Okay, so we will now discuss how metastasis actually occurs, which is the metastatic process. So how does metastasis actually happen? I have distilled the metastatic process into eight major steps, which is, primary tumor growth, angiogenesis, epithelial-to-mesenchymal transition or EMT, invasion, intravasation, survival in circulation, extravasation and dormancy and secondary tumor growth. We're going to go over each of these steps individually so you know what they are and in which order they occur and how they work. And at the end of this section we'll put it all together and hopefully it will be easier to see how they all work together. So first thing's first, there must be a primary tumor that grows to the point where it's large enough to metastasize. As you've heard from Doctors Emmond and Zarif in previous lectures, a normal cell generally becomes a cancer cell due to an accumulation of mutations that change it, so it begins to grow uncontrollably. These cells also gain other hallmarks of cancer, such as lack of growth suppression, changing metabolism, resistance to cell death, and avoiding immune destruction among others. All of these hallmarks allow for a tumor to get larger and also change itself over time to become more aggressive and more invasive. You can see here on the diagram, a single cell with a mutation begins to grow into something called hyperplasia, which is just an increase of proliferation and division in these cells. This is not yet cancer. As time goes on, cells begin to change enough so that they are referred to as cancer, but if it has not invaded yet then it is referred to as cancer in situ. Which means that it remains in its original place. This is a benign tumor, in other words. Eventually, the cancer becomes invasive and more and more aggressive until it metastasizes. However, it is important to point out here that some tumors reach metastatic capability. Some grow very large and never leave the primary tumor site and therefore be cured simply by removing the tumor surgically. But once the tumor metastasizes, it becomes much more difficult to treat. The next stop in the metastatic process is the angiogenesis, which is simply the formation of new blood vessels. Which are composed of vascular endothelial cells as you see in the picture. Tumor cells which in the picture are the cells with black nuclei divide and grow more quickly than normal cells, the ones with blue nuclei. And they often require more nutrients and oxygen as a result. Tumor cells also grow geographically farther away from blood vessels because of their fast growth rate and therefore they need more oxygen. So new blood vessels form from already existing blood vessels and reach to the tumor cells. This can continue as the tumor grows larger and larger. Because this process is not natural, these new blood vessels are often poorly made and leaky and therefore become easy for the tumor cells to get into. This provides an outlet for tumor cells to get into the bloodstream and become metastatic. So before I was talking about how tumor cells can become more aggressive over time. One way they do this is by becoming more mobile, and this is accomplished by the cells undergoing a transformation or transition, from an epithelial like cell, to a mesenchymal like cell. This process is called the epithelial to mesenchymal transition or EMT for short. EMT is natural during processes like development and wound healing because these processes require that cells become able to move. But most cells should not move, they should stay put and perform whatever function it is that they have. Most cancers originate from epithelial cells, which are cuboidal in shape, stationary and have strong intercellular interactions with the extracellular matrix and their neighboring cells. The extracellular matrix or ECM is a collection of secreted molecules that form a structure that provide support for epithelial cells, tissues, and organs. The ECM basically keeps everything in place where it should be, and protects cells from leaking out all over the place. Loss of contact with the ECM is one of the major ways for a cell to undergo EMT. This mesenchymal state that it turns into, causes cells to become more stretched out and allows them to move around. When cells lose contact with the ECM, they're able to move around, which is not desirable. Imagine liver cells that no longer stay put in their natural place, but start moving. And once cells start moving around they can start to invade neighboring tissue and get into the bloodstream. And that's actually the next couple steps here. So as cells become more aggressive and more mobile, they also gain cellular properties that allow them to break through the protective ECM and break out into neighboring tissue and gain access to blood vessels more easily. Please note that there is a difference between being able to move and being able to invade. Invasion requires the ability of a cell to secrete enzymes that actually chew through the molecules of the ECM to create a hole that mobile cells can go through. Importantly, invasion is that last straw to make a diagnosis of cancer. If invasion is seen in a histological test, cancer becomes the diagnosis because the cancer cell has the ability to possibly metastasize. So invasion pushes the severity of the cancer to a higher level. Once a cancer cell has invaded, the next logical step is for it to get into the bloodstream. This process is called intravasation. In other words, cancer cells intravasate into a blood vessel usually by pushing their way through the vascular endothelial cells. Depending on the leakiness of the blood vessel, some cancer cells can slough off or basically fall into the blood stream through small holes in the blood vessel. In general, cells that are passively sloughed off are likely less aggressive than the ones that actively push their way into the blood vessel. You can now understand how the process of angiogenesis could promote cancer cells to get into the blood stream more readily. I also need to tell you that cancer cells can get into the bloodstream through the lymphatic system. Cancer cells can intravasate into lymphatic vessels as well as blood vessels. Lymphatic vessels drain into lymph nodes as we've discussed already, but then they drain into the venous system. Veins carry blood to the heart which then pump blood to the entire body. So if a cancer cell gets into a lymph node, it could potentially get into the bloodstream without ever having intravasated into a blood vessel. Once cells get into the bloodstream they face an uphill battle. They have to survive the circuitous route of blood from the vein in which they entered, from whatever organ they may have originated from, to the heart, back out to the lungs, back to the heart again. And then through the arterial system where it could go anywhere and wind up anywhere in the body. Although sometimes a circulating tumor cell, or a CTC, may just stop in the lungs and never really enter the systemic arterial system, and these could develop lung metastasis. During the transit throughout the circulation, CTCs must survive various sources of death. Normally if an epithelial cell is sloughed off and accidentally enters the bloodstream, it dies. First, as we've discussed, epithelial cells are supposed to be attached to others cells and the ECM. If they lose this attachment, they are programmed to undergo cell death. So that's the first source of death that a CTC must avoid. But because it has undergone EMT, it is able to avoid this death. Secondly, the immune system is constantly surveying the blood in search for cells that are not supposed to be there. Epithelial cells fit this bill and they usually display certain molecular markers on the cell surface which tell immune cells to kill them under certain conditions. CTCs avoid this immune surveillance and survive and this is actually an active area of cancer research right now. How can we re-activate immune cells to kill cancer cells? All right, the third source of death that CTCs must avoid is the strong shearing force of the blood pressure. In the veins the amount of pressure is not as serious, but once blood is being pumped from the heart, it undergoes a significant increase in pressure which most epithelial cells would not withstand. CTCs however are able to survive this force and survive. Some cancer cells do indeed succumb to these three forms of death, but the ones that do not become viable CTCs and are the ones that form metastasis. One piece of evidence that shows that viable CTCs are the cells that eventually form metastasis is this. The more CTCs that are present in the blood of the cancer patient the worse that patient's prognosis is. This graph shows that when fewer than five CTCs on average were found in 7 milliliters of a breast cancer patient's blood, those patients median survival was about 22 months. However, when five or more CTCs were found, median survival dropped to only about 11 months. In fact, the detection of CTCs is a hot area of cancer research, because it can be used as a diagnostic, and potentially a screening marker of metastatic disease. You can imagine how difficult this is though. In 7 millilitres of human blood, there are about 50 million white blood cells on average. And cancer researchers are trying to find roughly five CTCs in that volume. It is quite difficult and it requires impressive biotechnological techniques to do. Okay, so moving on to the next step which is extravasation, which is the opposite of intravasation, where cells get into the blood. So extravasation is the process of cells getting back out of the blood at a secondary site. One way this happens is that cells get stuck in tiny capillaries, which are very small blood vessels inside an organ, and then they squeeze there way out into the surrounding tissue. Another way is that molecular factors that are secreted into the bloodstream by the cells in the tissue attract the CTC to exit the bloodstream and move towards those vectors. CTCs have protrusions that allow them to adhere to the side of a blood vessel wall and stick. Because blood keeps flowing, the cancer cell rolls along the interior surface of the vessel until it eventually is able to squeeze itself through the endothelial cells into the surrounding tissue. Once a cell has successfully extravasated, it is no longer referred to as a CTC but rather as a disseminated tumor cell, or DTC. So we've discussed that a CTC must traverse the entire circulatory system and is therefore able to wind up anywhere in the body. But sometimes cancer cells tend to wind up in the same location in many different patients. For example, breast cancer tends to metastasize to the bone, but never to the peritoneum. Stomach cancer on the other hand, almost always metastasizes to the peritoneum, but very rarely to the bone. So how can this be when it seems as though there should be an even chance for any type of cancer cell to wind up any where in the body. Well the answer to that question is actually not fully known at this point. And it's an active area of cancer research. But there is a lot of data that supports the seed and soil hypothesis made by Stephen Paget in a ground breaking paper in 1889, the distribution of secondary growths in cancer of the breast. This hypothesis says that cancer cells, which is the seed in this analogy, home to, or metastasize to certain secondary sites because they thrive better in that environment, or the soil, that that particular organ has to offer. So for example, if prostate cancer metastasizes, it is almost always found in the bone marrow. But you will never find a prostate metastasis growing in the pancreas for instance. So if this hypothesis is correct then the bone marrow is a more attractive environment or soil for the prostate cancer cells to grow in than the pancreas. Once a cancer cell extravasates and becomes a DTC, or disseminated tumor cell, it can begin to grow a metastatic tumor in a secondary site. This tumor follows many of the same hallmarks as primary tumors dividing uncontrollably, resisting cell death, avoiding immune to surveillance, changing metabolism, etc. The larger the secondary tumor becomes, the more problems it causes. And we discussed that previously when we talked about how metastatic cancer can kill people. It can cause bleeding, embolisms, hypercalcemia, etc. If there are multiple metastatic tumors throughout the body, this enhances all of those problems. Now one thing that can happen to a DTC in a secondary site is that it become dormant, which means that it can survive, but not proliferate or divide. And basically just stays put as a single cell or groups of cells for a long time. They basically hibernate. For how long? Well, there is evidence that suggests that a dormant DTC can reside in a patient's bone marrow for years and even decades. Sometimes these dormant DTCs don't cause any issues. A single cell can't really do much harm. But dormant DTCs can become activated and pushed toward proliferation and can become a metastatic tumor at any time. And what causes this reactivation from dormancy is unknown and is also an active area of cancer research. So this slide is just a summary of the metastatic process, and we've gone over all of these steps. And I'll just summarize it for you here. Cancer cells can grow into a tumor, cause new blood vessels to grow, which is angiogenesis, undergo physical changes, that's just EMT, and invade through the ECM. And get into the bloodstream, which is intravasation. CTCs must survive circulation until they extravasate into a secondary site. Once there as a DTC, they can either grow into a metastatic tumor or go dormant. I hope I have conveyed the metastatic process to you. And in the next section of the module we'll talk about cancer ecology as an analogy for the cancer metastatic process.
