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Additional file 3: Figure S3. Original blots are provided.

Additional file 1: Figure S1. TRAP IL-1 blocks proliferation and clonogenicity in CTX-resistant Caco-2 cell line. Caco-2 CXR cells (1 �� 103) were seeded in 96-well plate. The next day, Caco-2 CXR cells were treated with control medium supplemented with CTX (10 ��g/ml), only medium (FM 10% FBS), and TRAP IL-1 (20 ��g/ml). After 5 days, cell viability was assessed through AlamarBlue assay. Histograms show the average of two independent experiments quintuplicated (a). To measure clonogenicity, 3 �� 103 of Caco-2 CXR were seeded in 12-well plates in triplicate and treated after 24 h as described in a and b. After 10 days, cells were fixed in 4% PFA and stained with crystal violet for 30 min. The ability of cells to grow in a colony was determined by analyzing the covered area through ImageJ software. Percentage of covered area is shown (b). Statistical analysis was carried out using one-way ANOVA and significance calculated with Tukey���s multiple comparisons test *p

Epidermal Growth Factor Receptor (EGFR) activates a robust signaling network to which tumors often become addicted. Over the last three decades, EGFR targeting has been intensely pursued as a treatment strategy for metastatic colorectal cancer (mCRC). One approach uses monoclonal antibodies (mAbs) to inhibit the extracellular domain of EGFR, thus blocking natural ligands binding. Unfortunately, patients often develop resistance, with consequent tumor growth and relapse. Tumors heterogeneity has been addressed as the main culprit for multiple escaping mechanisms, reflecting the high level of molecular heterogeneity in each metastatic site. Mechanisms of cancer cell resistance include either acquisition of new mutations or non-genomic activation of alternative signaling routes and, in this context, a role of IL-1 is emerging. For example IL-1 expression proved to be elevated in human breast, colon, lung and head and neck cancers, and patients with IL-1 producing tumors have generally bad prognosis. Our studies, performed with a cohort of 150 colorectal cancer xenopatients, associate poor response to CTX with increased abundance of a set of inflammatory cytokines, including IL-1A, IL-1B and IL-8. Stemming from these observations, our working hypothesis assumes that resistance to CTX is acquired, in a subset of CRC patients, through cell plasticity and consequent rewiring of signalling networks, which confer dependency on the IL1 pathway. This hypothesis foresees an auto-stimulatory feedback loop dependent on the IL-1 produced by the tumors, with consequent immunosuppression and tumor progression. Thus, combining IL-1 and EGFR neutralization may synergistically impair CRC response to CTX in vivo.

// Valerio Gelfo 1, 2, * , Maria Teresa Rodia 1, * , Michela Pucci 1 , Massimiliano Dall’Ora 1 , Spartaco Santi 3, 4 , Rossella Solmi 1 , Lee Roth 5 , Moshit Lindzen 5 , Massimiliano Bonafe 1, 2 , Andrea Bertotti 6 , Elisabetta Caramelli 1 , Pier-Luigi Lollini 1 , Livio Trusolino 6 , Yosef Yarden 5 , Gabriele D’Uva 7, ** , Mattia Lauriola 1, 2, ** 1 Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Bologna, Italy 2 Center for Applied Biomedical Research (CRBA) S. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy 3 Institute of Molecular Genetics, National Research Council of Italy, Bologna, Italy 4 Laboratory of Musculoskeletal Cell Biology, IOR-IRCCS, Bologna, Italy 5 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel 6 Candiolo Cancer Institute-Fondazione del Piemonte per l’Oncologia (FPO), IRCCS, Department of Oncology, University of Torino, Candiolo, Italy 7 Scientific and Technology Pole, IRCCS MultiMedica, Milan, Italy * These authors have contributed equally to this work ** Co-last authors Correspondence to: Mattia Lauriola, email: mattia.lauriola@gmail.com Keywords: EGFR, transcriptional response, colon cancer, resistance, cetuximab Received: June 13, 2016 Accepted: September 21, 2016 Published: September 30, 2016 ABSTRACT Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A , IL1B and IL8 , which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro . This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting.