A novel mechanism for anti-EGFR antibody action involves chemokine-mediated leukocyte infiltration Thomas K. Hoffmann 1,2 * , Kerstin Schirlau 1 , Enik€ o Sonkoly 1 , Sven Brandau 2 , Stephan Lang 2 , Andor Pivarcsi 1 , Vera Balz 1 , Anja M€ uller 3 , Bernhard Homey 4 , Edwin Boelke 3 , Torsten Reichert 5 , Ulrike Friebe-Hoffmann 6 , Jens Greve 1,2 , Patrick Schuler 1,2 , Kathrin Scheckenbach 1 ,J€ org Schipper 1 , Murat Bas 7 , Theresa L. Whiteside 8 and Henning Bier 7 1 Department of Otorhinolaryngology, Heinrich-Heine-University, D€ usseldorf, Germany 2 Department of Otorhinolaryngology, University of Duisburg-Essen, Essen, Germany 3 Department of Radiooncology, Heinrich-Heine-University, D€ usseldorf, Germany 4 Department of Dermatology, Heinrich-Heine-University, D€ usseldorf, Germany 5 Department of Maxillofacial Surgery, University of Regensburg, Regensburg, Germany 6 Department of Obstetrics and Gynecology, Heinrich-Heine-University, D€ usseldorf, Germany 7 Department of Otorhinolaryngology, Technical University of Munich, Munich, Germany 8 Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PA Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of squamous cell carcinoma of the head and neck (SCCHN). Monoclonal antibodies (mAbs) against EGFR are cur- rently used for therapy of recurrent or metastatic disease; how- ever, their mode of action is not completely understood. To inves- tigate the immunological effects of anti-EGFR mAb, we generated a three-dimensional spheroid model of EGFR-expressing SCCHN and used this model to study the effect of anti-EGFR mAb on leu- kocyte migration toward tumors. Pretreatment with the blocking anti-EGFR mAb EMD 72000, its F(ab 0 )2 fragments or an EGFR tyrosine kinase inhibitor led to substantially increased leukocyte infiltration into EGFR overexpressing tumor spheroids, but not into those with low EGFR expression. Nonblocking anti-EGFR mAb or fibroblast-specific mAb did not affect leukocyte infiltra- tion, suggesting that the observed increase in leukocyte infiltration depends on interference with EGFR activation. Using a human cytokine macroarray, we demonstrated that the blockade of EGFR by anti-EGFR mAb in EGFR-overexpressing SCCHN cells leads to differential expression of several cytokines and chemo- kines, including the chemokine MCP-1/CCL-2. The significant upregulation of MCP-1/CCL2 on exposure to anti-EGFR mAb was confirmed by quantitative PCR and enzyme-linked immuno- spot analyses. Moreover, blocking anti-MCP-1 antibody inhibited leukocyte migration toward tumor cells induced by anti-EGFR mAb, pointing to an important role of MCP-1/CCL2 in anti-EGFR mAb-induced leukocyte migration. Our findings demonstrate that anti-EGFR mAb induces leukocyte infiltration to tumor spheroids by upregulating chemokine expression. This novel mechanism for anti-EGFR mAb action may contribute to the antitumor effects of anti-EGFR mAb in vivo. ' 2009 Wiley-Liss, Inc. Key words: epidermal growth factor receptor; head and neck cancer; spheroids; immune cells; PBMC; chemotaxis Cellular transformation is associated with aberrant or unregu- lated expression of growth factors, growth factor receptors and components of their intracellular signaling pathways. Conse- quently, these molecules are believed to represent key elements involved in the development and progression of cancer. 1,2 The epi- dermal growth factor receptor (EGFR) is a transmembrane phos- phoglycoprotein that has been identified in almost all adult tissues with the exception of hematopoietic cells, and overexpression and constitutive activation of EGFR is a hallmark of several solid human cancers, including squamous cell carcinoma of the head and neck (SCCHN). 3–6 Binding of peptide growth factors of the epidermal growth factor (EGF) family to the extracellular EGFR domain induces receptor dimerization and autophosphorylation of its cytoplasmic domain, hereby triggering a complex system of in- tracellular signals which culminate in the phosphorylation of cel- lular response proteins and transcription factors. 7,8 These EGFR- mediated events modulate a variety of biological phenomena known to be involved in the expression of the malignant pheno- type, such as proliferation, differentiation, apoptosis, adhesion, invasion and angiogenesis. 9,10 Hence, the EGFR is considered to be a primary target for antitumor treatment strategies. As the re- ceptor is located in the tumor cell surface membrane, monoclonal antibodies (mAb) raised against epitopes on the external domain of the human EGFR seem to be suitable agents for antitumor ther- apy. 9,11,12 Unconjugated mAb may act either through interference with receptor-ligand interactions, thus blocking EGFR tyrosine ki- nase activity, or by coating of tumor cells with immunoglobulin (opsonization), which facilitates the recruitment and activation of host immune cells. In a number of in vitro and in vivo models of SCCHN, EGFR mAb have been shown to exert multiple effects, including inhibition of tumor proliferation, induction of terminal differentiation and modulation of chemo- and radiosensitiv- ity. 11,13–15 Previously, we reported on the potential of EGFR mAb to induce antibody-directed cellular cytotoxicity (ADCC) in estab- lished cell lines of SCCHN. 16 This study, using a multicellular tu- mor spheroid model of SCCHN, extends the scope of EGFR mAb- mediated effects to include a novel mechanism. Here, we show a substantially increased infiltration of leukocytes into tumor sphe- roids following pretreatment with anti-EGFR mAb and identify molecular mechanisms responsible for anti-EGFR mAb-mediated infiltration of immune cells into tumors. Material and methods Cell lines and culture conditions Tumor cell lines (UD-SCC 4, 5, 6) and autologous fibroblast cultures were previously established from patients with SCCHN. 17 In addition, we used the SCCHN lines UM-SCC 14C and 22B established in the laboratory of Dr. Thomas Carey at the Univer- sity of Michigan (UM) (Ann Arbor, MI), and HLaC 79 isolated by H.-P. Zenner (Tubingen, Germany). Tumor cell lines and fibro- blasts were grown in plastic culture flasks (Greiner, Solingen, Ger- many) under standard conditions (37°C, 5% CO 2 , fully humidified atmosphere) in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (both Gibco, Eggenstein, Germany), 2 mM L-glu- tamine, 50 IU/ml penicillin and 50 lg/ml streptomycin (all ICN, Grant sponsors: University of D€ usseldorf, German Cancer Foundation. *Correspondence to: Department of Otorhinolaryngology, University of Essen, Hufelandstr. 55, University of Duisburg-Essen, 45147 Essen, Germany. E-mail: tkhoffmann@web.de Received 12 July 2008; Accepted after revision 16 December 2008 DOI 10.1002/ijc.24269 Published online 13 January 2009 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; MCP-1, monocyte chemotac- tic protein-1; PBMC, peripheral blood mononuclear cells; SCCHN, squa- mous cell carcinoma of the head and neck; T, tumor; T/F, tumor-fibroblast. Int. J. Cancer: 124, 2589–2596 (2009) ' 2009 Wiley-Liss, Inc. Publication of the International Union Against Cancer