 Hi, I am Dr. Tapastri Roy Sarkar. I am a faculty in the Department of Biology at Texas A&M University. Thank you so much for giving me a chance today to talk about my paper that recently published at Onko Target. The title of the paper is Carcinoma Cells that have undergone an epithelial to missing camel transition, differentiate into endothelial cells and contribute to tumor growth. So first I would like to say thank you to all my authors. We have graduate students, undergraduate students, post-doctoral scientists, and the faculties from Texas A&M University, the University of Texas, Indian Anderson Cancer Center, and also from Hamamatsu University School of Medicine in Japan. So they worked really hard to publish this paper. Now let me begin with the history of the research that leading up to this paper. So we know that angiogenesis is a normal physiological process that entails the development of new blood vessels through remodeling of a pre-existing vasculature that underpins by endothelial cells, sprouting, proliferation, and fusion. The ability of solid tumor to induce and sustain angiogenesis is termed as neo-angiogenesis, especially in the cancer context. And it has been recognized as one of the distinguishing hallmarks of cancer. Accordingly, the induction of this angiogenesis that helps the tumor growth beyond a certain critical size. That means when the pre-existing tissue's vasculature becomes inadequate to support the ever-increasing growth of this tumor demands, then this angiogenesis happens. In addition to ensuring the availability of essential nutrients and oxygen, the establishment of new blood vessels also facilitates the removal of cytotoxic metabolic by-products and carbon dioxide. And crucially, the most important thing, this angiogenesis process also helps the shaped tumor cells to disseminate. They travel through those blood vessels, go to distant locations, and they form secondary tumor or metastasis. Now, as they're rapidly developing tumors, they outgrow their blood supply. They begin to exhibit areas of localized oxygen deprivation. And this is known as hypoxia. This hypoxia initially induces EMT, which is known as epithelial to mesenchymal transition, that allows the cells to change their properties from epithelial and become more invasive and aggressive mesenchymal cells. And also, this hypoxia, in turn, it tilts the balance towards the pro-angiogenic factor, and then activating the angiogenic switch. And it's helping in this tumor or hypoxia-induced new angiogenesis process. Now, at the molecular level, an important nexus in tumor progression is the activation of this angiogenic switch that governed by the balance between multitude of pro and anti-angiogenic factor that regulate the endothelial cell proliferation and migration. So there are different ways this new angiogenesis can happen. I'm going to talk about four most important ones. So the dominance of pro-angiogenic factor that promotes the growth of new blood vessels from the pre-existing vessels through the so-called sprouting angiogenesis process. In addition to that, there is another process where the bone marrow-derived signals, or endothelial progenitor cells, that can be mobilized to initiate the de novo vessel formation in response to angiogenic signal. And that is known as vasculogenesis. However, there are some tumors. They can be a vascular tumor. They can nevertheless grow, at least initially, and without evoking any angiogenic response through the process that is known as vessel co-option. And what happened during that time is in this non-angiogenic mode, which mostly prevails in highly vascularized host tissues, the tumor cells they hijack the host vasculature. And they migrate along with the pre-existing blood vessels, thus invading the surrounding tissues. And the fourth option, which is known as vascular mimicry, that entails the de novo generation of micro vessels and that lined with very highly aggressive tumor cells embedded in a rich extracellular matrix. And they are mimicking the true vascular endothelium-like structure, but they are lacking the endothelial cell marker, such as CD31 or CD34. Therefore, the complex mechanism underlying this new angiogenesis differ from this physiological angiogenesis and lead to the formation of dysfunctional and disorganized blood vessels with a defective endothelial layer. Nevertheless, that fuels tumor progression and helping the tumor to grow. So as I just mentioned, that in addition to this angiogenesis hypoxia is also inducing epithelial to mesenchymal transition. Epithelial to mesenchymal transition is an elettant embryonic program that is misappropriated during this carcinoma progression. And it is a complex series of cellular reprogramming event that facilitates the conversion of immortal polarized epithelial cells into more intrinsically migratory, invasive, and aggressive mesenchymal counterparts. We previously identified that one transcription factor, foxy 2, during my postdoctoral study at MD Anderson. I work on with this foxy 2 transcriptional factor. And it is a key downstream effect of several converging EMT pathways. It functions to convert the mesenchymal and stem cell traits, underpinning the metastatic competence. And also interestingly, foxy 2 has been linked to angiogenesis, both in the context of normal development and tumor progression. It has the ability to transcriptionally regulate different pro-angiogenic factors, such as vascular endothelial growth factor, platelet derived growth factor, angiopoietin 2. So we also reported that the epithelial cells that induce to undergo EMT, they exhibit multilinear differentiation potential, similar to mesenchymal stem cells. So they display the ability to differentiate into major mesodermal lineages, such as osteoblast, adipocyte, and controsyte. So given those old facts together, we then decided to see or we sought to ascertain whether the cells that have undergone EMT in the hypoxic milieu can accurate these endothelial cell attributes, and therefore, augments tumor growth by directly contributing to the tumor vasculature. As I just mentioned, all those different procedure of endothelial angiogenesis process, as we know that also they are trying to mobilize the endothelial progenitor cells that are coming on the blood vessels. Here we are trying to say that one, the ansa cells which are induced this EMT procedure. So in this study, what we are trying to say that the cancer cells that undergone EMT, they can trans-differentiate into endothelial cells, and they can form the blood vessels, helping in this angiogenesis process. So they are not depending on the endothelial progenitor cells that can come from outside and form the blood vessels. So they can be independent. They can make their own blood vessels. So these are our study showed the direct evidence that the tumors, they can be independent. Next I'm gonna talk about the most notable part of my work. So we have done a lot of in vitro studies as well as in vivo studies. So in vitro studies, we have used triple negative breast cancer cell lines which has EMT induced cell lines. We have used epithelial cells which we use as a control. And we have also HMLE snail, HMLE twist cell lines which are also EMT induced cell lines. So for in vitro studies, we try to culture those cells in the endothelial media. And we try to see that one whether this incubation with the endothelial media, they are differentiating, trans-differentiating to endothelial cells and showing endothelial cell like property. For example, we try to see the expression pattern of CD31 which is a very commonly known endothelial marker. And yes, we observed that those EMT induced cells when we incubated them with the endothelial media, they were expressing high CD31. We have performed the tube formation of capillary like structure which is also a characteristic of endothelial cells. Indeed, we have seen that the formation of tube like structure increased when we incubated those cells, cultured those cells in endothelial media. We have also performed LDL uptake assay. We try to see the internalization of LDL or low density lipoprotein, the major carrier of cholesterol in the blood through the receptor mediated endocytosis. And we observed that these endothelial media, cultured mesenchymal cells, they exhibited significant uptake of those fluorescently tagged LDL. So that is showing that altogether that, yes, when we are disculturating those EMT induced cells with the endothelial media, they are trans-differentiating into endothelial cells. Next, we have performed in vivo studies and we have done two different types of tumor models. The first tumor models we used MCF7 cells and we are going the tumor of different size. And at the beginning, as I said that when the tumor is gradually growing at the core, there should be hypoxia. And we try to see that one how, during that hypoxia, the HIF1 alpha expression and the HIF1 alpha expression or hypoxia induces EMT. So the expression of EMT markers along with foxy to expression increased during that time. And once there is EMT happens, then that helps the trans-differentiation process. So we showed that this new angiogenesis at the core region of this outgoing tumors is enacted predominantly through this MCF7 cell trans-differentiation towards an endothelial phenotype. And we showed that how these EMT cells is trans-differentiating into endothelial cells. In our second mouse model, what we did is we used MCF7 with HMLE snail or HMLE twist cells and also we had a control cell. So what we did is we also try to develop the tumor and we showed that the study reinforce the assertion that the capacity to potentiate that endothelial trans-differentiation is predominantly associated with the mesenchymal phenotype of the cells input in this model. So both those models, it helped us to understand that how this EMT is helping the trans-differentiation process. EMT induced cells is trans-differentiating into endothelial cells for the new angiogenesis process. Can you try to see the molecular mechanism? And we observed that the foxy, as I mentioned, that which is one of the most important factor for the EMT process. And this foxy is playing a central role in regulating the endothelial trans-differentiation of cells that have undergone EMT. We did in vitro studies and also in vivo studies with control and foxy to silencer lines. And we have these beautiful experiments which showed that clearly that it is playing a very important role for regulating this endothelial trans-differentiation. So altogether in this present study, we demonstrated that the cells that have undergone EMT can promote tumor growth and neurovascularization and through this acquisition of endothelial like phenotype with the central EMT mediator foxy to that is playing a key role in this process. So our this finding it's basically linked the stem-ness conferred through EMT to the acquisition of endothelial cell traits and the augmentation of tumor angiogenesis in a foxy to dependent manner. So now what currently we are doing and what is our future plan is they say that we started doing the molecular mechanism. We showed that the role of foxy to but we want to study more. We want to know details about what is going on, how this EMT is helping this trans-differentiation process. So we try to find out the role of different factors. For example, how for the HIF-1 alpha and foxy to whether they're interacting directly. What is the role of we have we observed our preliminary study showed the role of GR1 MDS-ST that is myelitis and suppressor cells are also playing some role. And the EGFR2 or KDR they are also having some role. So we are trying to find out the interaction between them and they are full in this EMT mediated trans-differentiation endothelial trans-differentiation process. We are also doing RNA-seq using in vitro as well as in vivo tumor models. And one particular signaling path we are looking also is YEPTAS signaling pathway. We're trying to see that one or that has any role in this EMT mediated trans-differentiation. And at the beginning when I said that there is another one very interesting way to have this new angiogenesis process or these blood vessels in the tumor which is known as vascular mimicry where these aggressive breast cancer cell lines you can see that they are forming these blood vessel like structure which doesn't have endothelial like characteristics or they don't express CD31 or CD34. But I want to see that one or the EMT is playing any role in this vascular mimicry. How is there is any connection between this EMT and endothelial or this vascular mimicry process? So we are currently doing that one as well. And at the end of what I want to say that I want to thank so many people, the authors, all the researchers, they worked really hard and we have done this beautiful study where first time we are showing direct evidence that the cancer cells in that inside the tumor when the tumor cells are growing at the core of the tumor, they are experiencing hypoxia and once the hypoxia is there, these cells, the hypoxia induces EMT and this EMT cells, they can trans-differentiate directly into endothelial cells and they can form their blood vessels. They are making the tumors independent. They don't need any endothelial cells or peri-endothelial cells from outside to make blood vessels. They can make their own blood vessels making the tumor more independent. So I am really happy about this study and the outcome of this study. I think it's an amazing study and I want to thank many people, all the researchers here who helped doing all those amazing experiment. I want to thank Dr. Sendurai Mani who was in MD Anderson Cancer Center, was my previous postdoctoral mentor. He helped a lot with many reagents and also with many amazing suggestions. I also want to thank my department, Department of Biology, Texas A&M University for all the other helps they have provided me to finish up that paper. So thank you so much again, Ankut Aged, for giving me a chance to talk about my research, to explain what we did. Thank you so much.