Medulloblastoma Exosome Proteomics Yield Functional Roles for Extracellular Vesicles

Abstract

Medulloblastomas are the most prevalent malignant pediatric brain tumors. Survival for these patients has remained largely the same for approximately 20 years, and our therapies for these cancers cause significant health, cognitive, behavioral and developmental sequelae for those who survive the tumor and their treatments. We obviously need a better understanding of the biology of these tumors, particularly with regard to their migratory/invasive behaviors, their proliferative propensity, and their abilities to deflect immune responses. Exosomes, virus-sized membrane vesicles released extracellularly from cells after formation in, and transit thru, the endosomal pathway, may play roles in medulloblastoma pathogenesis but are as yet unstudied in this disease. Here we characterized exosomes from a medulloblastoma cell line with biochemical and proteomic analyses, and included characterization of patient serum exosomes. Further scrutiny of the proteomic data suggested functional properties of the exosomes that are relevant to medulloblastoma tumor biology, including their roles as proliferation stimulants, their activities as attractants for tumor cell migration, and their immune modulatory impacts on lymphocytes. Aspects of this held true for exosomes from other medulloblastoma cell lines as well. Additionally, pathway analyses suggested a possible role for the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A); however, inhibition of the protein’s activity actually increased D283MED cell proliferation/clonogenecity, suggesting that HNF4A may act as a tumor suppressor in this cell line. Our work demonstrates that relevant functional properties of exosomes may be derived from appropriate proteomic analyses, which translate into mechanisms of tumor pathophysiology harbored in these extracellular vesicles.

Immunosuppression of breast cancer cells mediated by transforming growth factor-β in exosomes from cancer cells

“…BT-474 and MDA-MB-231 cell lines were cultured in serum-free medium for exosome preparation. Exosome preparation was performed as described by Epple et al (26) with slight modification. After four days of culture, 100 ml of media were collected.…”

The regulation of cancer cell migration by lung cancer cell-derived exosomes through TGF-β and IL-10

“…Before the medium was collected for exosomes preparation, the HEK 293T, NCI-H2228 and NCI-H1688 cells were cultured in serum-free medium. The exosomes preparation was performed as previously described (18). Briefly, following 4 days in culture, 100 ml of medium was collected.…”

Label-free quantification proteomics reveals novel calcium binding proteins in matrix vesicles isolated from mineralizing Saos-2 cells

“…Moreover, after incubation in mineralizing buffer for 12 h MVs that were isolated from stimulated cells were able to continue Ca 2+ uptake ( Figure 1E) and form mineral HA, a finding that was confirmed using FTIR ( Figure 1D). Proteins including CD63, CD9, and Hsp70, which are known to be reliable biomarkers of MVs and exosomes (6,19), were present in both types of MV preparations (Figure 2). …”