Absorbance was then measured at 570 nm in a 96-well spectrophotometric microplates reader (Bio-Rad, Hercules, CA, USA). genes. Results obtained showed that gabexate mesilate significantly inhibited the growth, invasive potential and tumour-induced angiogenesis in all the CRC cells employed in this study (including those ones harbouring dual KRAS/PIK3CA or BRAF/PIK3CA mutation), while cetuximab affected these parameters only in CRC cells with KRAS, BRAF and PIK3CA wild-type. Notably, the antitumoral efficacy of gabexate mesilate and cetuximab in combination was found to be not superior than that observed with gabexate mesilate as single agent. Overall, these preliminary findings suggest that gabexate mesilate could represent a encouraging therapeutic option for mCRC patients, particularly for those harbouring KRAS, BRAF and PIK3CA mutations, either as mono-therapy or in addition to standard chemotherapy regimens. Further studies to better elucidate gabexate mesilate mechanism of action in CRC cells are therefore warranted. Introduction In the last fifteen years, the introduction of at least six key drugs (oxaliplatin, irinotecan, capecitabine, bevacizumab, cetuximab and panitumumab), after the era of 5-fluorouracil as a single agent, has improved median overall survival of metastatic colorectal malignancy (mCRC) patients up to 24 months , . The employment of the anti-epidermal growth factor receptor (EGFR) antibodies panitumumab and cetuximab, either as mono-therapy or in addition to standard chemotherapy regimens, represents a backbone of the therapeutic options for the treatment of mCRC. However, randomized controlled trials have provided persuasive evidence that EGFR-targeted therapy is usually poorly effective or ineffective in unselected mCRC patients. In recent years, activating mutations at codons 12 and 13 in the KRAS oncogene (KRASG12V and KRASG13D) have emerged FCRL5 as the best predictive factors of low/absent response to anti-EGFR therapy in these patients, either in the first-line or subsequent lines of treatment C. For this reason, mCRC patients are now profiled for KRAS mutation and the employment of cetuximab and panitumumab is currently restricted only to those ones bearing the KRAS wild-type, as recommended by the European Medical Agency and the American Society of Clinical Oncology . Although the presence of wild-type KRAS seems to be a condition for response to EGFR-targeted therapy, up to 50C65% of mCRC patients fail to benefit from this treatment, due to additional intrinsic resistance mechanisms . In this regard, the involvement of BRAFV600E and PIK3CAH1074R at exon 20 mutations in Citiolone the failure of such therapy has recently emerged C. Citiolone Due to the lack of an effective targeted therapy, the discovery of new therapeutic options for mCRC patients with mutated KRAS, BRAF and PIK3CA genes represents therefore an intense area of investigation. The protease inhibitor gabexate mesilate has been Citiolone shown to exert a significant antitumoral activity in CRC cells, both in vitro and in vivo . However, the effect of this drug, alone or in combination with cetuximab, in human CRC cells harbouring Citiolone a different expression pattern of wild-type/mutated KRAS, BRAF and PIK3CA still remains unsettled. The present study aims at investigating this hypothesis. Results We preliminary selected a panel of human CRC cells harbouring a different expression pattern of wild-type/mutated KRAS, BRAF and PIK3CA genes. To this purpose, based on the Catalogue of Somatic Mutations in Malignancy (COSMIC) database (http://www.sanger.ac.uk/genetics/CGP/cosmic/) and on the study of Jhawer et al. , CACO-2, SW48, HT-29, Colo205, SW480, SW620, RKO, LS174T and HCT-116 CRC cells were chosen (observe Table 1 for their corresponding KRAS, BRAF and PIK3CA status). Notably, no CRC cell collection with co-occurring KRAS and BRAF mutations was found and included in the present study, according to previous reports showing a pattern of mutual exclusivity for KRAS Citiolone and BRAF mutation in human CRC . The effect of gabexate mesilate, alone and in combination with cetuximab, was then investigated on CACO-2, SW48, HT-29, Colo205, SW480, SW620, RKO, LS174T and HCT-116 cell growth, invasive potential and tumour-induced angiogenesis, as they represents three fundamental processes in malignancy onset and progression. Table 1.