Purpose:The lack of secreted biomarkers measurable by non-invasive tests hampers the development of effective targeted therapies against cancer. Our hypothesis is that cetuximab (an anti-EGFR mAb) induces a specific secretome in colorectal cancer (CRC) cells that could be exploited for biomarker discovery. Experimental Design:Considering the strong correlation between mutated-KRAS and a lack of response to cetuximab therapy, we addressed whether performing secretome-based proteomics on isogenic CRC cells sharing the KRAS-mutations found on patients would yield candidate secreted biomarkers useful in the clinical setting. Since 2D-culture did not optimally model the sensitivity/resistance to cetuximab observed in CRC patients, we moved to 3D-spheroids, developing a methodology for both cell-based assays and quantitative proteomics. Results:A large comparative quantitative proteomic analysis of the 3D-secretomes of CRC isogenic cells treated with cetuximab uncovered an EGFR pathway-centric secretome found only when cells grow in 3D. The validation of the secretome findings in plasma of CRC patients, suggests that phosphorylated-EGFR (pEGFR) is a candidate secreted biomarker of response to cetuximab. Conclusions:We have proved that 3D-spheroids from CRC cells generate secretomes with a drug sensitivity profile that correlates well with CRC patients, illustrating molecular connections between intracellular and extracellular signaling. Furthermore, we show how the secretion of pEGFR is associated with the sensitivity of CRC cells to cetuximab and the response of CRC patients to the drug. Our work could allow the non-invasive monitoring of anti-EGFR treatment in CRC patients.
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