Epithelial derived cells are typically too large in static dimensions to fit through the static dimensions of capillaries, and so would be expected to be cleared the first time the entire blood volume is circulated, i.e. in about a minute.
I think neither Pimiskern ·nor Troyer answered the question. If I understand correctly, the point raised was that a tumor cell is too large to go through a capilalry vessel. Thus, even if a tumor cell in the primary tumor invades into a postcapillary venule larger than a capillary, it should then stop once it is transported to the capillary bed of another organ. This is actually what happens with tumors that metastasize to the lungs or to the liver, i.e. the first capillary beds encountered. Circulating cells are found, though. They either derive from the tumor and are detected in venous blood (the blood routinely collected for analysis) before they stop in a capillary bed, or are able to squeeze through a capillary and go for another round in the venous circulation. Tumor cells have the abillity to squeeze in between endothelial cells or even through an endothelial cells - this is how they extravasate and form metastases. They also make their way through breaks they create enzymatically in basement membranes and basal laminae that are (likely) smaller than the diameter of a capillary vessel. Thus, it is not surprising that a very small number of tumor cells can be detected in the venous circulation.
CTCs are very rare, often less than 1 per million cells. They are identified by markers such as EpCAM or cytokeratins characteristic of epithelial cells, and lack of markers such as CD45 chararteristic of leucocytes. Epitethelial cells in the tumor undergo epithelial mesenchymal transition, begin to crawl and invade and thus gain access to the circulation.
I think neither Pimiskern ·nor Troyer answered the question. If I understand correctly, the point raised was that a tumor cell is too large to go through a capilalry vessel. Thus, even if a tumor cell in the primary tumor invades into a postcapillary venule larger than a capillary, it should then stop once it is transported to the capillary bed of another organ. This is actually what happens with tumors that metastasize to the lungs or to the liver, i.e. the first capillary beds encountered. Circulating cells are found, though. They either derive from the tumor and are detected in venous blood (the blood routinely collected for analysis) before they stop in a capillary bed, or are able to squeeze through a capillary and go for another round in the venous circulation. Tumor cells have the abillity to squeeze in between endothelial cells or even through an endothelial cells - this is how they extravasate and form metastases. They also make their way through breaks they create enzymatically in basement membranes and basal laminae that are (likely) smaller than the diameter of a capillary vessel. Thus, it is not surprising that a very small number of tumor cells can be detected in the venous circulation.
I see no real problem. Epithelial cancer cells can spontaneously squeeze through small channels. At least in vitro, many cancer cells can move through channels as small as 3 by 3 microns. If you have doubts, watch these breast epithelial MDA-MB231 cells moving spontaneously through 12 by 12 micron channels, at speeds up to 200 um/hour :
https://www.youtube.com/watch?v=FPLbs-bV1L4.
It is very well possible that circulating tumor cells squeeze through and come out of capillaries as well, slower through smaller capillaries, faster through larger ones.
It has been known for long that there is a change in surface charge of cancer cells, which can cause them to detach from the original source and can move in circulation without being adsorbed to the endothelial cell, or say slightly repelled till it get a site which is either physically tight, like micro-vessels or get adsorbed to opposite charge cells and reside to start a new tumor.
I have a lateral thought to offer which could help to accommodate the differing views expressed above. There is evidence to suggest that CTCs may be derived from bone marrow spread of the original tumour. This is implied from the finding that patients with CTCs have a great propensity to develop bone metastases. An inference one can derive from this is that the initial micro-metastatic spread to the bone marrow ( which speculated to occur in ~30% of breast cancer patients) if it survives and becomes established as a nidus of viable tumour deposit, it may remain latent until a clone evolves with characteristics of bone marrow derived cells (i.e. blood cells). Such cells may have the capacity to survive when travelling through the blood circulation as well as circulate through the capillary bed like ordinary blood cells.
I have just started working of CTC in breast carcinoma. My method of detection is to use epithelial marker (A45B/B3) and those cells in circulation with positive epithelial marker and tumour cell like characteristics like high NC ratio are presumed to be tumour cells. To answer Christers question, the cells I have been seeing are the size of lymphocytes or slightly larger with a very high NC ratio. Carcinomas have multiple clones and those patients with CTC probably have already developed a clone that can circulate and survive in the blood vessels. The larger cells / cells that cannot survive are probably cleared by the cappilary / immune system as rightly pointed out.
Perhaps pathologist could tell where in capillaries (most) tumor cells are arrested: at the onset or along it's length. Adaptations include increased motility, fluidity of shape. Furthermore, modifications from the detachment status at the primary site to attachment at the site of micrometastasis is essential, similar to other alterations that influence the interaction of tumor cells with their environment (e.g. Int J Dev Biol 55, p823). Clinical experience further includes patients with carcinomas from organs from which metastatic cells' first capillary bed is the lung, but that demonstate only mets in organs such as liver of long. Secondary spread from micromets in the lung may offer an explanation, but I would not exclude passage through the lung directly without arrest.
One factor omitted in consideration of CTCs is time. And CTCs are REPLENISHED continuously from the growing tumor. It is such a crucial component of the consideration of spread of CTCs. The earliest onset of cancer is most likely to have no CTCs. As cancer progresses, CTCs increase, and conversely, as the cancer regresses due to treatment etc., the numbers decrease. So the process happens over time.
The very early blood stream entrants must be cleared easily. But one or few cells with the right genetic makeup - metastatic potential? - can lodge in a capillary bed and cause some "damage" to the wall and initiate angiogenic events to embed - even lightly - in the bed. They may be cleared, but some will stick tighter and deeper and the process has been initiated. Remember the tumor is growing, the probability of the cells increases. Along comes another few cells, and dig deeper, and the process goes on. Since the process takes time most cells entering the circulation are "cleared" but some will eventually make it to the initiated site. The "hole" is now getting large enough to hold a colony. THIS COLONY can also put out its own daughter cells. They are now actually MUCH BETTER prepared to do this process.
No matter what, the number of CTCs is always very low and does not "explode" or something over time. It is thus a difficult process to accomplish, but a few persistent cells OVER TIME can do the damage. But since it is inherently so difficult to start up and maintain, the number of CTCs are never so large as to be macroscopically obvious.
As we all know tumor cells are bigger in "static" dimensions than the diameter of capillaries. Even red blood cells, although small cells, are slightly bigger than capillaries and use their shape and elasicity to go through without forming microthrombi everywhere. From experimental metastasis (If I remember right), probably some 40 years ago, we know that one out of thousand or ten thousand incected B16 cells can form a lung metastasis, indicating cells may not survive or find the best "soil". About 15 years ago, Ann Chambers pointed out at a Metastasis Research Society meeting in San Diego, that these cells are bigger and just trapped in lung capillaries. I remember the videos from intra-lung microscopy, but I had developed my own model for B16 cells to roll and attach under even high shear forces on endothelial monolayers - comparable to rolling and sticking lymphocyte during homing processes. I could completely stop B16 melanoma cell rolling on both, endothelial monolayers as well as on immobilized counter-receptors, VCAM1-fusion proteins, using function-blocking antibodies against VCAM1 or VLA4, respectively. Therefore I still am convinced that cell adhesion receptors not only play a role in post-adhesion events, like transmigration. Others showed a that anti-VLA4-antibodies could reduce the B16 metastasis to the lung, which may support to some extend my idea of rolling and sticking as the important step in metastasis.
Circulating tumour cells represent a sub-clone of tumour cells that exist in the primary tumour. Those cells may possess a special properties that enable them to across blood vessels to be transported for other sites. Almost all metastatic tumour have tumour cells which possess common chromosomal changes them resemble of that of primary tumour. Therefore potentials cells can escape from primary site seeking further sites to establish metastatic tumour by using blood stream for sailing.
Please don't forget that a capillary isn't a cast polymer tube, but is a lumen surrounded by highly flattened cells wrapped in a manner reminiscent of a crepe around the filling-- the first electron micrograph I took showing a lymphocyte crawling through the capillary wall was stunning. I can imagine that even with the dynamic of a shape-shifting epithelial cell becoming highly elongated, the nucleus will become the occluding factor, and if the cell becomes stuck, it would be probing the capillary walls for the extracellular gap to give it a toehold into an escape route-- into the new tissue home.
Death/necrosis of circulating cancer cell with free circulating tumor nucleic acid could be an additional source of free circulating tumour nucleic acid!!
Interesting new point. The shape and elasticity of the nucleus appear to allow leukocytes and tumor cells to go through a capillary. Nuclear lamins A/C could be relevant.
Another underestimated point is, that all circulating cells may need an anti-adhesive or repellant surface to prevent the binding to other cells and to endotheliium.
All this makes me wonder how cellular "texture" as in digital images, independent of cell or nuclear shape, would reflect different features of circulating tumour cells. Kristine Atkinson, is there anything visually outstanding that you would say you have seen about the "texture" of lymphocytes moving or preparing to migrate through a wall?
I remember some Electron micrographs from over 20 years ago from Werner Risau's lab - it is fascinating to see a leukocyte sqeezing or migrating through an endothelial cell.
Audrey, as with any cell in motion the peripheral cytoplasm is devoid of organelles, and having done cytoskeletal staining on tissue culture cells I also know that the actin bundles that would anchor the cell at the edge would also have been reduced or at least disengaged-- so the image in TEM is of rather clear cytoplasm and a smooth membrane surface in the cytoplasmic extension passing between the endothelial surfaces. Robert Day Allen's lab at Dartmouth did wonderful videomicroscopy showing movement of living organelles, indicating a role for fine and intermediate filaments. If surface texture has to do with tension, then I'd think that living tissue would be more informative than EM images. For instance, an active uptake surface making many small vesicles during pinocytosis we would expect to be quite fluid, although the image may look pitted (once a membrane is fixed and stained with osmium tetroxide and lead citrate it looks like a charcoal drawing). Perhaps Robert can remember whether Risau's lab looked at the surfaces?
I have a mathematical background, so I can't add to the biological discussion. My personal conception of this is: Yes, nearly all of them are cleared in about a minute. But, that minute is enough to detect it. The term "circulating tumor cell" does not mean that the same tumor cells are really circulating through the body. It means that tumor cells can be find circulating within the blood. Nevertheless, it is an intersting question whether real circulation through the body actually occurs.
Agnes is right: Most cells released from the primary tumor are cleared from blood within hours. Primary tumors release millions of cells each day but you only find small numbers of circulating cells. Cells that have undergone epithelial mesenchymal transitions with drastic changes in cell size and shape can survive for longer times when they also have gained resistance ot anoikis (death due to loss of anchorage). These cells are the seeds for metastasis.
Building on Ulrich's comment about anoikis, we sometimes see cell lines (which must be considered cancerous in nature for failing to terminally differentiate into tissue) that become anchorage-independent, and perhaps this transition calls upon a part of the genome from an ancient past where the cell operated independently without forming a cohesive tissue. We know that cancer cells show a reversion to earlier programming that includes expression of markers made earlier in develpment ("fetal antigens") and also that they escape G-zero of the cell cycle. It would be most interesting to characterize this transition to anchorage independence-- how does the cell get smaller? What exactly is lost, cytoskeleton, organelles for differentiated manufacturing, such as rough ER?
They may be few, but the circulating seeds that survive from a large population cast adrift appear to be the tail of a bell curve of survival, and maybe it is only size that permits them to continue their travels unimpeded. That they preferentially seed certain places may be a behavior that can be manipulated-- many parasites do the same thing, wandering to specific tissues on specific days of development before finally settling into their final tissue home.
Kristine, do you know of any research into your proposed question? I would like to know more about this relationship amongst shape, size, motility, and preferential seeding, including and beyond issues related to circulation (the pipes themselves).
In theory there are two "strategies" for a tumor cell: to survive in the blood stream as long as possible or to penetrate into the target tissue as soon as possible. The former is mediated by anoikis resistance that probably relies on EMT, the latter is mediated by tethering to the endothelium of the vessels of target tissues which relies on the expression of proteins such as Lewis' antigen on the surface of the cancer cell and of the receptors such as E-selectin on endothelial cells as well as on the cell's capacity to transvasate using Matrix-metalloproteases (MMPs). See for example: http://www.annualreviews.org/doi/abs/10.1146/annurev-bioeng-061008-124949
Audrey, I don't offhand, but even if there are old references, it's time to dust them off and do new work. There must be programming that permits a cell to do the opposite of ramping up size and organelles for protein manufacturing: we see this in yeast and other cells that can go dormant or withstand adverse conditions by creating a small form with characteristics better suited to tolerate conditions such as desiccation or high pH; the production of gametes is another case where organelles may be stripped down. Nuclear/cytoplasmic ratio has always been an important criterion-- without giving much thought to whether the nucleus got bigger or the cytoplasm smaller. What I'm saying is that atrophy may be an actively chosen process, not just deterioration. I think finding those rare cells and comparing them to the source (structurally is probably the best bet, as there may not be enough material for much diagnostic work-- and you can do ultratructural immunocytochemistry in situ) should be done de novo.
Ulrich's theory is interesting, if we want to think like a cell. But the "penetrate as soon as possible" scenario doesn't agree with what we know about metastasis, which isn't haphazard or neighboring the source. Different types of tumors have different destinations. The question in my mind is whether the small forms are dying or if they are dormant (stealth) invaders. I think we can accept one thing for sure: a tumor seeds other tumors via the circulation, and navigating a capillary bed has to be tricky. Are the lucky invaders those that quickly found a vein or arteriole and didn't get filtered out? It may be helpful to look at the blood supply to organs we know are secondary targets and see whether a neat path can be mapped from the original tumor site. You would think everything would get caught in the lungs after once pass through the heart... but that surely is where receptors come into play, so the invader recognizes its target.
I agree with Mahmoud Sohail, the tumour cells change according to the extracellular signals and they are intelligent enough to change depending on the environment or that particular situation by both genetic and epigenetic programmes. The cell-cell comunication is disrupted, ECM dissolution or proteolysis takes place by secretions, a diverse number of changes in motility and contractility is acquired by static cells. So they are able to develop lamellipodia, or pseudopodia like structures, and myosin filaments are used to make to moves and allow them step by step detach and travel in groups, clusters, cohorts or with amoeboid movement or fibbrolast like movement as single cells. Epithelial cells as discussed above acquire EMT, epithelial-mesenchymal transition thus gaining mesenchymal properties and loosing static characteristics allowing them to move, locally invade, and migrate through the bloodstream and also same time tumour cells are resistent to apoptosis and acquire proliferative advantage and immotile characteristics. A lot is unknown, yet among some known molecular mechanism include the involvement of Rac-Rho GTPases belonging to the Ras superfamily. The review below nicely delineates the various mechanisms involved in acquiring such plasticity by tumour cells. A Figure has an easy illustration too. Hope this will be helpful.
I remember reading about the assumption that late-stage tumours continuously shed thousands of cells into the blood circulation. Indeed, most of those inevitably end up trapped in capillary beds and would enter an apoptotic state. Yet, some of these cells, albeit an absolute minority, will end up in the capillary system of a suitable organ, adhere to the luminal side of the endothelium and subsequently extravasate towards the foreign tissue of arrival.
Epithelial-Mesenchimal transition involves intravasation, i.e., cancer cells entering the vessel's tubes, and not only Cancer cells can enter vessels, bur Red Blood Cells, whn in good metabolic condition, have the potential of deformating its shape when going through narrow places; equally as Leukocytes go out of vessels when an inflammatory process releases the cytokines that make them extravasate and go to the inflammatory foci, Cancer cells can make the reverse trip, from out to inside the vessels' lumen, I always wondered if UPA and PAI1, that are markers of tumor aggresiveness, but also equivalents to the Fibrin Degradation Products, thus pointing to intravascular activation of coagulation cascades, may just reflect the changes in the inner walls of vessels induced by the Cancer cells while intravasating, or other cancer cell related vascular endothelium lesion.
Cancer cells have a huge range of mechanisms which allow them to metastasize. These include increased adhesion molecules and enzymes such as MMPs and collagenases which allow them to burrow through just about any tissue. In fact, patients may die from a catastrophic hemorrhage as they degrade the vascular epithelium to penetrate surrounding tissues from within a blood vessel.
I think as the circulating tumor cells come from primary tumor through metastasis and metastasis usually happen at the end stage of tumor progress, therefore many of these cells are different in their properties compared to their precursors. In fact when tumor microenvironment is not suitable for tumor cells due to lack of nutrient or oxygen ( hypoxia) some of them start these differences and try to leave their origin, so they can not be compared to the primary counterparts. Circulating tumor cells can survive and establish new tumor site in this way.
Generally primary epithelial tumors do not grow beyond 2 mm in size and are restricted in their place. When conditions are conducive (such as rapidly acquiring mutation, expose to growth factor blasts, etc.) some of the cells from these primary epithelial tumors acquire mesenchymal phenotype. They become fluidic in nature, start developing structures such as invadopodia, invade capillaries, enter the bloodstream, and create havoc (metastasis).
Recently the talk about circulating tumour cells has taken a considerable attention. Almost all researchers refer to the metastasis as the source of these cells. Tunour cells from a primary site find their way through blood stream to be settled in a specific organ. Will we stuck on that idea and rely on completely? What is the destination of these cells? What is the fate of these cells. If such cells take blood for transportation to other organs , they must be shortly exist in the blood in away that they cannot be detected easily. Therefore all of us should think again to study the significant of the presence of tumour cells in blood. If we go to understand the process of inflammation in the wound then we may find access to understand the reason for their existence.
Cancer is undifferentiated heterogeneous in nature so every cells has the different characteristics. Therefore not everyone but some has the characteristic and integrated built up to exhibit the metastatsis. Meanwhile their undifferentiated smooth surface form assist them to show leukocyte rolling type movement to transverse the capillary.
Professor J Garcia-Foncillas, now head of the Oncology dept of the Jimenez Diaz Foundation (FJD) in Madrid, once remarked during a lecture, that in some Cancer cells, Chromosomes are not at all recognizable, pointing to the huge separation from a normal Cell that the continued process of addition of replication-promoting changes in Cancer cells may reach.
Circulating tumor cells have an altered cytoskeleton and microtentacles that protect these cells from deformation caused by shear stress. Both carcinogenic and non carcinogenic circulating breast cells have microtentacles but those in tumorigenic cells are extra long and motile compared to those in non-tumorigenic cells (Cancer Res. 68, 5678-88, 2008; Cancer Res. 70, 8127-37, 2010). Microtentacles, which are formed by microtubules and also may contain intermediate filaments, are required for efficient attachment and act in the metastatic spread of cancer. Interestingly the common chemotherapeutic paclitaxel, which acts by stabilizing microtubules, strengthens the microtentacles resulting in increased attachment of circulating tumor cells to secondary sites (Breast Cancer Res Treat. 121, 65-78 (2010) so this type of one highly successful therapy for primary tumors may set the stage for a greatly increased risk of metastasis.
I think that this is a very interesting question and is perhaps even more complex. For a CTC derived directly from a primary tumour to be detected in a sample of venous blood taken from an arm vein, it would need to have passed through two sets of capillaries (or avoided them via shunts).
For example a breast cancer CTC would need to have left the primary tumour and entered the venous circulation ,either directly or via the lymphatics, then passed into the vena cava and right heart, from which it would then reach the pulmonary artery and the pulmonary capillary system, then it would enter the pulmonary venous system, pass into the left ventricle and enter the systemic arterial system and via the brachial artery find its way into the arm, pass through the capillary system in the arm and enter the venous system, allowing it to be plucked from the antecubital fossa by a venepuncturist. It is indeed remarkable that we can find any CTCs in peripheral blood!
Epithelial cells attain high rate of migration ability once they attain transition state, mainly known as EMT (epithelial to mesenchymal transition). This phenotypic cells are easy to migrate and invade to surrounding tissue, and this transition mechanism enables the cells to pass through the capillary system and reach its next location. Once it reaches it destination, again it will transform back to epithelial phenotype through the process MET (mesenchymal to Epithelial transition), which is essential to make the secondary tumors at distant site.
this EMT to MET to EMT state is the major factor helping the cancer cell to enter in circulation system.
Have drugs been developed that target the cytoskeleton or microtentacles of CTC's? If so, to what success? Is there a target specific to the altered cytoskeleton that is unique to CTC's?
My interest, exercise, should in theory increase shear stress. Is there enough significance that this may be what contributes to reduced recurrence of breast and colorectal cancers among those that begin exercise at diagnosis?http://jama.jamanetwork.com/article.aspx?articleid=200955http://jco.ascopubs.org/content/24/22/3527
http://www.ncbi.nlm.nih.gov/pubmed/18593799
If 90% of cancer deaths are from metastatic cancer, this would appear to be an important area to focus.
It is interesting to notice that we need to specify which type of tumour can release circulating tumour cells. Do all tumour types release such cells or just only few specific tumours. This need high attention and big study to clarify this task. Then we can find the clue of the presence circulating tumour cells in blood.
@Nahi Yaseen: CTCs as currently studied are identified based on epithelial marker expression. I suppose, in general, tumours of epithelial origin (breast, prostate, colon,...) will yield CTCs during their progression to a metastatic state. To my knowledge, tumours like glioblastoma multiforme that primarily occur in the brain do not metastasize.
I agree with you with those tumour types. I found that circulating cells associated with some types of lung cancer (small cell carcinoma) and with rhabdomyosarcoma. Therefore such findings need to be re-evaluated by adding more studies on circulating tumour cells.
The issue is that more of the attempts to identify and even isolate CTCs is based on positive selection. But what happened if a negative selection method can be applied? In that case we will not miss CTCs that are in EMT and they do not expresses epithelial marker and they have less epithelial phenotype. Then we will find that CTCs are heterogenous etc. Also another issue is that in advance stage of the disease we may not know from where the CTCs may arrise from. Do they arise from the rpimary? From the secodnaries? Do they coe from a depositor organ like bone marrow (well knwon in breast carcinoma cases). These are main issue that we need to consider a dnresove.
Circulating tumor cells (CTCs) are cells that have shed into the vasculature from a primary tumor and circulate in the bloodstream. CTCs thus constitute seeds for subsequent growth of additional tumors (metastasis) in vital distant organs, triggering a mechanism that is responsible for the vast majority of cancer-related deaths. The detection of CTCs may have important prognostic and therapeutic implications but because their numbers can be very small, these cells are not easily detected. It is estimated that among the cells that have detached from the primary tumor, only 0.01% can form metastases. Any useful method for isolation of CTCs must allow (i) their identification and enumeration and (ii) their characterization through immunocytochemistry, fluorescence in situ hybridization (FISH) DNA and RNA assays, and all other relevant molecular techniques using DNA and RNA. When circulating tumor cells are captured from blood using filtration devices (such as ScreenCell isolation device), further morphological and molecular characterization is required to reveal important predictive information and report changes in CTC biology, for example during tumor relapse
One question is, how can they move through capillar vessels. Answers s. above. Another important question is, how can they survive in a functioning immuno system. The answer is, they hide and cover themselves by special surface proteins, which prevent the attack from the immuno system.
The pathogenesis of systematic tumour progression and distant metastasis has not been clearly understood uptil now. Yet recent studies are trying to understand the cell types involved in the CTCs. The studies below show that from TAM or tumor associates macrophages, Disseminated TAMs, or DTAMs, large macrophage like giant cells may have a role in CTC's invasion, existence in circulation and extravasation to the metastatic site in epithelial tumors studied. I hope the refs. will of help in understanding.