A Subset of Host B Lymphocytes Controls Melanoma Metastasis through a Melanoma Cell Adhesion Molecule/MUC18-Dependent Interaction: Evidence from Mice and Humans Fernanda I. Staquicini, 1 Anita Tandle, 2 Steven K. Libutti, 2 Jessica Sun, 1 Maya Zigler, 1 Menashe Bar-Eli, 1 Fabiana Aliperti, 3 Elizabeth C. Pe ´rez, 3 Jeffrey E. Gershenwald, 1 Mario Mariano, 3 Renata Pasqualini, 1 Wadih Arap, 1 and Jose ´ Daniel Lopes 3 1 The University of Texas M. D. Anderson Cancer Center, Houston, Texas; 2 Surgery Branch, National Cancer Institute, Bethesda, Maryland; and 3 Sa ˜o Paulo Federal University, Sa ˜o Paulo, Brazil Abstract Host immunity affects tumor metastasis but the corre- spondingcellularandmolecularmechanismsarenotentirely clear. Here, we show that a subset of B lymphocytes (termed B-1population),butnototherlymphocytes,hasprometastatic effects on melanoma cells in vivo through a direct hetero- typic cell-cell interaction. In the classic B16 mouse mela- noma model, one mechanism underlying this phenomenon is a specific up-regulation and subsequent homophilic inter- action mediated by the cell surface glycoprotein MUC18 (also knownasmelanomacelladhesionmolecule).PresenceofB-1 lymphocytes in a panel of tumor samples from melanoma patients directly correlates with MUC18 expression in mela- nomacells,indicatingthatthesameproteininteractionexists in humans. These results suggest a new but as yet unrecog- nized functional role for host B-1 lymphocytes in tumor metastasis and establish a biochemical basis for such observations. Our findings support the counterintuitive central hypothesis in which a primitive layer of the immune system actually contributes to tumor progression and metas- tasisinamousemodelandinmelanomapatients.Giventhat monoclonal antibodies against MUC18 are in preclinical development but the reason for their antitumor activity is notwellunderstood,thesetranslationalresultsarerelevantin thesettingofhumanmelanomaandperhapsofothercancers. [Cancer Res 2008;68(20):8419–28] Introduction Studies addressing the role of the immune system in tumor growth and metastasis have yielded conflicting and often counterintuitive results. Over the 1970s, Prehn and colleagues proposed that the immune response mediated by lymphoid cells could paradoxically lead to tumor cell stimulation (1–3). To date, the interplay of immunity, inflammation, and cancer is still not entirely understood (4, 5). To add a further level of complexity— depending on the experimental model used—it is evident that host immunity can actually lead to enhancement, suppression, or even no effect at all on the metastatic potential of tumor cells, so that no global generalizations can be easily made (6). Specifically in the B16 mouse melanoma model, previous reports show that melanoma cells can be stimulated by lymphocytes (7) and that melanoma progression can indeed be delayed if tumor- bearing mice are rendered immunosuppressed (8). However, the basis for these intriguing experimental observations remains elusive. In particular, the relevance of cell subpopulations from the more primitive layers of the immune system such as B-1 lymphocytes (9–12) on tumor phenotype has not been fully elucidated, although clues for such a role have recently emerged (13, 14). Here, we have evaluated the cellular and molecular cross-talk by which B-1 lymphocytes affect melanoma growth and metastasis. First,weusedtheclassicB16mousemelanomamodeltoshowthat one mechanism accounting for this observation is the up- regulation and subsequent homophilic interaction of the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule). Next, we show that B-1 lymphocytes are also present in human tumors and directly correlate with MUC18 expression in melanoma cells, indicating that the same functional mechanism is conserved across species and likely active in human disease. Together, our results strongly suggest an important role for host B-1 lymphocytes in melanoma-derived metastasis and its corresponding biochemical basis in tumor-bearing mice and in patients. Materials and Methods Animals. Female mice were purchased and housed in the animal facilities of the University of Texas M. D. Anderson Cancer Center, Federal University of Sa ˜o Paulo, or University of Campinas. All animal procedures were approved by the respective Institutional Animal Care and Use Committee. Human specimens. Incidental human melanoma samples were obtained, through written informed consent, from patients treated at the Surgery Branch of the National Cancer Institute (NCI) or at The University of Texas M. D. Anderson Cancer Center. Reagents. Anti-MUC18 (mouse and human) antibodies were purchased from Santa Cruz Biotechnology and Zymed. Antibacteriophage (Sigma) and FITC-conjugated anti-human IgM, allophycocyanin-conjugated anti-human CD5, and phycoerythrin (PE)-conjugated anti-human MUC18 (BD Bio- sciences) were commercially obtained. MART-1 antibody was purchased from BioGenex and labeled with FITC by using EZ-Label FITC Protein Labeling kit (Pierce) and Zeba desalt spin columns (Pierce). Horsera- dish peroxidase (HRP)-conjugated anti-rabbit, PE-conjugated anti-mouse (Pharmingen), Cy3-conjugated anti-rabbit antibodies were purchased from Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requestsforreprints: Wadih Arap and Renata Pasqualini, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3873; Fax: 713-745-2999; E-mail: warap@mdanderson.org and rpasqual@mdanderson.org or Jose ´ Daniel Lopes, Federal University of Sa ˜o Paulo, Rua Botucatu 862, Sa ˜o Paulo 04023-062, Brazil. Phone: 55-011-5576-4532; Fax: 55-011- 5571-1095; E-mail: jdlopes@unifesp.br. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-08-1242 www.aacrjournals.org 8419 Cancer Res 2008; 68: (20). October 15, 2008 Research Article Research. on February 25, 2016. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from