Page 28 - Read Online

Page 24 Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10

miRNA) unique to their cell of origin and deliver them to recipient target cells thereby mediating cell-
cell communication. We have investigated whether exosomes from high metastatic HCCH (liver cancer)
cells can confer growth and metastatic properties to HCCL (low metastatic). Exosomes were isolated from
the supernatant culture media of cancer cells and a correlation was found between elevated CPE mRNA
levels in exosomes from high vs. low metastatic cell lines across various cancer types. Content analysis of
exosomes derived from HCC97H cells revealed CPE-WT mRNA and protein. We showed that exosomes
released from HCC97H cells were able to enhance invasion of HCC cells with poor metastatic ability
(HCC97L) in Matrigel invasion assay and proliferation in MTT assay. However, when CPE expression was
suppressed in the HCC97H cells before exosome isolation, the exosomes had no effect on proliferation and
invasion. These data demonstrate the ability of exosomes to confer metastasis in cancer cells and the role of
exosomal CPE in driving the process. We then utilized the inherent property of exosomes to act as efficient
delivery tools to carry a therapeutic agent such as shRNA. Previously it was shown that down-regulation
of CPE expression by shRNA can reverse tumor growth and metastasis in an HCC mouse model. We
therefore loaded CPE-shRNA into exosomes by infecting HEK293 (Human Embryonic Kidney) cells with
adenovirus carrying CPE-shRNA-GFP. These modified exosomes were harvested from the cell medium,
purified and then used to transfer CPE-shRNA to HCC97H cells. The exosomes taken up by the recipient
cells resulted in significant reduction of CPE mRNA levels and decrease in colony formation of these cells.
Thus, these studies demonstrate the ability of exosomal CPE to enhance invasion in low metastatic HCC
cells and the potential to use shRNA loaded exosomes to target CPE as a therapeutic strategy to treat liver
and other cancers. Finally, in a pilot study we measured CPE mRNA copy numbers in serum exosomes of
patients with cancer (glioma, breast, ovarian, colon, cervical, pancreatic or prostate cancer) and healthy
controls. Significantly elevated levels of CPE mRNA copy numbers were found in serum exosomes of
cancer patients versus healthy controls, suggesting that exosomal CPE mRNA could potentially be used as
a biomarker in an initial screen for diagnosing cancer.

4. Bacterial vesicles: vehicles for Inter-Kingdom communication and modulators of plant
immune response

2
3
1
2
Authors: Hannah M. McMillan , Sophia G. Zebell , Jean B. Ristaino , Xinnian Dong , Meta J. Kuehn 4
E-mail: hannah.mcmillan@duke.edu
Affiliations:
1 Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA.
2 HHMI, Department of Biology, Duke University. Durham, NC, USA.
3 Department of Entomology and Plant Pathology, NC State University, Raleigh, NC, USA.
4 Department of Biochemistry, Duke University, Durham, NC, USA.
Abstracts: Bacterial outer membrane vesicles perform a variety of functions in bacterial survival and
virulence. In mammalian systems, vesicles activate immune responses and have been exploited for use
in vaccines. However, little work has focused on the role vesicles play in dry-land environments or in the
context of plant-microbe interactions, and research has only just begun on their role as natural nanoscale
vehicles. We show that vesicles from the plant pathogen Pseudomonas syringae pv tomato DC3000 and
the plant beneficial Pseudomonas fluorescens activate plant immune responses that protect against future
bacterial and oomycete challenge. Interestingly, our results also expose differences in vesicle packaging
between pathogens and beneficials and in vesicles isolated from various environmental conditions that
lead to different sensitivities to biochemical stressors. Importantly, these studies reveal unique differences
in pathogen- versus beneficial-induced immune activation and support the use of vesicles as a tool to