Imaging Cholesterol Metabolism and Trafficking by Stimulated Raman Scattering Microscopy
Abstract
Cholesterol is an essential component of mammalian cells which is tightly regulated. However, our understanding of cholesterol transport and metabolism is still incomplete, partly due to lack of suitable tools for studying cholesterol dynamics in living cells and organisms with spatio-temporal information. My dissertation work applied spectroscopic imaging of cholesterol in human tissues, living cells, and model organisms to unravel new insights of cholesterol metabolism and trafficking. Using stimulated Raman spectroscopic analysis of lipid droplets in human prostate cancer patient tissues, we observed an aberrant accumulation of cholesteryl ester in metastatic lesions. Inhibition of cholesterol esterification in prostate cancer cells significantly suppresses the development and growth of metastatic cancer lesions in both orthotopic and intra-cardiac injection mouse models. Gene expression profiling shows that cholesteryl ester depletion suppresses the metastatic potential through upregulation of multiple regulators that negatively impact metastasis. Additionally, Wnt/β-catenin, one of vital pathways for metastasis, is downregulated upon cholesteryl ester depletion. Mechanistically, we found evidence suggesting that inhibition of cholesterol esterification significantly blocks secretion of Wnt3a through reduction of monounsaturated fatty acid levels, which limits Wnt3a acylation. These results collectively validate cholesterol esterification as a novel metabolic target for treating metastatic prostate cancer. My thesis work also developed a new biocompatible cholesterol analog, which enabled real-time imaging of cholesterol metabolism and trafficking in living cells and organisms. Based on quantum chemistry calculations, we designed and synthesized phenyl-diyne cholesterol (PhDY-Chol), which has an extremely large Raman scattering cross section. The phenyl-diyne group is biologically inert and provides a Raman scattering cross section that is 88 times larger than the endogenous C=O stretching mode. Stimulated Raman scattering microscopy offers an imaging speed that is faster than spontaneous Raman microscopy by three orders of magnitude, and a detection sensitivity of 31 μM PhDY-Chol (∼1,800 molecules in the excitation volume). Inside living cells, PhDY-Chol mimics the behavior of cholesterol, including membrane incorporation and esterification. In a cellular model of Niemann-Pick type C disease, PhDY-Chol reflects the lysosomal accumulation of cholesterol, and shows relocation to lipid droplets after HPβCD treatment. In living C. elegans, PhDY-Chol mimics cholesterol uptake by intestinal cells and reflects cholesterol storage. Together, this work demonstrates an enabling platform for study of cholesterol trafficking in living cells and organisms.
Degree
Ph.D.
Advisors
Ratliff, Purdue University.
Subject Area
Molecular biology|Cellular biology|Biomedical engineering|Pathology|Physiology
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