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Chronic lymphocytic leukemia cells induce anti-apoptotic effects of bone marrow stroma

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Abstract

The prolonged life span of chronic lymphocytic leukemia (CLL) cells in vivo is assumed to depend on the surrounding microenvironment since this biologic feature is lost in vitro. We studied here the molecular interactions between CLL cells and their surrounding stroma to identify factors that help CLL cells to resist apoptosis. Sorted CLL cells from 21 patients were cultured in vitro on allogenous, normal bone marrow stromal cells (BMSCs) in the presence/absence of CD40 ligand or in culture medium alone. Surface and mRNA expression of interaction molecules, cytokine production, and apoptosis rate was measured by flow cytometric, real-time PCR and standard immunologic assays. The interaction between CLL cells and BMSCs rescued CLL cells from apoptosis. BMSCs co-cultured with CLL cells showed a strong increase in IL-8 and IL-6 secretion and up-regulated the expression of ICAM-1 and CD40 mRNA. The mRNA expression of CXCL12 and VCAM1 remained unchanged. In turn, CLL cells in interaction with BMSCs significantly up-regulated the expression of CD18 and CD49d that are ligands for the critical adhesion molecules on BMSCs. As a validation of the in vitro data, we found a significant higher expression of CD49d on CLL cells in bone marrow aspirates compared to peripheral blood CLL cells in patient samples. Up-regulation of adhesion molecules and their ligands in CLL–BMSCs interaction along with the increased cytokine production of BMSCs indicate a strong effect of CLL cells on BMSCs in favor of their apoptosis resistance.

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Acknowledgements

We would like to thank Prof. Kreuser, Department of Oncology and Hematology, Barmherzige Brüder Hospital, Regensburg for providing the patient samples. We thank Birgit Wilhelm and Harry Isslinger for their excellent technical assistance.

Funding

This work was supported by grants from the the Catholic Academic Exchange Service (KAAD), the Bavarian Research Foundation (BFS), the German Research Foundation SFB Transregio-13 Project, the Fraunhofer Project Group Regensburg and the LipidomicNet (FP7-HEALTH 2007-2.1.1.6-202272) project.

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Authors and Affiliations

  1. Institute of Pathology, University of Regensburg, 93053, Regensburg, Germany

    Márk Plander, Silvia Seegers, Ferdinand Hofstädter & Gero Brockhoff

  2. Institute for Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, 93053, Regensburg, Germany

    Peter Ugocsai, Evelyn Orsó & Gerd Schmitz

  3. Department of Hematology and Clinical Oncology, University Hospital Regensburg, 93053, Regensburg, Germany

    Albrecht Reichle

  4. Department of Hematology, County Hospital of Vas, 9700, Szombathely, Hungary

    Márk Plander

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  1. Márk Plander
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Correspondence to Peter Ugocsai.

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Márk Plander and Peter Ugocsai contributed equally to the manuscript.

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Supplemental Fig. 1

Characterization of BMSCs. a Immunohistochemistry of BMCSs is positive for vimentin, but not for CD68 b mRNA expression in stromal cells. Bar graphs indicate high expression of VCAM-1, CXCL12, ICAM1, IL-6, and IL-8 and less, but detectable IL-1β, CD40, TNF-α. c Surface expression of antigens on stromal cells; mAb (black line), isotype control (broken line). d Secreted cytokines in supernatant. IL-6 and IL-8 is actively secreted, IL-1β and TNF-α to a lesser extent. Results for each mRNA expression and cytokine concentration are presented as mean ± percentiles of at least five independent experiments (PDF 130 kb)

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Plander, M., Ugocsai, P., Seegers, S. et al. Chronic lymphocytic leukemia cells induce anti-apoptotic effects of bone marrow stroma. Ann Hematol 90, 1381–1390 (2011). https://doi.org/10.1007/s00277-011-1218-z

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