ORIGINAL ARTICLE TOLL-LIKE RECEPTOR AGONISTS AS THIRD SIGNALS FOR DENDRITIC CELL–TUMOR FUSION VACCINES Edward I. Cho, MD, 1 Chunrui Tan, BS, 2 Gary K. Koski, PhD, 3 Peter A. Cohen, MD, 4 Suyu Shu, PhD, 5 Walter T. Lee, MD 6 1 Head and Neck Institute, Cleveland Clinic, Cleveland, Ohio 2 Division of Otolaryngology–Head and Neck Surgery, Duke University Medical Center, Durham, North Carolina 3 Center for Surgery Research, Cleveland Clinic, Cleveland, Ohio 4 Hematology/Oncology, Mayo Clinic, Scottsdale, Arizona 5 Laboratory of Molecular and Tumor Immunology, Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Portland, Oregon 6 Division of Otolaryngology–Head and Neck Surgery, Duke University Medical Center, Durham, North Carolina. E-mail: walter.lee@duke.edu Accepted 16 July 2009 Published online 11 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/hed.21241 Abstract: Background. The aim of the present study was to evaluate the therapeutic efficacy of dendritic cell (DC)–tumor fusion hybrids with Toll-like receptor (TLR) agonists. Methods. DC–tumor fusion hybrids were generated by electrofusion and injected into the inguinal lymph nodes of C57BL/6 mice with 3-day established pulmonary metastases. Paired TLR agonists polyinosine:polycytadilic acid [poly(I:C)] and cytosine–phosphate–guanine (CpG) were then injected in- traperitoneally. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate interleukin (IL)-12 production from the DC–tumor fusion hybrids in vitro. Results. Fusion þ TLR agonists (60 metastases) had signif- icantly fewer metastases than did the untreated control (262 metastases, p ¼ .0001) and fusion alone (150 metastases, p ¼ .02). ELISA showed that the DC–tumor fusion hybrids yielded 90 pg of IL-12 after TLR stimulation compared with 1610 pg from dendritic cells alone. Conclusions. CpG and poly(I:C) administered as a third signal with fusion hybrids as described significantly reduce melanoma metastasis compared with fusion hybrids alone. Fusion hybrids do not appear to be a significant source for IL- 12 secretion. V V C 2009 Wiley Periodicals, Inc. Head Neck 32: 700–707, 2010 Keywords: tumor immunology; dendritic cells; Toll-like receptor agonists; electrofusion; third signal Despite advances in chemotherapy, radiation, and surgical therapies, treatment of advanced head and neck melanoma continues to be associ- ated with high morbidity and mortality. As a result, other modalities of treatment have been Correspondence to: W. T. Lee This work was presented at the 2009 Combined Southern and Middle Section Meetings, Triological Society, Bonita Springs, FL, January 11, 2009. Contract grant sponsor: Lerner Institute Research Program Council; contract grant sponsor: Cleveland Clinic; contract grant sponsor: Triological Society Career Development Award; contract grant sponsor: Flight Attendants Medical Research Institute Young Clinical Scientist Award. V V C 2009 Wiley Periodicals, Inc. 700 Toll-Like Receptor Agonists and Tumor Fusion Vaccines HEAD & NECK—DOI 10.1002/hed June 2010