Pictograph 2 shows one of those rarer events where a vesicle is released from a non-protruding part of the cell. Like in Figure 1 the cell is labeled with 5ht3 receptor ligand. The image sequence shows the speed of the release (left, 12min after Cytochalasin addition, center, 1 minute later and right, 3 minutes after that. In general, after application of cytochalasin, the cells adapted a round form. Microfilaments contract and condense to local aggregates in the cell cortex. The otherwise continuous actin cytoskeleton became fragmentary. Supported by the cytoplasmic pressure, this effect leads to an expansion of the endoplasm in these regions. As a result, budding of the cell membrane occurs at these locations. These buds are either bulbous (as shown here) or -in most cases- pedunculate (See Figure1) in form. Again, for scale comparison, see Figure 3.
Pictograph 3 shows size and intensity of ligand labeling of those vesicles exposed for 70 s to the receptor ligand (labeling can clearly be seen after 15 s, not shown here). The size of the vesicle is directly in the confocal plane and therefore can be assessed by the scale bar on the right panel (1 micro m). Scale bar is (20 micro m left, 1 um right)
Our results open the unexpected avenue that filopodia (so called invadopodia) might be not only the feet cancer cells use to move from their origin to distant locations in the body, they might actually convey their metastatic property as well by shedding microvesicles. In this way, metastasis might be just two sides of the same coin: Invadopodia translocate cancer cells and at the same time shed vesicles which have been associated by numerous investigators with metastatic processes.
Our results show the release of micro vesicles from the plasma membrane of physiologically compromised immortal cells. Please note that the cytochalasin dosage we used was not discriminate enough to differentiate between glucose transport inhibition and cytochalasin B’s action on the actin structure. Further research is needed to verify whether those protrusions are same as or similar to invadopodia as formed by cancer cells under hypoxic or immune cell rich environment exposed conditions (HS Leong et al, 2014) . As we pointed out in the introduction, such invadopodia are used as escape route for primary tumors from their original location. Any block by cytochalasin of the glucose transporter could mimic such adversarial conditions. Yet, cytochalasin is known to affect the action filaments already from a concentration of 2 µM on, lower than the one we used (20-40 µM).
The elegance of our study is that we can mimic stress conditions for cancer cells in vitro in a way which allows to verify the identity of their released particles and their origin from the cell membrane. As summarized by Clancy JW and (2012) , recent advances in the study of tumor-derived microvesicles reveal new insights into the cellular basis of disease progression and the potential to translate this knowledge into innovative approaches for cancer diagnostics and personalized therapy. They have been thought to deposit paracrine information and create paths of least resistance, as well as be taken up by cells in the tumor microenvironment to modulate the molecular makeup and behavior of recipient cells. The complexity of their bioactive cargo-which includes proteins, RNA, microRNA, and DNA-suggests multipronged mechanisms by which microvesicles can condition the extracellular milieu to facilitate disease progression.
The formation of these shed vesicles likely involves both a redistribution of surface lipids and the vertical trafficking of cargo to sites of microvesicle biogenesis at the cell surface. We want to point out multifold effects of extracellular vesicles (which include microvesicles and exosomes) that promote metastasis by a) their physical translocation b) triggering molecular events supporting metastasis and/or c) transforming target cells by fusion / take up. Our model system is perfectly suited to investigate such processes in vitro and should be of interest to labs which do care about biological effects and cancer pathogenesis. We are currently seeking collaboration with laboratories which have FLIM microscopy capabilities.
We want to point out recent results published by Gomzikova M et al (2017)  that cytochalasin B-induced membrane vesicles convey angiogenic activity of parental cells. Brilliantly written, this paper brings to mind that cancer cells not only might metastasize away from challenging microenvironment, but bring a positive environment to them. Instead of escaping from hypoxic conditions they can bring oxygen supply to them in form of angiogenesis.
DePalma’s group has shown that two breast cancer antagonists have resulted in increasing metastasis concomitant with EV release (Keklikoglou I, et al, 2018) . Yet the group was not able to definitely say what type of EVs were released. As there are reports that EVs can also inhibit cancer, the situation is complex (, 2014). We might speculate that the anti- or prometastatic effect of EVs depends on whether microvesicles or ectosomes have been investigated in those experiments, and how much cross contamination between both has occurred. Of course, the anti- or prometastatic potency of various systems will depend on the type of cancer and type of chemotherapeutics used, among other conditions. Please note that signal molecule location can be tricky - for example it has been shown RNA can be located on the exterior of peroxisomes via the noncovalent contacts with the membrane proteins (Yarmishyn A et al, 2016) .
An obvious and crucial mechanism for extracellular vesicles to facilitate metastasis would be, of course, their direct interaction with target “host” cells. As mentioned above, a row of live microscopic techniques is required to touch on that question. As mentioned above, we recommend FLIM microscopy -and FRET- but other powerful tools have indeed been employed by other labs. They involve live or cryo-light microscopy, and powerful electron microscopic techniques. For example, B16 BL6 tumors in mice have shown to accelerate their growth by uptake of their own exosomes, and their uptake was demonstrated by confocal microscopy of showing the fluorescent PKH26 signal spread within the cells after 24 hours in comparison to 4hrs incubation (Matsumoto, et al. 2017) . Please be aware that these experiments used exosomes and not microvesicles. Electron microscopy was used to show the size of the exosomes. The role of microvesicles in targeting host tissue remains more obscured. Their importance in metastasis facilitation, however, has triggered increasing interest by electron microscopy groups as well, moving from architecture and their release mechanism from the plasma membrane towards the analysis of their direct interaction with the target tissue. In this respect Nanou A, et al (2018)  show exciting SEM images of circulating tumor cells and tumor derived extracellular vesicles on the surface of unidentified human cells. More works remains to be done to characterize the nature of such extracellular vesicles.
Arusu et al (2019) employed correlative light and electron microscopy and showed that extracellular vesicles (EV) with GFP tagged plasma membrane protein, HAS3, were indeed lying on the recipient cell's plasma membrane, while the level of EV-derived intracellular signal was low. Immunoelectron microscopy supported this finding. Furthermore, hyaluronan oligosaccharides decreased the numbers of bound EVs, suggesting that CD44 (a surface protein) participates in the regulation of their binding. We agree with their assessment that it will be up to live cell imaging at high resolution to obtain definite answers on the detailed mechanisms of binding, fusion and endocytosis of EVs. It is interesting that this group remains cautious and does refrain from calling their EVs microvesicles, despite them being labeled with GFP tagged plasma membrane protein, HAS3.
A step further towards the identification of EVs venture Antonyak, et al. (2018) . Using SEM and fluorescence microscopy they show that microvesicles shed from the surface membrane of two different human cancer cells, MDAMB231 breast carcinoma cells and U87 glioma cells, are capable of conferring onto normal fibroblasts and epithelial cells the transformed characteristics of cancer cells (e.g., anchorage-independent growth and enhanced survival capability) and that this effect requires the transfer of the protein cross-linking enzyme tissue transglutaminase (TTG) cooperating with fibronectin.
ultrahigh resolution live confocal microscopy such as STED or particle membrane
interaction observing FLIM, FRET is needed to address the precise mechanism of
how and under which conditions individual microvesicles contribute to
metastasis. Again, our system is superbly designed to address this question.
Last not least, it is no surprise that cytochalasin has a unique antineoplastic
activity that could potentiate a novel class of chemotherapeutic agents
(Trendowski, et al. 2015) . This underlines the importance of our work: We
use cytochalasin to generate microvesicles from stress fibers, yet cytochalasin
itself is a rather “benign” agent. Its arrest of the actin filament system may
inhibit tumor growth but actually facilitate metastasis. As mentioned at the
begin of our discussion, further research has to be done to differentiate
between cytochalasin’s glucose transporter inhibition and its ability to affect
the actin system and their probable cross talk. In this respect it should be
mentioned that cytochalasin, depending on concentration, can have a reversible
effect on cell actin filament organization. While a definite proof of the
cooperativity -or complicity- of microvesicles and invadopodia or stress fibers
for the facilitation of metastasis was beyond the scope of our work, we have
certainly opened the avenue for that possibility.
7. Wurm F, Jordan M (1997) Methods for calcium phosphate transfection. USA.
16. in vitroin vivo