Cell culture metabolomics in the diagnosis of lung cancer - The influence of cell culture conditions

Lung cancer is the leading cause of cancer deaths. Unfortunately, lung cancer is often diagnosed only when it becomes symptomatic or at an advanced stage when few treatment options are available. Hence, a diagnostic test suitable for screening widespread populations is required to enable earlier diagnosis. Analysis of exhaled breath provides a non-invasive method for early detection of lung cancer. Analysis of volatile organic compounds (VOCs) by various mass spectral techniques has identified potential biomarkers of disease. Nevertheless, the metabolic origins and the disease specificity of VOCs need further elucidation. Cell culture metabolomics can be used as a bottom-up approach to identify biomarkers of pathological conditions and can also be used to study the metabolic pathways that produce such compounds. This paper summarizes the current knowledge of lung cancer biomarkers in exhaled breath and emphasizes the critical role of cell culture conditions in determining the VOCs produced in vitro. Hypoxic culture conditions more closely mimic the conditions of cancer cell growth in vivo. We propose that since hypoxia influences cell metabolism and so potentially the VOCs that the cancer cells produce, the cell culture metabolomics projects should consider culturing cancer cells in hypoxic conditions

Ell3 Enhances Differentiation of Mouse Embryonic Stem Cells by Regulating Epithelial-Mesenchymal Transition and Apoptosis

Ell3 is a testis-specific RNA polymerase II elongation factor whose cellular function is not clear. The present study shows that Ell3 is activated during the differentiation of mouse embryonic stem cells (mESCs). Furthermore, Ell3 plays a critical role in stimulating lineage differentiation of mESCs by promoting epithelial-mesenchymal transition (EMT) and suppressing apoptosis. Mouse ESCs engineered to stably express Ell3 were rapidly differentiated compared with control cells either under spontaneous differentiation or neural lineage-specific differentiation conditions. Gene expression profile and quantitative RT-PCR analysis showed that the expression of EMT markers, such as Zeb1 and Zeb2, two major genes that regulate EMT, was upregulated in Ell3-overexpressing mESCs. Remarkably, knockdown of Zeb1 attenuated the enhanced differentiation capacity of Ell3-overexpressing mESCs, which indicates that Ell3 plays a role in the induction of mESC differentiation by inducing EMT. In contrast to Ell3-overexpressing mESCs, Ell3-knock down mESCs could not differentiate under differentiation conditions and, instead, underwent caspase-dependent apoptosis. In addition, apoptosis of differentiating Ell3-knock out mESCs was associated with enhanced expression of p53. The present results suggest that Ell3 promotes the differentiation of mESCs by activating the expression of EMT-related genes and by suppressing p53 expression

Interstitial fluid: the overlooked component of the tumor microenvironment?

Background: The interstitium, situated between the blood and lymph vessels and the cells, consists of a solid or matrix phase and a fluid phase, together constituting the tissue microenvironment. Here we focus on the interstitial fluid phase of tumors, i.e., the fluid bathing the tumor and stromal cells. Novel knowledge on this compartment may provide important insight into how tumors develop and how they respond to therapy. Results: We discuss available techniques for interstitial fluid isolation and implications of recent findings with respect to transcapillary fluid balance and uptake of macromolecular therapeutic agents. By the development of new methods it is emerging that local gradients exist in signaling substances from neoplastic tissue to plasma. Such gradients may provide new insight into the biology of tumors and mechanistic aspects linked to therapy. The emergence of sensitive proteomic technologies has made the interstitial fluid compartment in general and that of tumors in particular a highly valuable source for tissue-specific proteins that may serve as biomarker candidates. Potential biomarkers will appear locally at high concentrations in the tissue of interest and will eventually appear in the plasma, where they are diluted. Conclusions: Access to fluid that reliably reflects the local microenvironment enables us to identify substances that can be used in early detection and monitoring of disease

Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis

<p>Abstract</p> <p>Background</p> <p>Lipid-rich tumours have been associated with increased cancer metastasis and aggressive clinical behaviours. Nonetheless, pathologists cannot classify lipid-rich tumours as a clinically distinctive form of carcinoma due to a lack of mechanistic understanding on the roles of lipids in cancer development.</p> <p>Methods</p> <p>Coherent anti-Stokes Raman scattering (CARS) microscopy is employed to study cancer cell behaviours in excess lipid environments <it>in vivo </it>and <it>in vitro</it>. The impacts of a high fat diet on cancer development are evaluated in a Balb/c mice cancer model. Intravital flow cytometry and histology are employed to enumerate cancer cell escape to the bloodstream and metastasis to lung tissues, respectively. Cancer cell motility and tissue invasion capability are also evaluated in excess lipid environments.</p> <p>Results</p> <p>CARS imaging reveals intracellular lipid accumulation is induced by excess free fatty acids (FFAs). Excess FFAs incorporation onto cancer cell membrane induces membrane phase separation, reduces cell-cell contact, increases surface adhesion, and promotes tissue invasion. Increased plasma FFAs level and visceral adiposity are associated with early rise in circulating tumour cells and increased lung metastasis. Furthermore, CARS imaging reveals FFAs-induced lipid accumulation in primary, circulating, and metastasized cancer cells.</p> <p>Conclusion</p> <p>Lipid-rich tumours are linked to cancer metastasis through FFAs-induced physical perturbations on cancer cell membrane. Most importantly, the revelation of lipid-rich circulating tumour cells suggests possible development of CARS intravital flow cytometry for label-free detection of early-stage cancer metastasis.</p

Broadband infrared photometry of comet Hale-Bopp with ISOPHOT

Comet Hale-Bopp was observed five times with ISOPHOT, the photometer on board ESA's Infrared Space Observatory (ISO) between 4.6 and 2.8 AU. Each time, broadband photometry was performed using 4 different detectors, 5 apertures and 10 filters covering the range between 3.6 and 170 μm. Background observations were performed with identical instrument settings at the same positions on the sky several days after the comet observations. The observation strategy and the data reduction steps are described in some detail, including the techniques to correct for variable detector responsivity. The resulting inband power values of the Hale-Bopp observations and their uncertainties are given. The mean uncertainty is 25%. The final fluxes were computed, taking into account the zodiacal background, possible offset of the comet's position from the center of the aperture, the brightness distribution within the coma, and the spectral energy distribution of the comet's emission. Strong thermal emission from a broad size distribution of dust particles was detected in all of the data sets, even at r = 4.6-4.9 AU pre-perihelion and 3.9 AU post-perihelion; the total thermal energy varied as r-3. The 7.3-12.8 μm color temperature was ~1.5 times the blackbody temperature, higher than that observed in any other comet. Silicate features at 10 and 25 μm were prominent in all 5 data sets, the largest heliocentric distances that silicate emission has been detected in a comet. The presence of crystalline water ice grains is suggested from the 60 μm excess emission at 4.6-4.9 AU, consistent with the observed QOH if the icy grains were slightly warmer than an equilibrium blackbody. The average albedo of the dust is higher than that of comet P/Halley, but lower than other albedo measurements for Hale-Bopp nearer perihelion. There is no evidence for a component of cold, bright icy grains enhancing the scattered light at 4.6 AU. Simple models for a mixture of silicate and absorbing grains were fit to the ISO spectra and photometry at 2.8 AU. The observed flux at λ >100 μm requires a size distribution in which most of the mass is concentrated in large particles. Dust production rates of order 1.5 x 105 kg s-1 at 2.8 AU and 3 x 104 kg s-1 at 4.6 AU have been found. They correspond to dust to gas mass ratios of 6 to 10

Reelin Is Involved in Transforming Growth Factor-β1-Induced Cell Migration in Esophageal Carcinoma Cells

Reelin (RELN), which is a glycoprotein secreted by Cajal-Retzius cells of the developing cerebral cortex, plays an important role in neuronal migration, but its role in cell migration and cancer metastasis is largely unclear. Here, we showed that cell motility was significantly increased in KYSE-510 cells by TGF-β1 treatment. Moreover, TGF-β1 decreased RELN mRNA expression and overexpression of Reelin at least partly reversed TGF-β1-induced cell migration in KYSE-30 cells. Furthermore, this negative regulation of Reelin expression by TGF-β1 was through Snail, one transcription factor which was induced by TGF-β1 in KYSE-510 cells. RELN promoter activity was reduced in parallel with the induction of Snail after TGF-β1 treatment and Snail suppressed both RELN promoter activity and expression through binding to E-box sequences in the RELN promoter region in ESCC cells. Knockdown of RELN induced cell migration in KYSE-510 cells, together with the increase of mesenchymal markers expression. Taken together, Reelin is an essential negative regulator in the TGF-β1-induced cell migration process, and is suppressed by TGF-β pathway at the transcriptional level through Snail regulation. Therefore, the correlation of Reelin and TGF-β pathway was critical in cancer metastasis, and Reelin could be one potential anti-metastasis target in future clinical practice

The putative Tumor Suppressor VILIP-1 Counteracts Epidermal Growth Factor-Induced Epidermal-Mesenchymal Transition in Squamous Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2′,3′-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner