0041-1337/03/7612-1735/0 TRANSPLANTATION Vol. 76, 1735–1741, No. 12, December 27, 2003 Copyright © 2003 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. MYCOPHENOLATE MOFETIL INCREASES ADHESION CAPACITY OF TUMOR CELLS IN VITRO 1 ROMAN A. BLAHETA, 2,5 HARILAOS BOGOSSIAN, 2 WOLF-DIETRICH BEECKEN, 3 DIETGER JONAS, 3 CHRISTOPH HASENBERG, 3 JASMINA MAKAREVIC, 3 HENRY OGBOMO, 3 WOLF O. BECHSTEIN, 4 ELSIE OPPERMANN, 4 KERSTIN LECKEL, 4 AND JINDRICH CINATL,JR. 2 Background. The immunosuppressive drug myco- phenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leuko- cytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the im- munoglobulin superfamily which, however, act as ho- mophilic binding elements. Because decrease of ho- mophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger de- velopment or recurrence of neoplastic tumors. Methods. The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell mono- layer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, West- ern blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tu- mor cell binding was proven by treating NB with NCAM antisense oligonucleotides. Results. MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 M, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to en- hanced tumor cell adhesion. Conclusions. MMF decreases NCAM receptors, which is associated with enhanced tumor cell inva- siveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins. The novel immunosuppressive drug mycophenolate mofetil (MMF) is the morpholino ethylester prodrug of mycophenolic acid. The active compound mycophenolic acid inhibits T- and B-cell proliferation by blocking the production of guanosine nucleotides required for DNA synthesis. Another mechanism that contributes to the efficacy of MMF in preventing allo- graft rejection is focused toward membranous adhesion re- ceptors, expressed on activated endothelial cells. Several in vitro studies demonstrated that MMF decreases dose-depen- dently two members of the immunoglobulin (Ig) superfamily such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 (1, 2). Both receptors serve as heterophilic binding elements. In the context of cell-mediated organ rejection, alloactivated leukocytes recognize and bind to ICAM-1 and VCAM-1 by means of their specific adhesion li- gands, leukocyte function-associated antigen (LFA)-1 or very late antigen (VLA)-4, respectively. Consequently, decrease of ICAM-1 and VCAM-1 on endothelial cells has a desired benefit because heterophilic cell-cell contact is prevented and leukocyte infiltration into the donor graft is diminished. Other members of the Ig superfamily have been described to possess distinct homotypic adhesive properties such as cell-to-cell adhesion molecule (CAM), the epithelial V-like antigen, activated leukocyte CAM, the colon-carcinoma asso- ciated glycoprotein Ep-CAM, or the neural cell adhesion mol- ecule (NCAM). These receptors play a significant role in the regulation of intercellular cluster formation and aggregation of homotypic cell populations. Disappearance of these mole- cules is accompanied by a loss of homophilic cell-cell contacts and deaggregation of cell conglomerates (3–7). We hypothesized that MMF might also decrease CAM re- ceptors with homotypic characteristics. This property of MMF might be of high clinical relevance in the process of neoplastic tumor recurrence and de novo cancer develop- ment, which are serious complications of immunosuppressive therapy (8). Several publications emphasize that tumor cell dissemination depends on the ability of malignant cells to decrease homotypic binding receptors as a necessary prereq- uisite to allow their escape from the primary tumor or from circulating tumor cell colonies (7, 9). Therefore, reduction of CAM receptors on tumor cell membranes triggered by MMF might enable more tumor cells to leave the tumor aggregate easily and to start the transendothelial invasion program. To support our hypothesis, we analyzed the influence of MMF on the expression of homotypic adhesion receptors and on the capacity of cancer cells to bind to an endothelial cell monolayer. NCAM (CD56) has been chosen as the represen- tative homotypic adhesion molecule because it has been well 1 This work was supported by the Paul und Ursula Klein-Stiftung, the Heinrich und Erna Schaufler-Stiftung, the Gisela Stadelmann- Stiftung, the Matthias Lackas-Stiftung, and the foundation Hilfe fu ¨r krebskranke Kinder Frankfurt e.V. 2 Zentrum der Hygiene, Institut fu ¨ r Medizinische Virologie, Jo- hann Wolfgang Goethe-Universita ¨ t, Frankfurt am Main, Germany. 3 Zentrum der Chirurgie, Klinik fu ¨ r Urologie und Kinderurologie, Johann Wolfgang Goethe-Universita ¨ t, Frankfurt am Main, Germany. 4 Zentrum der Chirurgie, Klinik fu ¨ r Allgemein- und Gefa ¨ chirurgie, Johann Wolfgang Goethe-Universita ¨ t, Frankfurt am Main, Germany. 5 Address correspondence to: Dr. Roman Blaheta, J. W. Goethe- University Hospital, Zentrum der Chirurgie, Klinik fu ¨ r Urologie und Kinderurologie, Wissenschaftliches Labor der Chirurgie, Bldg. 23A, EG 7, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: blaheta@em.uni-frankfurt.de. Received 16 May 2003. Revision requested 9 July 2003. Accepted 10 July 2003. 1735 DOI: 10.1097/01.TP.0000092004.83992.B2