Sergio Lage

Analysis of the Lipidome of Xenografts Using MALDI-IMS and UHPLC-ESI-QTOF

AbstractHuman tumor xenografts in immunodeficient mice are a very popular model to study the development of cancer and to test new drug candidates. Among the parameters analyzed are the variations in the lipid composition, as they are good indicators of changes in the cellular metabolism. Here, we present a study on the distribution of lipids in xenografts of NCI-H1975 human lung cancer cells, using MALDI imaging mass spectrometry and UHPLC-ESI-QTOF. The identification of lipids directly from the tissue by MALDI was aided by the comparison with identification using ESI ionization in lipid extracts from the same xenografts. Lipids belonging to PCs, PIs, SMs, DAG, TAG, PS, PA, and PG classes were identified and their distribution over the xenograft was determined. Three areas were identified in the xenograft, corresponding to cells in different metabolic stages and to a layer of adipose tissue that covers the xenograft. FIGUREᅟ

Identification of Biomarkers of Necrosis in Xenografts Using Imaging Mass Spectrometry.

AbstractXenografts are commonly used to test the effect of new drugs on human cancer. However, because of their heterogeneity, analysis of the results is often controversial. Part of the problem originates in the existence of tumor cells at different metabolic stages: from metastatic to necrotic cells, as it happens in real tumors. Imaging mass spectrometry is an excellent solution for the analysis of the results as it yields detailed information not only on the composition of the tissue but also on the distribution of the biomolecules within the tissue. Here, we use imaging mass spectrometry to determine the distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and their plasmanyl- and plasmenylether derivatives (PC-P/O and PE-P/O) in xenografts of five different tumor cell lines: A-549, NCI-H1975, BX-PC3, HT29, and U-87 MG. The results demonstrate that the necrotic areas showed a higher abundance of Na+ adducts and of PC-P/O species, whereas a large abundance of PE-P/O species was found in all the xenografts. Thus, the PC/PC-ether and Na+/K+ ratios may highlight the necrotic areas while an increase on the number of PE-ether species may be pointing to the existence of viable tumor tissues. Furthermore, the existence of important changes in the concentration of Na+ and K+ adducts between different tissues has to be taken into account while interpreting the imaging mass spectrometry results. Graphical Abstractᅟ

Lipid fingerprint image accurately conveys human colon cell pathophysiologic state: A solid candidate as biomarker

Membrane lipids are gaining increasing attention in the clinical biomarker field, as they are associated with different pathologic processes such as cancer or neurodegenerative diseases. Analyzing human colonoscopic sections by matrix assisted laser/desorption ionization (MALDI) mass spectrometry imaging techniques, we identified a defined number of lipid species changing concomitant to the colonocyte differentiation and according to a quite simple mathematical expression. These species felt into two lipid families tightly associated in signaling: phosphatidylinositols and arachidonic acid-containing lipids. On the other hand, an opposed pattern was observed in lamina propria for AA-containing lipids, coinciding with the physiological distribution of the immunological response cells in this tissue. Importantly, the lipid gradient was accompanied by a gradient in expression of enzymes involved in lipid mobilization. Finally, both lipid and protein gradients were lost in adenomatous polyps. The latter allowed us to assess how different a single lipid species is handled in a pathological context depending on the cell type. The strict patterns of distribution in lipid species and lipid enzymes described here unveil the existence of fine regulatory mechanisms orchestrating the lipidome according to the physiological state of the cell. In addition, these results provide solid evidence that the cell lipid fingerprint image can be used to predict precisely the physiological and pathological status of a cell, reinforcing its translational impact in clinical research.

Optimized Protocol To Analyze Changes in the Lipidome of Xenografts after Treatment with 2-Hydroxyoleic Acid.

Xenografts are a popular model for the study of the action of new antitumor drugs. However, xenografts are highly heterogeneous structures, and therefore it is sometimes difficult to evaluate the effects of the compounds on tumor metabolism. In this context, imaging mass spectrometry (IMS) may yield the required information, due to its inherent characteristics of sensitivity and spatial resolution. To the best of our knowledge, there is still no clear analysis protocol to properly evaluate the changes between samples due to the treatment. Here we present a protocol for the evaluation of the effect of 2-hydroxyoleic acid (2-OHOA), an antitumor compound, on xenografts lipidome based on IMS. Direct treated/control comparison did not show conclusive results. As we will demonstrate, a more sophisticated protocol was required to evaluate these changes including the following: (1) identification of different areas in the xenograft, (2) classification of these areas (necrotic/viable) to compare similar types of tissues, (3) suppression of the effect of the variation of adduct formation between samples, and (4) normalization of the variables using the standard deviation to eliminate the excessive impact of the stronger peaks in the statistical analysis. In this way, the 36 lipid species that experienced the largest changes between treated and control were identified. Furthermore, incorporation of 2-hydroxyoleic acid to a sphinganine base was also confirmed by MS/MS. Comparison of the changes observed here with previous results obtained with different techniques demonstrates the validity of the protocol.

Influence of the Cation Adducts in the Analysis of Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry Data from Injury Models of Rat Spinal Cord

Imaging mass spectrometry (IMS) is quickly becoming a technique of reference to visualize the lipid distribution in tissue sections. Still, many questions remain open, and data analysis has to be optimized to avoid interpretation pitfalls. Here we analyze how the variation on the [Na+]/[K+] relative abundance affects the detection of lipids between sections of spinal cord of (uninjured) control rats and of models of spinal cord demyelination and traumatic contusion injury. The [M + Na]+/[M + K]+ adducts ratio remained approximately constant along transversal and longitudinal sections of spinal cord from control animals, but it strongly changed depending on the type of lesion. A substantial increase in the abundance of [M + Na]+ adducts was observed in samples from spinal cord with demyelination, while the intensity of the [M + K]+ adducts was stronger in those sections from mechanically injured spinal cords. Such changes masked the modifications in the lipid profile due to the injury and only after summing the signal intensity of all adducts and corresponding monoprotonated molecular ions of each detected lipid in a single variable, it was possible to unveil the real changes in the lipid profile due to the lesion. Such lipids included glycerophospholipids (both diacyl and aryl-acyl), sphingolipids, and nonpolar lipids (diacyl and triacylglycerols), which are the main lipid classes detected in positive-ion mode. Furthermore, the results demonstrate the sensitivity of the technique toward modification in tissue homeostasis and that the [M + Na]+/[M + K]+ ratio may be used to detect alterations in such homeostasis.

Abstract B32: Role of membrane lipids in colonocytes maturation revealed by mass spectrometry-base imaging techniques

The epithelial layer of the human colon form finger-like invaginations into the underlying connective tissue of the lamina propria to establish the basic functional unit of the intestine, the crypt. Adult stems cells, located at the crypt base, proliferate and differentiate into the mature lineages of the surface epithelium. It is know that any alteration of the pathways regulating stem cell renewal leads to tumor formation. In this context, little is know about how processes as cell differentiation or tumorigenesis affect one of the critical components of a cell: the plasma membrane. Thus, plasma membrane by regulating its lipid composition determines the membrane physical properties, which in turn modulates the activity of receptors, the starting point in many signaling pathways. Mass spectrometry-based imaging (MSI) techniques allow mapping each detected analyte within a tissue section providing spatial information that is crucial to understand the biochemical complexity occurring in living organisms. There are different MSI techniques but MALDI-MS (Matrix Assisted Laser Desorption Ionization Mass Spectrometry) turned out to be particularly suitable for lipid analyses. We took advantage of this fact and we established by MSI techniques the distribution of human mucosa lipid species in 10 μm thick sections obtained from human colonoscopic biopsies. The high spatial resolution achieved, 5 μM, allowed to discriminate the lipid composition of epithelium, lamina propria and muscularis mucosa. A first statistical analysis by K-means clustering showed three clusters that were easily associated to each tissue. However, further analysis revealed additional clusters with plausible biological meaning. We analyzed the lipid composition along the crypt in healthy mucosa, pixel by pixel. Forty lipid species showed extremely regular patterns according to colonocytes differentiation state. Interestingly, they grouped into phosphatidylinositol- and arachidonic-containing lipid species, reinforcing the role that these lipids and its derivatives have in cell proliferation and cell differentiation. It is clear that this regular lipid distribution could be only achieved by coordinating the expression and/or activity of the enzymes involved in the metabolism of these lipid species. To address this issue we analyzed the expression levels of Ca2+-dependent and Ca2+-independent phospholipase A2, cyclooxygenases (COX1 / 2) and fatty acid desaturase (FAD2) by immunofluorescence. Whole crypts were divided in three areas: bottom, middle and top, and the fluorescence intensity for each area was quantified. The statistical analysis of the results showed that the expression levels of some of these enzymes displayed a gradient consistent with the results in lipid composition. Importantly, regular distribution of both lipid species and enzymes was missing in adenomatous polyps. Therefore we can conclude that there is a strict control along the colonic crypt of certain lipid species, particularly those containing arachidonic acid and of the enzymes involved in their metabolism suggesting that they play a role in colonocyte differentiation. Citation Format: Joan Bestard-Escalas, Garate Jone, Albert Maimo-Barcelo, Fernandez Roberto, Sergio Lage, Daniel Horacio Lopez, Rebeca Reigada, Sam Khorrami, Daniel Ginard, Jose Andres Fernandez, Gwendolyn Barcelo-Cobliijn. Role of membrane lipids in colonocytes maturation revealed by mass spectrometry-base imaging techniques. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B32.