Abstract B32: Role of membrane lipids in colonocytes maturation revealed by mass spectrometry-base imaging techniques
Authors: Bestard-Escalas Joan , Jone Garate , Maimó-Barceló Albert , Roberto Fernández , Lage Sergio , Lopez Daniel Horacio , Reigada Rebeca , Khorrami Sam , Ginard Daniel , Fernández José Andrés , Barceló-Cobliijn Gwendolyn 
Published: May 2016

Abstract
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 Maimó-Barceló, Fernández Roberto, Sergio Lage, Daniel Horacio Lopez, Rebeca Reigada, Sam Khorrami, Daniel Ginard, José Andrés Fernández, Gwendolyn Barceló-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.