Please notice that this presentation aims at summarizing currently available data only, without the claim of representing established knowledge. Thus, information presented in this course may be subject to change in the future, due to the rapid evolvement of the field of extracellular vesicles. So the title for this talk is going to be oncology, shared functional aspects of EVs in oncology. So my name is Dave Carter. I'm from Oxford Brookes University, and you can see me there with my cool exosomes cup. So here's the outline of the lectures in this particular section. So in Part 1, we'll talk about EVs and how they can contribute to cancer. In Part 2, I'm going to present Janusz's slides on EVs and how they can transfer material in the tumor microenvironment, and also, thanks to Michiel Pegtel who has helped also with these slides in this presentation. We'll start with a little overview to cancer, to give an idea of the scale of the problem. So cancer is still a major global problem. There more than 8 million deaths per year globally. The 10-year survival rate for all tumors is around about 50 percent, which is really quite poor. It's the second leading cause of death in the world. So cancer is really not cool, but trying to understand how cancer works, in my opinion, is cool. The reason for that is that it's important to be able to tackle cancer because we need better fundamental understanding of the causes and mechanisms of the disease, and how it progresses. One aspect that's only recently been studied, relatively speaking, is the role of extracellular vesicles, which by the way, are also very cool. So to help give a little bit of context of the roles of EVs in cancer, I'm going to tell you a bit about the concept of the hallmarks of cancer. So the hallmarks of cancer is a set of features that characterizes the behavior of most tumors. The framework to think about these hallmarks of cancer was developed by Hanahan and Weinberg in their very famous review article which has been cited like a gazillion times, and it's a brilliant article. So I recommend that you go read it. So these hallmarks are sustaining proliferative signaling or sustaining proliferation, evading growth suppression, replicative immortality, resisting cell death, angiogenesis, and invasion and metastasis. There are also some emerging hallmarks which are deregulating cellular energetics, and avoiding immune destruction. There are also some things called enabling characteristics, which if you go read the article, you can learn more about that. So I'm going to go through these different hallmarks, and we'll give examples of how EVs can contribute to these different hallmarks. So the first hallmark we'll talk about is the sustained proliferative signaling hallmark. Now, cells as part of normal homeostasis have to regulate cell division, and they can respond to signals that stimulate cell division. This involves a range of signaling ligands, receptors, intracellular signaling pathways, the activation of transcription factors, and subsequent transcriptional responses. In theory, any of these steps can potentially go wrong in cancer. Now, there is evidence that EVs can be involved in this process, and the aberrant signaling from EVs can also lead to proliferative signaling. So for example, we can have uptaken delivery of cargo which leads to proliferative signaling, or we can have interaction at the cell's surface, and which then stimulates signaling. So for example, it's been shown that stimulation of the PI3 kinase and AKT pathway, but also the ERK pathway can lead to increased proliferation. We'll talk a bit more about transfer of material between cells and tumor microenvironment later on in the second part. So just as there are signals that promote the growth of cells, there are also signals that normally suppressed proliferation, and cancer has evolved ways to evade these growth suppressors. So P53 is a classic example of a tumor suppressor which normally arrest growth into various conditions, including stress. So P53 is often mutated in a high proportion of tumors. But we know a lot less about how EVs could act in the context of suppressing growth. However, there are some early studies and some interesting nice studies that have given us some insights into how this could be. So one example is that EVs carry microRNAs that repress tumor suppressors can be released by sense cells, and some cancer cells can take these up. When they take these up, the microRNAs that repress these tumor suppressors now repress those tumor suppressors, and that means that those tumor suppressors are gone or reduced. So the brakes are effectively broken on the cell, which means it can continue to grow. Another possible mechanism is for tumor cells to actually jettison microRNAs with tumor suppressive capability, allowing them to relate the repression on oncogenic expression, and thus grow more rapidly. So if a microRNA is normally suppressing growth by stopping those tumor suppressors, let's just get rid of it. As cells, we can potentially just get rid of the tumor suppressors altogether. So potentially, we can jettison tumor suppressors, and there's evidence for example, the P10, unimportant tumors suppressor can be released in EVs, therefore evading the growth suppression. But we do need to do a lot more work to establish the extent to which tumors use these mechanisms to help them grow. Now, you've got a tumor suppressive microRNAs, and tumor suppressors can be jettisoned. When cells achieve the ability to stimulate their own growth and evade growth depressors, then you think that they are basically set to grow and grow indefinitely. However, they do first face some other barriers. So with each cell division, their telomeres, the protective DNA caps at the ends of the chromosomes, they get shorter. When they reach a critically short length, this can trigger the cell to become senescence. This is a state where the cell is metabolically reactive, but cell division has been halted. Interestingly, senescence cells release a variety of molecules in a process known as the senescence associated secretary phenotype or SASP, and these molecules we now know can include EVs. This process of SASP can lead to inflammation in the surrounding tissue, which in itself can promote the tumor growth. So cells that are able to actually escape senescence, and this can be through mutation in various genes that control the senescence phenotype, then continue dividing. As their telomeres continue to shorten, they face a process called crisis, in which chromosomal integrity can't be maintained during cell division, and this leads to widespread cell death. But any mutations that allow the telomere length to be restored, the telomere structure to be restored can reactivate and allow continued growth. This is often by the reactivation of the enzyme telomerase. So again, when this enzyme is reactivated, the cells can continue to grow because their telomeres are restored. The role of EVs in this particular hallmark are less clear. That we do know that senescent cells release EVs that can cause other problems to cells, as we've mentioned already with SASP. We also know now that the EVs released under stress conditions can affect telomere activity in bystander cells. So this is something that potentially is affecting this hormone. We also know that EVs can carry telomeric non-coding RNAs, suggesting that there's yet incompletely understood role of EVs in this hallmarks. So further work is required. Normal cells are able to detect abnormal conditions, such as DNA damage and excessive proliferation, and respond by initiating different types of programmed cell death. So these major problems would normally kill the cells. Tumor cells however, or cancer cells have developed mechanisms which helps prevent this cell death from occurring. Tumor cells can be killed, but it's harder to kill them. They're not as readily responsive to these problems. In terms of the role in EVs, EVs have been shown to be linked with cell death, and have been shown to be involved in helping tumor cells to develop drug resistance. So they are involved. But not just in the development of the cancer, but also dynamically in response to therapies. One example has been shown is bone marrow derived EVs can inhibit the JNK signaling pathway, and that can lead to the down-regulation of pro-apoptotic BCL2-like protein bin, which leads to reduced caspase activity, and therefore reduce cell death. EVs from cancer cells can also up-regulate the anti-apoptotic BCL2 proteins when added to other tumor cells. Again, this helps to resist cell death. When we try and treat tumors with chemotherapeutics, it's known that there are dynamic responses that involve EVs. So cytotoxic drugs such as cisplatin which are known to induce apoptosis. But some tumor cells have been shown resist treatment, and there are different ways in which that can happen. One of those ways is that these tumor cells can effectively load their chemotherapeutic agent molecules into EVs, and eject them from the cells, so that the tumor cells can use these EVs to carry the drug back out of them. Cells treated with various types of therapeutic, including ionizing radiation and chemotherapeutics, it's been shown that they cause the release of vesicles that when taken up by naive bystander cells, can induce a stress response, and that can help prime the cells to resist cell death in future. Also, it's been shown that EVs can act as decoys carrying molecules like HER2, which means that these EVs can act as a decoy for the drug. For example, trastuzumab or herceptin. So this protects the tumor cells because the monoclonal antibodies like trastuzumab, and are going after the EVs rather than the cells. So this can act as a way of allowing the tumor cells to become more resistant. So we still need to do more experiments to understand mechanisms by which EVs regulate cell death, and the way in which they contribute to resistance to cell death. But there is clearly a role that's been demonstrated for EVs in resisting cell death. Angiogenesis is a dynamic and normal process which allows new blood growth to service cells that are experiencing hypoxic conditions. Rapid growth of tumor cells leads to hypoxia which, as mentioned in itself, triggers angiogenesis, and this is needed for the tumors to grow to macroscopic levels. However, tumor cells can release EVs with the capability of inducing angiogenesis, and the ways in which these can induce angiogenesis are various and complex. So to give you some examples of various findings in the fields. So direct interaction of EVs from glioblastoma cells, EVs derived from glioblastoma cells and endothelial cells and the interaction between them, leads to increased and endothelial proliferation, for example. You can also get transfer of pro endothelial proteins such as VEGF or FGF and angiopoietin, can lead to increased angiogenesis. Also Delta-like 4, can be transferred by EVs and induce angiogenesis by inhibiting the notch signaling pathway. MicroRNA's can also be transferred via EVs, which then target specific genes for repression, through that repression leads to increase angiogenesis. In another example, lung cancer EVs have been shown to transfer EGFR into endothelial cells, leading to autocrine signaling via VEGF and the activation of the VEGF receptor. So that EGF are released by the tumor cells leads to an autocrine loop within the endothelial cells. So all of these different mechanisms can contribute to the angiogenic process and angiogenesis that supports tumor growth. The hallmark of invasion and metastasis is probably the best studies when it comes to the role of EVs, and arguably it's the most important as it's ultimately the metastatic tumors that tend to cause death in patients. The journey that a tumor cell has to make is long and complex, from its original site to the metastatic sites. So it involves various steps including, migration through and breakdown of the extracellular matrix, they have to pass through basement membrane, they have to interact with endothelial cells and pass through into the circulation either in the bloodstream or in the lymph, they then have to attach to secondary sites, endothelial locations, break back through extravasation, back into the recipient, into the secondary organ and again breakthrough that matrix and established and colonize new tumors. So this is a highly complex process, it's not straightforward. A lot of labs have tried to look at what is the role of EVs in this highly complex process. So several labs have now shown that when you take EVs from highly metastatic cells, and take those EVs and put them onto less metastatic cells that, that metastatic ability that, phenotype can to some extent be transferred between cells through EVs. But we know that EVs are also involved in lots of different steps along the process. So for example, it's known that EVs carrying enzymes like matrix metalloproteinases, can contribute to the breakdown of the extracellular matrix and that assists the cells as they invade from the primary side that helps to break down that matrix and clear a path for one. So we get matrix breakdown. We also know that EVs released by cells can potentially act as a foothold. So integrins and other proteins that are in the EV's when they interact with the extracellular matrix, they can provide a hand-hold or a foothold, if you like, to allow guided migration of the EVs through the extracellular matrix. EVs can also be transferred from tumor cells to immune or endothelial cells leading to a wider range of changes in the wider tumor micro-enviroment that ultimately are are prometastatic. So for example, we can have transfer EVs from tumor cells carrying microRNA's to endothelial cells leading to the repression of various genes, including for example, a tight junction protein and that leads to increased probability that the tumor cells can pass through those gaps between those endothelial cells. Epithelial to mesenchymal transition is also an important process in this metastatic cascade, as it allows more epithelial cells to become more vital and invasive. So EVs have been shown to carry various types of cargo such as, TGF beta and microRNA's that can induce EMT and that leads to increased potential for motility of invasiveness. Another way which EVs can contribute to metastasis is via preparation of a premetastatic niche. So we know that EVs released by tumor cells and can potentially get into the circulation, they can also get into secondary organs and sites and the interaction of those EVs with cells in that tumor microenvironment or the pre-metastatic niche can essentially seed the soil and alter the characteristics of these cells at that location, change the transcriptional profile et cetera. Which ultimately, establishes a more receptive site for the future arrival of tumor cells. It prepares the recipient organ, and that increases the probability ultimately that these tumor cells will be able to colonize. So we know that invasion of metastasis is a big role for EVs in various steps along the metastatic cascade. There's the tumor cell colonizing. In addition to the six primary hallmarks of cancer, Hanahan and Weinberg identified in their updated version of the hallmarks of cancer paper, and they identify two more emerging hallmarks of cancer. These are, reprogramming of energy metabolism and evading immune destruction. So first, I'll say a little bit about reprogramming of energy metabolism. Excessive use of the glycolysis pathway, something that cells tend to do under hypoxic conditions but, tumor cells have a tendency to rely more heavily on glycolysis for fuel even though there's usually enough oxygen around, and this is known as the Warburg effect. It effectively gives the cancer cells like a sweet tooth. But it's unclear what role EVs might play in this process, but it is known that EVs contain many glycolysis enzymes involved in the glycolytic pathways and also numerous metabolites. So it's possible they could play a role in modifying the metabolic function of recipient's cells, so they have metabolites and metabolic enzymes. The other is the avoiding of the immune destruction, so the cancer cells that present clinically, have ultimately managed to avoid destruction by the immune system. We know that immune system can clear a lot of tumors before they ever present. EVs released by these tumor cells, can play a role in this process of immune evasion. There are several examples that have now been described and it's known that EVs can carry many molecules that affect immune function. For example, tumor cells can release EVs that affect natural killer cells. Natural killer cells are lymphocytes and normally kill early tumor cells. But EVs released from the tumor cells, can suppress natural killer cell function, so that's one example. Another example is that EVs can suppress T-cell function. So they can induce T-cells to function as suppressive regulatory T-cells rather than cytotoxic T-cells. By altering those different functions, they can effectively prevent the immune system or reduce the chances that the immune system will be clearing those T-cells. Tumor cells can therefore use EVs to subserve another escape of clutches of the immune system. Adam Weinberg also described two enabling characteristics, these are features of tumor cells which enable the other hallmarks to happen. These are genomic instability and tumor promoting inflammation. Genomic instability, is the feature that allows the genome of a tumor to change and evolve relatively quickly and gives rise to a heterogeneous tumor mass. It's not exactly clear what all the different roles are that EVs can play in this. The one intriguing study has suggested a shine that there's a link between genomic instability and EVs. So in this study, we're shown that cells exposed to ionizing radiation, release EVs that can have effects or naive bystander cells. These effects include, DNA damage by standard DNA damage and also genomic instability suggesting the potential transfer of this phenotype between cells via EVs. Inflammation has also been shown to be important in promoting all the different hallmarks of cancer, particularly proliferation angiogenesis and metastasis. This ultimately helps the tumor to develop and progress. EVs can play a role in regulating this inflammations. For example, it's been shown that mass cells can transfer EVs with various proteins into tumor cells, that induce proliferation and migration, so we see this can contribute to the development of that tumor. Signaling through toll-like receptors can also lead to tumor promoting inflammation. So EVs from tumor cells, can be taken up by tumor associated macrophages and microRNA 21 that's been shown to be transferred via EVs, connects as ligands to activate toll-like receptor in these tumor associated macrophages leading to the activation of the NF-kappaB pathway resulting in a pro-inflammatory signaling phenotype that promotes Migration. Tumor is not just a homogeneous mass of cancer cells, but a diverse ecosystem with many different cell types. Interspersed among the tumor cells, are an army of stromal cells with various functions. So for example, there are cancer-associated fibroblasts, there are tumor associated macrophages. There's lots of other cell types and stromal cell types too and ultimately, they are able to interact with tumor cells sometimes via EVs and this can lead to approaching agentic phenotype. These cells are normally supporting organs in a healthy way. So for example, endothelial cells, blood vessels, fibroblasts involved in structural aspects and support an organ, macrophages can pick up dying cells etc. So they have various normal functions. But in cancer cells, their presence supports the tumor mass and their function is further corrupted to the benefit of the cancer. This corruption of function is in part mediated by EV's, the transfer cargo from cancer cells to stromal cells or vice versa. It's this communication which ultimately helps the tumor to grow and to become more aggressive, and this communication is ongoing throughout normal tissues, but also is a key aspect of supporting the tumor microenvironment. If you're interested in learning more about the tumor microenvironment, which is becoming increasingly appreciated as a key drive and a key factor of tumor growth, then there will be another lecture in this [inaudible] of series.The important thing is EV mediated cross talk is actually key to the behavior of the tumor and the tumor microenvironment. So just to conclude then, I've told you that EVs can contribute to every aspect of cancer and cancer progression and that they can do so by tapping into every one of the various hallmarks of cancer and emerging hallmarks and enabling characteristics. EVs are cool, let's get this straight. So EVs are cool, but in cancer always they can be naughty, they go wrong. So understanding particularly how EVs go wrong in tumors, is important. We need that better understanding of what they do and how they do it, in order for spell to potentially target them therapeutically. So we do need a better understanding here so that we can potentially, hopefully in future, be able to target EVs as a therapeutic option. Better understanding is absolutely key here, and that's why we need to strive to move this forward as workforce. In the second part of the lecture, we are going to be talking about transfer of cellular components to the tumor microenvironment by EVs. So we're going to a bit more specific detail and give some more specific examples. This slide was prepared by Janusz Rak, from McGill University in Montreal, in Canada. I was rather hoping that we could have [inaudible] in writing this. But alas, you have me. But it's going to be very exciting all the same. In this lecture, we are going to cover a number of different topics including cancer cell vesiculation. How EVs can be used to transfer material between cells with a particular focus on oncoproteins and oncogenic RNAs and so on and how they impact on metastasis oncogenic transformation and so on. So you can see the list of topics, I won't read through them, if you want to see them in more detail before you start which just means pause the presentation here. But those are the topics that we'll be covering. Copyright licenses, no copyright licenses which are included here. Sources referenced as indicated. Cancer cells release their fragments in a form of several subtypes of extracellular vesicle. These EV subsets are distinguished by different sizes, properties and mechanisms leading to their biogenesis. Process of the EVs released from cells are often collectively referred to as vesiculation. Fundamentally, there are three different scenarios that may lead to the release of EVs from cancer cells. Firstly, EVs may form as a result of cellular fragmentation following apoptotic cell death, and these EVs are relatively large and often referred to as apoptotic bodies. Apoptotic body formation is controlled by genes regulating cell deaths, such as caspase. Secondly, EVs may form my external budding of the plasma membrane, fiber cells. These EVs are usually larger than a 150 nanometers in diameter and are often described as microvesicles, ectosomes or microparticles. Now, this process is controlled by several molecules as well that regulate membrane dynamics, such as aphasics, right GTPase, scramblase and several others. Distinct mechanisms lead to shedding of larger EVs with diameter greater than one micro meter. That might includes so called large oncosomes, migrasomes and other EVs. The third mechanism of EVS release, begins with the formation of small vesicles of 30-150 nanometers. Which bud inside the living of the cellular organelles known as the lay endosome. Endosome is filled with such small intraluminal vesicles, known as multivesicular bodies and it may be directed to the plasma membrane where the ILVs or the intraluminal vesicles are released to the extracellular space. Such EVs are typically referred to as exosomes. Exosome bio-genesis is controlled by several molecular mechanisms, including a multi-protein endosomal sorting complexes required for transport or esco proteins. Other regulators include tetraspanins, Rab proteins and lipids, as indicated on the left side of the diagram here in red. Cells may release several subtypes of exosome simultaneously e.g, large and small. As well as smaller solid particles known as exomeres. Cancer cell vesiculation may also be affected by different processes as listed on the right of the screen here. Such as influences of the microenvironment or oncogenic transformation, which will be discussed in more detail in the later slides. The process of the EV release from cells is thought to serve two fundamental functions. First, for the EV releasing cell or EV donor cell. This process provides the opportunity to rapidly and efficiently remove large amounts of molecular ballast store and once it's superfluous activity that may interfere with the cellular homeostasis, overwhelm or evade intracellular degradation machinery such as the lysosome. Unfortunately, in cancer, this explosion may also mean the removal or dumping of tumor suppressor activities such as PTEN or microRNAs, thereby potentially accelerating tumor growth. Secondly, for the cells exposed to the cancer derived EVs and their microenvironment, also referred to as the recipient cells. Such EVs represents a unique mechanism, whereby they can acquire new properties. EV transfer may enable intercellular transfer of molecules that may or may not be secreted through the conventional pathways, including integral cellular proteins such as cytokines, transmembrane receptors and nucleic acids. Such intercellular transfer may be exemplified by the export oncogenic epidermal growth factor receptor or it's mutant form, known as epidermal growth factor receptor variant III from dynacells to that indolent or normal neighbors. Once transferred to recipient cells, EGFR variant III, oncoprotein may elicit stimulatory signals that could lead to activation of growth pathways, functional reprogramming of recipients cells, and changes in their phenotype. EV mediated intracellular transfer has been studied in the case of several cancer-related macromolecules. Some of which represents product transforming oncogene such as EGFR variant III as we've mentioned, but also KRAS AND HER2. While others possess accessory functions as mediators of adhesion and invasions such as integrons or mediate vascular and blood-clotting abnormalities, such as tissue factor which are frequently observed in cancer. Expelled tumor suppressors could also be transferred to recipient cells, but their effects on normal recipients are presently unclear and could be limited by intracellular degradation. EVs may interact with and stimulate the recipient cells in several different ways. For example, EVs may simply bind to the cellular surface of recipient's cells and stimulate or inhibit their responses through interactions with membrane receptors. EVs may also release their soluble or bio active content such as growth factors near target cells and facilitate ligand receptor interactions. EVs may fuse with cellular membranes delivering that cargo into the interior of the recipient cell or modify their surfaces. EVs may also undergo a full internalization by recipient cells through processes of endocytosis, macropinocytosis, or phagocytosis etc. In this manner proteins, lipids, RNA, DNA all these different cargos, contained within EVs could be transferred into recipient cells. There in, these molecules may either exert biological effects as parts of a cell to cell interaction network throughout the tumor microenvironment or the cargo may undergo degradation by the recipient cells. EVs mediate communication between cancer cells and components of the tumor microenvironment. There are several examples of such interactions. EVs may impact donor cancer cells in the autocrine manner by interacting with their surfaces and promoting cell migration or other processes. EVs may also play a role in communication between tumor cells and the angiogenic vasculature. Several cancer-related processes involve such vascular interactions including angiogenesis, new blood vessel formation mediated by EV's associated receptors, growth factors and other cargo. Thrombosis is abnormal activation of blood-clotting systems resulting from transmission of procoagulant proteins such as tissue factor. Vascular permeability, it's induction of vascular leakage and edema locally, and in distant organs by these that alter endothelial cells by transferring permeability mediators such as proteins and nucleic acids. Cancer derived EVs are known to educate blood cells, such as myeloid cells and platelets, thereby contributing to inflammation and metastatic permissiveness. EVs may also directly contribute to amino modulation or immuno suppression, for example, by transferring checkpoint molecules, eg PDL 1 between cancer cells and immune cells. EVs contributes a paracrine communication between different cancer cell populations. One example of such interactions is the previously mentioned transfer of the oncogenic receptor, EGFRE3 from malignant to glioma cells, resulting in phenotypic changes. Metastatic dissemination of certain cancers may entail long-range interactions between EVs, released from the primary tumor and distant tissues. Such EV homing to distant sites, is stromer and vasculature, is mediated by integrons on the surface of cancer Evs. Evs may also promote tumor invasion due to their ability to carry matrix metalloproteinases, and these enzymes may cause degradation of extracellular matrix and promote tumor invasion as has been mentioned earlier. A particularly starting example of EV mediated effects in cancer is their role in long-range intercellular communication during metastatic dissemination of tumor cells to distant organs. In so doing, EVs exert important effects in the primary tumor, eg during invasion, during their passage in the circulation. For example, on effects on thrombosis medication blood cells, and also at the metastatic side. Effects of cancer EVs at the metastatic site is frequent in linked with the concept of the pre metastatic niche. It's long been observed that specific populations of metastatic cancer cells have a preference to colonize specific organs, such as lung, liver or brain. However, prior to arrival of cancer cells, these organs must be conditioned to support metastatic growth, a process described by lightens group and referred to as the pre metastatic niche. Evidence suggests that primary tumors, flood the blood circulation with exosome like EVs, the home to these specific organs size, and activate very cellular components such as stromal cells, phagocytes and blood vessels. These organs seeking exosomes recognize their targets through specific surface integrant. For example, Alpha six Beta four integrin mediates harming of EVs to fiber nekton in the lung while Alpha five Beta three integrin mediates adhesion of cancer EVs to laminin in the liver. Other EVs may also condition brain for metastasis. You can see the top panel. Subsequently, dissemination of cancer cells from the primary tumor enables them to preferentially colonized organ signs, These premature setting niches to which they previously sent there activating exosomes. Tumor-related exosomes may also simply damage the endothelial lining of distant organs, enabling the entry of circulating cancer cells. This occurs in the brain where the blood brain barrier is compromised by EV mediated transfer of micron A 1, Micron A 5 from cancer cells to endothelium as depicted on the bottom right section of the image here of the slide. While in certain target organs, cancer cells may orchestrate their niche, in others the niche may influence the cancer cells. For example, in the brain exosomes from brain tissue astrocytes transfer micron 990A to cancer cells, which results in down-regulation of the tumor suppressor P10, and increase in metastatic aggressiveness. You'll see that again in the bottom of my image. EVs are a part of the cellular communication network orchestrated by cancer cells within the tumor microenvironment. Oncogenic mutations often represent a triggering event for these interactions, as can be exemplified by several non cell autonomous consequences of the expression of meets EGFRvIII in glioma cells. As expected, the oncogene expressing cells, those with EGFRvII become aggressively tumorigenic upon intracranial inoculation. At the same time, EGFRvIII triggers up-regulation of angiogenic factors such as vascular endothelial growth factor VEGF, coagulation proteins like tissue factor and inflammatory mediators such as interleukin aids. Notably, EGFRvIII profoundly altered cellular vesiculation, in terms of EV emission profile, protein content diversity and biological activity. EVs play a role in cancer-related thromboses. Certain cancers provoke blood-clotting throughout the vascular system including sites distant to the tumor, such as deep veins and likes. This serious condition is known as venous thromboembolism, VTE, and is associated with poor prognosis. The left side, the left panel here on the slide, illustrates how EVs may contribute to VTE in cancer, by acting as carriers of coagulant proteins such as tissue factor. This could happen in at least two different ways. First, circulating and TF tissue factor carrying EVs could directly interact with clotting factors in blood. This effect could be transient due to the limited half-life of EVs in blood, and secondly EVs can interact with blood cells and endothelial cells, rendering them TF positive and pro coagulant. The right panel just shows examples of experimental data supporting the ability of cancer derived EVs to trigger tissue factor activity in endothelial cells at the top or leukocytes at the bottom there. In both cases the effect of EVs is measured by testing the catalytic activity of tissue factor against exogenous clotting factors, factors 710 and this is known as transcription factor coagulant activity I will say or TF-PCA. The top of endothelial cells [inaudible] are shown to up regulate TF-PCA after the uptake of tissue factor containing EVs from mesenchymal cancer cells harboring oncogenic EGFRvIII, and in the bottom panel here you can see cancer cells harboring Rasoncogene, as shown to induce TF-PCA in cultured leukocytes. While various effects of EV-associated clotting and angiogenic factors may also occur in disease states other than cancer, such as injury or inflammation, cancer EVs may also exhibit vascular effects that are relatively unique. One such effect occurs through the transfer of oncogenes from cancer cells to angiogenic endothelium. The upper portion of the diagram here illustrates how transfer of oncogenic EGFR could impact endothelial cells by triggering abnormal expression of VEGF. VEGF is central to vascular growth in endothelial homeostasis, but it normally acts in a paracrine manner by being produced by cells other than endothelium. The bottom panel here shows some of the data illustrating how oncogene containing these referred to as oncosomes transfer EGFR to endothelial cells triggering signaling pathways and VEGF expression. So on the left, the facts analysis shows the acquisition of surface EGFR expression by endothelial cells, the HUVEC that had been incubated with EGFR positive cancer cell derived EVs. Middle panel shows Western blot analysis showing that phosphorylation of EGFR downstream pathways. The Erk pathway and Akt in endothelial cells that have taken up EV-associated EGFR. The next panel shows that the transfer of EGFR triggers the expression of VEGF, mRNA in endothelial cells in which also the main VEGF receptor becomes phosphorylated. These results show that endothelial cells can produce and respond to VEGF following EV-mediated transfer of oncogenic EGFR. A number of compounds listed in the right bottom panel there may interfere with vascular effects of EV-mediated oncogenic transfer. Their therapeutic utility in cancer is worth considering in that line. Functional proteins like tissue factor or oncoproteins like EGFR are not the only cargo that EVs could shuttle from cancer cells to various cells in the tumor micro-environment. In fact, the pioneering recent work of several labs has documented a role for EVs in extracellular emission and intracellular transfer of RNA often referred to as exoRNA. The various RNA bio-forms found in cancer EVs are listed in the left panel in red notably including several regulatory and non-coding species as well as mRNA. RNA enters the cargo of EVs through several mechanisms including share over-expression, molecular modifications, association with RNA binding proteins or sequence zip codes. Once released, RNA containing EVs enter the recipient cells through different mechanisms such as membrane fusion, endocytosis, phagocytosis, macropinocytosis, etc. The transferred exoRNA may then be expressed and exert regulatory effects or simply undergo degradation in the recipient cells. There are several examples shown in the right section of the image here of exoRNA expression in recipient cells. This includes detection of transcription factors like OCT4, activity reporter molecules like luciferase and cre- recombinase or microRNA in cells that have taken up the receptive EVs. For instance, EV-mediated microRNA transfer was shown to result in suppression of canonical microRNA targets such as PTEN. Lying colleagues who've directly documented the translation of mRNA transferred to recipient's cells through the EV uptake mechanism. In this case, GlucB and mCherry protein levels were then regulated by treatment of EV-recipient cells with protein synthesis inhibitors cycloheximide. One of the most tantalizing aspects of EV-mediated communication is the intercellular transfer of DNA especially genomic sequences containing oncogenic mutations. It's presently unclear how genomic sequences in chromatin enter the EV compartment in the **** of cancer cells. Among the possibilities that are being explored is the formation of apoptotic bodies by dying cells or EV-mediated release of cytoplasmic chromatin from viable cells. EV-associated DNA may contain full length oncogenic sequences. Whether this material is efficiently transferred to normal cells and can cause that permanent transformation by horizontal transformation is under debate. Some data, shown in the top right panel here illustrate the EV-mediated transfer of oncogenic DNA from cancer cells harboring mutant H-ras to a normal rodent fibroblasts. This transfer causes morphological changes and increases foci formation which is often regarded as a sign of malignant transformation. However, this effect is transient and reversible in nature with foci disappearing after 3-4 weeks of treatment with the H-ras DNA containing EVs. In this case, the H-RAS DNA persists but eventually disappears and doesn't integrate into the genome of the recipient cells. Large-scale horizontal transformation through intercellular transfer of oncogenic DNA between different cellular populations seems unlikely. Such a scenario would also be inconsistent with several tenants of cancer genetics as listed in the bottom section of the right panel. So if you're interested in more details of this please have a look at the references listed on the last page at the bottom here. This image here summarizes some of the links between oncongenic transformation and EV-mediated processes of cell-to-cell communication in cancer. These include, one, EV-mediated release and transfer of oncogenes in all their molecular forms such as mutant proteins, mRNA, and DNA. We've illustrated this point here using the EGFR variant three and RAS just as an example. Two, oncogenes reprogram multiple components of the EV cargo, thereby changing their contents and biological activity. Three, oncogene can also impact expression of genes that regulate biogenesis and release of EVs resulting in distortions of the vesiculation process as such. Four, changes in the vesiculation mechanisms may result in oncogene dependent alterations in the EV repertoire, composition, and heterogeneity among and within EV subtypes such as exosomes. Five, oncogenes impact on the transferability of EVs between the cells. They may alter the surface properties of EVs and their ability to interact with a given cell type. Six, oncogene transformed into cancer cells often exhibit an increased or altered ability to uptake EVs. So in this presentation we have described selected examples of how cancer in general and oncogenenic transformation in particular exploits vesiculation pathways as a mechanisms to communicate with a tumor micro-environment and alter it. The EV may trigger local and systemic responses that create states permissive disease progression, therapeutic resistance, or metastasis. Here for a reference, for interests, I acquire a lot of references that you can have a look into if you're interested in following up on any of this.
