Nanodynamic Imaging of Leukemic Cell Adhesion
Dr. Michel Aurrand-Lions
Director’s research unit:
Cancer Research Centre of Marseille (CRCM)
Description of the PHD thesis project:
Cell junctions play a key role in the integrity of biological tissues, via Cell Adhesion Molecules (CAMs). In particular, in the bone marrow, interactions between hematopoietic and stromal cells allow the mutual transmission of signals involved in the development and homeostasis of both cell types. This crosstalk also involves adhesion mechanisms, with a major impact on the physiology of hematopoietic and stromal cells (development, maintenance, proliferation). Partner I (A. Sergé & M. Aurrand-Lions, Leuko/Stromal Interactions team, CRCM) is specialized in these interactions, especially for integrins and Junctional Adhesion Molecules (JAMs, De Grandis et al. Cell Mol Life Sci 2015). In physiological context, the team reported contacts between JAM-C-expressing hematopoietic stem cells and JAM-B-expressing stromal cells (Arcangeli et al. Blood 2011). These interactions are deeply revised in tumor context. Preliminary data show that JAM-C-mediated adhesion of leukemic stem cell (LSC) to the stroma is involved in Cell-Adhesion-Mediated Drug Resistance (CAM-DR), suggesting that JAM-C constitutes a potential therapeutic target in leukemia.
Recent advances in optical nanoscopy completely revisited the classic pattern of static adhesion structures in cells (Rossier et al. Nat Cell Biol 2012; Paszek et al. Nature 2014). Major players, such as integrins and their adapters, are tightly regulated by association/dissociation mechanisms, modulated according to pathophysiological conditions and signals received by the cell. Brief episodes of confinement or colocalization can reveal molecular events leading to cellular pathway activation. Hence, discrete events can lead to critical outcomes, thanks to non-linear amplification, as often reported in cell signaling. Moreover, integrin-mediated adhesion to collagen, a major component of the extracellular matrix (ECM), is profoundly implicated in tumor evolution. Analysis of molecular trajectories with our homemade software MTT (Serge et al. Nat Met 2008; Rouger et al. JoVE 2012) in combination with simultaneous monitoring on the fine distribution of collagen in the close environment of the living cell will identify interactions during the onset and stabilization of leukemic cells/stroma contacts. Ultra-resolved imaging will document the role of CAMs in the dynamic establishment of cell/cell and cell/ECM adhesion in real time. We will notably study cells weakly or strongly expressing JAM-C, to assess the impact of blocking antibodies in LSC/stroma interaction.
Simultaneous visualization of collagen fibers and CAM dynamics requires developing a specific, multi-modal imaging system on live cells. Partner II (S. Monneret, biophotonics group, Fresnel Institute) will conduct such an instrumental development thanks to its expertise both in nanoscopy (Bon et al. Nature Comm. 2015) and in phase imaging (development of a now commercially available phase imaging system in close collaboration with a SME). Partner II already proposed a fluorescence/ phase bimodal microscope (Bon et al. JBO 2012) and more recently a modality particularly adapted to enhance real-time visualization of collagen fibers (Aknoun et al. Opt Exp 2014). In this project, we propose to improve the system so as to combine single molecule fluorescence imaging in the visible wavelengths range, with both phase and intensity imaging in the infrared (IR) range. Indeed, cancer cells exhibit remarkable IR signatures through collagen and lipids, especially in IR wavelengths, which could be exploited. Partner III, First Light Imaging, is a company that provides world fastest cameras with single-molecule sensitivity, initially developed for astronomy. We plan to integrate this new generation of cameras into the phase imaging system. EMCCD has already been integrated for nonlinear phase imaging (Berto et al. PRL 2012). We will use it either in 2D, plating cells on coated coverslips, or in 3D, cultivating cells within collagen spheroids, to obtain more physiological cell geometries.
3I dimensions and other aspects of the project:
This biophysical project is fundamentally interdisciplinary. Indeed, on one side, it concerns the biology of cell adhesion molecules in leukemic context (expertise from Partner I). This includes molecular biology, cell biology, cell culture, and possibly transgenic animal use, such as JAM-C KO mice, available in the team. On the other side, the project addresses the physics of Brownian motion and its repercussions on the organization of the cell membrane (shared expertise between Partners I & II), in combination with a need to develop optical instrumentation and innovative imaging techniques (partners II & III). The project will also use different existing imaging modes to compare/validate the new system, including fast videonanoscopy (based on spinning disc confocal microscopy), Total Internal Reflection Fluorescence (TIRF, particularly suited to observe the basal cell membrane coated on collagen) and Fluorescence Recovery After Photobleaching (FRAP, an interesting alternative to measure diffusion within cell membranes). These technics are all limited by detection sensitivity and acquisition speed. This limit will be challenged by the potential of the cameras of Partner III. We will also develop dedicated innovative tools for automated single molecule tracking, analysis of molecular trajectories and collagen fibers in phase images. Such experimental and analytical developments are expected to ultimately beneficiate to the whole academic community. Notably, Partner II is member of France Bio Imaging and France Life Imaging French national structures, implying that technological results will be shared within at least French national bio-imaging facilities.
At the international level, we have a long-standing collaboration with the team of S. Nourshargh in London (Leinster et al. FASEB J 2013; Scheiermann et al. ATVB 2009; Scheiermann et al. Science 2007). Together we have addressed questions related to the dynamic of CAMs during leukocyte trans-endothelial migration in inflammatory conditions. The imaging system used in this particular setting consists in fast spinning disk confocal video-microscopy and acquisition of time-lapse z-stacks on exteriorized tissues of living animals (Woodfin et al. Nat Immunol 2011). Here again, the major limitation is the acquisition speed since the transmigration event by itself may take less than one minute and sequential remodeling of adhesive structures is in the frame of seconds. The PhD student will have the opportunity to join the team of S. Nourshargh in order to translate observations made in vitro using our imaging modality to in vivo situations.
As described above, the imaging system we are proposing needs to be very efficient in the visible as in the IR spectral domains, in order to reach single molecule as well as single collagen fiber imaging and cancer signature, respectively. It must also be very rapid to allow large averaging in order to improve phase sensitivity. Both limits are technologically challenging, calling for intersectoral collaboration with industry to gain access to top-of-the-art cameras. Partner III develops high-speed low-noise EMCCD cameras (able to run up to 3500 frames/s with sub-electron readout noise), and starts to extend their applications to biology. They agree to borrow cameras so as to reach both required sensitivity and rapidity that should simultaneously allow exhibiting cancer signatures and resolving collagen fibers in phase microscopy. Access to such outstanding cameras will be a key advantage for monitoring adhesion dynamics of molecules in their complex environment.
Of course, we plan to communicate our major results at international conferences, before or upon publication in peer-review journals. We notably plan to attend the European or American Biophysical Meetings. Project valuation is also clearly expected by industrial Partner III.