Please note 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 involvement of the field of extracellular vesicles. Welcome to the MOOC chapter Oncology communication within a tumor micro-environment. My name is Jason Webber from Cardiff University. I have produced this lecture in collaboration with Dr. Dan Lambert from the University of Sheffield. In this lecture, we will highlight key findings, demonstrating the importance of extracellular vesicles and regulation of processes such as angiogenesis, stromal cell activation, tumor growth metastasis. It is designed to compliment other lectures within the MOOC to online lecture series, on extracellular vesicles in health and disease produced by the International Society for Extracellular Vesicles. Let's begin. The tumor micro-environment or TME is extremely complex and highly dynamic. It consists not only of cancerous cells, but also other residents cells, including fibroblasts, endothelial or vascular cells, pericytes, adipocytes, [inaudible] immune cells that have infiltrated the local environment. To understand the functions of various cells within the TME, many studies have focused on the role of soluble growth factors and extracellular matrix components as modulators of the TME. There has however, been a recent growing interest in the role of extracellular vesicles or EVs as regulators of the TME. All cells are capable of secreting EVs into the extracellular space. Vesicle secretion is known to become elevated in response to cellular stress. This is particularly relevant in cancer, where stress can be a direct result of disease-related changes. This includes a remodeling of the extracellular matrix, nutrient deficiency, hypoxia associated changes to the local environment and other factors. Furthermore, such stimuli can also trigger alterations to the molecular cargo contained within secreted EVs. In this lecture, we describe several key functions of extracellular vesicles within the tumor micro-environment. Key examples include the regulation of angiogenesis, the activation of stromal fibroblasts and tumor metastasis. While there is still much to be learnt, it is becoming increasingly obvious that EVs play a major role in regulation of the tumor micro-environment and therefore, subsequent tumor growth and disease progression. A major hallmark of cancer is the ability of tumors to generate their only vasculature in order to meet growing demands for oxygen and nutrients. The formation of new blood vessels from pre-existing vasculature is termed angiogenesis. Whilst angiogenesis is vital for numerous physiological and pathological responses within a healthy individual, it is also a feature of tumor growth and disease progression. As the tumor grows, it will become hypoxic and deprived of nutrients. This will trigger an angiogenic switch regulated by both anti-angiogenic and pro-angiogenic cytokines such as endothelial growth factor, fibroblast growth factor, a pericyte growth factor, and vascular endothelial growth factor. This results in the formation of new blood vessels in order to supply the tumor with oxygen and nutrients. However, continued remodeling of this vasculature network results in poor structural organization and leaky blood vessels. Not only does this lead to an irregular blood flow, but it also allows tumor cells to invade into the circulatory system, which can result in metastasis to distant sites within the body. Whilst many anticancer therapies look to block angiogenesis, it has been reported that cancer cells can become non-responsive to antiangiogenic therapy over time. Furthermore, the vessel abnormality is associated with tumor angiogenesis, can render tumors non-responsive to other forms of therapy such as radiotherapy. Therefore, there exist an argument that therapy is designs to repair blood vessels, and therefore restore vessel normalization, may actually improve tumor response to chemotherapy or radiotherapy, and therefore improve patient outcome. Whilst this remains to be seen, it is clear that an improved understanding of the mechanisms that regulate angiogenesis, will be critical for developing future strategies to treat patients with cancer. Cancer cell derived EVs have been shown to promote angiogenesis in several different cancer types. The expression of tetraspanins is a key feature of a variety of different EVs including [inaudible]. The Tetraspanins have been implicated in various biological processes including artisan and motility of endothelial cells. It is therefore, perhaps unsurprising that pancreatic cancer cell derived EVs, baring the tetraspanin CO-029, also known as Tetraspanin 8, have been shown to promote vessel branching. Tetraspanin 8 has also been shown to modulate the binding and uptake of cancer cell derived EVs by endothelial cells. EVs have been shown to promote angiogenesis by our activation of specific signaling pathways. Prostate cancer EVs have been shown to contain the CSEC tyrasine kinase, insulin-like growth factor receptor 1, IGFR, and focal adhesion kinase, FAK. As crosstalk between FAK and IGFR has previously been shown to promote angiogenesis. It is believed that the prostate cancer EVs also have a pro-angiogenic function. A major finding by Al Nedawi et al , demonstrated that lung cancer EVs could deliver mutated endothelial growth factor receptor protein to endothelial cells. This results in continuous activation of the EGF receptor and signaling through the MAP kinase and AKT pathways. Ultimately, misled to autocrine VEGF secretion and an enhanced endothelial cell response to VEGF. The same group provided evidence that interfering with EV binding to recipient endothelial cells could reduce the angiogenic response in [inaudible]. This suggests that targeting of cancer cell derived EVs, may be an effective anti-angiogenic therapy. Evs from several cancer types are known to be enriched with messenger RNA transcripts relating to pro-angiogenic function. These can be delivered by EVs and then translated by the recipient cell. A study Skong et al , examines the mRNA content of glioblastoma EVs. Here they demonstrated EV mediated to delivery of angiogenesis related mRNA. This was then translated to protein within the recipient cell. Similar observations have also been made for colorectal cancer and a study by Hong et al demonstrated that EV mediated delivery of angiogenesis related mRNAs resulted in enhanced endothelial cell proliferation and subsequent increase in tubule formation within the 3D culture systems. Collectively, these findings suggest that cancer cell derived EVs, can play a major role in regulation of tumor growth and metastasis by facilitating angiogenesis related to processes. In addition to mRNAs, EVs have also been shown to encapsulate microRNAs and may deliver these microRNAs as a means of regulating the transcription of the recipient cell. Delivery of microRNAs by EVs to endothelial cells has been shown to enhance endothelial cell migration and subsequent tubule formation within 3D cultures. The transfer of microRNAs such as miR-92 and on miR-17-92 have previously been reported to enhance angiogenic function. Furthermore, the transfer of the miR-17-92 cluster from EVs to endothelial cells was shown to regulate each endothelial expression of integrating Alpha five. Lots of this integrating resulted in enhanced endothelial cell migration and tubule formation, thus demonstrating that EV mediated delivery of microRNAs also play a role in regulation of angiogenesis. As the tumor continues to grow, the diffusion distances from the existing vasculature to the center of the tumor increase. This results in a decreasing oxygen gradient and tumor hypoxia. It is widely known that hypoxic conditions have an aberrant impact on the cellular secretome. This includes an enhanced secretion of cancer EVs with an altered molecular cargo. EVs derived from solid tumors cultured in hypoxic conditions have been shown to become enriched with hypoxia regulated mRNAs and proteins such as caveolin 1, interleukin 8, various matrix metalloproteinases and PDGF. Furthermore, these hypoxic EVs were shown to have a greater propensity to promote angiogenesis compared to EVs from normoxic cells. The microRNA cargo of EVs have similarly been shown to become altered in response to hypoxia. Both breast cancer and leukemic cells cultured in hypoxic conditions have been found to secrete EVs containing elevated levels of microRNA-210. These EVs demonstrated a greater capacity to drive endothelial cell tubule formation compared to EVs from cells grown under normoxic conditions. Whilst many studies have focused on the direct activation of endothelial cells i.e EVs, Evs have also been shown to promote angiogenesis in an indirect manner by interactions with a variety of other cell types. Such studies have shown that EVs can activate primary stromal cells including fibroblasts. This causes elevated secretion of multiple pro-angiogenic factors that can drive endothelial cell migration and vessel formation, therefore facilitating in vivo tumor growth. Further studies have demonstrated that cancer EVs can stimulate the secretion of angiogenesis related factors such as HGF, VEGF, and MMPs from bone marrow derived mesenchymal stem cells. EV activated MSCs were also shown to support the formation of endothelial vessel-like structures. Similarly, EVs from metastatic melanomas have been shown to interact with bone marrow progenitor cells via the tyrosine kinase MET. This results in increased tumor vascular density in vivo. Numerous studies have shown that EVs are promoters of angiogenesis, either by direct or indirect interaction with endothelial cells. Such activities can be enhanced with the modulation of the EV cargo in response to the dynamic conditions within the tumor microenvironment. It is therefore clear that EVs play an important role in tumor angiogenesis and the subsequent tumor growth. The tumor microenvironment is very complex, consisting of multiple cell types. Cancer Associated Fibroblasts, CAFs, have frequently been shown to be the most prominent cell type within the microenvironment of solid tumors. CAFs are often identified by their expression of the smooth muscle protein, Alpha smooth muscle actin, shown here in the inset to picture as a brown stain contrasting with the blue nuclei of both cancer cells and CAFs. This is normally only expressed by activated fibroblasts that have undergone differentiation to gain a myofiberblast like phenotype. As a result, CAFs are often referred to as myofibroblasts. It's becoming clear that CAFs are actually a complex mixture of different phenotypes. Although the origin of CAFs is contentious and may include cells derived from bone marrow stem cells and other diverse sources including endothelial cells and parasites, it is generally accepted that at least a proportion of CAFs arise from resident fibroblasts corrupted by cancer cell derived factions. Regardless of their origin, the presence of CAFs is associated with resistance to treatment and poor patient prognosis in a number of cancer types. Soluble factors released by cancer cells have been known for many years to promote changes in normal resident fibroblasts towards a cap-like phenotype. Several media issues of this affect have been reported, with transforming growth factor Beta, TGF Beta particularly prominent for amongst these. Multiple studies have demonstrated that EVs secreted by cancer cells are also able to promote the acquisition of CAF-like properties and a variety of stromal cell types. The mechanisms responsible for this effect remain to be fully elucidated, but several studies have demonstrated that EVs for multiple cancer types can deliver TGF Beta, resulting in differentiation of stromal cells such as fibroblasts or MSCs to an Alpha smooth muscle actin positive phenotype. It has been established that EV-associated TGF Beta can activate smart, dependent signaling pathways, resulting in a stromal cell phenotype that is capable of facilitating tumor growth in vivo. Further studies have demonstrated that cancer EVs can drive TGF Beta dependent MAP-kinase signaling. EV transfer or various RNA species including messenger RNA and micro RNA have also been implicated in the initiation of CAFs. Multiple EV sub-groups have been shown to regulate stromal cell phenotype. Whilst many studies have focused on the role of small EVs often referred to as exosomes, a recent study has highlighted the uptake of large EVs termed oncosomes, which was shown to result in elevated conscriptional activity of MYC and fiberglass differentiation, resulting in enhanced tumor growth. Stromal cells activated by cancer cells secreted EVs can in turn release their own EVs into the tumor microenvironment, which can then be internalized by cancer cells. Stromal cell derived EVs have been shown to contain a variety of RNAs and proteins which can be delivered to recipient cancer cells, resulting in a two-way communication mechanism. Uptake of these stromal EVs has been linked to enhanced tumor growth in vivo. A study by Luga et al, highlighted to the actions of stromal cell derived EVs. Here, it was demonstrated that CD81 positive EVs from activated fibroblasts could in turn activate the one-two signaling pathway within breast cancer cells. This resulted in an increase in cancer cell motility and subsequent tumor metastasis. Furthermore, it has also been revealed that stromal EVs can confer chemo resistance to surrounding cancer cells via the transfer of RNAs. Boelens et al demonstrated that the transfer of RNA from stromol EVs to recipient breast cancer cells resulted in the activation of rig one antiviral signaling This ultimately led to the induction of [inaudible] signaling, and subsequent resistance to both radiotherapy and chemotherapy. The secretion of EVs from activated stromal cells and subsequent activation of cancer cells suggests that EVs play a complex role in reciprocal cross- communication between cancer cells and the surrounding stroma. This complex mechanism of cross-communication results seem progression to aggressive therapeutic resistance disease. Cancer cell invasion and disease progression, has been linked to remodeling of the Extra Cellular Matrix, ECM. The major physiological role of fibroblasts is to regulate the composition and physical characteristics of the ECM. They do this by the secretion of a wide variety of proteins and glycoproteins. These are proteins with large sugar groups attached to them. This provides the scaffold in which cells sit and helps to establish the tissue architecture. This is vital for normal cell-to-cell communication and cellular responses to physical cues. Maintaining the ECM, also requires regulated turnover of its constituents which is predominantly performed by a family of enzymes termed Matrix metalloproteinases, MMPs. Changes in ECM structure and function are key feature of solids tumors and contribute to tumor growth and the spread into the surrounding tissues. EVs are rich in ECM components such as heparan sulfate proteoglycans which are thought to be important in their interactions with the ECM and with cells in binding to paracellular matrix proteins such as fibronectin. In addition, they have been shown to harbor MMPs, and these can contribute to remodeling of the ECM in order to facilitate invasion by cancer cells. The concept that cancer cells release circulating factors to prepare a distant sites for colonization by metastatic cells entering the circulatory or lymphatic site has existed for decades. In recent years, evidence has emerged for a role of EVs in this seed and soil hypothesis. Several groups have reported that circulating tumor derived EVs released by cancer cells or possibly even cells from within the tumor micro-environment, invoke changes in specific organs preparing a niche for metastasis. Indeed, evidence has emerged that this mechanism may account for a phenomenon that has puzzled cancer researchers for decades. That is the predilection of cancers to metastasize to specific organs. For example, prostate cancer preferentially colonize into the bone, an effect known as organotropism. There are numerous studies demonstrating a variety of immune regulatory functions of EVs. These will be covered in much greater depth, and an additional lecture titled, EVs in Immunity, within these series. EVs play an important role in regulating the host immune response to tumors. Here we will briefly summarize the key actions of cancer EVs that result in immune suppression. Many studies have described an immune activating role of EVs from monocyte to precancer cells which were capable of T-cell stimulation. However, various other reports have also demonstrated that cancer cell EVs, can suppress T cell activation through the delivery of heat shock proteins as well as RNA. It has been demonstrated that cancer EVs can skew differentiation of dendritic cells away from an antigen presenting phenotype towards that of a TGF-Beta producing myeloid cell that can suppress T-cell responses. Further evidence has shown that cancer EVs can activate monocytes causing them to gain an M2 type macrophages phenotype that is capable of supporting tumor growth. One of the major problems facing the treatments of cancer patients is the propensity of tumors to acquire resistance to therapy, leading to relapse. EVs have been reported to contribute to resistance to therapy in a number of ways. Firstly, EVs may directly block cancer toxin drugs by acting as decoys. For example, by expressing antigen targeted by antibodies and immunotherapies such as checkpoint inhibitors. In addition, they can sequester chemotherapeutic agents reducing the penetration of tumors. EVs have also been implicated in transferring resistant phenotype between cancer cells and their contribution to corrupting fibroblasts of the TME results in an altered ECM which reduces the ability of therapies to reach cancer cells. Finally, EVs are known to play a role in the so-called bystander effect in which the effects of tumor directed radiotherapy spread resulting damage to surrounding healthy tissue. These effects are sometimes severe enough to force treatment to be halted. Please also refer to the MOOC chapter; Cancer Proliferation and Survival, for further information on drug resistance. As research into EVs continues to evolve, it is becoming increasingly obvious that the action of EVs within a Tumor Macro Environment are very complex. EVs exert multiple effects across a highly diverse range of different cell types. In this chapter, we focused on specific roles of EVs. We have discussed the role of cancer cell derived EVs, in terms of promoting angiogenesis. EV mediated delivery of a variety of proteins mRNA and microRNA, can increase endothelial cell proliferation and migration, leading to enhanced formation of endothelial tubules. This is supported by various studies showing that cancer EVs can trigger vessel formation and enhanced tumor growth in vivo. We have also described mechanisms of activation of stromal fibroblasts by cancer cell EVs. Stimulation of fibroblasts with EVs results in an activated phenotype, often referred to as a cancer-associated fibroblasts. CAFs play a key role in remodeling the extracellular matrix and further supports tumor angiogenesis. CAFs also secrete their own EVs and can confer therapeutic resistance to surrounding cancer cells. Overall, the secretion of EVs from both cancer cells and activated fibroblasts results in a complex mechanism of cross-communication that leads to enhanced tumorgenic activity and aggressive disease.
