© 2016 Nature America, Inc. All rights reserved. ARTICLES NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1 The management of patients with metastatic colorectal cancer (mCRC) has greatly improved over the past two decades, and the median overall survival has increased from 12 months to approximately 30 months 1 , owing mainly to the introduction of new chemotherapeutics and targeted therapies, including cetuximab 2 . Cetuximab is a chimeric monoclonal antibody directed to the EGFR and is effective in the treat- ment of mCRC, either alone or in combination with chemotherapy 3,4 . Cetuximab blocks the interaction between EGFR and its ligands, thus inhibiting downstream RAS-signaling cascade and ERK activation. Hence, activating mutations of RAS affect the efficacy of cetuximab 5,6 , and treatment is presently tailored to patients who carry a wild-type (WT) form of RAS 3 . Panitumumab is another EGFR-neutralizing antibody, and it has an efficacy very similar to that of cetuximab 7 . However, panitumumab, a fully human IgG2 antibody, is unable to promote antibody-dependent, cell-mediated cytotoxicity (ADCC) 7 by natural killer (NK) cells. Therefore, the major mechanism of action of EGFR-monoclonal-antibody targeted therapy seems to be medi- ated by the antibodies’ capacity to block EGFR–ligand interaction and downstream signaling 8 . Yet individuals who carry certain mutations in the Kirsten rat sarcoma viral oncogene homolog KRAS (KRAS G12D and KRAS G13D ) 9,10 occasionally show some clinical benefit of EGFR- targeting antibodies. This suggests that other mechanisms are involved in mediating the efficacy of EGFR-targeting treatments. Most anticancer agents kill tumor cells in a nonimmunogenic fash- ion, whereas some drugs, such as anthracyclines and oxaliplatin, or ionizing irradiation induce an immunogenic modality of tumor cell death (ICD) 11 . ICD makes the cells ‘visible’ to the immune system and, in particular, to dendritic cells (DCs) that phagocytose dying tumor cells and that initiate a strong antitumor response. Cells that are undergoing ICD upregulate the expression of ‘eat me’ signals, such as calreticulin (CRT), on the cell surface, which enables the phagocytosis of such dying cells by DCs 12 . The release of high-mobility-group box 1 (HMGB1) and ATP are also observed during ICD induction 13 . Here we show that cetuximab, when used in combination with FOLFIRI (a standard chemotherapy regimen for mCRC that does not induce ICD 14 ), induces ICD in CRC cell lines and in a mouse model of CRC. RESULTS Cetuximab triggers ER stress response and CRT translocation We analyzed the effects of antibody treatment on human DiFi CRC cells, which are highly sensitive to cetuximab 15 . These cells are WT for KRAS, NRAS, BRAF and PIK3CA and therefore recapitulate the molecular features of CRC that are likely to respond to anti- EGFR therapies. We treated DiFi cells with FOLFIRI, cetuximab or the combination of the two (F + C) in a dose-dependent fashion. We observed apoptosis upon treatment with F, C or F + C, as measured by the expo- sure of phosphatidylserine (Fig. 1a). One described event of ICD is the pre-apoptotic translocation of CRT and of the ER-associated protein 1 Department of Experimental Oncology, European Institute of Oncology, Milan, Italy. 2 Unit of Gastrointestinal and Neuroendocrine Tumors, Division of Medical Oncology, European Institute of Oncology, Milan, Italy. 3 IFOM, Fondazione Istituto FIRC (Fondazione Italiana per la Ricerca sul Cancro) di Oncologia Molecolare, Milan, Italy. 4 Candiolo Cancer Institute–Fondazione Piemontese per l′Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Candiolo, Italy. 5 Department of Oncology and Hemato-oncology, University of Milan, Milan, Italy. 6 Department of Oncology, University of Torino, Torino, Italy. Correspondence should be addressed to M.R. (maria.rescigno@ieo.eu). Received 14 December 2015; accepted 7 March 2016; published online 2 May 2016; doi:10.1038/nm.4078 The EGFR-specific antibody cetuximab combined with chemotherapy triggers immunogenic cell death Chiara Pozzi 1 , Alessandro Cuomo 1 , Ilaria Spadoni 1 , Elena Magni 2 , Alessio Silvola 1 , Alexia Conte 3 , Sara Sigismund 3 , Paola Simona Ravenda 2 , Tiziana Bonaldi 1 , Maria Giulia Zampino 2 , Carlotta Cancelliere 4 , Pier Paolo Di Fiore 1,3,5 , Alberto Bardelli 3,6 , Giuseppe Penna 1 & Maria Rescigno 1,5 Cetuximab is a monoclonal antibody that is effective in the treatment of metastatic colorectal cancer (mCRC). Cetuximab blocks epidermal growth factor receptor (EGFR)-ligand interaction and inhibits downstream RAS–ERK activation. However, only some activating mutations in RAS affect cetuximab efficacy, and it is not clear what else mediates treatment success. Here we hypothesized that cetuximab induces immunogenic cell death (ICD) that activates a potent antitumor response. We found that cetuximab, in combination with chemotherapy, fostered ICD in CRC cells, which we measured via the endoplasmic reticulum (ER) stress response and an increase in phagocytosis by dendritic cells. ICD induction depended on the mutational status of the EGFR signaling pathway and on the inhibition of the splicing of X-box binding protein 1 (XBP1), an unfolded protein response (UPR) mediator. We confirmed the enhanced immunogenicity elicited by cetuximab in a mouse model of human EGFR-expressing CRC. Overall, we demonstrate a new, immune-related mechanism of action of cetuximab that may help to tailor personalized medicine.