Why+is+it+that+some+leukemias+form+secondary+tumors?

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Cancer Biology 179: Final ProjectBy Eduardo Bent and Carlo Juarez

 ** Purpose: ** =Introduction =
 * To comprehend why some types of leukemias, which do not form solid primary tumors, are able  to invade a tissue and form se condary tumors at specific locations?
 * Our project has evolved significantly as the quarter progressed. First, our question was why doesn't leukemia metastasize in humans? But through some research we found that indeed leukemia metastasis, making our initial question irrelevant. Metastasis is seen primarily in Acute Lymphocytic leukemia at about 20% (ALL). So after this finding, we narrowed our project to focus on why there was only 20% of ALL metastasis in humans, but little information was found, leading us to reinstating our principal question. All the previous research led us to be interested in the mechanism by which leukoblast, premature leukemic leukocytes (a type of white blood cell), form secondary tumors after metastasizing in specific locations. In order to understand the process, we need to take into consideration that leukemia is essentially a type of blood cancer, and that blood runs via veins through our entire body. But before getting to the details, let us take a step back to gain some initial background information both about leukemia and metastasis, as well as some theories that are postulated to explain how in general cells metastasis occur in humans.

=What is Metastasis? = > the circulatory system and successfully colonize itself in a new location (tissue). Once installed in the new location it must form a colony by cell proliferation, also known as a secondary tumor. The latter step of infiltration, seems to be the most difficult step in metastasis. In a study of metastatic melanomas made by the Winship Cancer Instititute, more than 80% of cells survived up to this point and traveled to the liver. In this experiment only 1 out of 40 cells formed micro-metastases, while 1 cell in 100 formed macrometastasis. This occurs because the new mass has entered a hostile environment which it is not used to. It has to make the environment friendly enough for it to support its growth and survival and if this is not so the newly installed tissue mass will become dormant and not form a secondary tumor. Unfortunately, if by chance new mutations occur around the tissue which favor the new masses survival then the tissue will seek angiogenesis and grow into a secondary tumor. The accurate mechanism for metastasis is not known yet, but there are several postulated mechanisms that are believed to be the process by which tumor cells leave and colonize other tissues. Some of these mechanisms are : EMT, diapesis, Myofibroblast basal membrane degradation, and Tumor-Leukocyte Hybridization.
 * Metastasis comes from the greek words of µETá meaning next and στάσις meaning placement. It is literally the movement of Cancer within the body. In other words, metastasis is the spread of tumors via the circulatory system. One of the six hallmarks of Cancer is invasion and metastasis (the difference between a benign and malignant tumor). Thus, cancers are categorized as tumors which spread from a primary tumor (initial tumor) and even in some cases from secondary tumors also (tumorsthat have formed due to metastasis). Cancers metastasize by infiltrating the basal lamina and penetrating the lymphatic or blood vessels. Once this occurs, the tumors are able to spread by circulating in the blood and implanting themselves in other locations around the body (i.e mainly organs). Thus, patients in the late stages of cancer can have many secondary tumors spread through out their body if the metastatic tumor cells succeed the colonization of other tissues. The secondary tumors are still considered the same type of cancer as the initial or primary cancer, regardless of where it implants. For example, if an individual has developed a metastatic form of breast cancer, which then establishes in the lung causing the formation of another tumor, it is said that the latter tumor is formed of breast cancer cells, therefore, it will consider a secondary breast cancer tumor, not a long cancer tumor. It is unknown why certain tumors have a higher probability of metastasizing in comparison to others. Also, it is not very clear why certain areas of the body are more prone to the formation of secondary tumors than others. In this project we will try to tie everything together if enough data is gathered.
 * [[image:islaslab/metastasis.jpg width="454" height="221" align="left" caption="Figure 14.4 The Biology of Cancer (© Garland Science 2007) "]]Metastasis itself is a very rare process. There are multiple things that have to occur for a tumor to become metastatic and for it to form a secondary tumors. If one of these processes fail, then metastasis will not occur. First of all, the tumor must detach itself from the primary tumor and form a mass of its own. Once this occurs, the tumor must become mobile in order to be able to enter the circulatory system. Once in the circulatory system, the mass must survive in this new medium and be able to exit

=What are secondary tumors and how do they form? =
 * Secondary tumors form after the primary tumor has become invasive and metastasized. Tumors metastasize, or move around as explained above, within the body through lymphatic or blood vessels. Tumors that have broken through the basal lamina have the capacity of also penetrating the walls of the lymph/blood vessels and thus allowing others tumor cells to break from the primary tumor. These tumor cells are coined secondary tumors or metastasized tumors after they infiltrate in a new tissue and make colonies. Unfortunately, once metastasize happens, it is very difficult to combat the cancer since it has started to invade other parts of the body with out a specific destination. In some other rare cases secondary tumors can also metastasize, but the mechanism is still unknown to this day.

=Theories for Tumor Cell Metastasis =
 * ** Diapedesis: **
 * The process by which both white blood cells leave the blood vessels andinfiltrate into the extravascular compartment to go to some other tissues, without harming the vessels themselves. White blood cells squeeze media type="youtube" key="I9zSe0qmXGw" height="279" width="339" align="right"in between the epithelial cells, by a process induced by various chemical signals--which will be discussed more in depth later on -- in conjunction with integrins, receptors in the surface of the leukocytes, that facilitate the leukocyte-endothelial cell interaction Eventually, the leukocyte will change its morphological structure to infiltrate in between the endothelium cells (i.e. High Endothelial Venules) towards the extravascular compartments, so that it can reach their respective infected targeted destinations. Trafficking regulator receptors and venules are extremely important to this process because it has been suggested that diapedesis of leukocytes only occurs at specific locations.
 * Diapedesis might be a very important aspect of our project because it might explain why some types of leukemia metastasize. Maybe it is that leukoblast cells express high numbers of this important receptors that are used for cell trafficking, creating stronger cell-to-cell binding interactions, which ultimately result in leukemic cells' diapedesis. However, it is important to note that we have not found any information that specify specific tissues that have demonstrated some form of diapedesis.
 * **Myofibroblast Basal Membrane Degradation**
 * <span style="font-family: 'Times New Roman',Times,serif;">The process by which Matrix Metalloprotinases (MMPs) eat through the basal lamina membrane, subsequently allowing tumor cells from a primary solid tumor to enter the blood stream through the lymphatic or blood vessels. These MMPs disrupt the cell-to-cell and cell-to-matrix adhesion interactions, facilitated by transmembrane receptors such as CD44, however, the mechanism by which these enzymes are activated is unknown. The disruption of the adhesion ability of this tumor cells allow such cells to wander off into the circulatory or lymphatic system. Additionally, MMPs induce growth factors in the cancer cells themselves, so once tumor cells have colonized new tissue they will support and strive for angiogenesis in order to grow. If angiogenesis is not achieved by these tumor cells, or if cell proliferation is not induced for "X" reason, the cells will become dormant until they detect some type of stimulus.
 * <span style="font-family: 'Times New Roman',Times,serif;">**Tumor-Leukocyte Hybridization**
 * <span style="font-family: 'Times New Roman',Times,serif;">It is believed that once the cancer cells enter the blood stream, either through the MMP process or another process, the cancer cells bind and hybridize to white blood cells. The mechanism by which the cancer cells hybridize with the white blood cells is unknown, but Dr. Pawelek, a Cancer biologist at Yale University, is certain that is the process by which tumors spread. Once hybridized, the white blood cells continue on their normal path and end up entering in tissues due to chemical signals. Once the white blood cells implant themselves in the tissue, the cancer cells de-hybradize from the white blood cells and start forming a tumor.
 * <span style="font-family: 'Times New Roman',Times,serif;">You can read more on the article we found and some interesting ideas we found in this [|blog] entry.
 * <span style="font-family: 'Times New Roman',Times,serif;">**Epithelial-Mesynchymal Transition**
 * <span style="color: #231e20; font-family: 'Times New Roman',Times,serif; font-size: 1.03em;">[[image:islaslab/emt.gif width="459" height="302" align="left" caption="http://www.nature.com/nrc/journal/v2/n6/fig_tab/nrc822_F2.html"]]Epithelial Mesynchymal Transition: is a biological process that allows polarized epithelial cells, which interacts with basal lamina membrane, to gain migratory capacity, invasiveness, and elevated resistance to apoptosis through various internal, biochemical changes, eventually enabling to gain mesynchymal cell phenotype.The completion of this process is signaled by the disruption of underlying basal lamina membranes, allowing the cell to live the tumor tissue and get into the bloodstream. It is important to note that activation of transcription factors that leads to eventual expression of specific transmembrane domain, reorganization and expression of additional cytoskeletal protein, production of ECM, degrading enzymes, and many more biological changes is essential for the epithelial cell to gain mesynchymal phenotype, allowing it to gain migratory abilities. After being in lymph or blood vessels, this tumor cells that adopted mesynchymal abilities find a new tissue and extravasate forming secondary tumors through a reverse process called Mesenchymal Epithelium Transition.


 * =<span style="-webkit-border-horizontal-spacing: 0px; -webkit-border-vertical-spacing: 0px; border-collapse: separate; color: #0000ff; font-family: 'Times New Roman',Times,serif; font-size: 16px; line-height: 38px;">**What are White Blood Cells and Where are they Formed?** = ||
 * <span style="font-family: 'Times New Roman',Times,serif;"> White blood cells, also known as leukocytes, are part of our bodies immune system. They are cells that help us fight off infections through a variety of ways. Mainly, there are five different [|types of white blood cells]: granulocytes, neutrophils, eosinophils, basophils, and monocytes. They travel along with red blood cells and infiltrate to areas that are affected or injured. As explained above, through signaling induction white blood cells undergo diapedesis as an auto-immune defense response to injuries and foreign particles [3] . There are, on average, 7,000 leukocytes/µL of blood roaming around in our circulatory system plasma, which is made of about 90% water and 10% nutrients, proteins, hormones, and waste products. However, in the presence of an infection or foreign virus, white blood cells are over-regulated in the body in order to fight against the infection. Most white blood cells are made in the bone marrow's hematopoietic stem cell location, which is the soft part of the bone. In toddlers and young kids, the leukocytes are made in many different bones in the body, but as humans grow older the number of bones were white blood cells are made decreases. At this latter stage, the bone morrow in the vertebras, ribs, pelvis, skull, sternum, femur, and the humerus are the only producers of leukocytes in our body.
 * <span style="font-family: 'Times New Roman',Times,serif; font-size: 14px;">So how is it that white blood cells work for the immune system?
 * <span style="font-family: 'Times New Roman',Times,serif;">There are two types of immunity, innate and adaptive. Innate is lessspecific and attacks a broader range of bacteria's and infections. In essence, [[image:islaslab/immune_system.jpg width="400" height="507" align="right" caption="http://uhaweb.hartford.edu/BUGL/immune.htm"]]innate is basically the first line of defense to foreign viruses and to injuries. Adaptive immunity is more specific and is activated through chemical signals. The recognition of invader of the human body activate a cascade of chemical signaling pathways that eventually allow white blood cells to either disrupt the functions of the invader or to destroy it. To fight these infections in the human body, we have two main types of white blood cells: T cells, which mature in the thymus, hence their name, and B Cells produced in the bone marrow. T cells compose about 70-75% of the adaptive immune system ([|cell-mediated immunity]), while B cells only account for about 10-20% ([|humoral immunity]). Additionally, T cells can be subcategorize in two main types: helpers and killers. Helpers T cells are in charge of activating B cells in order to kill off the invader, while killers T cells take care of the intruder themselves (killer T cells also attack cancerous cells). The previous cells have receptors that are specific to each kind of invader, so when they bind to the invader they recognize it and get rid very rapidly. On the other hand, B cells have to go through a recognition process for antigens that match its receptor. Once B cells encounter the proper antigen-receptor match, they use T cells to induce replication of both plasma and memory cells. The plasma and memory cells will eventually assist in the kill off of foreign antigens. Interestingly, plasma cells also express the same receptor as B and T cells, which allows them to detect foreign antigen molecules that need to be fought against . "Memory cells" are made in order to "remember" the intruder, so that if next time the human body happens to encounter the same invader, a quick auto-immunune defense response can disrupt the foreigner infections. So with this information, we can clearly see why is it that the human body can rarely get sick twice with the same flu. A mutated type of infection is the one to blame when you get sick twice. &

<span style="color: #0000ff; font-family: 'Times New Roman',Times,serif; font-size: 18px; line-height: 27px;">**What is Leukemia?**
 * <span style="font-family: 'Times New Roman',Times,serif;"> [|Leukemia], as reported by the National Library of Medicine, is a cancer of the white blood (Leukocytes) cells produced by the bone marrow. White blood cells are essential for humans because they help fight infection. So Leukemia is the production of abnormal white blood cells by the bone marrow, which eventually out-number normal white and red blood cells, causing several diseases such as anemia and respiratory problems. There are four [|types of leukemia] : Acute and Chronic Lymphocytic Leukemia (ALL or CLL), and Acute and Chronic Myeloid Leukemia (AML or CML)
 * <span style="font-family: 'Times New Roman',Times,serif;">According to a study done in 2006, the rate of success in the treatment for ALL has increased steadily since the 1960's, stating that rates of "cure (defined by the absence of evidence of disease for at least 10 years) with the use of modern treatments are about 8- percent for children and 40 percent for adults" (Pui, Relling, and Downing 2006)
 * <span style="font-family: 'Times New Roman',Times,serif;">An important fact is that, according to statistics obtained from the National Cancer Institute, about 50% of patients with leukemia died in 2010 in the United states.
 * <span style="font-family: 'Times New Roman',Times,serif;">Since we are focusing on Acute Lymphoblastic Leukemia, we would like to go more into detail about definitive characteristics of this type of leukemia:
 * <span style="font-family: 'Times New Roman',Times,serif;">Acute Lymphoblastic (Lymphocytic) Leukemia (ALL)
 * <span style="font-family: 'Times New Roman',Times,serif;">Facts
 * <span style="font-family: 'Times New Roman',Times,serif;">Expresses a poor prognosis and account for more than 30% of adult cases.
 * <span style="font-family: 'Times New Roman',Times,serif;">Makes about 80% for childhood acute leukemias (most cases in kids 3-7 years old)
 * <span style="font-family: 'Times New Roman',Times,serif;">Worsens very quickly by crowding out the circulatory system with cancerous WBC that replaced normal WBC cells
 * <span style="font-family: 'Times New Roman',Times,serif;">Possible risk factors: chromosomal problems, radiation exposures, past chemotherapy treatments, benzene toxicity, and bone morrow transplants are commonly seen in developmental stages for acute lymphoblastic leukemia
 * <span style="font-family: 'Times New Roman',Times,serif;">Symptoms
 * <span style="font-family: 'Times New Roman',Times,serif;">Usually a person with ALL would be very likely to bleed because of the low percentages of normal blood cells and platelets in the blood stream. (Note: it is important to know that some of the symptoms mentioned bellow are also commonly seen in other types of diseases. Further medical procedures should be taken for analysis)
 * <span style="font-family: 'Times New Roman',Times,serif;">Bone and joint pain/tenderness
 * <span style="font-family: 'Times New Roman',Times,serif;">Easy bruising
 * <span style="font-family: 'Times New Roman',Times,serif;">Feeling weak and tired
 * <span style="font-family: 'Times New Roman',Times,serif;">Fever
 * <span style="font-family: 'Times New Roman',Times,serif;">Loss of appetite and weight loss
 * <span style="font-family: 'Times New Roman',Times,serif;">Paleness
 * <span style="font-family: 'Times New Roman',Times,serif;">Night sweats
 * <span style="font-family: 'Times New Roman',Times,serif;">Test to detect ALL
 * <span style="font-family: 'Times New Roman',Times,serif;">Physical Exam: Through this kind of test bleeding could be seen, as well as enlarged organs such as liver, spleen, and lymph nodes.
 * <span style="font-family: 'Times New Roman',Times,serif;">Blood Testing: Could be used to confirm ALL by detecting abnormal white blood cell count, low platelets and red blood cells count, which could eventually lead to respiratory complications or anemia.
 * <span style="font-family: 'Times New Roman',Times,serif;">Bone morrow aspiration: will show abnormal levels of cells produced in the bone marrow
 * <span style="font-family: 'Times New Roman',Times,serif;">Treatment
 * <span style="font-family: 'Times New Roman',Times,serif;">Chemotherapy: For the first round, called induction, hospital staying for 3-6 weeks may be needed. Then, chemotherapy will be given upon schedule visits to the doctor. Even isolation in a sterile room might be necessary, if white blood cells count is very low, which essentially means that the body is very susceptible for infections.
 * <span style="font-family: 'Times New Roman',Times,serif;">Radiation: many chemotherapy drugs do not reach the brain or spinal cord, so radiation may be used to prevent metastasis to this areas.
 * <span style="font-family: 'Times New Roman',Times,serif;">Bone morrow/stem cell transplant: highly recommended to replace the production of abnormal white blood cells
 * <span style="font-family: 'Times New Roman',Times,serif;">Preventive Measures:
 * <span style="font-family: 'Times New Roman',Times,serif;">Since the cause for ALL is unknown prevention, it is not very possible to prevent most cases. However, by reducing exposure to radiations, toxins, and chemicals changes may decrease significantly.

=<span style="color: #0000ff; font-family: 'Times New Roman',Times,serif; font-size: 16px;">Types of Tests Used to Detect Leukemic Metastasis: =
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif; line-height: 20px;">X-rays: a form of electromagnetic radiation, are used at wavelengthin the range of 0.01 to 10 nanometers, allowing energy beam to go through the body and onto film, making a picture of areas inside the body.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif; line-height: 20px;">Lumbar puncture is a procedure used to collect cerebrospinal fluid from the spinal cord. Cerebrospinal fluids flows in and around the hollow spaces of the brain and spinal cord, and between two of the meninges, so if leukemia metastasizes, it can be detected upon analyzing the cerebrospinal fluid.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif; line-height: 20px;">Ultrasound exams can also be used to get sonograms through the use of high-energy sound waves that bounce off internal organs, creating echoes, which are translated to sonograms.

=<span style="color: #0000ff; font-family: 'Times New Roman',Times,serif;">Experimental Research and Data <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Infiltrates || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Specific Location || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Severity || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">*Notes || <span style="color: #000000; font-family: 'times new roman',times,serif;">Table 1. Leukemic Infiltrates in distinct locations of 9 Guinea Pigs sampled. Intensity of leukemic infiltrates was scored from 0 being no infiltrates to + 4 being severe. Animals tested showed very similar lesions with minimal variations in intensity; thus they were described together. <span style="color: #000000; font-family: 'times new roman',times,serif;">**Introduction:** =<span style="color: #0000ff; font-family: 'Times New Roman',Times,serif;">So... What is the Significance? = =<span style="color: #0000ff; font-family: 'Times New Roman',Times,serif;">Proposed Metastasis Mechanism = <span style="color: #000000; font-family: 'times new roman',times,serif; font-size: 12px;">So now that we have learned some background information about the different theories of tumor cell metastasis, as well as the way in which leukocytes move in the bloodstream, we would like to show you how everything ties together. As we stated in our purpose statement, some leukemias form secondary tumors at specific locations. Through the case study we learned that lymphocytic leukoblast might have certain chracteristics that creates very high affinity for high endothelial venules (HEVs). But remember CD44? We learned that CD44 is a receptor on the surface of leukocytes, which is in charge of cell trafficking and lymph node homing [14]. Moreover, we learned about CXCR4 and its relation to SDF-1 for leukocyte extravasation. And lastly, we learned about what diapedesis is and how through chemical signaling the leukocytes infiltrate in between endothelial cells. So now is time to put all our knowledge together. By now, you probably already know how is that everything is intercorrelated. So lymphocytic leukoblast metastasis is a multistep process that requires of many events to happen. Let us recall that leukemic leukocytes over-express CXCR4 and CD44. Under conditions of flow through the vessels, leukocytes-endothelium cell adhesion interaction occured because of CD44 (integrin) binding to hyaluronic acid on the surface of HEVs (1st stage of cell-endothelium interaction). Then the moving cells are stimulated by chemokines or as in the picture bellow, extravascular stimulus, (SDF-1) produced by endothelium or stromal cells, signaling that increases the affinity of integrins (CD44) to their ligands. SDF-1 activates the leukocytes by binding to its receptor CXCR4, and strengthening the binding between HEVs and leukocytes. Upon the previously mentioned signaling, the leukoblast goes through a morphological/structure change (spheric --> hemispheric; 2nd stage of cell-endothelium interaction). Lastly, the leukoblast infiltrates into the new tissue or extravascular compartment through diapedesis (3rd stage of cell-endothelium interaction). Note: Migration of leukocyte across the endothelial surface is also integrin-dependent. However, we must note that metastasis is not completed until the leukemic cell is able to proliferate and survive in the new tissue after colonization.
 * <span style="font-family: 'Times New Roman',Times,serif;">**Overal Introduction:** t he study was done in 1989 by researchers from the Indiana University Medical Center. The purpose of the study was to analyze the correlation between the pattern of metastasis and the homing sites of the normal progenitor cells. Additionally, the researchers were interested in tracking over time the interaction between leukemic (L2C) cells and High Endothelial Venules, which are specialized postcapillary venules found in lymphoid tissues that support high levels of lymphocyte extravasation from the blood and leukocytic trafficking in the bloodstream, in guinea pigs by carrying out two different experiments. What these researches found is that non-random tumor metastasis is a possibility. In other words secondary tumor implanting location could be assessed and even predicted by examining cell-endothelium binding sites. L2C cells, leukemic cells as mentioned above, where inoculated in type II Guinea Pigs and leukemic cell infiltrates were observed mainly around veins in specific locations mostly in lymph node tissues. These venules or small blood vessels showed signs of diapedesis and cell binding. In contrast, capillary and sinusoidal levels, at distinct organs, did not show any sign of diapedesis, instead extravasation, or vein fluid leakage, occurred due to intravascular proliferation and secondary damage to the vessel itself. (Azzarelli pg 45).
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">**Microscopic Morphometry:**High endothelial venules in the lymph node were identified by typical tall appearance of the endothelium. Central veins of the liver were identified by their location at the center the liver lobule. The leptomeningeal veins were easy to identified because of their thin wall, which lacks lastic membrane. The previous two types of veins are not associated with lymphatic vessels, excluding a possible source of error because it can be hard to differentiate venules from lymphatic vessels.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">**Experiment 1:**
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">4.7 million leukemic cells were injected under the skin of 13 type II Guinea Pigs weighing a little under half a kilogram (300 to 360 grams each). 2 additional Guinea Pigs were injected with saline solutions to serve as a control group. Blood sample of all Guinea Pigs tested were taken at intervals of 1 to 15 days after inoculation. Samples were counted and examined for total and differential white blood cell counts. Finally, the animals tested were then sacrificed by perfusion fixation (which kills the tissue, but preserves the morphology just as if the specimen was alive) 8 and 15 days after inoculation.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">In order to distinguish the injected leukemic cells from the normal WBC of the Guinea pigs, scientists noted that the L2C cells had small amounts of cytoplasm, their surface was covered by numerous, widely spaced microvilli, large numbers of ribosomes and the nuclei were indented and contained ordinary amounts of dispersed chromatin with some condensation on the nuclear membrane.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Guinea Pigs 1 to 3 were perfused fixed 6 to 8 days after inoculation and no leukemic cells were found. Guinea Pig 4 was perfused on day 9 after inoculation and minimal leukemia infiltrates were spotted, due to insufficient evidence this data was not recorded. Guinea Pigs 5 to 13 were perfused 12 to 15 days after inoculation. The number of WBC varied from 4.0 x 10 ^ 4 to 2.8 x 10 ^ 5 / mm ^ 3. Pigs 13 to 15 were found to have severe leukemic infiltrations as stated in the chart bellow. Leukemic infiltrations " refers to leukemic cells in the extravascular compartment" or the space that surrounds the cells in a tissue mainly near the venules.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Leukostasis, a white blood cell count above 100000/μL, was observed in the kidney, lung capillaries, adrenal glands and the brain of the Guinea Pigs with leukemia. It was also noted that in regions with severe leukemic infiltrates, veins showed leukemic cell migration, binding and diapedesis.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">The most interesting part of this experiment is the fact that tumors might actually grow by the accumulation of leukemia cells in the extravascular compartment. This was observed in abdominal lymph nodes of the Guinea Pigs with leukemia. Leukemia cells piled up and were covered completely by a thin cytoplasmic layer of HEV origin. The leukemia cells continue to divide and promptly the endothelium stretches over the cells. Simultaneously the endothelium cells are undergoing hyperplasia or cell proliferation and more leukemia cells are binding to the HEV. Our end product are "large intravascular nodules" or a cluster of cells which block the vein.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">**We then hypothesize that the leukemia cells continue to undergo mitoses until it gets large enough and once engulfed by the endothelial cells, breaks through the venule and implants itself in the extravascular compartment.**
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Organ/Tissue
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Brain || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Leptomeningeal Veins || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+4 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Arachnoid || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+2 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Virchow-Robin spaces || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">0 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Subependymal veins || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+ ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Choroid Plexus Stroma || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+ ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Brain Parenchyma || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">0 || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">One Guinea Pig showed signs of nodules in brain parenchyma. ||
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Lung || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Peribronchial Veins || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+4 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Parenchyma || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">0 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Subpleural || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+2 ||  ||
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Liver || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Capsule || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+2 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Portal Spaces || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+4 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Liver Plates || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+2 ||  ||
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Kidney || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Outer and Medullary Cortex || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">0 || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">One Guinea Pig shows foci of leukemic infiltrates in the outer cortex. ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Medulla || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+ ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Renal Columns || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+4 || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Around interlobar arteries and veins ||
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Spleen || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Diffuse || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+4 ||  ||
 * || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Abdominal Lymph Node || <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">+4 ||  ||
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">**Experiment 2:**
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">For this experiment, 8 leukemic pigs with white blood cells levels above 8x104/mm3 were used as donors. After being given some body weight (0.5 mCi/gm and 150 units of heparin/kg) for anticoagulant purposes, the animals were decapitated and whole bloodstream collected (approx. 20ml). L2C cells were isolated by Ficoll Hypaque technique, a density-gradient centrifugation technique used to separate lymphocytes (WBC) from other formed elements in the bloodstream. After isolation, cells were suspended in a phosphate-buffered saline solution to keep a constant pH in the cells and cells’ osmolarity (isotonic and non-toxic to cells), and then they were used within one hour with Strain II guinea pigs (inbred strains).
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Additionally, exact heparin amount as above was injected to recipient Strain II animals, using a number 18 catheter through the jugular veins to reach the ear. Then 3.4x108 to 1.6x109 of working L2C cells were injected in a period of 10 minutes to an 8 guinea pigs. Each animal received a volume of approximately 2 to 4 ml of cell suspension. A thoracotomy, an incision into the pleural area of the chest, was done after intervals of 10, 60, 75, 80, 100, 200, 300, and 385 minutes of the injection of L2C cells. In four animals, the vascular system is cleared by perfusion with gassed (95% O2, 5% CO2) medium for 2 minutes. The other four animals were perfused with only fixative, a chemical substance used to preserve biological material prior to microscopy or other type of examination.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">**Results:**
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">The vascular perfusion resulted in good clearing or all examined organs. Only lymphocytes were observed marginated and in diapedesis at the HEV in lymphoid tissues.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Retention of L2C cells in the inside wall of the vessels was most prominent in lung capillaries and liver sinusoids. However, in other tissues including the brain, lymph nodes, kidneys, adrenal glands, skeletal muscle, retention of L2C cells was uncommon.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Infiltration into the extravascular compartments and diapedesis was observed in: cervical lymph nodes, bronchus associated lymphatic tissue (BALT), pulmonary veins with lymphoid associated tissue, and veins of the portal triads.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">It was seen that L2C cells significantly marginated to HEV in lymphoid tissues when compared with central veins of liver and leptomeningeal.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Highest degree of margination (136) took place in the abdominal lymph node, only within 10 min of insertion of L2C cells.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Under electron microscope marginated cells contacted the endothelium either through villous (covered with villi) processes or through more extensive membrane contacts.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Diapedesis was never observed in animal number one. However, in animals 2 to 8 (perfused-fixed from 60 to 385) show infiltration, binding, and diapedesis in all LYMPHOID tissues (i.e. cervical and abdominal lymph nodes, Peyers patches and BALT).
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">**Discussion:**
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Usually tumor dissemination in the blood is study by injecting one single cell in the circulatory system, previously suspended from fragmentation of a solid tumor. The way by which these tumor cells are obtained commonly alter the metastatic properties of the cells, which raises questions as to the relevance of the data to the natural processes of neoplastic dissemination. Nonetheless, the study that we analyzed here allows us to analyze in a time sequence the propagation of the L2C cells. The previous comparison is an important one for the understanding of out study, since we do not clearly understand the mechanism by which tumor cells lose adhesion to solid tumors in other cancer cases.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">The artificial dissemination of L2C cells resulted in a predictable pattern of metastasis. The researchers of the study knew beforehand where leukemic cells would infiltrate. Their findings suggest that leukemic cells infiltrate in guinea pigs at specific locations in each organ:
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">This suggests that the leukemic infiltrates were the result of transvenular traffic in these regions. However, it could not be determined whether cells in traffic were in the process of extra- or intravasation because the L2C cells were found in both the intra- or extravascular compartments. [[image:islaslab/Picture_table_3.jpg caption="Picture_table_3.jpg"]]
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">//In vitro// binding assays strongly suggest specificity of lymphoma cell-HEV binding. However, the significance of these results to the actual process of tumor invasion is not completely known, but some people argue and strongly believe that in other cases cells follow the EMT theory, which is discussed above.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Data gathered in the study that we analyzed show morphologic evidence that clearly demonstrate not only that leukemia cells can bind to endothelium tissue, but also invasion.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Leukemic cell extravasation through the HEV in lymphoid tissues and selected veins of lung and liver is accomplished by diapedesis. This finding would support data gathered on various studies, which shows high percentages of metastasis for Acute Lymphocytic Leukemia (ALL).
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Some authors postulate that lymphocytes exiting of the lumen are through transcytoplasmic routes, and others believe that it is through the cells junction.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">A recent report states that lymphocytes initiate a transcytoplasmic pathway, followed by diapedesis.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">The previous study done by researchers at Indiana University Medical Center suggest the following stages for cell-endothelium interaction:
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">First stage: is the development of cell contact through villi from leukemic cells. Similar contacts have been previously described between HEV and normal, functioning lymphocytes. As we may know, contact between cells should not be seen according to physical laws because the cell surface of eukaryotic cells bear a net negative charge and cell repulsion should happen. However, it has been postulated that probes, with low radius of curvature, may reduce the repulsion energy between two approaching cells. It is thought that these probes interact with focal areas, exposing less surface charge than average, and that facilitate the interaction between leukocytes and low anionic groups, such as in areas near HEV (Note: Distribution of negative charge is not equal in the surface of endothelial vascular cells).
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Second stage: is the change of cell morphology from spheric to hemispheric, which is evidence to suggest specific cell-to-cell binding. Note: In lungs capillaries and liver sinusoids the typical morphological change was never seen, interestingly this finding may tell us that there is something different about such structures.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Third stage: Is the penetration of the basal lamina by strong fibrillated podocytes.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">Interestingly, this studies shows that leukemic cell migration through HEV’s is very slow and this can be seen even at the 385 minutes thoracotomy done to animal number 8 where L2C cells were found under the endothelium.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">The previous study, although old, was very informative and useful for our research process. In both experiments large number of leukemic cells were observed undergoing diapedesis and margination at lymph node tissues after strongly interacting with HEV's at specific locations. For example, in Experiment 2, it was observed that a significant amount of leukemic cells were located at high endothelial venules at lymph node tissues, such as the abdominal lymph node where 136 cells were marginated. We could compare the previous results to brain or liver tissue where 0 and 1 marginated cells, respectively, were observed. As mentioned above in our purpose statement, we wanted to analyzed why some types of leukemias form secondary tumors at specific locations.
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">As we research more in depth to analyzed HEVs and their interaction with leukemic cells, we found a couple articles that were very interesting. We found that CD44, which is a very important transmembrane glycoprotein receptor expressed in all types of cells, is involved in lymph node homing, matrix adhesion, and T lymphocyte activation. CD44 is an important receptor, over-expressed in lymphocytic leukoblast, that acts as an integrin to mediate attachment to the surface of high endothelial venules at lymph node tissues. CD44 has very high affinity for HEVs at lymph tissue because it binds to hyaluronic acid found at the surface of lymphatic HEVs. It is though that "because normal lymphocytes are distinct and traffic between the lymph and blood circulating systems throughout most of their life cycle, it is reasonable to assume that the metastasis of malignant lymphoid tumours may reflect expression of their homing receptor (CD44)" (Hsieh et.al 1999, 26). For leukocytes to travel through the blood vessels, leukocyte-HEV adhesion interaction is needed. CD44 is responsible for binding and trafficking of leukocytes through the lymphatic system. &
 * <span style="color: #000000; font-family: 'Times New Roman',Times,serif;">[[image:islaslab/graph.jpg align="left" caption="Fig 4. Correlation between CXCR4 expression and the SDF-1- induced transendothelial migration of ALL lymphoblasts. A positive correlation of r = 0 ́73 (P = 0 ́001) was found between the expression level of CXCR4 (measured as median fluorescence intensity) and the percentage of ALL lymphoblasts transmigrating in response to SDF-1 (MS-5 conditioned medium in the lower chamber of the transmigration system) (n= 17)."]]Additionally, as we did some more research we found another article that postulate a potential mechanism in the trafficking of leukemia cells in the lymphatic vessels. It is thought that CXCR4, an over-expressed transmembrane chemokine receptor in the surface of lymphocytic leukoblast, interacts with its ligand (chemokine) stromal-cell derived factor-1 (SDF-1). A chemokine is a molecule that can induced chemotaxis in nearby responsive cells. Chemotaxis is the process by which cells direct their movements in relation to chemical signals in their nearby environment. SDF-1 is constitutively produce by stroma cells, endothelial cells, bone morrow, an other types of tissues. According to the study "chronic as well as acute lymphocytic leukaemia B cells have been reported to express the chemokine receptor CXCR4 and to migrate after stimulation with the stromal cell-derived factor-1" (Crazzolara et. al 2001, 551). Interestingly, they found that //in vivo// studies of breast cancer metastasis that by inhibiting the chemokine receotor CXR4 by antibodies, significantly reduced metastasis of tumor cells to other tissues, suggesting that the SDF-1 induce infiltration of lymphocytic leukoblast into extravascular compartments and other tissues by a concentration gradient driving force. So in a few words, this study suggest that cell migration occurs by a concentration gradient force that induces leukocytes to infiltrate into the extravascular compartments.

=<span style="color: #0000ff; font-family: 'Times New Roman',Times,serif;">Conclusion = <span style="color: #000000; font-family: 'times new roman',times,serif; font-size: 12px;">After extensive research, we have gotten to the conclusion that metastasis is a multistep process that is very difficult for any type of tumor cell to successfully achieve. Nonetheless, we have comprehend a potential mechanism by which some types of leukemias metastasize at specific locations. This project has made us more aware about the complexity of cancer in general, but specially in lymphocytic leukemia that show possible metastasis through integrin interaction, chemotaxis induced by SDF-1, and diapedesis. It is very important to understand that the previous mechanism is just an idea of what the possible mechanism for leukemic metastasis is, but based of our research we believe that this mechanism seems reasonable because of the extensive evidence and data suggested on various studies.

<span style="color: #000000; font-family: 'times new roman',times,serif; font-size: 12px;"> We have definitely enjoyed doing this project because it was not only challenging and informative, but it was also fun!

<span style="color: #0000ff; font-family: 'times new roman',times,serif; font-size: 12px;"> ** References: **