Raman spectroscopic imaging reveals lipid metabolism as a marker and therapeutic target of cancers

Cancer cells have been known to have reprogrammed metabolism since 1920s, when Otto Warburg observed increased aerobic glycolysis in cancer cells, named as Warburg effect. Due to advances in the past decades, cancer metabolism is increasingly recognized as a hallmark of cancer. Altered metabolic pathways, including glycolysis, glutaminolysis, nucleotide and lipid synthesis, have provided new targets for cancer therapy. Among these metabolic changes, lipid metabolism in cancer is the least studied area, partially due to lack of suitable tools to study lipids. In my dissertation work, we applied Raman spectroscopic imaging techniques to study lipid metabolism in cancers, specifically de novo lipogenesis, lipid composition and lipid desaturation. Firstly, we developed an imaging method to directly visualize de novo lipogenesis in single living cells. Using isotope labeled glucose (glucose-d7), we were able to trace the dynamic metabolism of glucose in single living cells with high spatial-temporal resolution. As the first direct visualization, we observed that glucose was largely utilized for lipid synthesis in pancreatic cancer cells, which occurs at a much lower rate in immortalized normal pancreatic epithelial cells. By inhibition of glycolysis and fatty acid synthase (FAS), the key enzyme for fatty acid synthesis, we confirmed the deuterium labeled lipids in cancer cells were from de novo lipid synthesis. Interestingly, we also found that prostate cancer cells exhibit relatively lower level of de novo lipogenesis, but higher fatty acid uptake compared to pancreatic cancer cells. Secondly, using SRS imaging and Raman spectroscopy, we unveiled an underappreciated role of cholesterol esterification in pancreatic cancer growth and metastasis. By employing label-free Raman spectromicroscopy, we found an aberrant accumulation of cholesteryl ester (CE) in human pancreatic cancer specimens and cell lines. Abrogation of cholesterol esterification activity, either by inhibiting the acyl-CoA cholesterol acyltransferase-1 (ACAT-1) enzyme activity, or by specific shRNA knockdown of ACAT-1 expression, significantly reduced pancreatic tumor growth and metastasis in an orthotopic mouse model of pancreatic cancer. Furthermore, we showed that ACAT-1 inhibition disturbed cholesterol homeostasis, by increasing intracellular free cholesterol level, which induced endoplasmic reticulum (ER) stress and caused apoptosis. Increased free cholesterol was further showed to downregulate MAPK pathway mediated by caveolin-1. Finally, combining hyperspectral SRS imaging and Raman spectral analysis, we identified an unexpected increased level of lipid unsaturation in ovarian cancer stem cells (CSCs). In two ovarian cancer cell lines, we observed that lipid unsaturation level was significantly higher in flow cytometry sorted CSCs, when compared to non-CSCs. Higher lipid unsaturation level was also found in CSC enriched spheres when compared to monolayer cells. The same phenomena was confirmed in two primary ovarian cancer cells isolated from patient ascites. Furthermore, we showed that lipid unsaturation was positively correlated ALDH1 expression, as ALDH inhibition significantly decreased unsaturation level, suggesting a regulatory role of ALDH on lipid desaturation. Inhibition of lipid desaturases (Δ9, Δ5, and Δ6) by inhibitors significantly suppressed sphere formation and reduced the percentage of ALDH positive cells. Inhibition of lipid desaturation was also found to downregulate AKT and NF-κB pathway. Collectively, this study demonstrates increased lipid unsaturation as a potential marker for ovarian CSCs and the possibility to target lipid desaturases as a CSC-specific therapy.