Month: February 2020

Extracellular vesicles (EVs) regulate many processes in the healthy body. They also play a role in cancer, sending signals between cells in the tumor microenvironment. EVs can stimulate tumor cell migration, invasion, blood vessel growth, immune response, and cell survival, as well as metastasis. However, we know little about the cargo of these EVs that play such diverse roles. Analysis of vesicle cargo can shed light on the molecular mechanisms of vesicle biology and be helpful in disease diagnosis and prognosis.

I am lucky to be a member in Jan Lötvall’s lab in Gothenburg, Sweden, which pioneered the field of extracellular vesicles with the early discovery of exosomes shuttling RNA between cells. An exciting collaboration with Yong Song Gho from POSTECH in South Korea led us to develop a new approach to isolate vesicles from human tumor tissues. Using this technology, we were able to isolate and characterize subpopulations of extracellular vesicles from melanoma metastatic tissue. We just published our findings in the Journal of Extracellular Vesicles. Jan Lötvall also discussed them in a recent ERCC webinar.

Finding extracellular vesicles in tumor tissue with TEM

Our first challenge was to find vesicles in metastatic melanoma tumor tissues. Using transmission electron microscopy, we showed that the tumor microenvironment is a complex world composed of different types of cells and structures with vesicles present between them.

TEM of extracellular vesicles in tumor tissue
Transmission electron micrograph of melanoma metastatic tissue showing a large tumor cell and two lymphocytes. Black stain, possibly melanin, is clearly visible inside the melanoma cells, which are recognizable from their characteristic cell membrane. The higher magnification image shows vesicles (red arrows) in the extracellular space.

In this study, we performed a detailed proteomics analysis of EVs isolated from metastatic melanoma tissues from 27 patients. We identified numerous new EV proteins, including potential biomarkers for metastatic melanoma.

Tumor tissue vs. Cell lines

Studying extracellular vesicles in tumor tissues is important, because, compared to cell lines, tumor tissues more closely approximate the situation in vivo. EVs from tumor tissue are more likely to represent the full array of vesicle behaviors and populations in the tumor microenvironment. Furthermore, to develop a non-invasive test for cancer, we must use biofluids such as circulating plasma, where vesicles from all over the body intermingle. A proteomic snapshot of vesicles isolated directly from tumor tissue can help target the search for disease-specific biomarker in that complex mixture. We trust the tools and experiments developed in this work will contribute to our field’s understanding of EV function in complicated tissues such as the metastatic melanoma tumor.

Reference
Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög J.L, Johansson I, Fuchs J, Thorsell A, Gho YS, R, Olofsson Bagge R, Lötvall J Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. Journal of Extracellular Vesicles 9:1, 1722433 doi: 10.1080/20013078.2020.1722433.

This work was supported by the Swedish Research Council (K2014-85X-22504-01-3), the Swedish Heart and Lung Foundation (20120528), the Swedish Cancer Foundation (CAN2014/844), and the Knut och Alice Wallenberg Foundation (Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden).

Flow cytometry (FC) is a powerful method for counting single cells and measuring their molecular components. There is increasing interest in applying flow cytometry to the analysis of extracellular vesicles (EV), but EVs are orders of magnitude smaller than the cells for which FC instruments and protocols were originally designed. To catalyze the development of new instruments and assays for EV flow cytometry, three scientific societies came together to form the EV Flow Cytometry Working Group (evflowcytometry.org):

  • ISEV, the International Society of Extracellular Vesicles
  • ISAC, the International Society for Advancement of Cytometry, and
  • ISTH, the International Society for Thrombosis and Haemostasis.

The working group first performed two standardization studies, distributing standards and samples to EV-FC laboratories worldwide to enable an objective comparison of methods, instruments, controls, and analytical tools. Those initial studies led to the realization that a standard framework for reporting experimental results is essential.

The working group has now published that standard in the Journal of Extracellular Vesicles. It is called MIFlowCyt-EV, the Minimum Information to report for Flow Cytometry studies of Extracellular Vesicles. The MIFlowCyt-EV reporting framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a Flow Cytometry experiment (MIFlowCyt) standard.

The figure above outlines the 7 main categories of information included in the framework. Not all EV-FC experiments will involve all seven areas, but any area touched on by an experiment should follow the MIFlowCyt-EV reporting guidelines.

MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not mandate the use of specific instruments or protocols, since the field of EV flow cytometry is still rapidly evolving. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Consistent reporting of the results of EV flow cytometry studies will improve the ability to quantitatively compare results from different laboratories and support the development of new instruments and assays for improved measurement of EVs.

Reference
Welsh JA, et al. MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. J Extracell Vesicles (2020) doi: 10.1080/20013078.2020.1713526