Abstract LB-139: Laser scanning cytometry for the imaging and quantitation of membrane lipid rafts in cancer cells

Abstract

Abstract The role of lipid trafficking in the physiological and patho-physiological processes is a subject of intense investigation. The enrichment of a large array of signal transduction components in caveolae and lipid rafts provide testimony to their role as signal portals. Similarly, the formation of intracellular lipid droplets, which were originally thought to be lipid storage organelles, now are known to play an important role in signaling and lipid trafficking. While these cellular components can be assessed using TPLC, mass spectrometry or western blotting, they can also be identified based on the staining of specific lip ids and proteins and analyzed by flow cytometry or using a microscope. We have employed fluorescent based techniques with the laser scanning cytometry (LSC), which allows quantification and localization of fluorescence signals as well as generating digital based images, to investigate lipid rafts and lipid droplets in 3 different cell culture models: 1) B cell lymphomas(Raji and DG75 cell lines) in which we previously reported mislocalization of the apoptosis adaptor protein APAF-1 in the lipid rafts. Nystatin, methyl-beta cyclodextrin and cytocholasin B were used to validate the labeling technique, 2) macrolipophagy, induced during autophagy, in the lung cancer cell line A549 and the cervical cancer cell line HeLa (ECACC) to evaluate the changes in lipid droplets accompanying the induction of autophagic vesicles, and 3) the formation of lipid droplets and raft structures in A549 cells following exposure to a panel of anti-cancer agents. To monitor these events, a sequential multi-colored labeling strategy was employed that allowed for the labeling of the plasma membrane using an antibody to pan-cadherin or by staining with Alexa Fluor 647 conjugated wheat germ agglutinin, prior to fixation, in combination with the labeling of lipid rafts using biotin cholera toxin B coupled with avidin conjugate Q dot 655 and antibodies to caveolin and the pro-apoptotic protein APAF-1. This labeling protocol was used in both cell cultures and in tissues. To detect the formation of lipid droplets, nile red and oil red were used to characterize the lipid content and where possible the number and localization of droplets. A threshold based segmentation strategy was employed that allows for the detection of changes to the total amount of lipid rafts, droplets, or vesicles, together with the digital maxpixel which permits monitoring of clustering or diffusion of events, and the correlation with the cell cycle. Using the profile and segmentation tools, the distance from the plasma membrane or nucleus was calculated and the localization of the events in the cytoplasm, nucleus and plasma membrane were determined using peripheral contouring and masking filters. Furthermore the digital generated images were processed using a set of image filters to generate a “3D” image of the cell or tissue giving a better resolution of events. Results provide a novel imaging strategy for the detection of lipid raft-like structures in cells and tissues, and given their importance in diverse biological systems and pathways could have potential clinical implications Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-139.