Tumor-derived microvesicles are a critical determinant for successful metastatic colonization in colorectal cancer

Sivaloganathan, S; Blazquez, R; Pukrop, T; Wlochowitz, D; Beißbarth, T; König, S; Schulz, M; Binder, C; Menck, K; Bleckmann, A

Abstract

Introduction: The formation of distant metastases is the major obstacle in treating cancer. Metastatic spread requires a cancer-induced modulation of the surrounding environment, which can be mediated via the release of tumor-derived extracellular vesicles (T-EV). Using a syngeneic mouse model for colorectal cancer (CRC), we identified two variants of the murine CRCcell line CMT93, which differed significantly in their metastatic colonization in vivo. The aim of this study was to identify themolecular mechanisms responsible for the effect. Methods: The two CMT93 variants were characterized regarding their basic characteristics using proliferation, invasion, migration and adhesion assays. T-EV released by the cells were collected via differential ultracentrifugation at 17,000 g for larger microvesicles (MV) and 143,000 g for small EV. The obtained EV were characterized by immunoblotting, nanoparticle tracking analysis, transmission electron microscopy and proteomics. Results: Analysing the cell-intrinsic basic characteristics of the cells revealed no major difference between the two CMT93 vari-ants. Thus, we extended our analysis to the released secretome, which revealed a major difference in the amount and composition of released T-EV. While T-EV released by the malignant CMT93 variant induced tumor invasion, this effect was not observed with T-EV from the less aggressive CMT93 variant. Intriguingly, this functional difference was more prominent with MV than with small EV. To identify the molecular characteristics responsible for this difference, the MV released by both CMT93 variantswere compared by proteomic analysis. The results revealed that the MV released by the malignant CMT93 variant were enriched in proteins associated with adhesion. Using immunoblotting, we were able to validate an upregulation of the proteins Itga3 and Fascin1 on MV released by the malignant variant. Analysing microarray data, we identified that these proteins were upregulated in human primary and metastatic CRC in contrast to normal colon tissue, highlighting the translational relevance of our findings. Summary/Conclusion: Conclusively, we identified MV as the critical determinants for successful tumor invasion in CRC andattributed this effect to the enrichment of tumor-supporting adhesion proteins on the vesicles. These results shed further lighton the molecular mechanisms underlying EV-mediated metastatic colonization and open new options for targeted therapy.