Mass-spectrometry profiling of extracellular matrix identifies citrulline as marker of colorectal cancer liver metastasis


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Arseniy Yuzhalin1,Su Yin Lim2,Alex Gordon-Weeks2,Rebecca Konietzny3,Benedikt Kessler3,Ruth Muschel2
1,2Cancer Research UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK,3TDI Mass Spectrometry Laboratory, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK



One of the important hallmarks of cancer metastasis is its extracellular matrix (ECM). We aimed to identify ECM signatures of colorectal liver metastasis using an advanced mass-spectrometry-aided proteomics technique.


We developed a method to isolate the ECM from human liver colorectal cancer metastases and adjacent, uninvolved liver (N=5) using detergent treatment followed by biochemical enrichment. Tissues were decellularised using detergents and enzymatic treatments. Quantitative mass-spectrometry was done using the Thermo LTQ Orbitrap Elite mass spectrometer. Liver metastases were developed in mice by intrasplenic tumour cell injection.  The PAD4 inhibitor BB-Cl-amidine was administered at 7.5 mg/kg every 48 h by IP injection.


Mass-spectrometry identified 70 proteins which were differentially expressed between liver metastasis and adjacent uninvolved liver. One of the most prominent candidates, PAD4, modifies protein arginine residues to citrulline, and was 11 times more abundant in the liver metastases as compared to uninvolved livers. In keeping with this finding, citrulline was expressed at significantly higher levels in human liver metastases compared with matched uninvolved liver. We identified high levels of PAD4 in metastatic cancer cells and their associated exosomes.  Furthermore, inhibition of exosome production led to a reduction in PAD4 levels in cancer cell-conditioned media implicating the exosome pathway in PAD4 ECM deposition. BB-Cl-amidine treatment reduced liver metastasis growth and citrulline levels in murine xenogenic liver metastases. To address PAD4 function, we screened the resultant metastases for mesenchymal and epithelial markers, which revealed that liver metastases from BB-Cl-amidine-treated mice do not effectively progress from EMT to MET, suggesting that PAD4-mediated citrullination promotes metastatic growth in the liver through MET.


Our results implicate PAD4-mediated citrullination of the ECM in the progression of liver metastasis through the promotion of MET. This novel mechanism identifies a mechanism through which cancer cells modulate the ECM for further metastatic progression.