


S1: let me remind you, one more time about the discussion sections tomorrow those of you who happen to be in the one o' clock, discussion session we're moving from fourteen hundred, to thirteen hundred Chemistry Building, uh if by any chance you go to fourteen hundred Chem tomorrow i think you'll end up in a P chem class, which i assume most of you would, prefer not to be at, some of you might wanna be there but, uh if you end up in a chemistry class remember we're in the lecture room on the opposite side, of the building. also remember for tomorrow's discussion that i gave you kind of a thought exercise, at the end of last Wednesday's discussion after we had critiqued, um that article that i read you from the Ann Arbor News found all the things that were wrong with that study, i asked you to think, in this intervening week about the kinds of experiments you might do. uh, and do a better job of trying to find out whether there's a relationship between birth control pill usage, and cancer risk, and i think i said there is no single experiment it's not like there's one right answer it's a very complicated question how scientists go about, establishing cause and effect, uh so i would hope to hear a variety of approaches which you might come up with, in terms of trying to experimentally address that question so we'll discuss those tomorrow, and see both the strengths and weaknesses of the various experimental approaches. today as you know from looking at the lecture schedule we're gonna to be talking about metastasis, which uh, the process refers to the spread of, tumor cells from their primary site of origin where they originated, to another organ via body fluids and most of the time we're talking about the blood stream the circulatory system. but you should keep in, mind that there are other body fluids there's the lymphatic system there's the cerebral spinal fluid, there's also the peritoneal fluid in the abdominal cavity. anywhere there's a fluid in the body that cancer cells can get into, those cancer cells could float through that fluid and end up somewhere else in the body. so basically it's uh, getting the cancer cell into a fluid somewhere and transporting it to a distant site. and usually we're talking about, the blood stream. uh by now you have a pretty good idea of the importance of metastasis, um, as a phenomenon first of all it's part of the inherent definition of cancer. remember the only defining characteristic of cancer, was the ability to spread by the process of engaging in metastasis. that's the only thing that distinguishes it unequivocally from a benign tumor. so metastasis is part of the defining, features, of a cancer. also last time you learned that uh, metastasis is one of the main ways that, cancer uses to kill people. uh the main cause of death is usually, not the primary tumor, but metastases spreading throughout the body and often getting into the one one of the vital organs, such as the, brain or liver or kidney, and this is often what cancer patients will die of. so this means that if we could do anything to interfere with the process of metastasis, in essence we could cure people of cancer, or we could at least cure people of the most debilitating and threatening life threatening, aspect of cancer which is the ability, of these cancer cells to metastasize. so today we're gonna focus on metastasis. and in essence we'll be focusing on the question of what is it about cancer cells, that in fact, allows them, to metastasize, while the benign tumor cells, can't do this nor do other, normal kinds of cells do this. in addressing this question, first of all you have to realize that metastasis is not a single event. and we talk about metastasis it's not really, a single, process that we're talking about it's actually, a sequential series of events, all of which must take place, in order for this phenomenon, of metastasis to occur. we commonly we therefore divide, metastasis into a series of stages and i'm gonna use three major steps, to divide this process, today. <:13 PAUSE WHILE WRITING ON OVERHEAD> the first, step, in the process of metastasis, involves the ability of cancer cells, to invade surrounding tissues, and vessels <:16 PAUSE WHILE WRITING> perhaps i should remind you that the definition of cancer remember was, the tumor that had the capability of invading and metastasizing, really, the first step in the process of metastasis is invasion. <P :04> so the first part of the definition of a tumor cell, is in fa- of a cancer cell is in fact this, process that is the first step, in metastasis. why do cancer cells do this invade surrounding tissue why do they tend to wander off and infiltrate, penetrate surrounding tissue while normal cells don't do that. actually there's several factors that appear to be involved, first of all, cancer cells don't stick together. uh, if a group of you in this in this room wanted to think of yourselves as a cluster of cancer cells, if you were all holding hands and joined to each other clearly it'd be very difficult to metastasize you couldn't very, easily invade, uh you'd all hafta, you know move as a as an attack unit, and if all the cells in a tumor, were attached to each other, it would be virtually impossible. so the first thing that cancer cells exhibit to get over this, problem is decreased adhesiveness... they don't stick together. they don't hold hands. unlike normal cells. um, if you were to take, a clump of, tumor tissue take a biopsy a specimen of tumor tissue and, put those cells into a test tube and, put 'em in, some kind of a, a nutrient solution, and shake that test tube very very vigorously, you'd find that the cancer cells would readily come apart. and you'd soon have a solution a suspension of individual largely individual cancer cells. now you might not think that's unusual, until you hear what happens with normal cells if you took a normal, chunk of tissue, a biopsy from a normal region of tissue, uh and put them in a test tube with a nutrient solution and try to shake them up, they would not come apart. there are very very strong structures, that hold adjacent cells together, in most tissues of the body. especially in epithelia, which remember are the main tissues in which, cancers arise. so cancer cells don't, stick to each other they're not structurally joined to each other, the way normal cells are. and since they're not stuck to each other, individual cells can easily wander off... the second thing that cancer cells, exhibit that allows them to invade is increased motility <:09 PAUSE WHILE WRITING> and again, picture yourself as part of a clump of cancer cells, if you were all, holding hands if you let go, but you just stood there, again, you're not gonna invade, you're not gonna go anywhere. you gotta be able to walk you gotta be able to move. most cells in the body don't move. most cells are not motile there are some exceptions, some cells as part of their normal functions need to move, but in an adult organism very few cells actually do this. and even cells in the_ blood cells you might think of them moving but it's really the fluid that's moving more than the cells themselves. so most cells in the body don't tend to wander off, walk away, so to speak, from their site of origin. untrue of cancer cells. cancer cells tend to exhibit increased motility they have, uh, contractural proteins within them, now most cells have these but in cancer cells they're being used, to actively move the cancer cell from one location to another. the final thing you gotta worry about, yo- you've seen we we've gotten rid of the links that hold cells together we've given them the ability to move, the final thing is you've got to_ you're not just moving through open space, tumors are surrounded by tissue, and if cancer cells are gonna move they have to somehow get through that tissue. and the way they do this is through the secretion, of a family of enzymes called proteases, and presumably from your biology background you know what this word protease means it's any protein, digesting enzyme <:06 PAUSE WHILE WRITING> and cancer cells use these proteases because, much of the supporting tissue around the tumor, has protein structures and protein fibers that represent a barrier. so in essence what these proteases do is digest a path, through the surrounding tissue. <:06 PAUSE WHILE WRITING> and especially the tissue that we call, the stroma. the stroma is the supporting tissue. perhaps connective tissue you may have heard that term the supporting tissue, of the body. and it consists of, cell types called fibroblasts, which secrete, protein fibers, and these protein fibers come together to form a structure called the extracellular matrix <:05 PAUSE WHILE WRITING> a dense network of intertwined protein fibers, and finally so we've got fibroblasts we've got these protein fibers to form the extracellular matrix, and we also have blood vessels, in the stroma. and we talked about the importance of those, uh to some extent last time. so this supporting tissue contains a mixture of, cells of protein fibers forming this matrix, and blood vessels. and if cancer cells are gonna wander off and invade and infiltrate surrou- surrounding tissues they've gotta digest a path, through this stroma. and as i said they do it through the secretion of proteases, and there's actually, two main proteases that they use, one is called plasminogen activator <:07 PAUSE WHILE WRITING> and plasminogen activator, actually catalyzes a reaction. it catalyzes the conversion of a protein called plasminogen... and converts it to a protein called plasmin. and plasmin is an active protease... while plasminogen represents an inactive precursor of that protease. <P :06> now remember i told you that plasminogen activator itself is a protease what is a protease by definition it's something that breaks peptide bonds. so plasminogen activator breaks one of the peptide bonds in this precursor called plasminogen which is normally inactive, by breaking that, peptide bond (and) cleaving off a fragment from its inactive precursor, it converts it to plasmin, which now, in turn can also function as a protease. now you might wonder what is the purpose of, cancer cells secreting one protease whose sole function seems to be, to create a second, different protease. in essence this is an amplification mechanism. cancer cells don't have to, secrete very much plasminogen activator, cuz this is in essence an enzyme. remember enzymes being catalysts required in very very small quantities, so a small amount of plasminogen activator can convert large amounts of plasminogen, to large amounts of plasmin. and plasminogen is found in very high concentration, in the stroma. okay so most of the body tissues and the supporting tissues of the body in the stroma, contain large amounts of plasminogen. it's normally not doing anything it's just sitting there, as an inactive precursor. but when the cancer cells secrete plasminogen activator, it only takes a few molecules they can convert millions of molecules of plasminogen into plasmin, till you get a very very high local concentration of protease, from secreting just a few molecules from the cancer cells. now another protease that's active in, cancer tissue is a, family of protei- proteases called, matrix metalloproteases <:06 PAUSE WHILE WRITING> or M-M-Ps <:04 PAUSE WHILE WRITING> and these matrix metalloproteases are often, actually produced by the stroma itself the cancer cells somehow stimulate the stroma, to again do something rather, dumb from the point of view of, of the host organism remember last week i told you that, cancer cells cause the normal tissue to produce blood vessels which is sort of dumb in terms of the host because, the blood vessels supported the tumor. now the ho- normal host tissue is doing another thing that's pretty dumb from the point of view of the host, which is producing matrix metalloproteases, on the existence of these cancer cells, and these matrix metalloproteases again help to break down the extracellular, matrix. it appears that both of these families of proteases they do somewhat different things and both of them are needed in order to get efficient, invasion by cancer cells through the stroma. now once, these enzymes have allowed the cancer cells to digest a path through the stroma the, cancer cells can migrate away from the primary site of origin, until they encounter a blood vessel, usually it's a small capillary, a large bus- blood vessel would be harder for cancer cells to get into. but small capillaries which have a wall that's only a single layer thick, cancer cells can actually penetrate that wall, again with the aid of these proteolytic enzymes helping to break down, the structure of that capillary wall, the cancer cells can penetrate and now get into these small blood vessels. so the cancer cells end up doing three things, that allow them, to start off the process of metastasis. first of all they don't stick together, so you don't have_ you know remember i told you you can have enormous benign tumors. benign tumors can weigh fifty or a hundred pounds. and the reason they are benign, is that those tu- cells are all stuck to each other. no matter how, big the tumor gets, you still have just one mass of cells, surgeon goes there removes it no matter how big the tumor, uh, and you're cured. the only case where that's really a problem is if that tumor happens to be growing in your brain, uh, where, clearly if the surgeon doesn't get to it quickly it can cause significant brain damage. but in benign tumors if the cells stick together, you have no problem. malignancy the cells don't stick together, the cells are mobile they wander off, finally they secrete these enzymes and stimulate the stroma to produce more enzymes, which ends up digesting a path through the, surrounding tissues and into blood vessels. so that's the first, of the three steps of metastasis the, uh invasion, of cancer cells in the surrounding tissues and in the blood vessels. once they've gotten into blood vessels, we'll go to roman numeral two now, the second major stage in the process of metastasis, once the cancer cells get through the walls of these small blood vessels they're now in the circulatory system. and at this point, cancer cells are transported, <:04 PAUSE WHILE WRITING> through the circulatory system, to distant place. so through the bloodstream <:11 PAUSE WHILE WRITING> in essence the cancer cells are now free to circulate everywhere in the body. <P :05> now you might think, intuitively that once, cancer cells, had entered into the circulatory system the game is sort of over. that there'd be s- no problem getting these cancer cells transported everywhere around the body uh to set up distant metastases. but actually it turns out that the blood stream is not a particularly hospitable place, for cancer cells and in fact very few cancer cells, actually survive the trip. how do we know that? uh, we make lots of statements how do we know these things? well clearly you can't do experiments on how cancers cells survive in the blood stream by injecting cancer cells into the, veins and arteries of humans right? that would not only be unethical, it actually wouldn't work because of the immunological rejection problem we talked about, last week so, you can't study it in humans, again you can't study it in test tube experiments because test tubes don't have circulatory systems, so the only way you can test this is in animals. and the way it's tested in animals, you've gotta have a way, once you've injected the cancer cells into the bloodstream, to follow them, to find out where they are to follow their fates and the way this is done, is by creating populations of radioactive cancer cells, <:04 PAUSE WHILE WRITING> so you could take a transplantable tumor for a mouse for example, and grow it in a mouse for a while in the presence of radioactive isotopes put some isotopes into the mouse's diet, or do this in culture, expose these cells to radioactive isotopes, making the rad- the cancer cells radioactive. now it's easy to spot these cells because they're radioactive. so now you isolate some of these radioactive cells, inject them into another animal's, bloodstream <:12 PAUSE WHILE WRITING> and you wait to see what happens and you can use a Geiger counter or some other, mechanism for monitoring radioactivity, to find out what's happened to these cancer cells. what you find is that ninety-nine-point-nine percent of those cancer cells are quickly destroyed in the bloodstream... less than zero-point-one percent, will typically survive the trip... a few days later most of radio- most of those radioactive cells, most of the radioactivity has been, uh, discharged from the body. broken down gotten rid of. so most cancer cells, and this is_ remember, with inbre- inbred strains of animals, these animals should be able to genetically accept these tumor cells that we're working with, yet very few of the cancer cells survive. so it's been concluded that in fact only a tiny, fraction of the cancer cells of a typical tumor, can in fact survive in the bloodstream. and therefore, lead to the process of metastasis. now what determines, whether or not a cancer cell in fact can do this, if they're supplied in the blood stream, ultimately set up a distant metastasis. is it random, is the one cell or so out of a thousand that survives the trip and ends up setting up a metastasis, is that just a lucky cell that somehow escaped all the, pitfalls of traveling through the bloodstream? or was there something special about that cell something unusual, that allowed it, to survive the trip? well if you open your coursepack to page twenty-nine, uh sorry page twenty-one, let's talk about this experiment, which addresses that question, of whether there's something special about the cells, that metastasize. <P :07> these experiments involve a uh, a melanoma cell line, remember this is a malignancy of pigmented cells of the skin, uh a melanoma called, B-sixteen. it's a mouse, transplantable tumor. and it rarely metastasizes. if you want to implant some of these cancer cells under the skin of a typical mouse, um, and you wait a few weeks for it to grow into large tumors eventually you will find, a few metastases in the lungs. you might find one or two or three if you wait several weeks but not very many. but let's take, some of the cancer cells out of one of those successful metastases, isolate those cancer cells, put them under the skin of another mouse, and repeat the process a second time. wait a few weeks, look in the lungs, you'll find this time there may be four or five metastases a few more than you got the first time. again isolate those cells, inject them into a third mouse. and repeat this over and over, ten successive times. after ten repeats we have a cell population which we refer to as melanoma, B-sixteen they're still the cell same, cancer cell type, but we call it the F-ten generation it's the tenth, transplanted generation. where you've selected each time, four cells that metastasize, and reinject the dose. the melanoma B-sixteen F-ten line, metastasizes very very frequently. you inject this into an animal, and in a few weeks you'll find hundreds and hundreds of metastases in the lungs. so it is metastasizing very very frequently. so what can you conclude from this? although it might not be apparent immediately to you in fact these experiments show you that cancer cells vary in the frequency, with which they metastasize. <:15 PAUSE WHILE WRITING> now how do we know that cell populations vary, that they're not all the same? let's go back and think about this experiment again. what you're obviously doing is selecting each time, for those cells that su- successfully metastasize. if it was sheer luck, just a random fluke that they managed to do it, then the next time you injected those cells in the whole population shouldn't do any better at metastasizing, than the first population did, if it were sheer luck. but that's not what's happening each time it's getting better, because you are selecting, specifically for those cells that know how to metastasize. and by the end you have a highly enriched population you've taken a, cancer cell line where initially, those cells represented a very very tiny fraction of the total population, now those cells capable of metastasizing represent a very high percentage of the population, because you have been selecting for that particular type of cell. so the cancer cells vary in the frequency with which they metastasize and in this experiment we are selecting preferentially for those cells, that do have that capability. now although these experiments involve, what i might call a gradual change, in the cancer cell population induced by an artificial selection, the experimenter is selecting for this, generation after generation, there is something similar, that actually occurs, in normal situations in people that have cancer. and we refer, to this as tumor progression. <P :07> and this simply refers to the process by which, the cellular composition of the tumor changes with time. <:26 PAUSE WHILE WRITING> and this concept is based on the notion that i've just introduced you to that cancer cell, tumors cancer cell populations are heterogeneous, populations of cells they're not all the same. in the experiment we just saw, we selected, for the ability of cells to metastasize. um, in cancer patients sometimes you get that kind of selection for example, you have a primary tumor, and maybe very few of those cells can metastasize, but some of them do say you have a- a primary melanoma in the skin somewhere, and it metastasizes to the liver a couple of them, well those tumors that now grow up on the liver, are cells that, have the ability to metastasize. so those secondary tumors can in turn, metastasize and they'll do it at a much higher frequency, because you'd already selected once, so now you may get cancer cells all over the body, that are quite different from the initial population in the primary tumor. you've gotten a gradual, progression change in the characteristics with time, as metastasis has taken place. uh, this phenomenon may be especially important for understanding what happens, uh during attempts to treat cancer with some of the cancer chemotherapeutic drugs that we're gonna talk about, later in the semester. and what you're gonna tr- learn later in the semester but let me, just give you a preview today, is that cancer patients who are treated with chemotherapeutic drugs often go into what we call a remission. and this word remission simply... refers to a temporary disappearance... of the disease. temporary disappearance. and we know it's temporary because that's what the word remission means. if by some, chance, the drug actually cured the person and it never came back, then we would call it a cure. uh, when it initially happens you don't know whether it's cure or remission, doctors to be safe will usually initially say, well the patient's gone into remission. there's no sign of the tumor now. we don't know whether it's a cure or not we'll have to wait you know five ten years maybe, depending on how fast that tumor was growing and spreading, to know whether it was actually a cure, or just a remission. unfortunately for people who already have metastases and were treated with chemotherapeutic drugs, uh, most of the time, it's actually a remission that occurs. it's rare that a single treatment of chemotherapeutic drugs will completely cure a patient of cancer. why does the disease come back? well what happens is, with the drug you kill maybe ninety-nine-point-nine-nine percent of the cancer cells, but the point-oh-oh-one percent of the cells left behind, again it's not a random, group that's left behind, the cells that are left behind that weren't killed by the drug, are cells that are inherently resistant, to the effects of the drug. so when that, cancer cell population eventually grows back, it's usually, uh can't be successfully treated with that same initial drug. so it's often, when people have remission and then the cancer comes back it's often much much more difficult to treat them the second time, because that population has undergone progression, the cells are more aggressive these are now resistant to the drug, cuz those are the ones you selected for. remember at the end of the exper- uh, class last time, uh we were looking at Folkman's experiment with endostatin and i said the remarkable thing about that experiment, was remember he treated, the cancer cells once in the animal with endostatin, the tumor went away, he removed the treatment for a while and let the tumor come back, when the tumor came back the second time it was just as sensitive to treatment as it was the first time. it wasn't resistant. that's very unusual. with most, cancer drugs, in fact the second time the tumor comes back it is resistant, and it's because this phenomenon, of, uh, tumor progression, over time you are selecting for either the body or with drugs or with metastasis, you are selecting for a more aggressive (composition.) are there any questions on this, phenomenon? it's often misunderstood, so i wanna make sure it's clear to you yeah in back? 
S2: um, is tumor progression, necessarily uh, come with uh an enhanced grading, remember how you graded the tumor, it's microscopic 
S1: that's a good question. it's not exactly the same thing. remember i told you that tumor grading, was based, solely on the way cancer cells look under the microscope what size of nuclei, metallic index lots of things that you see in the microscope. those traits don't have to change, in order for you to select, for a population that's resistant to a drug or can metastasize more readily, those things tend not to show up under the microscope. so often the tumor has progressed and it's it's quote unquote more aggressive, and yet to you look at it under the microscope and it looks pretty much the same. that's not always the case. sometimes it will look different. it would, it would be great if it always looked different because then you could recognize it. unfortunately it doesn't usually look different, we're looking at different properties here. <P :05> anything else? <P :04> okay, well we've seen that the tumors are heterogeneous populations of cells they're not uniform but heterogeneous populations of cells, only a tiny fraction of which are capable, of carrying out this process of metastasis. but you can select for those cells over time, and get a population that eventually, is enriched in those cells that carry out metastasis. the next question i wanna address is, how are these cells different? if we are selecting for cells, that are more efficient at the process, of metastasizing, uh what makes that possible? how are they different at the fundamental molecular cellular level, that they can in fact, uh metastasize more readily? as i just indicated from my answer to that question, it's not the way they look. it's obviously gonna be something else. well although we are far from being able to answer this question in detail, it appears that surface properties properties of the surface membrane of a cancer cell have a lot to do, with whether or not it can efficiently metastasize. now let me describe for you one, simple experiment, which makes this point. this is on the next page i believe of the coursepack. <P :05> page, twenty-two. <P :04> this experiment sort of, picks up where the last one took off remember the experiment on page twenty-one, ended up with this, cell line melanoma B-sixteen F-ten, that metastasizes frequently. so we've selected for these cells, that are very very efficient in metastasizing, now we're trying to find out why are we so good at it. what is it about these cells? well if you take these cells and rupture them put 'em into a big blender and, you know, or grind them or something like that to rupture the cells, break them apart into their individual components, isolate out the membranes, using centrifugation to separate the membranes from the rest of the cell, now remember_ not from the rest of the cell from the rest of the cell components we have no cells left at this point. we have only isolated membranes. clearly these by themselves are not cancer they can't grow, these are not cells they're just membrane fragments. but under certain experimental conditions, we can kind of, promote the fusion, of these membrane fragments to intact cells. so let's take these membrane fragments and under these conditions fuse them, to melanoma B-sixteen F-one cells. remember that B-sixteen F-one cells were the first transplant generation from the preceding experiment. these are cells that rarely metastasize. so we're taking cells that are very inefficient at metastasizing, and we're fusing, into their plasma membranes membrane fragments pieces of membrane, from cells, that readily metastasize. what do you end up getting? you end up getting cells that frequent metastasize. so this clearly shows that the plasma membrane, influences the ability, to metastasize. <P :13> so you change the membrane composition, you change the ability of the cell to metastasize. now why is that? what's going on here? well we don't know for sure, uh but if you read the article that i assigned that you were supposed to read prior to today's lecture, you know that we think that interaction between the immune system and this plasma membrane is somehow involved. now for you to thoroughly understand these experiments you need to know, a few basic things, about the principles of immunology, i assume that most of you know these but let me quickly review them, just to make sure we're all on even footing here. if you were to attempt, to graft an organ, a kidney or a heart or even a skin graft something like that, from one person or another, uh the recipient's immune system obviously this is a problem with heart transplants all the time with kidney transplants, the person receiving the organ their immune system recognizes that organ, as being foreign, as coming from somewhere else. and the immune system wants to attack that invading tissue in essence. in order for heart transplants and kidney transplants to work the patients have to be, treated with drugs so-called immunosuppressant drugs, which uh inhibit the immune system suppress the immune system, so that their immune system will not in fact reject those tissues as being foreign. the way the immune system normally, attacks foreign tissues is using a special cell type called the T-lymphocyte. <P :05> the T-lymphocyte is is the main component of the immune system, which attacks, foreign tissues foreign cells, when they're introduced into the body. and when they attack, a foreign cell, what signals them that they should be doing this, is a composition of that foreign cell's plasma membrane. it's something about the plasma membrane that the T-lymphocytes recognize as being foreign. and part of what rec- part of what it recognizes, not completely, but part of what it recognizes, is a plasma membrane glycoprotein. <P :05> i assume you know what this term glycoprotein means it's a, a protein that has some sugar (roots) attached to it, the T-lymphocyte recognizes a plasma membrane glycoprotein, called the major, histocompatibility complex. <:12 PAUSE WHILE WRITING> or M-H-C, as we usually abbreviate it. and again you should be familiar with this from the reading that you did for today's lecture. in mice, uh where the experiments i'm, about to talk about took place in mice, several different genes, for M-H-C exist and depending on the cell type or of the inheritance uh, how that mouse was bred, who his parents were, it may get different forms of the gene for M-H-C. and these have been used, to study the process of metastasis. uh the experiments i'm about to describe to you actually i think were pretty well described in that coursepack article, so i will pretty quickly, go over them with you and make sure you completely understand them. remember in that article you read about, two, mouse cancer cell lines yeah, turn to the page in the course pack yeah right uh, this is now on page twenty-two... remember you read about, two cancer cell populations in mice. the D-one-twenty-two cells, and the A-nine cells. remember that these two cancer cell lines differ in their ability to metastasize when you inject them into animals. D-one-twenty-two cells metastasize very very frequently, A-nine cells metastasize quite rarely. these cell lines also differ in the ability of, their ability to elicit an immune response. if we just measure the ability of the recipient mouse's, immune system see whether its T-lymphocytes, are attacking mounting a response against those cancer cells, you find that the D-one-twenty-two cells when injected, elicit only a weak immune response, while the A-nine cells, elicit a very, strong immune response. this goes along with the fact that these two cancer cell lines D-one-twenty-two and A-nine, have different M-H-C genes being expressed. so the D-one-twenty-two cells have only the H-two-D form, of M-H-C in their plasma membranes, while the A-nine cells have both the H-two-D form, and the H-two-K form, of the M-H-C molecule in their plasma membranes. now if you were to just look at these experiments, all you could really conclude, is that a strong immune response <:05 PAUSE WHILE WRITING> is correlated <:05 PAUSE WHILE WRITING> is correlated with... a low rate of metastasis. <:10 PAUSE WHILE WRITING> in other words the A-nine cells that metastasize rarely that's correlated with a strong immune response vice versa, if a weak immune response that's correlated with a high rate of metastasis. but the key word here is correlation. you cannot prove cause and effect here, from this kind of observation. if i were to have jumped to the conclusion that the immune strong immune response elicited by the A-nine cell, is responsible for their low rate of metastasis if i concluded that, solely based, on these data i would be committing the post hoc fallacy. the kind of fallacy we were talking about in discussion section last week. the fallacy that when you see two things that go together, A goes with B therefore concluding that A causes B. clearly that's a logical fallacy. it might be something else, that's causing, this low rate of metastasis other than the strong immune response. so this is just a correlation. how can we move from a correlation, to a cause and effect, proving that there really is a cause and effect relationship. this is an important issue, and something hopefully you've been thinking about this past week because i sort of challenged you, at the end of last week's discussion to think about, how could you design a better experiment, to show the relationship between birth control pill usage and cancer, if there was in fact a relationship how could you unequivocally show one way or another, whether there was a cause and effect relationship so you've hopefully been thinking about this, and, one of the things that you might have been thinking about is doing some experiments. these are just passive observations. you gotta do some experiments, if you wanna prove cause and effect. one of the experiments, you again should have read about, in the coursepack article, was this experiment. <P :04> the D-one-twenty-two cells remember, metastasize, very very frequently and that's the_ correlated with a weak immune response and we thought maybe the immune system, has something to do with it. well if the immune system does have something to do with it, we could test that, by changing the M-H-C gene being expressed in the D-one-twenty-two cell. so let's take these D-one-twenty-two cells and transfect them with H-two-K D-N-A. transfection is a technique if you haven't heard it about it from your biology background let me just quickly say it's a, it's a laboratory method for introducing D-N-A into cells i'll talk more about it next week in m- in more detail, but it's just a way it was it was briefly described in the coursepack article as well, just a way to get foreign D-N-A into cells so we're getting this, D-N-A coding for the H-two-K, M-H-C gene, into the D-one-twenty-two cells. as soon as we do that and inject those cells now, back into mice, now they metastasize quite rarely. they're suddenly behaving like A-nine cells. in terms, of their ability to metastasize. and they now elicit a strong immune response. just like, the A-nine cells. so this is much more direct, evidence to the fact that the immune system does in fact influence <:07 PAUSE WHILE WRITING> the ability to metastasize. <:08 PAUSE WHILE WRITING> when we use the word influence we're talking cause and effect. well we know now it's cause and effect because we've done an experiment. we've changed the immune response by changing the M-H-C gene that's being expressed, on the surface of those cells, the M-H-C protein that's being expressed, once we do that the immune system now recognizes the D D-one-twenty-two cells, it attacks them just like it attacked the A-nine cells, and drives down their rate of metastasis. so the immune, response the ability of the immune system to recognize and potentially destroy, cancer cells, clearly can interfere with their ability to metastasize. these experiments, clearly show that. but is the immune system, the only, factor involved? or are there other variables that influence the ability to metastasize? well there was one more, very clever experiment described in the coursepack article, involving the use of immunosuppressed animals. if you wanna find out, whether or not, the immune response, differences in the immune response is the only variable, influencing the rate of metastasis the way to test that, is to inject cancer cells, by comparison into immunosuppressed animals. these are animals that have been treated with drugs that suppress their immune system, or there are strains of animals that have an inherently defective immune system, either way we're dealing with immune- immunosuppressed animals which cannot, mount an immune response. if the immune response were the only thing determining whether cancer cells can metastasize or not, then the difference between the D-one-twenty-two behavior and the A-nine behavior in terms of metastasis oughta be completely obliterated, in immunosuppressed animals right? if the only, reason that they're behaving differently, in rates of metastasis is because they elicit different immune responses, then you get rid of the immune response, you should get rid of the difference. and those two cell lines should metastasize at the same rate. notice that that's not what happens. the D-one-twenty-two cells still metastasize at their initially high rate as you would expect, this tells you the immune system is, basically not capable, under these conditions of attacking, the D-one-twenty-two cells. the A-nine cells in the immunosuppressed animals, remember, in normal animals they gave you a low rate, of metastasis, now they go to a medium, rate of metastasis they metastasize more frequently, telling you that the immune system was in fact inhibiting their ability to metastasize, but they still don't metastasize as well as D-one-twenty-two. fo- so this suggests that there are additional factors that the immune system yes is one factor but there are other factors as well, that can differentiate between different cell populations in terms of their ability, to metastasize. any questions on, this set of experiments? <P :05> clear? if you have any, any trouble at all when you go over your notes trying to follow what's going here again this set of experiments is pretty well described, in the coursepack article that i, assigned (to take home.) <P :04> okay. so we've now talked in detail about the first two steps in the process, of metastasis. we talked about the ability of cancer cells to invade through surrounding tissues in terms of penetrating the vessels, and now we just talked about the transport of cancer cells via the bloodstream, uh to distant sites of the body, and we've seen that most of the cancer cells die along the way most of them don't make it, and clearly the immune system is part of the explanation, for why, all the cells don't make it the immune system can clearly attack cells along the way, but there must be some other factors involved as well. this now brings us to the third, step in the process of metastasis, and that is the ability of cancer cells to reinvade and grow at various sites. <:18 PAUSE WHILE WRITING> now i'm gonna say at specific sites and you'll see why i use this particular word, in the next few minutes. what determines, where cancer cells will actually end up, reinvading into the tissue somewhere else setting up housekeeping and forming an actual metastasis. turns out that there are two different factors that play a role here. one of 'em is, pure topography. it's based on the anatomy, of the circulatory system. <:15 PAUSE WHILE WRITING> and if you'll turn to page twenty-four, in the coursepack i've got a diagram here, to help you understand... what's going on... this is pure geography we're talking about. and we're gonna talk about three potential locations, where primary tumors might arise there are, only three, i should say or three major categories of places where tumors can arise. now let's start off with the broadest category which is category one down here, which is called body tissues and organs, and that, literally means every tissue and organ in the body, where cancer cells can arise with the exception of the lungs, and the stomach and intestines cuz the lungs and stomach and intestines behave a little differently, than we'll see in a few moments. so let's talk about (with the) majority of the organs in the body let's say, an individual had an osteosarcoma in the leg bone for example. where are those cells likely to metastasize to? well as you saw in step one, they're gonna_ cancer cells as they invade through the bone are eventually gonna, encounter blood vessels somewhere, and, the blood vessel they're, li- most likely to invade into is gonna be a very tiny capillary cuz it's got the thinnest wall. so it invades into the capillaries and the capillaries immediately_ now the fluid flow the blood flow will be going towards the small veins, and the small veins feed into the larger veins which feed into the larger veins and finally end up, in the right chamber of the heart. so we're going into bigger and bigger plumbing. okay we're starting out with cancer cells in a very very narrow tube, going into bigger and bigger plumbing where there's more and more room for them, and so we end up in the right side of the heart. from the right side of the heart, these cancer cells in along with the blood are gonna be pumped into the lungs. in the lungs the, pulmonary arteries break up into a series of veins so the blood, uh in a series of capillaries, so that the blood can become oxygenated. so we go back down to very very very tiny, vessels again. which is the way we started. well when we started down here in capillaries of course the cancer cell was burrowing a hole into the vessel, uh through its protease and so forth, when it gets back, here back into capillaries, it's possible_ certainly the cancer cell's gonna be, slowed down and often, the diameter of a cancer cell is such that it's gonna ver- have a very hard time fitting through a capillary. and if you have a happen to have a couple of cancer cells sticking together, it will be almost impossible for them, to pass through the capillary. so this is the first place, where the cancer cells are gonna have a hard time getting through the plumbing, because the plumbing has gotten so small so narrow. so for most cancers that arise in the various body tissues and organs other than the lung and the stomach and intestines for most cancers, the first place they're likely to get hung up, is in the lung. so the first place you would tend to look for metastases in th- is in the lungs. and the lungs are in fact a major site for metastases, for many kinds of cancers. what about for tumors that start in the stomach and intestines, the so-called gastrointestinal tract? here again the cancer cells will infiltrate into very tiny capillaries which will feed in the small veins which go into larger veins which go into yet larger veins, but these veins will then enter the liver. and in the liver, they will break up back into a capillary bed. uh the purpose of this being for the exchange of the, the nutrients that have been taken up and detoxification and so forth all kinds of things have to happen in the, in the liver, and the blood supply therefore breaks stuff into these little tiny capillaries in the liver, so you can have the various exchange of molecules that needs to take place there, but lo and behold you have the same problem that you had before in the lungs. these cancer cells now are likely to be as big as or bigger than the diameter of these capillaries, and therefore likely to get hung up and stuck. so the most common site or one of the most common sites for stomach cancer and colon cancer to metastasize to, is the liver. because that's the first capillary bed, those cells encounter. finally what about lung cancer? in ways lung cancer's the worse scenario of all, we've talked about the terrible prognosis for lung cancer, and here's one of the reasons that lung cancer has such a terrible prognosis. kills so many people within five years. a cancer starting out in the lungs will go into these capillaries. the, blood flow will then push them into the, vessels they get bigger and bigger, as they go into the, left chamber of the heart, from the left chamber of the heart, uh, the vessel gets even bigger it's pumped out into the aorta, the huge vessel, cancer cells have no problem, they're gonna be pumped all over the body, through all the arteries of the body they'll be distributed everywhere, and, everywhere, they go they will eventually encounter a capillary bed. okay? they could end up in here they could end up in the stomach and intestines they could end up in the liver they could end up in all the body tissues and organs so lung cancer is a very very nasty actor. because its cells immediately are pumped out into the entire circulatory system, and get access to capillary beds where they get stuck, all over the body. so anatomy is the first factor the basic anatomy of the circulatory system is the first factor, that determines, where cancer cells are gonna metastasize. but those simple rules those three rules i just gave to you, aren't the whole story. how do i know they're not the whole story? let's look at another experiment. and there's, an experiment on page, twenty-five in the coursepack. <P :08> if you take mouse melanoma cells like the ones we've been talking about today, inject them into the tail vein, of a mouse, what's likely to happen? well from the diagram on page twenty-four you know from the veins you get eventually pumped into the, uh right side of the heart, from the right side of the heart you get uh, the cel- the cells are pumped into the lungs where it breaks up into the capillary bed, so we do expect these cells initially to get hung up in the lung. and in fact if you look, one or two days after you've injected those cancer cells in the lungs, you will find lots of cancer cells, lodged in the lungs. if you wait two to three, weeks for actual metastases not just individual cells but, metastases to grow up solid tumor modules that you can see and, actual tumors, in fact you get lots of these tumors in the lung. as you would expect, from the rules that i just gave you. but you're gonna see now when we looked at the right side of this, experiment that those rules, weren't the whole explanation for what just happened. let's do another experiment with the same exact cell type, but inject them into the left, ventricle of the heart. as you see from the diagram on twenty-four if you inject cells or cells cancer cells that are in the left, chamber of the heart, from there they are pumped out into the aorta, which means that they are pumped out from the arteries all over the body, and so like lung cancer cells themselves you would expect those to get lodged all over the body. and in fact within the first day or two if you look under the microscope at various tissues, you can find cancer cells lodged, in various tissues throughout the body. but if you come back two or three weeks later to see well where are tumors actually growing, where are metastases actually occurring, they're occurring almost predominately almost entirely in the lung. so this tells us it's not just the anatomy of the circulatory system. there's something else going on here. okay not just the, circulatory system there seems to be something, about these mouse melanoma cells, don't overgeneralize there seems to be something about these mouse melanoma cells, that allows them, to preferentially grow in the lung even though, they've lodged everywhere in the body, they only seem to grow well, in the lung. there's some kind of affinity, some kind of hospitality going on there. these melanoma cells like the lung. it's not just that the_ it was first place they stopped, it wasn't just the first motel they checked into, there was something, nice about that place, so whether it was the first place or not they end up, occupying the lung. so this gives us, a second, principle, that determines, the sites at which cancer cells are gonna grow in terms of metastases. the second principle, which we can conclude from that experiment, is that some cancer cells... prefer to grow... at specific sites... that's why i put the specific sites in this third stage. some cancer cells prefer to grow at specific sites certain sites. some kind of, we think biochemical affinity there's just something about the molecules and environment there, in certain tissues that certain kinds of tumor cells like to grow there. now if that's true it raises a very interesting question. is this the same for all cancer cells? i've only shown you experiments thus far for melanoma, and we haven't even looked at all melanoma cells, we've just looked at melanomas in in this gross sense and found that, as a, total population they seem to, preferentially like the lung. but is that true for all melanoma cells do all melanoma cells prefer the lung? or do some, melanoma cells like other places? now there's been some very clever experiments carried out to address this question if you'll turn to page twenty-six, in the course pack <P :06> you'll see an experiment here which is a variation, on the theme of the experiment described on page twenty-one. i urge you to make a note in your notes not to confuse, experiments on page twenty-one and twenty-six. they look very similar to each other they both involve these sequential, selections and transplantations but the conclusions that are drawn are significantly different, because the experiment is done in a somewhat different way. so make sure you, don't confuse, the experiment on twenty-one, the experiment i'm now about to go over. in this experiment, they took_ some some mouse melanoma cells were taken and injected into the tail vein of the mice, just (male) mice just like, the experiment on twenty-one, on twenty-one we focused on the lung. we saw that, metastases occurred, initially few in the lung and we selected those and enriched for those. here we're gonna do something a little different. it's true, that metastases occur, mainly in the lung. remember, when you start off, i said you only maybe got one or two of those. so, you get many many more metastases in the lung than you will in the brain and ovary. and you may have to look at dozens and dozens of mice, before you'll find one where there's a single metastasis in the brain, or a single metastasis in the ovary. so these metastases to these organs are much less frequent, than to the lung. is this just a random fluke, did they just happen to get, stuck occasionally in the brain and and it ended up growing there, or was there something special about those cells? well let's do now our sequential transplantation with, cancer cells from these two different organs. start out with the brain. we'll take that one brain metastasis that we found by scouring, you know through thirty or forty mice. take that one brain metastasis remove it, inject it into another mouse. into the tail vein. again you'll find metastases predominantly in the lung, but every once in a while you'll find one in the brain, or the ovary. this time you might find them a little more frequently in the brain than you had the first time. let's take the metastases from the brain again take those cancer cells isolate them inject them into another mouse. and repeat this over, ten times in a row. you repeat it ten times in a row and you will end up with a cell population, that, metastasizes mainly to the brain. metastasizes more frequently to the brain than to the lungs... you do the parallel set of experiments with the ovarian metastases you get a comparable result. eventually you can select for cells that metastasize mainly to the ovary. they metastasize more frequently to the ovary than they do to the lung, and certainly way more frequently to the ovary these would metastasize to the brain. so this tells you that cancer cells vary, in the sites to which they preferentially metastasize. again not all cancer cells are not the same. we saw earlier that all cancer cells were not the same in terms of their, ability to metastasize the frequency with which they metastasize. now we see that they're not all the same in terms of the sites to which they like to metastasize. so from a single, cancer cell population, you can isolate, subsets subpopulations of cells that preferentially metastasize, to different organs. now remember when we talked about the question of why some cells metastasize more frequently than others, uh i described to you some experiments which indicated that properties of the plasma membranes, appear to be involved in determining the frequency or influencing the frequency with which cancer cells metastasize. although the evidence isn't quite as good, in this case, there's also some evidence suggesting that differences in the plasma membrane, help to determine, which organ a cancer cell likes, to metastasize to. so we've seen with the melanoma we can isolate, uh, cancer cells that metastasize to the lung those that like the liver uh those that like the ovary those that like the brain, and if you look at the plasma membranes, of those three different subsets, of cancer cells in that population, their plasma membranes look somewhat different not under the microscope in terms of their biochemical makeup. so it appears to be interactions between the plasma membrane, and components of these different target tissues be it brain ovary or lung, that causes cancer cells of different types to preferentially metastasize, to one, organ or another. so the plasma membrane appears to influence not just the frequency, with which cells metastasize, but also where <AUDIO DISTURBANCE> (generally.) now once cancer cells, have picked the appropriate site, either from, just being lodged there, to the anatomy of the circulatory system, or through some more preferential mechanism some kind of biochemical affinity which, helps them to grow at a certain site, once this has happened, uh there are again some other factors, that come into play that determine whether or not extensive growth will take place... extensive growth of metastases requires a couple things <:09 PAUSE WHILE WRITING> the first thing it requires is angiogenesis. the process we talked last time. just like primary tumors which must, somehow trigger, switch the balance between angiogenesis stimulators and inhibitors, tip that balance towards stimulation to trigger the formation of blood supply, in order to grow beyond a millimeter or two in diameter, the same thing is true of distant metastases. they must tip that balance in the favoring of angiogenesis at the distant site, before they can grow, beyond a millimeter or two in diameter. and remember you have this complicating factor that if there's a large primary tumor somewhere else in the body, if that large primary tumor, is producing large amounts of angiostatin which can spill over into the bloodstream, unlike these other regulators of angiogenesis, that angiostatin can circulate throughout the body, and show up at one of these sites where there's a little tiny tumor nodule, and that little tumor nodule with this massive amount of angiostatin, in the cirulat- circulation will not necessarily be able to overpower that inhibition, and so you may have these silent nodules of a millimeter or two, for long periods of time, until something triggers, that balance for net angiogenesis... so the molecules that regu- regu- regulate angiogenesis clearly play a role. another family of molecule, that appear to play a role, are growth factors. i'm not saying that_ unlike angiogenesis, which is always required, i'm not implying that growth factors are always required but they may play a role. they may play a role. now just to make sure that you're clear on what a growth factor is if you'll turn to page twenty-seven... of the coursepack... i've briefly outlined for you here, what a growth factor is, uh, first of all, these are non-nutritional. you think, think of things making you grow often you'll think of nutrients these are not nutrients. these are not nutrients. these are, protein molecules, but very specific kinds of protein molecules, that stimulate the growth and division of particular cell types. they don't non-descriminately just stimulate the growth of any cell type but there are different growth factors, which selectively, stimulate the growth, of certain kinds of cells. for example, there's a growth factor called epidermal growth factor or E-G-F, protein that stimulates the growth of epithelial cells. there's a growth factor called platelet-derived growth factor, P-P-G-F, plays an important role in wound healing. uh stimulates predominantly the growth, growth of fibroblasts. uh you've not heard of either of those before, i'm not gonna hold you responsible for those two yet at this part of the course later in the semester later we're gonna come back, talk about these in some detail so at that point i'll hold you responsible for them, but notice that there are a couple, fibroblast growth factor F-G-F vascular endothelial growth factor V-E-G-F, these are ones you already know about. these are ones i will hold you responsible for knowing about, in this part of the course, because those are ones, that stimulate the growth of blood vessels, amongst other things, and therefore these are the growth factors that regulate the process, of angiogenesis. so the only two you read in this list that you really, have to know about right now are these two that regulate, angiogenesis. how do different growth factors manage to selectively, turn on the growth and division of specific cell types? the answer is that different cell types have different kinds of receptor molecules, on their plasma membrane surfaces. these are proteins expressed on the surface of cells, depending on the receptor, that you have on a cell it will bind to different kinds of growth factors so i've tried to illustrate this, by having a complementary shape, between a surface on this growth factor, and this receptor on the surface of this cell. so this growth factor will only bind to cells that contain this particular kind of receptor. the receptor for that specific growth factor. and the binding, of the growth factor to that receptor will, send a signal through the cell, and by the way later in the semester we're gonna talk an awful lot about all the steps involved in transmitting that signal, because the transmission of that signal is intimately associated, with the loss of control of growth in cancer cells so, we're gonna come back to this later in the semester and talk a lot about this pathway, in detail. but for the moment, it'll be just be sort of a black box we won't talk about the individual steps. growth factor binds to a specific receptor, it sends a signal into the cell which ultimately triggers, the division, of that cell. now, if you read the, coursepack article closely, you will, perhaps recall or maybe not but, you should go- be able to go back and find it, the D-one-twenty-two cell line, behaves a little different regarding growth factor from the A-nine. remember the D-one-twenty-two that was the one, that metastasized more frequently than the A-nine, remember although the immune system played some role, that when we, injected D-one-twenty-two and A-nine cells into immunosuppressed animals to get rid of the immune response as a variable, even though we got rid of the immune response as a variable, D-one-twenty-two still metastasized somewhat better, than A-nine. uh there wasn't as great a difference as before, but it still metastasized somewhat better. so there must be an additional difference, between D-one-twenty-two and A-nine, other than the difference in the M-H-C which enhances the immune response. i can now tell you that this other difference is the fact, that there's a gene called the FMS gene F-M-S <:06 PAUSE WHILE WRITING> which has become activated in the D-one-twenty-two cells, but not in the A-nine cells. now what is the significance of this gene? this particular gene <:05 PAUSE WHILE WRITING> codes for a growth factor receptor.<:05 PAUSE WHILE WRITING> remember the receptor is the molecule on the surface of the cell to which a growth factor binds, therefore triggering that cell to grow and divide. so the D-one-twenty-two cells, have a higher concentration of growth factor receptor, on the cell surface. this means that, for a given concentration of growth factor in the tissue, the A-nine cell, which has fewer growth factor receptors, isn't gonna be influenced as much, isn't gonna have its growth stimulated as much, as the D-one-twenty-two cells, because they have more, a higher concentration of growth factor receptor, therefore their growth will be stimulated more, in the presence of the same concentration of growth factor. any question on, this set of observations? <P :07> uh-uh, okay? okay, then, i wanna remind you, that there are three steps, as we've seen in the process of angiogenes- uh, sorry, process of metastasis, uh there's penetration, uh, of surrounding triss- tissue infiltration invasion of surrounding tissues and penetration of the blood vessels, was step one, uh, transport by the circulatory system to distant sites influenced by the immune system and other factors are step two, reinvasion and growth, finally of the distant site was step three. what i want to emphasize to you about the existence of these multiple steps not only do we have multiple steps, but remember we have multiple variables, influencing, each one of those steps impacting each one of those steps. so we have a very complex cascade of events taking place in the process of metastasis, and, only a small number of cancer cells can successfully go through every one of those stages and exhibit all of those properties, that you need in order to get through all the steps in the process of metastasis. and that's why very very few cancer cells actually successfully metastasize. a very very tiny fraction, of a can- cancer cell population successfully metastasizes. i mean if a person, had, you know a hundred metastases that would be considered to be an enormous number. but you've got tumors that have millions and millions if not billions of cells in them. so only a very very tiny fraction of cells, successfully gets through that complex, series of events. the important point, the important take-home lesson here, is that if we could successfully interfere with just one of these steps, just one of these things that i talked about today, if you could stop the motility of the cancer cell in stage one, if you could inhibit the production of proteases or interfere with their action, uh if could promote the interaction of the immune system, if you could influence any one of those steps, to the detriment of the cancer cell to the detriment of the cascade, then metastasis would not, take place, and if metastasis didn't pla- take place, cancer wouldn't be a disease that we'd have to worry about. okay that concludes what i wanted to say about metastases and i promised you i would occasionally let you go early this semester and today's one such day, but next time we'll talk about more properties, of cancer. 
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