Kyungpook National University of School of Medicine, Korea
Identification of Developmental Endothelial Locus-1 on Circulating Extracellular Vesicles as a Novel Biomarker for Early Breast Cancer Detection
Currently, there are no molecular biomarkers for the early detection of breast cancer. This study focused on identifying surface proteins found on circulating extracellular vesicles (EVs) for detecting early-stage breast cancer. Circulating EVs, isolated from the plasma of 10 patients with breast cancer (stages I and II) and 5 healthy controls, were analyzed using LC-MS/MS. Developmental endothelial locus-1 protein (Del-1) was selected as a candidate biomarker. Two different ELISAswere used tomeasureDel-1 in plasma samples fromhealthy controls (n = 81), patients with breast cancer (n = 269), breast cancer patients after surgical resection (n = 50), patients with benign breast tumors (n = 64), and patients with noncancerous diseases (n = 98) in two cohorts. Plasma Del-1 levels were significantly higher (P <0.0001) in patients with breast cancer than in all controls and returned to almost normal after tumor removal. The diagnostic accuracy of Del-1 was AUC, 0.961 [95% confidence interval (CI), 0.924–0.983], sensitivity of 94.70%, and specificity of 86.36% in test cohort and 0.968 (0.933–0.988), 92.31%, and 86.62% in validation cohort for early-stage breast cancer by one type of ELISA. Furthermore, Del-1 maintained diagnostic accuracy for patients with early-stage breast cancer using the other type of ELISA [0.946 (0.905–0.972), 90.90%, and 77.14% in the test cohort; 0.943 (0.900–0.971), 89.23%, and 80.99% in the validation cohort]. Del-1 on circulating EVs is a promising marker to improve identification of patients with early-stage breast cancer and distinguish breast cancer from benign breast tumors and noncancerous diseases.
Korea Institue of Science & Technology and KU-KIST School, Korea University, Korea
Exosome for Membrane Protein Therapeutics
Major targets for antibody therapeutics are membrane associated proteins including growth receptors, cytokine receptors and immune check points. In addition, most of protein therapeutics are known to target membrane proteins and a number of major human diseases are related to defects in membrane proteins. Here, we raised two questions, 1) Can we make membrane protein therpapeutics in their natural form? 2) Can we edit membrane with membrane proteins? Exosomes are vesicles with membrane structures released from most of cells and potentially provide a perfect microenvironment for membrane proteins in terms of activity and distribution on the membrane. Based on these charateristics, exosomes can be engineered to be useful for the development of vaccines, therapeutics and editing cell membranes. Here I present a few evidences showing how we could engineer exosomes for these purposes.
Exosome engineering for protein delivery via optogenetic approach
In this presentation, an opto-genetically engineered exosome system, named ‘exosomes for protein loading via optically reversible protein–protein interaction” (EXPLOR) that can deliver soluble proteins into the cytosol via controlled, reversible protein–protein interactions (PPI) will be introduced. By integrating a reversible PPI module controlled by blue light with the endogenous process of exosome biogenesis, cargo proteins can be loaded into newly generated exosomes. Treatment with protein-loaded EXPLORs was shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in both a time- and dose-dependent manner. Previously, it has been shown to delivery mCherry, Cre enzyme, Bax, and Super repressor IκB proteins as functional proteins in the target cells and in vivo. In this presentation, the results for follow-up studies will also be discussed.
Functional proteome transportation via exosomes in the tumor microenvironment
The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. We further performed highly optimized MS analyses on the exosomal and cellular proteins isolated from human colorectal cancer SW620 cells. With stringent data quality control, 313 phosphoproteins with 1091 phosphosites were confidently identified from the SW620 exosome, from which 202 new phosphosites were detected. Exosomal phosphoproteins were significantly enriched in the 11q12.1-13.5 region of chromosome 11, and had a remarkably high level of tyrosine-phosphorylated proteins (6.4%), which were functionally relevant to ephrin signaling pathway-directed cytoskeleton remodeling. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit.
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