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Anti-cancer drugs designed to target specific molecular pathways have shown an excellent therapeutic potential but also very poor long-term durability of tumor responses, mainly due to the outbreak of resistant clones among the residual neoplastic cell population. For that reason, understanding the molecular mechanisms underlying the onset of anti-cancer drug resistance (ACDR) is one of the major goals of clinical research. ACDR has been widely studied by DNA/RNA sequencing of primary human samples and several culprits identified. We have previously developed an approach based on lentiviral vector (LV)-induced insertional mutagenesis that allowed to identify the genes involved in lapatinib and erlotinib resistance on HER2+ human breast cancer cell lines and EGFR+ pancreatic cell line respectively. Here we took advantage of this platform to investigate ACDR genes in colorectal cancer (CRC). Cetuximab, anti-EGFR monoclonal antibody, is used as first line therapy in metastatic CRC, which results in prolonged survival of treated patients. However, nearly all patients relapse due to ACDR. We thus selected CRC cells sensitive to cetuximab deriving either from five microsatellite stable cell lines or from eight Patient Derived Xenografts (PDX), primary human CRC cells implanted subcutaneously into immunodeficient mice (NSG). To induce insertional mutagenesis we generated a luciferase-expressing LV harboring the SFFV enhancer/promoter in the long terminal repeats able to perturb the expression of genes nearby the integration site. As control, we used a non-genotoxic SIN-LV. We set up a collagenase IV-based disaggregation protocol that allows single-cell suspension and a serum-free culture condition to maintain the stemness of in vitro cultured cells. This protocol allowed to efficiently disaggregate and expand CRC cells in vitro as well as reach a LV copy number per cell ranging from 0.25 to 5.6. Luciferase gene expression was stable and allowed live-animal monitoring for up to 30 weeks after transplant. CRC-0069 and -0077 PDXs and NCI-H508 and HDC82 cell lines were transduced ex vivo and kept in vitro and/or transplanted in NSG mice. After in vitro or in vivo expansion of the transduced CRCs cetuximab treatment was applied. After an initial shrinking of the tumor mass in mice we observed ACDR in 3 out of 10 mice transplanted with NCI-H508 cells transduced with SFFV-LV and in none of the controls. Genomic DNA from resistant cells is being used for insertion site (IS) analysis to identify common IS, ACDR gene candidates. IS obtained from SIN-LV groups will be used to filter LV integration biases, whereas IS from SFFV-LV transduced cells but not treated with cetuximab will be used to filter mutations that provide a proliferative advantage unrelated to cetuximab treatment. We will validate the most promising candidates by LV-mediated overexpression and knockdown techniques. This approach could pave the way to perform insertional mutagenesis-based forward genetics studies on primary human samples.