Christophis, C.

Grunze, M.

Hanke, M.

Ho, A.

Rosenhahn, A.

Taubert, I.

KITopen

Shear Stress induced Rolling of CD44 expressing Leukaemic CellsMaximilian Hanke1*, Christof Christophis1, Isabel Taubert2,Anthony Ho2, Michael Grunze1,3, Axel Rosenhahn1,31: Applied Physical Chemistry, University Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany2: Department of Medicine V, University Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany3: Institute of Functional Interfaces, Karlsruhe Institute of Technology *E-Mail: m.hanke@stud.uni-heidelberg.det = shear stress; μ = viscosity; Q = volumetric flow; h = channel height; w = channel widthPrinciple of hydrodynamic shear force in a parallel plate flow channel and flow program for the detachment experiment)2(62whQ)1(dydu0 100 200 300 400 5001E-30,010,1110100100010000shear stress [dyn/cm2]Time [s]Program of a time dependent shear stress220 230 240 250 260 2701234shear stress [dyn/cm2]Time [s]Program of a time dependent shear stressIntroduction:Cell adhesion is important as it affects the cell cycle, proliferation and differentiation. Studying the interaction of cells with artificial surfaces can serve as models for biological systems. In leukaemiatherapy such knowledge is important as adhesion is connected with stem cell homing and thus with renewal of the hematopoietic system after leukemia therapy. Recent experiments undercontrolled microfluidic contidions[1] showed that leukaemic progenitor cell lines such as KG-1a and HL-60 are capable of rolling on Hyaluronan surfaces when a shear flow is applied.[1] This rollingphenomenon is similar to that of leukocytes in the extravasation process[2]. Antibody experiments reveal that binding of leukemic cells is CD44 and not selectin mediated. It could be shown that amaximum of cell adherence and thus activation of the catch bonds occurs at a shear stress of ~ 1dyn/cm².[1]The setup: incubation microscope with syringe pump, microchannel and liquid reservoir.References[1] C. Christophis, I. Taubert, G. R. Meseck, M. Schubert, M. Grunze, A.D. Ho, A. Rosenhahn, "Shear stress regulates adhesion and rolling of CD44+ leukemic and hematopoietic progenitor cells on hyaluronan.", BiophysicalJournal, 2011, 101(1)[2] K. Ley, C. Laudanna, M.I. Cybulsky, S. Nourshargh, Nature Reviews Immunology, 2007, 678-689Only the cell lines expressing the CD44 receptor are able to attach from flow. Blood carcinoma cell lines are suspension cells used as an alternative for hematopoietic stem cells to investigate the behavior of primary stem cells. Some types express the CD44 receptor (green stain), others don’t.KG-1a cells Jurkat cellsCellular RollingAn important step in leukocyte extravasation is the rolling of the leukocyte across the endothelium. This phenomenon is selectin mediated.Leukocyte extravasation is a well known process. A similar rolling effect can be observed for cells, such as haematopoietic stem cells, expressing the receptor CD44 on hyaluronan surfaces.. KG-1asuspension cells proved to be a potent reference cell line for experiments adressing the importance of CD44 receptors for cellular rolling on hyaluronan surfaces.0 1 2 3 402004006008001000cell countattachment flow time [min] KG-1a (CD44 ~100%) K-562 (CD44 ~100%) HL-60 (CD44 ~100%) Jurkat (CD44 ~0%) Kasumi-1 (CD44 <5%)The rolling of KG-1a cells is CD44 mediated, unlike leukocyte which is induced by selectins.0 1 2 3 4 505101520cell rolling speed [µm/s]wall shear stress [dyn/cm2]0,000,250,500,751,000,01 0,1 1 10 100 1000Fraction of adherent cells KG-1a cells on alginateshear stress [dyn/cm2] KG-1a cells on hyaluronanCell adhesion strength assay: CD44 receptor expressing KG1-a cells barely stick on alginate but show flow induced cell adhesion and rolling on  hyaluronan.KG1-a cells respond actively to applied flow. The rolling speed reaches a maximum of ~ 15 µm/s at ~1 dyn/cm2. CD44 mediated Rolling and its CharacteristicsAccumulation and detachment assays are the two most commonly applied measurement techniques in our microfluidic system. They allowthe calculation of cell rolling speed and the comparison of different cell lines or differently treated cells. The flow induced adhesion can beobserved beyond a critical shear stress of ~ 0.2 dyn/cm² up to a maximum at ~ 1 dyn/cm².The rolling of cells expressing CD44 is flow induced and can only be observed beyond a critical shear stress.Accumulation assay: KG-1a cells accumulate on a hyaluronan surface at a constant flow of ~ 0.2 dyn/cm²(~ 0.00014 ml/min).Attachment can be blocked  by antibody blocking of  CD44. Two different techniques of measurement show the same results concerning the adhesion maximum of KG-1a cells on hyaluronan.Conclusions Flow induced rolling of CD44 expressing cellscan be observed after a critical shear stress of~ 0.2 dyn/cm². Rolling can be suppressed by incubation withthe antibody BU52 proving CD44 to be thereceptor responsible for rolling. Rolling speed increases up to a maximum of~ 15 µm/s at ~1 dyn/cm2. Further studies of the CD44-Hyaluronaninteraction, such as Hyaluronan modification. Analogous studies shall be performed withhuman material such as cord blood cells andhaematopoietic stem cells to be able to comparetheir rolling characteristics. Behavior of stem cells under a chemokinegradient shall be studied to further understand thestem cell homing process.Acknowledgments• Natalia Baran for helpful input on the workWith this setup the adhesion strength, cell rolling speed and cell attachment rates can be studied. It is also possible to modify the surface properties to be able to study the surface s effect on cellularadhesion.

Shear stress induced rolling of CD44 expressing leukaemic cells

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Shear stress induced rolling of CD44 expressing leukaemic cells

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