Greening - Extracellular vesicles, exosomes, cancer biology and uterine biology

Within the past decade, extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication, particularly involved in the transmission of biological signals and select cargo between cells, thereby regulating various pleiotropic biological processes. EVs exert diverse physiological and pathophysiological functions by horizontal transfer of protein, DNA, and RNA species between cells. There is now a growing awareness that predominant EV subtypes; exosomes from endosomal origin, and shed microvesicles from plasma membrane budding, can be further stratified into distinct subtypes. We investigate the mechanisms of distinct EV biogenesis and release, defining select EV classes (and subpopulations), which will be crucial for development of EV-based functions and clinical applications. The advent of quantitative mass spectrometry-based proteomics, in conjunction with advances in chemical labelling, cross-linking, molecular cell biology, and EV purification strategies, has contributed significantly to our improved characterization and understanding of the molecular composition and functionality of these distinct EV subpopulations.

Research areas

Extracellular Vesicle (EV) cancer biology

This project focuses on utilising an integrated proteomic/genomic strategy directed towards characterisation and understanding the role of the extracellular environment (specifically membrane vesicles; exosomes) in cancer progression. We utilise cell culture, functional assay development, lipophilic-labelling, cell sorting, molecular biology, microscopy (confocal, live-cell), and transmission/cryo electron microscopy, mass spectrometry-based protein profiling for discovery and targeted strategies, miR/mRNA profiling and qRT-PCR validation.

This project investigates the role of EVs in cancer progression and metastasis, utilising various in vitro and in vivo cancer/developmental models, including established cell models, primary cells, and animal models. The research will further contribute to understanding how EVs and their cargo regulate the tumour microenvironment.

This project will investigate the following questions:

  1. What are the different methods to purify the secretome and distinct EV subtypes?
  2. What are the distinct functions of the secretome and EVs during cancer initiation, development, epithelial-mesenchymal transition, and metastasis?
  3. What cargo/surface components (protein/nucleic acids) are contained within EVs?
  4. Can distinct cargo components in EVs be perturbed using molecular biology?
  5. Develop key methods for characterisation and quantitation of EV cargo.

Exosomes and their role in regulating embryo implantation

This research investigates how exosome cargo functionally regulate early-stage pregnancy of the embryo and endometrium. Exosome-mediated transfer of cargo (particularly mRNA and proteins) to either the blastocyst or adjacent endometrium, is hypothesized to directly modulate the implantation process. This project focuses on the importance of defining the precise contribution of endometrial and trophoblast cell-derived exosomes within the uterine cavity.

This function-focused and clinically-relevant intercellular signalling approach will provide unique information regarding endometrial and trophoblast influences on the preimplantation embryo, specifically mediated by exosomes. The outcomes of this research will establish how exosomes and their cargo regulate the extracellular uterine environment, specifically of the endometrium and trophectoderm, and elucidate cellular and molecular events associated with implantation and receptivity. This work will have significant implications in understanding exosome biology, the uterine microenvironment, fundamental causes of implantation failure, and promotion of healthy pregnancy.

This project will investigate the following questions:

  1. Determine how endometrial exosomes influence peri-implantation?
  2. Investigate trophectoderm exosomes under menstrual-cycle phase hormones in context of uterine receptivity.
  3. Delineate whether exosomes functionally act independently or synergistically with soluble growth factors/ hormones.
  4. Demonstrate the functional effects of endometrial exosome transfer to human trophoblast cells.

Contribution of extracellular vesicles (EV) in epithelial-mesenchymal transition

The metastatic cascade describes the process by which tumour cells escape their primary site and colonize secondary locations. Tumour angiogenesis facilitates passage, and cells at the leading edge of the primary tumour, are thought to undergo epithelial-mesenchymal transition (EMT) to acquire increased motility and invasiveness.

Whether oncogenic cells that have undergone EMT directly promotes endothelial cell recruitment remains largely unknown, and the role of extracellular vesicles (EVs) (30-1,000nm diameter) in this process has not yet been definitively explored.

Conventional biological assays for cell proliferation, motility, migration, and invasion are already established in our lab. Using global profiling approaches, including proteomic (mass-spectrometry based profiling) and genomic (miR/mRNA profiling) analyses, we intend to catalogue and identify the contribution of EVs during EMT, especially induced expression of signalling pathway receptors/ modulators in recipient cells to modify their function.

We currently employ a multi-omics and multidiscipline approach integrating cancer biology, molecular biology, genomics, proteomics, and bioinformatics to explore EVs and their functions.

Preliminary results demonstrate that oncogenic cells undergoing EMT can communicate with endothelial cells via specific EVs, and establish angiogenic promoters that may function during the initial stages of metastasis.

This project will investigate the following questions:

  1. What are the different methods to purify the secretome and distinct EV subtypes?
  2. Can distinct cargo components in EVs be perturbed using molecular biology?
  3. Investigate the contribution of secreted (soluble and vesicle components) to modifying recipient cell function.
  4. How do vesicles selectively package and transfer soluble cargo such as cellular mediators directly into endothelial and epithelial cells?

Meet the team

Group members

Dr David Greening GroupGroup leader

Dr David Greening

Research fellow

Dr Alin Rai

PhD students

Bethany Claridge
Qi Hui Poh
Adnan Shafiq (co-supervised with Professor Richard Simpson)
Wittaya Suwakulsiri (co-supervised with Professor Richard Simpson)

Honours student

Monique Fatmous

Publications

See a full list of publications [external link], Scopus [external link], ORCID [external link] or view Dr David Greening's profile.