Abstract 3947: Mass-spectrometry analysis of metastatic matrisome from MC38 experimental liver metastasis
Authors: Arseniy E. Yuzhalin, Dihua Yu, Ruth J. Muschel
Journal: Cancer Research (2020)

Abstract Prognosis of patients with colorectal cancer liver metastasis is generally dismal due to unavailability of effective therapeutic modalities. Thus, more basic research is required to extend our knowledge about mechanisms underlying development of hepatic metastasis. The extracellular matrix (ECM) represents a backbone scaffold supporting tissue integrity and establishing cell-cell communications; tumor stroma is highly enriched in certain ECM components which have been suggested as drivers of metastatic dissemination. We performed a label-free quantitative mass-spectrometry (MS) analysis of the ECM isolated from experimental liver metastasis generated by intrasplenic injection of MC38 colon cancer cells. As a group of comparison, we used unaffected mouse liver tissue. Extraction of the ECM was performed utilizing an original method of detergent decellularization, fractionation, followed by biochemical enrichment (i.e. depletion of polysaccharides and nucleic acids) developed in our lab. Label-free MS analysis of resultant ECM samples revealed 2328 proteins, of which 140 were classified as murine matrisome components. Inter-sample variability between ECM molecules was insignificant. Among 140 identified ECM molecules, 13 matrix proteins were dramatically upregulated in metastasis tissues in comparison with tumor-free liver ECM. A combination of these 13 ECM proteins significantly predicted overall survival from colorectal adenocarcinoma based on assessment of 486 patients (P&amp;lt;0.01). One of the most prominent ECM candidates was Annexin A1 (ANXA1), an ECM-affiliated protein consistently overrepresented in the murine metastatic tissue by 7 fold with the highest significance score (P&amp;lt;0.0001). We further validated ANXA1 overexpression in human hepatic metastasis tissues. Higher concentrations of tumor ANXA1 tended to predict colorectal cancer prognosis with borderline significance (P=0.06). Using immunohistochemistry, we identified major sources of ANXA1 in the tumor microenvironment, which were the cancer cells, myeloid cells and T cells. ANXA1 has been previously linked to chemoresistance and altered TGF-beta signaling in cancer cells. To explore the functional role of ANXA1, we performed a stable knockdown of this molecule using shRNA transfection. In vitro, ANXA1-deficient cancer cells displayed slower growth rates but elevated migratory capabilities. In vivo, ANXA1 knockdown cells grew significantly slower as compared to vector control cells when injected subcutaneously, suggesting its role in tumor outgrowth. To conclude, here were report a comprehensive proteomics-aided ECM characterization of MC38 murine colorectal liver metastasis and suggest ANXA1 as a possible target for this disease. Citation Format: Arseniy E. Yuzhalin, Dihua Yu, Ruth J. Muschel. Mass-spectrometry analysis of metastatic matrisome from MC38 experimental liver metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3947.