Extracellular Matrix and Metastasis
K. Aardema
First let's look at some definitions:
What is the extracellular matrix?
The extracellular matrix (ECM) is a structural component of tissues and is described thoroughly on our first page. One pertinent form of the ECM is the basement membranes. The basement membranes surround the extracellular surfaces of epithelial cells and endothelial cells (such as of blood vessels) and envelop nerves and entire organs. The basement membranes are permeable during tissue development or repair and the inflammatory response (Brooks et al., 2008; Brietkrutz, Mirancea & Nischt, 2009).
Collagens are a major component of the ECM, and one major change in ECM in the disease state of cancer is increased collagen deposition, or fibrosis. Specifically, collagen helps regulate cancer cell polarity, migration, and signalling (Gilkes, Semenza & Wirtz, 2014).
Collagens are a major component of the ECM, and one major change in ECM in the disease state of cancer is increased collagen deposition, or fibrosis. Specifically, collagen helps regulate cancer cell polarity, migration, and signalling (Gilkes, Semenza & Wirtz, 2014).
What is cancer?
The Gale Encyclopedia of Cancer defines cancer as "a disease of unregulated growth" (Longe, 2006). Cancer cells may have mutations of tumor suppressor genes or genes involved in DNA repair (Longe, 2006). Cancer cells are usually less differentiated than normal tissue cells and have characteristics of rapidly growing cells (Molecular). Cells normally have "checks and balances" (such as tumor suppressors) that keep them from proliferating uncontrollably, but tumors form when these regulations are ignored.
In culture, cancer cells require fewer growth factors, indicating production of their own growth factors. The cells also lack the normal signal to stop growing when the culture becomes crowded (Longe, 2006). To form a tumor, one mutated cell proliferates abnormally, so multiple mutated cells make up the majority of the tumor. The newly formed mutated cells under-go clonal selection, where advantages due to mutations cause the specific cells to survive and multiply. The final tumor may have cells with various mutations. It's a cellular version of "Only the Strong Survive".
Below are two helpful diagrams showing primary tumor (the first tumor of each specific cancer) formation. An adenoma is the first benign mass of cells formed (Longe, 2006), and it can grow in size. The adenoma can eventually form a malignant carcinoma (cancer of epithelial cells), but the process can take more than twenty years (Longe, 2006). In the last step of the 2nd figure, the primary tumor is beginning to metastasize. As the video says, the cells move to a new location through the blood vessel.
In culture, cancer cells require fewer growth factors, indicating production of their own growth factors. The cells also lack the normal signal to stop growing when the culture becomes crowded (Longe, 2006). To form a tumor, one mutated cell proliferates abnormally, so multiple mutated cells make up the majority of the tumor. The newly formed mutated cells under-go clonal selection, where advantages due to mutations cause the specific cells to survive and multiply. The final tumor may have cells with various mutations. It's a cellular version of "Only the Strong Survive".
Below are two helpful diagrams showing primary tumor (the first tumor of each specific cancer) formation. An adenoma is the first benign mass of cells formed (Longe, 2006), and it can grow in size. The adenoma can eventually form a malignant carcinoma (cancer of epithelial cells), but the process can take more than twenty years (Longe, 2006). In the last step of the 2nd figure, the primary tumor is beginning to metastasize. As the video says, the cells move to a new location through the blood vessel.
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| Primary Tumor Formation (Here) |
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| Colon Cancer Formation (Here) |
What is metastasis?
The movement of the cancerous cells to a new location is the basis of metastasis. If the words "primary tumor" are used clinically, the implication is that a secondary site of cancer exists. A primary tumor or cancer indicates the original location that the malignant cancer occurs (such as in the breast), and it can metastasize. Metastasis causes tumors in a secondary site (John's Hopkins) . The cancer in the secondary site will be composed of cells microscopically similar and with the same mutations as the primary cancer cells (National Cancer Institute), though the new environment of the cells may make some changes.
Like the above video says, primary tumors spread through the blood or lymph. The spreading of tumors from a primary site to a secondary site is called metastasis, and the cancer is called metastatic cancer. Metastatic cancer is associated with a poorer prognosis in the medical field, and the spread of cancer throughout the body makes treatment more difficult and is what makes cancer particularly dangerous. Although metastatic cancer can be treated, when people die of cancer it is usually the metastatic form (National Cancer Institute). Metastasis can be summed up by the following cascade, which is described further here.
Like the above video says, primary tumors spread through the blood or lymph. The spreading of tumors from a primary site to a secondary site is called metastasis, and the cancer is called metastatic cancer. Metastatic cancer is associated with a poorer prognosis in the medical field, and the spread of cancer throughout the body makes treatment more difficult and is what makes cancer particularly dangerous. Although metastatic cancer can be treated, when people die of cancer it is usually the metastatic form (National Cancer Institute). Metastasis can be summed up by the following cascade, which is described further here.
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| Metastatic Cascade (here) |
Using a common example of pancreatic cancer metastasizing to the liver (National Cancer Institute), the cancer cells would have to pass through their extracellular matrix, the basement membrane of the epithelial cells of the liver, the basement membranes of the endothelial cells of the blood vessel, back out through the basement membranes again, and finally through the basement membrane and extracellular matrix of the liver. That is a lot of work! In order for the cancer cells to get back out of the blood vessel, they must be able to adhere to the vessel walls. After extravasation (leaving the blood vessel), the primary cancer cells must colonize to form the secondary tumor in the liver.
What is angiogenesis?
Angiogenesis is the formation and growth of new blood vessels and is important during wound healing, embryonic development, and the menstrual cycle. More pertinently, angiogenesis is the first step of metastasis. Angiogenesis is stimulated when a tumor needs nutrients and oxygen (Nishida et al, 2006), so without angiogenesis tumors would only be able to grow to a diameter of about 2 millimeters (Brooks et al., 2008). First, the basement membrane of the local tissue of the tumor is damaged, and the tumor enters a state of hypoxia (Nishida et al, 2006). The damage occurs because the tumor stimulates the blood vessels to release proteolytic enzymes, creating a place for new vessels to grow and migrate to the tumor(Brooks et al., 2008). Then, angiogenic factors cause endothelial cells to migrate and proliferate, while angiogenesis inhibitors are down-regulated (Nishida et al, 2006). Here is a figure depicting the steps of angiogenesis:
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| Steps of Tumor Angiogenesis (here) |
Here is an interesting video about angiogenesis in disease and foods that may help to "starve" cancer through anti-angiogenesis. To sum it up, research shows that middle-aged people have many microscopic cancers, but they don't become dangerous because they do not have a blood supply. This means the body has a natural defense mechanism against cancer... until mutated cancer cells release angiogenesis stimulators. Treatment to cut off angiogenesis could stop cancer without harming other blood vessels, since cancer blood vessels are poorly constructed and vulnerable to drugs. FDA approved avastin stops angiogenesis in breast cancer. Eleven other anti-angiogenic drugs also exist, and they improve survival possibility. New research shows that some foods (such as Earl Grey tea or cooked tomatoes) naturally contain angiogenesis inhibitors, and potency increases when we eat multiple anti-angiogenic foods. This means that our diet can be used to prevent and to treat cancer.
What is a cell niche?
A cell niche is the area around the cell, or the microenvironment, that affects the cell's fate. Cancer cells have a metastatic microenvironment that includes: the extracellular matrix, secreted enzymes, growth factors, and cytokines (Descot and Oskarsson, 2013).Metastasis and the Extracellular Matrix:
Imagine you were sitting casually in a coffee shop eavesdropping on the conversation beside you, and you heard "Yeah, one of the kids got attacked and killed by a dog. I'm just glad we only lost one". Horrified, you start to look up a child protective services number, when you hear, "We tried taking her to the veterinarian" and realize the person is referring to a young goat. Context matters in life, and this includes the context of cells. Here is a TED Talk by Mina Bissell about the context of breast cancer cells:
Bissell reminds her audience that each cell has the same genetic material, but many different tissues are made from that genetic material. The diversity of tissues formed with the same genetic information indicates that context and architecture must affect cell differentiation and proliferation. Therefore, context and architecture affect cancer.
For example, Bissell and her lab decided they wanted to study the formation of the acinus of the mouse mammary gland. In Bissell's research pregnant mouse mammary cells formed milk and had a round nucleus, but after 3 days in vitro the cells did not produce milk and had an irregularly shaped nucleus. However, if the cells were placed in a dish with ECM they formed milk. Next, Bissell's lab examined normal and malignant human acinus cells to create a baseline, and then the malignant cells were reverted to normal with an inhibitor. Bissell's research indicates that malignancy is regulated at the tissue level by cell microenvironment.
The ECM is an important part of the cell's microenvironment. For example, the Bissell lab also showed that Rous Sarcoma Virus did not cause tumors if applied to a chick site with no wound infliction. Wound healing, which involves collagen deposition, changed the cell microenvironment.
The ECM also allows the cell to sense forces; integrins on cells bind to the ECM to form cellular matrix adhesion complexes (CMACs), which can sense forces created externally or internal actomyosin forces (Dijk, Goransson & Stromblad, 2013).
ECM and integrins also mediate growth factor (GF) signalling, which helps control cell proliferation, differentiation, migration, and invasion. For instance, ECM attachment regulates cell proliferation by causing the formation of cyclin D1 supports progression of the G1-phase of the cell cycle (Dijk, Goransson & Stromblad, 2013). ECM composition impacts what signalling pathways become activated, since different ECM ligands engage different integrins (Dijk, Goransson & Stromblad, 2013). Attachment is required in the G1-phase as well as in cytokinesis.
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| Klein et al., 2009 |
As seen in the previous figure, cyclin D mRNA levels depend on the stiffness of the ECM. Cyclin D mRNA levels were increased in MCF10A mammary epithelial cells, vascular smooth muscle cells, and mouse embryonic fibroblasts grown on high stiffness (a hydrogel at 24,000 Pa) rather than low stiffness (less than 2000 Pa). The hydrogels corresponded to a ECM within the normal physiological range (1500 - 150,000 Pa). The paper concludes, "Matrix remodeling associated with pathogenesis is in itself a positive regulator of the cell cycle through a highly selective effect on integrin-dependent signaling to FAK, Rac, and cyclin D1" (Klein et al., 2009). Because cyclin D is important for cell cycle progression, it is clear that the rigidity of the matrix affects cell fate. Interestingly, high expression of cyclin D and high tissue stiffness both occur in breast cancer (Klein et al., 2009).
Another example of the effect of the ECM rigidity on cell fate is found in the following figure that shows Madin-Darby canine kidney epithelial cells cultured on rigid or compliant gels and treated with transforming growth factor β (TGF-β).
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| Leight et al., 2011 |
TGF-β initially acts as a tumor suppressor, but as cancer progresses it promotes tumorigenic properties, like epithelial-mesenchymal transition (EMT). The cells cultured on a rigid gel showed EMT with TGF-β1 treatment. (The cells are elongated and have actin stress fibers shown by phalloidin and delocalization of the epithelial cell–cell adhesion markers E-cadherin and ZO-1.) The cells cultured on a compliant cell showed normal morphology and actin and increased apoptosis (shown by an increase in caspase-3). The rigidity of the ECM affects TGF-β signaling, so cell context matters for cancer!
Here are a couple of videos and a figure to explain EMT:
As the video explains, epithelial cells cannot migrate, but mesenchymal cells can aquire migration ability. The epithelial-mesenchymal transition is used in the formation of the embryo and by metastatic cancer cells to achieve migration ability and stem cell characteristics that maintain the tumor.
As the video states, EMT is frequently followed by the reverse transition (mesenchymal back to epithelial) during development of epithelial tissues (such as the lining of organs). Epithelial cells give a tissue structure because they are connected by epithelial calcium-dependent adhesion molecules (E-cadherin) and are connected to the ECM by integrins. Regulatory molecules bind to extracellular or intracellular receptors to initiate EMT by activating transcription factors that lead to EMT gene expression. The genes encode for proteins that degrade E-cadherins and integrins, proteins that restructure the cytoplasm, and proteins that form pseudopodia and help the cell migrate. The transitioned cells can migrate and invade the ECM.
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| Brooks et al., 2008 |
The figure shows E-cadherins joining two cells. Beta catenin connects the extracellular domain of cadherins intracellulary to actin in the cytoskeleton.
Which came first, intracellular or extracellular changes?
| what came first? |
There are many parts of life and science that are cyclic, making it difficult to figure out what the first step is or was. A relative example is Cell-ECM Reciprocity. Basically, mechanical changes to the ECM modify intracellular actomyosin contractility and cytoskeletal reorganization, changing intracellar tension and leading to remodeling of the ECM. The cycle continues. However, because cells create the ECM, it is thought that intracellular changes are the first steps of the cycle (Dijk, Goransson & Stromblad, 2013). Here is a figure to explain further:
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| Dijk, Goransson & Stromblad, 2013 |
The ECM remodeling seen in the figure can lead to exposure of normally hidden ECM proteins, such as a site on collagen IV that has a role in angiogenesis. ECM remodeling also causes abnormal fiber alignment and an increase in deposition of collagen, fibronectin, matrix metalloproteinases (MMPs), and lysl oxidases (LOXs). MMPs and LOXs are matrix remodeling enzymes (Dijk, Goransson & Stromblad, 2013).
MMPs cleave ECM proteins that are similar to epidermal growth factor (EGF) and stimulate signaling pathways. MMPs also release TGF-β. LOXs crosslink collagen fibers to stiffen the ECM, which leads to an EMT response to TGF-β, and a poor prognosis for cancer patients. Platelets can also induce EMT by releasing TGF-β, and platelets may access metastatic tumors due to their leaky vasculature (Descot and Oskarsson, 2013).
All of these changes affect adhesion receptor signalling. For instance, integrin levels vary in different types of cancer, and CD44 adhesion receptor and hyaluronan are extremely increased at the tumor attachment site. Attachment activates Rho and PI3K signaling, which improves the cell's ability to migrate and survive (Dijk, Goransson & Stromblad, 2013).
Cancer tissue ECM is similar to ECM of tissues during wound healing, tissue remodeling, and fibrosis. Specifically, hyaluronan is increased during tissue remodeling, fibronectin is part of the ECM during wound healing, and collagen I accumulates during fibrosis (Descot and Oskarsson, 2013).
How else does the metastatic niche promote cancer?
Migration and survival are certainly important for cancer metastasis, and so are other characteristics provided by the metastatic microenvironment. This environment promotes metastasis initiation and outgrowth through characteristics like hypoxic pockets, an invasive front, and endothelial structures. The invasive front, or moving intersection between tumor and ECM, is frequently highly vascularized, and cancer cells become highly motile through epithelial-mesenchymal transition (Descot and Oskarsson, 2013).
Angiogenesis is stimulated through hypoxia and through tumor and ECM cells that produce growth factors that bind to endothelial cells of vasculature near the tumor and promote synthesis of integrins and MMPs. The endothelial cells degrade the ECM and migrate to form immature vessels.
The vascularization is leaky, so the tumor is poorly oxygenated promoting EMT through hypoxia inducible factor. Hypoxia inducible factor (HIF) affects the chemokine receptors like CXCR4 associated with metastatic breast cancer. HIF also activates genes coding for anaerobic metabolism, cell motility, and apoptosis resistance. Mesenchymal cells remain undifferentiated under hypoxic conditions. Endothelial cells may also be an important part of promoting metastasis because microRNA have been used to prevent endothelial cell recruitment and metastasis to the lung in mice with breast cancer. Endothelial cells may use the Notch pathway to gain stem cell characteristics (Descot and Oskarsson, 2013, Brooks et. al, 2008).
As the video says, the Notch pathway is a developmental pathway and is cleavage-mediated. The Notch receptor is a single-membrane spanning protein, with most of the receptor being extracellular. The Notch receptor is important for cell-cell communication during neural and stem cell development. When a ligand from another cell binds, gamma-secretase cleaves the receptor, releasing notch intracellular domain (NICD). NICD transports to the nucleus and turns on the Notch signalling pathway by dissociating NcoR. CSL is activated, and target genes affecting development are turned on.
To sum up:
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As the figure indicates, the cancer cell is "fit" because of it's microenvironment, or metastatic niche. This niche is formed by many components, one of which is the extracellular matrix. The changes in extracellular matrix cause changes in signalling with cellular effects like EMT. The ECM is remodeled, and cryptic fragments become exposed. The ECM allows cancer cells to adhere during tumor formation. The ECM promotes cancer cell fitness.
Lastly, please enjoy the following overly dramatic video which explains extracellular remodeling during metastasis. Briefly, the tumor is signaled to produce matrix metalloproteinases, which break down the ECM, exposing cryptic ECM fragments that act as tumor growth signals. The open space allows the tumor to migrate, and a secondary tumor is formed. A possible cancer treatment could be to inhibit matrix metalloproteinases. Here is the video:
Lastly, please enjoy the following overly dramatic video which explains extracellular remodeling during metastasis. Briefly, the tumor is signaled to produce matrix metalloproteinases, which break down the ECM, exposing cryptic ECM fragments that act as tumor growth signals. The open space allows the tumor to migrate, and a secondary tumor is formed. A possible cancer treatment could be to inhibit matrix metalloproteinases. Here is the video:
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