narrow spectral linewidth (< 3 nm), making them ideal for multiplexed imaging. In the study, Rdots were used as immunostaining reporters to visualize cytoskeleton networks and surface markers in cell and tissue samples

Cancer Nanomedicine

This special issue brings together cutting edge research and insightful commentary on the currentl state of the Cancer Nanomedicine field

Skin Tissue Models

Skin Tissue Models provides a translational link for biomedical researchers on the interdisciplinary approaches to skin regeneration. As the skin is the largest organ in the body, engineered substitutes have critical medical application to patients with disease and injury - from burn wounds and surgical scars, to vitiligo, psoriasis and even plastic surgery. This volume offers readers preliminary description of the normal structure and function of mammalian skin, exposure to clinical problems and disease, coverage of potential therapeutic molecules and testing, skin substitutes, models as study platforms of skin biology and emerging technologies. The editors have created a table of contents which frames the relevance of skin tissue models for researchers as platforms to study skin biology and therapeutic approaches for different skin diseases, for clinicians as tissue substitutes, and for cosmetic and pharmaceutical industries as alternative test substrates that can replace animal models. Offers descriptions of the normal structure/function of mammalian skin, exposure to clinical problems, and more Presents coverage of skin diseases (cancer, genodermatoses, vitiligo and psoriasis) that extends to clinical requirements and skin diseases in vitro models Addresses legal requirements and ethical concerns in drugs and cosmetics in vitro testing Edited and authored by internationally renowned group of researchers, presenting the broadest coverage possible. © 2018 Elsevier Inc. All rights reserved.(undefined)info:eu-repo/semantics/publishedVersio

Photonic discrimination and specific targeting of vascular stem cells.

Cardiovascular disease remains the leading cause of death and disability world-wide. The current treatment options include balloon angioplasty and the deployment of drug-eluting stents (DES) coated with anti-mitotic drugs to prevent intimal-medial thickening (IMT). Despite this, an unacceptably high failure rate remains due to non-specific targeting of cells and drug-depletion over time. The source of the cells contributing to IMT remains controversial; one theory suggests a reprogramming of native differentiated vascular smooth muscle cells (SMC) while the other proposes myogenic differentiation of resident vascular and/or circulating stem cells. Resolution of this controversy through identification of the source of the contributing cells would greatly assist in the development of future drug targeting strategies using novel DES platforms. The use of photonics and vibrational spectroscopy is gaining popularity for disease diagnosis. Both platforms have the ability to yield cellular and molecular information about cells and tissues label-free, making them attractive technologies for analysing biological specimen. The first main objective of this work was to analyse individual cells from normal (healthy) and arteriosclerotic (diseased) vessels ex vivo using autofluorescence (AF) in response to broadband light and to compare their AF signatures to undifferentiated stem cells and their myogenic progeny in vitro. The second aim was to use vibrational spectroscopy (Raman and FTIR) to examine undifferentiated stem cells, their myogenic and osteogenic progeny and and further compare their spectra to re- programmed differentiated SMC. Finally, a novel therapeutic platform for targeting stem cell-derived myogenic progeny using magnetic nanoparticles was developed. Using pharmacological inhibitors of glycogen synthase 3 beta (GSK3β), the effects on Notch, a well known mediator of myogenic differentiation were first evaluated in vitro. Further to this, a prototype GSK3β inhibitor was incorporated into a novel drug delivery system consisting of polymer coated Fe304 magnetic nanoparticles which can be systemically administered and specifically targeted to bare-metal stents by an external magnetic field