 Good morning and I'd like to thank the organizing committee for inviting me concerning the question. This was not the answer I hoped to. I started my career in working on the immunological microenvironment but then there are other things that in my mind are possibly more important and this is the metastatic microenvironment. You all know the, I'm sure you all know the scheme of metastases when cells from the primary tumor leave the primary tumor, they invade the exocellular matrix, they interverse it into blood or lymph and they reach a secondary site. In the secondary site there are three scenarios, the cells could either die, could either stay as dormant micrometastases and micrometastases are the single cells or small classes of cells or develop into a micrometastases. The question is where do metastases arise and how do they get there? This gentleman 125 years ago provided an answer that is still valid for many of us. His name is Steven Peget, he published this article in the Lancet in 1889 and in his words he was very much concerned with site specific metastases. He wrote, when a plant goes to seed, its seeds are carried in all directions but they can only live and grow if they fall on congenial soil. If you translate this language into today's language, so today's the magnitude, the seed would be the tumor and the soil would be the metastatic microenvironment. How do cells get to the secondary site? There are probably several ways I just depicted two. One would be what we would call a targeted migration in which cells, tumor cells, express certain receptors or counter receptors for ligands that are present in a certain secondary organ. This could be as we and many other people have looked at chemokine receptors. The ligand in this case is present in the lungs. The tumor has receptors for this ligand and they would then be targeted to the secondary site and produce metastases in this place. The second scenario would be that is actually the topic of the lecture, which I changed a little bit, would be epithelial to mesenchymal transition. In this case, the cells would change the phenotype from epithelial to mesenchymal. Mesenchymal cells can migrate much more easily than epithelial cells and these cells would migrate into many organs but would make metastases only in a permissive microenvironment which in this case is the lungs. I was asked to talk about epithelial to mesenchymal transition. Now let me give you some description of this phenomenon which some of us think it is very crucial in the metastatic spread. This is the process by which epithelial cells lose their cell polarity and cell-to-cell adhesion and gain migratory and invasive properties. As undergoing EMT, epithelial mesenchymal transition, are characterized by phenotypic changes such as loss of the epithelial marker Eichadarine and a gain of the mesenchymal marker Vimentin. We'll come back to Vimentin a little later. Many of the EMT induces originate in the tumor microenvironment and as you could gather the tumor microenvironment is a basic theme of this lecture. Initiation of metastases requires invasion which is enabled by EMT. Now this is a schematic representation of EMT occurring in one of the cells in an epithelial monolayer. In this case there are signals that are delivered from the microenvironment. As you can see here, from various cells in the microenvironment, fibroblasts, lymphocytes, macrophages, neutrophils and so on and so on, these either by direct cell-to-cell contact or by soluble factors and receptors that are expressed on the cells that undergo EMT, deliver signals to the epithelial cell that is undergoing EMT and here is a list, incomplete list, of genes that are involved in EMT. For instance you'll see later on snail, zeb, slug, twist and so on. Now this is very schematically the way that cells metastasize using the EMT route. Some cells in the margin of the primary tumor undergo changes into cells that are expressed here as partial EMT. These cells then migrate, invade the blood, extravasate, form dormant micrometastasis which later on undergo progression to form a micrometastasis. Now there are quite a lot of, not a lot but some studies on the involvement of EMT in real Casinoma. For instance, snail which you have seen is a gene that is involved in cells undergoing EMT is a major regulator of EMT, is predominantly expressed in high grade renal Casinoma and high snail expression is a bad prognostic factor. I'm not going through all these studies, those who wish to see, I can show the slide later on in the lunch break. Those who want to look at some specific studies. Let me now go back to the secondary site in which as I told you the cells that arrive there could either die, could remain as dormant micrometastasis or progress into micrometastasis. We have evidence, other people too of course, is that the metastatic microenvironment, the microenvironment that operates in a certain organ site is responsible for these three things, either for death, for dormancy, or for progression. What is actually the tumor microenvironment? You have seen by the question that was posed before that you have seen some two or three different aspects of the microenvironment, angiogenesis, immunity and so on, inflammation, very important, EMT, another thing, so what is the tumor microenvironment? It's composed of resident cells that live in the metastatic organ all the time, such as endothelial cells of fibroblasts. Infiltrating cells and metastasis cause the influx of various types of cells into the metastasis, so do primary tumors by the way. These are lymphocytes, macrophages, fibroblasts and so on. Then you have the extracellular matrix with tens or hundreds of different proteins, some of them are collagen, fibronectin, release molecules, you have a lot of cytokines, chemokines, antibody, proteases, angiogenic factors and so on. Solid tumors are characterized by lack of oxygen and if the patient is treated as it is, the drugs obviously should arrive at the microenvironment. All these factors may interact with tumor cells and with non-tumor cells in the tumor microenvironment and these interactions regulate gene expression in non-tumor cells or in cancer cells. Either, and let me start from the right as we do in Israel, death, dormancy or tumor progression. I have one of the main emphasis of this talk is the fact that metastasis is an organ-specific phenomenon. This is very, very important and let me now just illustrate in a system that we are working on besides others and this is melanoma, primary melanoma, grows and migrates to other side to the region, the lymph nodes to lungs, liver and finally to brain. It should be I think born in mind that because of the different microenvironments in these different organs and the fact that microenvironmental factors regulate the many characteristics in the tumor cells including the malignancy phenotype that the different microenvironments can result in different types of tumors and I think that clinicians maybe the next phase of clinical research should be to ask whether metastasis in a certain organ is a different entity or sub entity of metastasis in another organ. Now there are several questions that can be asked. What directs tumor cells to different organ sites? If you have different organ sites, obviously different microenvironments and microenvironment signals differ from organ to organ. This is very simple. Do tumor cells in one metastatic site differ from tumor cells in other sites? We and others have evidence that this is the case. In other words, site-specific metastatic signature. What keeps micrometastasis in a state of dormancy? This is one of the questions, a key question that I'd like to give you some hints to and not in renal cell casinova but it may also be true in renal cell casinova and what awakens dormant micrometastasis. Site-specific soil and gold factor, both these questions are clinically very important because if you have something that restrains the growth of metastasis or micrometastasis, you can maybe think about the potential drug that could be maybe produced and administered. If you know what awakens dormant micrometastasis, you may be able to inhibit such awakens. Just a few words how our systems, we are working with human to mouse denographed models in which we generated non-metastatic and metastatic variants that originate from the same human tumors we are using neuroblastoma and melanoma. In other words, they have an identical genetic background. This means that genetic proteomic and transprectomic differences between such variants may therefore be attributed to their differential metastatic capacity. Let me now show you some interactions between neuroblastoma cells and a certain metastatic site and this is the lungs. Now the way we have done it is to inject human neuroblastoma cells into the renal gland of mice where a lot of a neuroblastoma starts. This is called an orthotopic inoculation. We got tumors obviously in the adrenal and spontaneous metastasis in the lungs. These cells were cultured. They were again put into mice and what we have seen that these cells from the adrenal produced micrometastasis in the lung whereas the micrometastatic cells produced as expected micrometastatic lesions in the nude mice. Now micrometastatic cells cannot usually be seen by ordinary methods like histochemistry or immunohistochemistry. Do I have 20 minutes or 15 minutes? You had 20 minutes in the program. I think it was only dialed to 15. We started the session a bit late as well. So we can detect these cells by a real time PCR using a probe for a human gene. In other words mouse which is not expressed by the mouse. So any human gene in the mouse would mean that we have neuroblastoma cells. Now as you can see here is a signal for a micrometastasis which is very large and a very small signal for micrometastasis but is there. So we have a very small number of human and neuroblastoma cells in the lungs that cannot be seen by ordinary histological or immunohistological methods. I talked about micrometastasis but I think it's important to be on the same page. Now micrometastasis are called by some people dormant tumor cells, disseminated tumor cells or micrometastasis. Several questions. Are micrometastasis the progenitor cells for metastasis? If yes, what keeps them dormant? Do dormant micrometastasis wake up? If yes, what wakes them up? Are genomic, epigenomic alteration in the dormant tumor cells involved? Is it the metastatic microenvironment? Is it both? Can the awakening be inhibited? One very important point is that at least in the neuroblastoma system and I can say that also in another system that they are working with and this is brain metastasis in melanoma. The dormant cells are only dormant in the secondary organ side. If you take these cells out, they would proliferate very nicely in vitro. If you put them again, the neuroblastoma cells into the adrenal, the human melanoma cells into the skin, they would grow tumors. In other words, the dormancy is limited to the metastatic side and this is not a loss of inherent ability to proliferate. What keeps micrometastasis dormant in the metastatic side? There was an article of George Klein a few years ago entitled, Why do we not all die of cancer cells at an early age? And his answer to that was that there is a phenomenon that he called microenvironment control or nonimmunological surveillance. In other words, he showed for instance that normal fibroblasts at very early stages of tumors could kill in vitro, could kill tumor cells. The fibroblasts lose their ability to kill the same tumor cells in later stages. Our hypothesis was that organ-specific factors restrained the proliferation of micrometastatic cells. And we have proof of that in neuroblastoma, for instance, also lately starting with melanoma. We are talking about lung metastasis. We take molecules that originate in the lung, mix them in vitro with either micrometastatic or micrometastatic neuroblastoma cells. We look at viability, we look at cell cycle kinetics and signal transduction in these target cells. Very briefly, these are the only results I'm going to show you today, is that viability in both types of cells in the micro and micrometastasis is reduced, more so in the micrometastasis, that phosphorylation of ERC is reduced in both types of cells, more in the micro than in the micrometastatic cells. Cell cycle arrest is higher in micrometastatic cells than in micrometastatic cells, and apoptosis in both types of cells is the same. We are now in the final stages to purify the inhibitory factor and to identify what it is. What's the significance? And I think it goes beyond neuroblastoma. We know it is also occurring in melanoma, probably in other tumors, that endogenous mitometastasis restraining bioactive factors could regulate tumor progression, may overcome drug resistance, we have some evidence for that, and serve as an anti-tumor agent. What's the emphasis here? The emphasis is that the tumor microenvironment, I'd like to end with that, has actually is a double-edged sword. Under certain circumstances, it can stop and restrain the proliferation of micrometastasis in other instances, it provides support for the proliferation of micrometastasis. Some people show the institute, I show my group, I'm endowed with a great group of young ladies, one male, and the names are here, and these are our collaborators. And I do thank you very much.