JHU - Understanding Cancer Metastasis

Last updated Mar 6, 2023

• Type note
• Status new
• Source course
• On biology
• On cancer

Metadata

• Source:: Understanding Cancer Metastasis | Coursera
• Instructor:: Kenneth Pienta, Jelani Zarif, Sarah Amend, Haley Axelrod, Kenneth Valkenburg
• Offered by:: Johns Hopkins University
• Platform:: Coursera
• Publish Date::
• Review Date::

About this Course
Everyone has been, or will be touched by cancer in some way during their lifetime. Thanks to years of dedication and commitment to research we’ve made enormous advances in the prevention and treatment of cancer, But there is still a lot of work to be done. In this course, physicians and scientists at the Johns Hopkins School of Medicine explain how cancer spreads or metastasizes. We’ll describe the major theories of metastasis and then describe the biology behind the steps in metastasis. The course also describes the major organs targeted by metastasis and describes how metastases harm the patient.

• We know that all solid tumors can metastasize.
• Liquid cancers like leukemia do not metastasize because there is no primary tumor.
• Most people who die of cancer will die because they have a metastatic tumor or metastatic tumors.

• Joseph Recamier, in his 1829 treatise, coined the term “metastasis” to describe the spread of cancer.
• From the Greek metastasis: “a removing, removal; migration; a changing; change, revolution”
• In 1858, Rudolf Virchow (father of modern pathology), suggested that metastatic tumor dissemination was determined by mechanical factors — the arrest of tumor-cell emboli in the vasculature.
• The true father of metastasis theory is Stephen Paget. He wrote a paper in 1889: “The Distribution Of Secondary Growths in Cancer of the Breast”.
  • Analyzed 735 fatal cases of breast cancer and argued that the distribution of metastases cannot be due to chance.
• The Seed and Soil Hypothesis
  • Paget recognized that metastatic patterns did not simply follow blood flow distribution as suggested by Virchow.
  • Paget referred to cancer cells as “seeds” and the way they distributed throughout the body must be understood by studying the “properties of the soil” of the secondary organs.
  • “When a plant goes to seed, its seeds are carried in all directions, but they can only live and grow if they fall on congenial soil.”
• In 1928, James Ewing challenged the “seed and soil” hypothesis by proposing that metastasis was determined by the anatomy of the vascular and lymphatic channels that drain the primary tumor.
• In a series of studies in the 1970s – present, Isaiah (Josh) Fidler merged and added to previous theories and work and demonstrated that metastasis occurred in a series of sequential steps that involve cancer cells with different metastatic capabilities interacting with their microenvironment and other cells: ultimately selecting for successful cancer clones in a stochastic manner.

The sequential steps of metastasis can be broken down into 8 steps:

1. Primary tumor growth
   • Cancer starts to grow at one site. This is termed the “primary tumor” or “primary cancer” of that organ, e.g., breast cancer, lung cancer, prostate cancer.
   • Even after the cancer spreads to other organs, it is still referred to as a cancer of that primary organ.
     • For example, prostate cancer that is found metastasized to the bone is still referred to as prostate cancer.
2. Angiogenesis
   • A tumor cannot grow to more than a millimeter in size unless it attracts new blood vessels.
   • New blood vessels provide the growing cancer with nutrients and oxygen and provide the cancer cells with a pathway to other sites in the body.
3. Epithelial-to-mesenchymal Transition (EMT) (motility)
   • Acquisition of a motile phenotype (a cell that can walk around and move versus one that can’t)
     • It is thought that cancer cells undergo a change in phenotype to make them more mesenchymal — cells that lack polarity and are more motile.
   • This allows them to be more invasive and “walk out” of the primary tumor.
4. Invasion
   • Cancer cells invade the tissue surrounding the cancer.
5. Intravasation
   • Cancer cells can break through the blood vessel wall and move into the circulation.
6. Survival in circulation
   • Cancer cells in the circulation must survive turbulent blood flow and escape detection by cells of the immune system that would recognize them as abnormal and try to kill them.
7. Extravasation
   • When cancer cells reach a target organ, they attach to the blood vessel wall and then invade into the tissue.
8. Dormancy and subsequent secondary tumor growth
   • Cancer cells in the new tissue or “soil” most likely survive in a dormant state for a period of time before they start to proliferate and form a detectable, clinical metastasis.

• Cancer is the continuous, uncontrolled division of a single cell
  • Different types of cancer, depending on the cell of origin
    • carcinoma: cancer originating from epithelial cells; 90% of all cancers
      • e.g. prostate, lung, breast, kidney, colon, stomach
    • sarcoma: cancer originating from connective tissue (like muscle or bone); very rare
      • e.g. osteosarcoma, fibrosarcoma
    • leukemia/lymphoma: cancer originating from blood cells and blood-forming cells; 8% of all cancers
• Cell division: one cell becomes two cells
  • In simple terms, cell division occurs when one cell divides and becomes two daughter cells
  • Cell division is called mitosis
• The Cell Cycle: cell division takes time and is controlled
  • Cell division is really a cycle
  • synthesis: cell’s entire genome duplicates
  • gap phases: cell conducts quality control, checks for damage, grows
  • mitosis: cell divides
  • 
  • Cell cycle checkpoints assure that damaged cells do not divide
    • During the cell cycle, a cell must pass three checkpoints, where cellular machinery detects cell/DNA damage.
    • If damage is detected, the cell cycle is halted:
      • cell repair
      • senescence
        • cell permanently stops dividing
      • cell death
        • Apoptosis: programmed cell death; damaged cells should die.
• Uncontrolled division of a damaged cell eventually turns into a tumor
  • In cancer
    • Cell/DNA damage (e.g. mutation) occurs but is not detected
    • Cell/DNA damage is detected, but programmed cell death machinery is faulty
• Cancer cells are quite damaged and are recognizable under microscope.
  • In fact, to this day, the way cancer is largely diagnosed is by a pathologist, studying the shape and the look of cells taken from a patient.
  • 

• A normal cell becomes a cancer cell through mutations in the DNA.
  • A mutation is a permanent alteration in the DNA sequence that is different from what is found in most people and that causes or increases risk of a disease.
  • In the case of cancer, the mutation contributes to a growth advantage.
• Oncogene: promotes cancer
  • Activating mutations (turns on gene improperly) in oncogenes are tumorigenic
• Tumor suppressor: protects against cancer
  • Inactivating mutations (turns off gene improperly) in tumor suppressors are tumorigenic
• Three main types of mutations/genetic changes
  • Point mutation
    • substitution
      • An individual DNA piece (“nucleotide”) is substituted with a different nucleotide.
    • insertion/deletion (“indel”)
      • Additional DNA is inserted into an existing sequence, or existing DNA is removed.
  • Amplification
    • Production of multiple copies of a sequence of DNA; could be whole chromosomes.
  • Translocation
    • Pieces of two chromosomes switch places
• Most of the time, a change in the DNA is generally either detrimental enough to a cell that it dies (recall cell cycle checkpoints) or harmless enough that nothing happens to the cell.
• Mutations are a problem because a cell not only survives in its presence, but thrives and divides in its presence.
• There are two broad types of mutations
  • Hereditary
    • Inherited in the egg or sperm cells from parents; present in every cell in the body
    • A person is born with this mutation
  • Somatic
    • Occur at some time during a person’s life and are present in only the cells in which the mutation occurred and that cells’l daughter cells. Parent cannot pass this mutation down to their children.
    • These arise randomly or through carcinogens.
• The two-hit hypothesis states that both copies of DNA need to be changed to become cancer-causing.
• Accumulation of mutations over time contributes to malignancy.
• Additional mutations can create heterogeneity within a tumor
  • Various mutations create multiple clones or versions of cancer cells
  • Each clone may behave and respond to therapy differently
  • Clonal expansion may happen as well, if growth of a specific clone or clones is favored

• Vasculogenesis: new blood vessels form out of stem cells during embryonic development
• Angiogenesis: new blood vessels form out of pre-existing vessels. This occurs
  • during growth and development
  • during wound healing
  • as a result of tumor growth (this is specifically called “neoangiogenesis”)
• Cardiovascular system = Vasculogenesis + Angiogenesis
  • The combination of vasculogenesis and angiogenesis creates all of the blood vessels in the body
  • These vessels bring oxygen and nutrients to all cells while carrying away CO_2 and cellular waste
• Capillaries are the smallest blood vessels that exist.
  • They are small enough to fit in between tissues and to reach all of the cells.
• Arteries carry oxygen and nutrients to the cells.
  • They connect to the veins through capillaries
• Neoangiogenesis
  • Tumor cells grow more quickly than normal cells and outgrow their source of oxygen and nutrients
  • In order to grow, tumors need to make new blood vessels to provide necessary nutrients and oxygen
  • Neoangiogenesis is one of the Hallmarks of Cancer
  • Cells that are too far away from a blood vessel undergo hypoxic conditions — this drives neoangiogenesis
  • More neoangiogenesis = more routes for cancer cells to metastasize
• Normoxic conditions: there is enough oxygen for the cells
• Hypoxic conditions: there is not enough oxygen for the cells
• [?] Can we stop tumors from growing and/or metastasizing by inhibiting neoangiogeneis?
  • Not a “home run” treatment for cancer
    • Inhibiting neoangiogenesis will only stop new blood vessels from being made
    • At the time of tumor detection, a tumor already has an established blood supply
    • Therefore, stopping new blood vessels would not stop existing tumor cells from getting oxygen and nutrients
  • Continues to be a popular and somewhat effective strategy, mostly in combination with other therapies
    • bevacizumab inhibits vascular endothelial growth factor A, which promotes neoangiogenesis
    • currently used in four cancer types in combination with chemotherapy

• A tumor is made up of more than just tumor cells
• Tumors exist in a microenvironment, which is composed of all the cells that normally exist in a tissue or in the blood, that then interact with and can influence the tumor
• Extracellular Matrix (ECM)
  • Secreted extracellular proteins and complex carbohydrates
  • Provides structure/support
  • Acts as a barrier to tumor invasion
  • Tumor invasion through ECM is an indicator malignancy
• Neurons
  • Present within many tumor types
  • Peptides and neurotransmitters secreted by neurons play an influential role in tumor progression
  • Tumor cells can use nerves as physical support to invade surrounding tissue
• Secreted Molecules
  • Tumors secrete molecules (proteins, DNA, and RNA) that cause immunosuppression, neoangiogenesis, and cancer cells sustainability
  • Other cells secrete molecules that promote tumor growth and mobility
    • This is not their normal function, but tumor cells commandeer these secreted molecules for their benefit
• Fibroblasts
  • Normal fibroblasts are a part of connective tissue and produce collagen and other fibers
  • Cancer-associated fibroblasts (CAFs) signal to cancer cells via secreted molecular factors, causing:
    • additional mutations
    • cell division
    • EMT
    • invasion/metastasis
    • inflammation
• Fat Cells (adipocytes)
  • Obesity has been linked to increased risk of many cancers
  • Buildup of white adipose tissue (WAT) produces varios secreted proteins that promote:
    • tumor formation
    • tumor progression
    • EMT
    • invasion/metastasis
• White Blood Cells (Immune Cells)
  • Many kinds of immune cells
  • Some immune cells kill cancer cells by recognizing abnormal proteins on cell surface
  • Other immune cells promote cancer by:
    • suppressing the killer immune cells
    • stimulating various Hallmarks of Cancer
    • assisting in the process of metastasis
• Different immune cells have different kinds of interactions with tumor cells
  • Blood stem cells produce many different kinds of immune cells that form the basis for innate and adaptive immunity
  • These cells respond to stress, infection, foreign antigens, abnormal cells, etc.
  • Due to the wide variety of functions of these cells, each cell may influence tumor growth differently

• Go versus Grow hypothesis
  • A cell can
    • proliferate
    • move
    • differentiate to a terminal cell
  • Within an individual cell, proliferation (growing) and cell movement (going) are typically spatio-temporally separated
• Epithelial-mesenchymal transition (EMT)
  • The reversible process in which epithelial cells lose characteristic epithelial properties while simultaneously gaining mesenchymal stem cell characteristics.
• Epithelial cells
  • These make up the epithelial tissues that line all outer and inner cavities of the body and are characterized by their strong structural integrity.
  • Exhibit apicobasal polarity, meaning that they have different characteristics on their top and bottom sides
  • Strongly adhere to the extracellular matrix
  • Form tight cell-cell interactions to form layers
  • Cuboidal morphology
  • Highly proliferative
  • Non-motile
  • Epithelial cells are characterized by tight cell-cell interaction
• Mesenchymal cells
  • They are essential for wound healing and embryonic development.
  • They can differentiate into many different cell types, can regulate immune response, and regulate wound healing through modulation of surrounding cell types.
  • Do not directly bind with other cells
  • Specialized cytoskeleton components
  • Spindle morphology
  • Front-to-back polarity
  • Highly migratory
• Cells attach to the ECM via focal adhesions
• How to distinguish a cancer cell EMT event?
  • First off, it’s important to remember that in cancer cells EMT just like go or grow is a continuum. Some cells will retain, some epithelial characteristics when they transition or cells may show different mesenchymal characteristics. It’s all context and cell dependent.
• EMT is induced by many factors and cells present in the tumor microenvironment.
• Mesenchymal-epithelial transition (MET)
  • Importantly, once metastatic cells reach a secondary site, they must re-gain the proliferative characteristics of epithelial cells to form a secondary tumor.
  • EMT is a reversible process regulate by gene expression (not gene mutation).

• Extracellular matrix (ECM)
  • The extracellular matrix, or ECM, are molecules outside of cells that form a web-like matrix to form structural support for surrounding cells.
  • To invade the extratumoral space, cancer cells must break through the ECM.
  • The ECM is made up of collagens, fibronecin, laminins, and many other non-collagenous proteins that are secreted by stromal cells.
  • The ECM is present throughout all the body, and it’s essential for structural integrity, cell signaling and migration.
  • There are two major components of the ECM
    • Basement membrane, which is the part that cells bind to and that separates tissues and cell layers.
    • Interstitial matrix, and that is the ECM that is found between cells and that provides tensile strength to tissues.
  • In order to invade, the cancer cell needs to use the ECM to anchor its movement while at the same time destroying the ECM to make room to invade the adjacent space.
  • ECM attachments: adhesion and migration
    • During migration, the cell undergoes dramatic morphology changes, made possible through cytoskeletal rearrangements within the cell.
    • Cell-ECM adhesions are made primarily via cell-surface attachment proteins (integrins) binding to form focal adhesions.
    • File:Four steps of cell migration.png - Wikipedia
  • Individual and collective cell migration
    • There is evidence of cancer cells moving both as individuals, or in clumps, and this is another active area of research. Individual movement may either be mesenchymal, as we described on the previous slide, or amoeboid gliding movement, which is mostly described as being like an amoeba. Collective cell movement requires cell-cell communication and cell-cell adhesion, in addition to cell matrix adhesion.
  • Deregulated ECM remodeling in the tumor microenvironment
    • In the tumor, that ECM remodeling is deregulated. There is increased expression and secretion of mini proteases that degrade the ECM, including matrix metalloproteinases and cathepsins.
    • In addition to the whole tumor, the cancer cells deregulate ECM remodelling. The host cells that are recruited to the primary tumor also contribute. This leads to patchy and unstable ECM. And this weakened ECM is more permissible to cancer cell invasion, allowing the cancer cells to secrete proteases and break through the ECM surrounding the tumor.
  • Once cells have disrupted the tumor ECM, they may invade local tissues.
    • Once cells have disrupted the tumor ECM, they may both invade the local area within their organ of origin. For example, a prostate cancer invading healthy prostate tissue. In addition, the cells may also invade local tissues. A good example of this is that bladder cancer often invades local fat tissue. This phenomenon of local invasion is important in tumor staging, in regard to invasion and to adjacent muscle. This brings us to the end of the invasion section. Next, we’ll discuss a specialized form of invasion, intravasation.

• Intravasation is the process by which a tumor cell invades through the vessel wall to enter either the circulation or the lymphatic system.
• In order to intravasate, the cancer cell must invade through the ECM surrounding the cell layers of the blood or lymphatic vessel.
• The cancer cell must also invade through the layers of endothelial cells.
  • To do this after remodeling the basement membrane surrounding the vessel. The cancer cell physically disrupts the cell-cell contact of the endothelial cells. It then deforms its shape to invade through the newly degraded space in the vessel wall.
• There is evidence about active and passive intravasation into both lymph and blood vessels.
  • In active intravasation, the tumor cell migrates into the capillary wall and actively remodels endothelial cell-associated ECM.
  • In passive intravasation the cells simply fall into the blood or lymph stream by chance. This is made possible by the rapid neoangiogenesis of the tumor that results in disorganized sinusoidal capillaries.

• Anoikis – A form of programmed cell death when cells detach from their native ECM.
• Circulating Tumor Cell (CTC) – A tumor cell that has entered into the bloodstream and lymphatics and is able to survive.
• Metastasis Suppressor Genes – is a gene that acts to slow or prevent metastases. These are lost in cancer cells.
• Extravasation – movement of cancer cells out of a blood vessel into tissue during metastasis
• Disseminated Tumor Cell (DTC) – A CTC that has exited out of the circulation into a distant, secondary site.
• Metastasis – The process by which cancer spreads from its origin to another part of the body.

• The Lymphatic System
  • Lymphatic vessels carry lymph fluid to lymph nodes and then to bloodstream
  • Purpose: to drain excess fluid to maintain volume and composition of tissue; to act as a defense against infection
  • Lymph is the interstitial fluid composed of
    • Water
    • White blood cells (leukocytes)
    • Proteins, fats, sugars, salts
    • Cellular waste
• Cells can enter into the lymphatics or then get into the bloodstream directly.
• Lymphatic capillaries are able to flow into venous capillaries.
• Cancer cells can enter the lymph nodes and this is observed clinically.

• Metastasis Suppressor Genes lost during intravasation
  • Genomic instability, one of the 10 cellular hallmarks of cancer, is a prerequisite not only for tumorigenesis but also for metastasis.
  • Metastasis suppressor genes are genes that can suppress a cell’s ability to metastasize. These genes are lost in cell that become metastatic.
  • Known metastasis suppressor genes: CD82/KAI1, PAKK4, PKIP, NM23-H1, NM23-H2, and KiSS1
• CD82/KAI1
  • This gene expression allows normal cells that may passively enter the bloodstream to bind to endothelial cells via the DARC receptor.
  • Since this gene is lost in several cancer types, cancer cells exhibit activated oncogenes (Src), are favorable to enter the bloodstream and travel to distant sites.
• Anoikis
  • The term “Anoikis” was first used by Steven Frisch and Hunter Francis in 1994.
  • Anoikis (“without a house”) is a form of programmed cell death that occurs when cells detach from their surrounding ECM. Normal cells die during this process.
  • Metastatic tumor cells undergo detachment but, are able to evade death, another hallmark of cancer.
  • How are cancer cells able to evade cell death?
    • Epidermal Growth Factor (EGF) and its receptor (EGFR) can lead to increased PI2-K signaling.
    • This signaling has an effect on downstream substrate AKT and its phosphorylation
    • This activates the mTOR (Target of Rapamycin) which allows cells to confer anoikis resistance.

• The bloodstream is a place where most detached cells die
• CTCs associate with several other cell types.
  • One of these cell types are platelets.
    • Platelets are nuclear cells that are involved with clotting vessel injuries. They are found in large numbers in the blood and contribute to angiogenesis.
    • CTCs can interact with platelets via tumor cell inducted platelet aggregation or TCIPA. TCIPA activates platelets and allows them to attach to CTCs and shield them from Immune destruction.
    • Platelets also have the ability to transfer Major Histocompatibility Complex (MHC) Proteins onto CTCs, so that the immune system does not detect them. This allows for evasion of the immune system.
  • Another cell type that has been shown experimentally to associate with CTCs are M2 Macrophages.
    • M2 Macrophages are a type of white blood cell that is important for wound healing. And these cell types have been shown experimentally to allow a cancer cell to undergo epithelial mesenchymal transition as well as intravasate.
    • In experimental models (in vitro), isolated CTCs have been shown to have the proclivity to recruit cells belonging to a myeloid-macrophage lineage using factors known as cytokines.
    • This association with myeloid cells allow CTC cells to suppress the cell proliferation of T-cells via programmed death ligand 1 (PD-L1) – a notable checkpoint ligand.
• Increased programmed death ligand 1 (PD-L1) on CTCs allows immune system evasion

• Cancer cell lifecycle:
• Seed and soil hypothesis
• Mechanism of homing: Chemokines aide CTCs to home into distant organs
  • Chemokines are a family of cell signaling proteins that are secreted by many cells types. These molecules can bind to receptors expressed on target cells to aid in chemotaxis.
  • There are largely two types of chemokines that are studied:
    • One type is called basal chemokines. Basal chemokines are secreted by cells in the thymus and in lymphoid tissues.
    • The other is known as inflammatory chemokines, and these are released during injury to recruit particular cells to the site of injury.
• Platelets aid in the extravasation process
  • CTCs first anchor on the luminal side of the endothelial cells (Dock and Lock) and then are aided by platelets.
  • First, platelets rush to the site of extravasation and induce the expression of CCL-5 on endothelial cells, which can recruit CTCs and leukocytes.
  • Second, platelets secrete platelet derived TGF-$\beta$ and platelet derived growth factor (PDGF) to aid induce EMT, which allows cells to pass through the sub-epithelial vessel wall.
  • Lastly, activated platelets are recruited by CTC’s ability to up-regulate CCL2 to promote monocyte recruitment and increase vascular permeabilization.
• Steps of extravasation
  • CTCs leave the bloodstream by becoming attracted to a secondary site via chemokines, docking and locking, and then platelet mediated extravasation.
  • Once CTCs extravasate into a secondary site, they become known as Disseminated Tumor Cells (DTCs)

• Why do different cancers metastasize to different organ sites?
  • Two theories
    • Cells specifically and preferentially home to those sites, as if they have an “address”
    • Cells non-specifically home to many sites, but only survive or grow at preferential sites (seed and soil hypothesis)
• Seed and soil hypothesis for secondary growth
  • “When a plant goes to seed, its seeds are carried in all directions; but they can only live and grow if they fall on congenial soil”
  • Seed = cell that left the primary tumor and has entered the circulation
    • Cells may travel to many organ sites
  • Soil = the secondary organ site
    • Composed of the cell types within that organ, and the surrounding microenvironment
    • Permissive or restrictive for growth of seed (proliferation of the cell)
    • The cell types and microenvironment at the secondary organ site determine if the disseminated cell is able to survive and proliferate
  • What are the fates of a cell upon landing on foreign soil?
    • Death – does not receive proper pro-survival signals from the microenvironment; no formation of metastasis
    • Proliferation – able to receive proliferation cues; eventually establishes a new mass of cells which represents a metastasis
    • Dormancy – neither death nor proliferation; potential for metastatic outgrowth
      • Thought to be the reason for cancer recurrence years after a patient has been “cured”

• Clinical dormancy is the period when you cannot detect residual tumor cells
• The notion of cancer dormancy first evolved from clinical observations
  • In 1952, the pathologist Rupert Willis first coined the phrase, dormant cancer cell, as the only plausible explanation for his observations. He noticed that for each of these primary tumors, the time to clinical relapse, which is when the patient presents with metastases, could be between 5 and 30 years. He thought that if even one cell started proliferating, it would not take as long as 5 years for it to proliferate into a detectable mass.
  • Then in 1954, a pathology professor named Geoffrey Hadfield agreed with this thought. Saying, when the interval is prolonged to six years or more it seems impossible to escape the conclusion that the cells of the dormant growth are in a state of temporary mitotic arrest, no matter how long the period may be.
  • Another observation for the existence of clinical cancer dormancy unexpectedly comes from a transplantation case study.
    • This study reported the unknowing transmission of a cancer to organ receivers.
    • Patients who die from central nervous system tumors, or CNS tumors, are allowed to be organ donors because of the low metastatic rates of CNS tumors. The organs that were donated by these patients were seemingly disease free, but once they were transplanted to the receiving immunosuppressed patients, those patients rapidly developed cancer of CNS origin in those organs. The table below displays these observations where the cancer was transmitted in almost all of the organs transplanted from these patients, and that the onset of disease was only a matter of months post transplant. This indicated that there must have been residual cancer cells present in the transplanted organs that had disseminated from the CNS tumor. Yet, they were undetectable because they have been residual cancer cells. Once they were placed into a patient without a functioning immune system, the cancer cell were able to proliferate and grow out.
  • More evidence for cancer dormancy comes from the fact that cancer cells disseminate early in primary tumor development.
    • This has been proven through clinical observations, where disseminated tumor cells, or DTCs, have been found to be present in the bone marrow of breast and prostate cancer patients at the time of surgery, which is usually just after the tumor has been detected. We also know that even complete removal of early-stage breast and prostate cancer, when it is found early, does not always prevent relapse either, meaning cells had already disseminated.
    • Additionally, early dissemination has been proven through genomic analysis where for example, one study found that bone marrow DTCs harbor fewer genomic aberrations compared to the primary tumor. This suggests that the primary tumor continued to evolve after the cells disseminated.
• Two main types of cancer dormancy
  • Tumor mass dormancy
    • Tumor mass dormancy as a concept refers to the balance of proliferation and apoptosis occurring within a mass of cancer cells. Some cells will be proliferating, but some cells will be dying. So there’s no net increase in tumor mass, and it remains undetected.
    • Two subtypes
      • Angiogenic dormancy
        • This is when the growth of the micrometastasis is restricted due to the lack of sufficient vascularization.
        • In order for a tumor mass to grow beyond 1 millimeter cubed, it needs to be able to promote angiogenesis which is the formation of new blood vessels.
          • These vessels are required because they allow for the delivery of growth factors and oxygen.
          • To control angiogenesis, the cancer cells can secrete pro or anti-angiogenic factors.
      • Immunologic dormancy
        • This is when the growth of the micrometastasis is restricted due to the immune system.
        • The immune system has the ability to recognize cancer cells as “foreign” and target them for elimination.
        • Cells that are able to escape this recognition and continue to proliferate are balanced by cells that are recognized and targeted by the immune system.
  • Cellular dormancy
    • This refers to individual cells that are growth-arrested and are not proliferating, but are also not dead.
    • The cellular dormancy follows the same principles as quiescence does in normal physiology, as it must be a reversible state in order for the cell to eventually develop into a metastasis.
    • Cellular dormancy therefore cannot represent senescent cells or the differentiated post-mitotic cells, which are most of the cells in your body, because those cells are all irreversibly growth-arrested.
    • Cellular dormancy is the most accepted explanation for the long period of clinical dormancy compared to tumor mass dormancy.
  • These are all considered “micrometastases” because they are too small to detect
• Quiescence in normal physiology is defined as a reversible cell cycle arrest and specifically refers to cells that exist in the cell cycle in a state called $G_0$.
  • They can sit in this state for years where they are always poised to reenter the cell cycle.
  • Cells may undergo quiescence in response to
    • deprivation of extracellular growth factors
    • anti-proliferative cytokines
    • contact-inhibition.
  • Some examples of healthy quiescent cells in the body include adult stem cells, progenitor cells, fibroblasts, and hepatocytes.
  • Quiescence is extremely important because
    • it was required for tissue homeostasis. For example, quiescence of hepatocytes allows for the ability of the liver to renew and regenerate because they are able to reenter the cell cycle and proliferate when they are needed.
    • it protects long-lived cells against stress and toxicity. And this is especially important for cells like hematopoietic stem cells, which are responsible for generating immune cells. With each cell division there’s a potential for mutations to occur, and cells are more susceptible to stress and toxicity. So it becomes crucial that such important and long-lived cells are able to go into a quiescent state to avoid this susceptibility.
• In contrast to quiescence and dormancy, senescence is an irreversible growth arrest.
  • And these cells have been reported to arrest in G1 or G2, as opposed to G0.
  • Senescence can be induced by many factors, but the main one is due to telomere shortening, called replicative senescence.
  • Any cell can undergo senescence, and this is a major factor that contributes to aging because these cells no longer have the functional capacity that healthy cells do.
  • Senescence is important because it prevents the outgrowth of non-healthy cells
    • Cancer evades this! This is a property of cancer cells
• Control of cellular dormancy in cancer
  • Lack of extracellular growth factors
    • Cells do not receive proliferation signals
    • Extrinisic microenvironment is not conducive for proliferation
  • Anti-proliferative cytokines
    • Actively tell the cell to stop proliferation
    • This is how quiescent stem cells can be regulated
  • Change in cell’s metabolism – Autophagy
    • Autophagy (autophagocytosis) is a controlled process that degrades and recycles cellular componenets that may not be absolutely necessary
    • Cells undergo autophagy in response to stress as a survival mechanism
    • Been shown to induce a quiescent state

• Property 1: Dormant cancer cells have decreased cellular activity
  • Decreased RNA and protein synthesis
  • Decreased metabolic activity
  • Do not express cell cycle-promoting proteins
• Property 2: Dormant cancer cells are thought to be “stem-like”
  • Stem cells are maintained in a quiescent state until they are needed
  • Must have the ability to self-renew
  • It is hypothesized that cancer stem cells are responsible for initiating tumors
• Property 3: Dormant cancer cells are more resistant to therapy
  • Standard chemotherapies target mechanisms used by proliferating cells
  • Activation of stress response mechanisms
  • Thought that some therapies can induce dormancy of the cells that were able to evade it
  • Tightly coupled with the idea that they are “stem-like”
• Property 4: Dormant cancer cells are clinically undetectable
  • Standard clinical tests are unable to precisely detect these rare cells
    • Imaging (MRI, CT, PET, X-ray)
    • Biopsy
    • Blood test
  • The insufficient sensitivity of standard assays does not catch residual cancer cells which have the potential to cause recurrence
  • If we had the ability to detect micometastases, maybe we could prevent lethal metastases altogether
• Property 5: Dormat cancer cells are in a benign state
  • Think about what fundamental property defines cancer – uncontrolled proliferation
  • Dormant cells are not proliferating
  • When does cancer become lethal? When it leads to a large mass that cause tissue damage, extreme pain, cachexia, etc.
  • If we were able to maintain the dormant state of residual cancer cells, the patient would not die of cancer
• Property 6: Dormant cancer cells are difficult to study in the lab
  • A lot of cancer research is done using cell line models
    • In order to propagate a cell line to repeatedly use it, cells must be proliferating
  • Standard, cheap experimental assays require many cells (which are hard to obtain if they are not proliferating)
  • In vitro, models are robut – how to make a cancer cell become quiescent? And not be senescent or dead
  • Animal models can be great, but are expensive (money, time, labor)
  • Poorly defined universal positive markers (we have great markers/assays for proliferating cells)

• Quiescent cells can be precursors to secondary outgrowth
  • A quiescent cell has the potential to re-proliferate
  • Resuming uncontrolled proliferation is what forms a metastasis
    • Clinically detectable
    • More aggressive than the primary tumor
    • Can occur at many sites
• How does a years-long dormant cell resume proliferation?
  • Not known exactly – mechanisms may vary depending on the type of “seed” or the type of “soil”
  • Some hypothesis
    • Receiving pro-proliferative cues from the microenvironment
    • Attachment to other resident cells through specific surface protein interactions
    • Activation of movement machinery
    • Extracellular matrix stiffening (fibrosis)
    • Inflammation
• Re-awakening of a dormant cell leads to a metastasis
  • Uncontrolled proliferation resumes, and a growing mass is formed
    • Must recruit and form new blood vessels – angiogenesis
    • Similar to the primary tumor
  • This growth is thought to be more aggressive than the primary tumor – already went through the metastatic process and were able to survive
  • Cells can re-enter the circulation and home somewhere else to potentially form a new metastasis
• ==Dormancy is emerging as another hallmark of cancer==
• There are many unanswere questions in this field
  • Do dormant cells exist in every patient who has had a primary tumor?
  • We dont know exactly what drives dormancy.
    • Is it universal across all types of cancer?
    • Are there multiple mechanisms?
  • When does dormancy occur? Can cells actually become dormant before they disseminate?
  • How do cells re-awaken and why don’t they all re-awaken?
  • Are dormant cells really just cancer stem cells?