Thuc T. Le

Detection of Lipid-Rich Prostate Circulating Tumour Cells with Coherent Anti-Stokes Raman Scattering Microscopy

BackgroundCirculating tumour cells (CTC) are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remain a technical challenge.MethodsCoherent anti-Stokes Raman scattering (CARS) microscopy was employed to examine the lipid content of CTC isolated from the peripheral blood of metastatic prostate cancer patients. CARS microscopy was also employed to evaluate lipid uptake and mobilization kinetics of a metastatic human prostate cancer cell line.ResultsOne hundred CTC from eight metastatic prostate cancer patients exhibited strong CARS signal which arose from intracellular lipid. In contrast, leukocytes exhibited weak CARS signal which arose mostly from cellular membrane. On average, CARS signal intensity of prostate CTC was 7-fold higher than that of leukocytes (P<0.0000001). When incubated with human plasma, C4-2 metastatic human prostate cancer cells exhibited rapid lipid uptake kinetics and slow lipid mobilization kinetics. Higher expression of lipid transport proteins in C4-2 cells compared to non-transformed RWPE-1 and non-malignant BPH-1 prostate epithelial cells further indicated strong affinity for lipid of metastatic prostate cancer cells.ConclusionsIntracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC.

Autophagy genes are required for normal lipid levels in C. elegans

Autophagy is a cellular catabolic process in which various cytosolic components are degraded. For example, autophagy can mediate lipolysis of neutral lipid droplets. In contrast, we here report that autophagy is required to facilitate normal levels of neutral lipids in C. elegans. Specifically, by using multiple methods to detect lipid droplets including CARS microscopy, we observed that mutants in the gene bec-1 (VPS30/ATG6/BECN1), a key regulator of autophagy, failed to store substantial neutral lipids in their intestines during development. Moreover, loss of bec-1 resulted in a decline in lipid levels in daf-2 [insulin/IGF-1 receptor (IIR) ortholog] mutants and in germline-less glp-1/Notch animals, both previously recognized to accumulate neutral lipids and have increased autophagy levels. Similarly, inhibition of additional autophagy genes, including unc-51/ULK1/ATG1 and lgg-1/ATG8/MAP1LC3A/LC3 during development, led to a reduction in lipid content. Importantly, the decrease in fat accumulation observed in animals with reduced autophagy did not appear to be due to a change in food uptake or defecation. Taken together, these observations suggest a broader role for autophagy in lipid remodeling in C. elegans.

Wnt interaction and extracellular release of prominin-1/CD133 in human malignant melanoma cells

Prominin-1 (CD133) is the first identified gene of a novel class of pentaspan membrane glycoproteins. It is expressed by various epithelial and non-epithelial cells, and notably by stem and cancer stem cells. In non-cancerous cells such as neuro-epithelial and hematopoietic stem cells, prominin-1 is selectively concentrated in plasma membrane protrusions, and released into the extracellular milieu in association with small vesicles. Previously, we demonstrated that prominin-1 contributes to melanoma cells pro-metastatic properties and suggested that it may constitute a molecular target to prevent prominin-1-expressing melanomas from colonizing and growing in lymph nodes and distant organs. Here, we report that three distinct pools of prominin-1 co-exist in cultures of human FEMX-I metastatic melanoma. Morphologically, in addition to the plasma membrane localization, prominin-1 is found within the intracellular compartments, (e.g., Golgi apparatus) and in association with extracellular membrane vesicles. The latter prominin-1-positive structures appeared in three sizes (small, ≤40 nm; intermediates ~40-80 nm, and large, >80 nm). Functionally, the down-regulation of prominin-1 in FEMX-I cells resulted in a significant reduction of number of lipid droplets as observed by coherent anti-Stokes Raman scattering image analysis and Oil red O staining, and surprisingly in a decrease in the nuclear localization of beta-catenin, a surrogate marker of Wnt activation. Moreover, the T-cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was 2 to 4 times higher in parental than in prominin-1-knockdown cells. Collectively, our results point to Wnt signaling and/or release of prominin-1-containing membrane vesicles as mediators of the pro-metastatic activity of prominin-1 in FEMX-I melanoma.

Disruption of uridine homeostasis links liver pyrimidine metabolism to lipid accumulation

We report in this study an intrinsic link between pyrimidine metabolism and liver lipid accumulation utilizing a uridine phosphorylase 1 transgenic mouse model UPase1-TG. Hepatic microvesicular steatosis is induced by disruption of uridine homeostasis through transgenic overexpression of UPase1, an enzyme of the pyrimidine catabolism and salvage pathway. Microvesicular steatosis is also induced by the inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme of the de novo pyrimidine biosynthesis pathway. Interestingly, uridine supplementation completely suppresses microvesicular steatosis in both scenarios. The effective concentration (EC50) for uridine to suppress microvesicular steatosis is approximately 20 µM in primary hepatocytes of UPase1-TG mice. We find that uridine does not have any effect on in vitro DHODH enzymatic activity. On the other hand, uridine supplementation alters the liver NAD+/NADH and NADP+/NADPH ratios and the acetylation profile of metabolic, oxidation-reduction, and antioxidation enzymes. Protein acetylation is emerging as a key regulatory mechanism for cellular metabolism. Therefore, we propose that uridine suppresses fatty liver by modulating the liver protein acetylation profile. Our findings reveal a novel link between uridine homeostasis, pyrimidine metabolism, and liver lipid metabolism.

Imaging Immune and Metabolic Cells of Visceral Adipose Tissues with Multimodal Nonlinear Optical Microscopy

Visceral adipose tissue (VAT) inflammation is recognized as a mechanism by which obesity is associated with metabolic diseases. The communication between adipose tissue macrophages (ATMs) and adipocytes is important to understanding the interaction between immunity and energy metabolism and its roles in obesity-induced diseases. Yet visualizing adipocytes and macrophages in complex tissues is challenging to standard imaging methods. Here, we describe the use of a multimodal nonlinear optical (NLO) microscope to characterize the composition of VATs of lean and obese mice including adipocytes, macrophages, and collagen fibrils in a label-free manner. We show that lipid metabolism processes such as lipid droplet formation, lipid droplet microvesiculation, and free fatty acids trafficking can be dynamically monitored in macrophages and adipocytes. With its versatility, NLO microscopy should be a powerful imaging tool to complement molecular characterization of the immunity-metabolism interface.

Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans.

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.

Uridine Prevents Fenofibrate-Induced Fatty Liver

Uridine, a pyrimidine nucleoside, can modulate liver lipid metabolism although its specific acting targets have not been identified. Using mice with fenofibrate-induced fatty liver as a model system, the effects of uridine on liver lipid metabolism are examined. At a daily dosage of 400 mg/kg, fenofibrate treatment causes reduction of liver NAD+/NADH ratio, induces hyper-acetylation of peroxisomal bifunctional enzyme (ECHD) and acyl-CoA oxidase 1 (ACOX1), and induces excessive accumulation of long chain fatty acids (LCFA) and very long chain fatty acids (VLCFA). Uridine co-administration at a daily dosage of 400 mg/kg raises NAD+/NADH ratio, inhibits fenofibrate-induced hyper-acetylation of ECHD, ACOX1, and reduces accumulation of LCFA and VLCFA. Our data indicates a therapeutic potential for uridine co-administration to prevent fenofibrate-induced fatty liver.

Letter to the Editor: An Intriguing Relationship Between Lipid Droplets, Cholesterol‐Binding Protein CD133 and Wnt/β‐Catenin Signaling Pathway in Carcinogenesis

We read with great interest a recent publication entitled “Lipid Droplets: A New Player in Colorectal Cancer Stem Cells Unveiled by Spectroscopic Imaging” by Tirinato et al. released in STEM CELLS (2014), which highlights by means of Raman microspectroscopy high levels of lipid droplets in colorectal-cancer stem cells (CR-CSCs) by comparison to the differentiated tumor cells and normal colon epithelial cells [1]. The authors propose that a cellular organelle (i.e., lipid droplets) might be considered, in addition to single molecular markers, as a new signature of CSCs. The relation between excess lipids as measured by label-free coherent anti-Stokes Raman scattering (CARS) and aggressive tumor behaviors is not restricted to colon cancers and earlier studies support the present hypothesis [2–4]. Indeed, intracellular lipid droplets as detected by CARS could serve as a biomarker for prostate circulating tumor cells [3, 4]. Along the same line, an aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases was recently reported [5]. Here, Tirinato et al. [1] also pointed out as “a remarkable correlation” the observation that the lipid droplet content in CR-CSCs (as measured by flow cytometry using hydrophobic dyes) is elevated in cells with higher expression of CD133 (prominin-1) and Wnt/b-catenin pathway activity. Unfortunately, it is not further substantiated and no link between CD133, Wnt/b-catenin signaling pathway, and lipid droplets is provided. It is now well accepted that CD133/1 and CD133/2 epitopes may be undetected despite the presence of CD133 protein or its mRNA, and that CD133 protein may be expressed beyond stem and CSCs [6, 7]. It would have been interesting to document the respective profiles of the cancer cell lines (HCT116 and RKO) used in this study as colon cancer cells (CCC) and how they correlate with their Wnt/b-catenin activity levels. Yet the observations made by Tirinato et al. [1] in colorectal cancers are in line with part of those reported earlier by our groups in melanoma. We have indeed demonstrated a link between CD133 and Wnt/b-catenin pathway and their incidence on the amount of lipid droplets in human melanoma cells as well as their impact on their metastatic capacity [8, 9]. We propose to develop here potential scenarios (Fig. 1), which are not mutually exclusive, and may help the readers of STEM CELLS to further dissect the current observation. In a first series of experiments published in STEM CELLS (2008), we demonstrated that downregulation of CD133 using short hairpin (sh) RNAs resulted in vitro a in slower cell growth, reduced motility, and decreased capacity of melanoma cells to produce spheroids under stem cell-like growth conditions [8]. In vivo, the lack of CD133 severely reduced the capacity of the cells to metastasize. Microarray analysis of CD133-downregulated cells identified a change in expression levels for 143 annotated genes among which 10 of the 76 upregulated ones coded for established or putative Wnt inhibitors (e.g., DKK1 and DACT1) indicating a link between CD133 and the canonical Wnt pathway [8]. In a second series of experiments published in Experimental Cell Research (2013), we were able to demonstrate that the suppression of CD133 prevents the nuclear localization of b-catenin and reduces Wnt pathway signaling through Tcell factor/lymphoid enhancing factor (TCF/ LEF) transcription factors. Nuclear localization of b-catenin was nevertheless restored upon addition of Wnt3a to CD133-knockdown cells, indicating that the Wnt/b-catenin pathway can still be activated by physiological ligands in the absence of CD133 [9]. Moreover, and readily pertinent to the observation of Tirinato et al. [1] this effect was confirmed in the human Caco-2 colon carcinoma cell-line [9]. As TCFLEF-binding sites are found in the PROM1 promoter [20], it could in turn be a direct target of the Wnt pathway. The relation between CD133 and the Wnt/b-catenin pathway is Cancer Research Center, Roseman University College of Medicine, Las Vegas, Nevada, USA; Tissue Engineering Laboratories (BIOTEC), Technische Universit€at Dresden, Dresden, Germany Contract grant sponsor: U.S. NIH; Contract grant number: R01CA133797. Contract grant sponsor: Deutsche Forschungsgemeinschaft; Contract grant numbers: SFB655; TRR83. Contract grant sponsor: S€achsisches Staatsministerium f€ ur Wissenschaft und Kunst; Contract grant number: 47531.60/29/31.

Uridine Affects Liver Protein Glycosylation, Insulin Signaling, and Heme Biosynthesis

Purines and pyrimidines are complementary bases of the genetic code. The roles of purines and their derivatives in cellular signal transduction and energy metabolism are well-known. In contrast, the roles of pyrimidines and their derivatives in cellular function remain poorly understood. In this study, the roles of uridine, a pyrimidine nucleoside, in liver metabolism are examined in mice. We report that short-term uridine administration in C57BL/6J mice increases liver protein glycosylation profiles, reduces phosphorylation level of insulin signaling proteins, and activates the HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. Short-term uridine administration is also associated with reduced liver hemin level and reduced ability for insulin-stimulated blood glucose removal during an insulin tolerance test. Some of the short-term effects of exogenous uridine in C57BL/6J mice are conserved in transgenic UPase1 −/− mice with long-term elevation of endogenous uridine level. UPase1 −/− mice exhibit activation of the liver HRI-eIF-2α-ATF4 heme-deficiency stress response pathway. UPase1 −/− mice also exhibit impaired ability for insulin-stimulated blood glucose removal. However, other short-term effects of exogenous uridine in C57BL/6J mice are not conserved in UPase1 −/− mice. UPase1 −/− mice exhibit normal phosphorylation level of liver insulin signaling proteins and increased liver hemin concentration compared to untreated control C57BL/6J mice. Contrasting short-term and long-term consequences of uridine on liver metabolism suggest that uridine exerts transient effects and elicits adaptive responses. Taken together, our data support potential roles of pyrimidines and their derivatives in the regulation of liver metabolism.

Observation-driven inquiry: Raman spectroscopic imaging illuminates cancer lipid metabolism

Metabolic reprogramming of cancer cells trades energy efficiency for biomass accumulation to support growth and proliferation (1). Indeed, cancer cells are known to have increased endogenous fatty acid synthesis, exogenous fatty acid uptake, and lipid droplet accumulation to support biosynthesis of membrane and signaling molecules (2). Targeting fatty acid biosynthesis has been shown to be an effective means to control cancer cell proliferation (3). Conjugation of drug molecules with lipids for enhanced tumor targeting is an ongoing strategy in drug delivery (4). In addition, intracellular lipid and cholesterol accumulation have been used as novel biomarkers for cancer aggressiveness (5), cancer stem cells (6), and circulating tumor cells (7). Together, lipid metabolism is emerging as an exploitable cellular process for cancer diagnosis and therapy.