CBIO4: Hallmark 6 - tissue invasion & metastasis Flashcards
What percentage of death by cancer is metastasis responsible for?
90%
What is haematogenous?
Cancer cells travelling through blood vessels
What is lymphogenous?
Cancer cells travelling through lymphatic vessels
What organs to tumour cells have a preference for metastasising?
Lung
Liver
Bone
List the key stages of the metastatic process
1) Primary tumour growth
2) Angiogenesis
3) Intravasation (blood vessel penetration)
4) Transport through circulatory and/or lymphatic system
5) Extravasation (escaping the blood vessel)
6) Growth in a secondary organ
What is cancer that has spread from the breast to the lung referred to?
Breast cancer of the lung
What is the difference between monoclonal and polyclonal metastasis?
Whether metastatic tumours form from one cell or multiple cells - different types of cells within a tumour can give rise to different types of metastasis
What fundamentally leads to the metastatic process?
Genomic instability, which can create many cell subtypes, also called clones, which may have metastatic properties
What does phenotypically heterogenic monoclonal mean?
A metastasis containing one clone which has undergone further genetic alterations
What does CTCs stand for?
circulating tumour cells
Why did it take a long time for CTCs to be realised?
- Screening for CTCs is technically challenging.
- Rare compared with other circulating cells and there is no universal surface marker to recognise them by. Apart from CTCs, human blood contains other material that can originate from primary tumours, including cell-free tumour DNA (ctDNA) and RNA (ctRNA), proteins, and vesicles (exosomes).
What is a liquid biopsy?
Liquid biopsy is a clinical test to detect circulating tumour cells or tumour-derived material in the blood and other fluids from patients with cancer
Apart from CTCs, what other material that can originate from primary tumours?
- Cell-free tumour DNA (ctDNA)
- RNA (ctRNA)
- Proteins
- Vesicles (exosomes)
Apart from blood, what other body fluid contain tumour-derived material?
Urine
Saliva
Pleural effusions
Cerebrospinal fluid (CSF)
Why is clinical screening of CTCs via liquid biopsy so challenging?
- ctDNA is fragmented and highly under-represented compared with tumour DNA
- Only a limited number can be isolated from a given blood sample.
- Low abundance of CTCs (approximately 1 cell per 1x109 blood cells).
How do you overcome the challenge of CTC screening?
The sample needs to be enriched for CTCs, the number of CTCs in the sample needs to be increased
How do you increase the relative number of CTCs?
Negative or Positive enrichment
Describe negative enrichment
Remove other blood cells based on shape, size or other biophysical properties
Describe positive enrichment
Select cells expressing specific markers on cell surfaces (surface markers). The surface markers can, for example, distinguish and epithelial cells from a blood cell, as in the case of epithelial cell adhesion molecules (EpCAM)
What is the limitation of using surface makers to positively increase CTCs in a sample? How is this problem overcome?
The surface markers do not distinguish between a malignant and non-malignant epithelial cell. To do so, the isolated CTCs can be molecularly characterised.
Analysis of liquid biopsies requires what?
highly sensitive assays
What is the current only FDA approvedplatform for the isolation and enumeration of CTCs in patients with metastatic breast, colorectal, or prostate cancer?
CellSearch platform
How does CellSearch work?
It uses positive enrichment based on positive expression of EpCAM and negative expression of CD45 (leukocyte-specific molecule)
By what process are cells provided with nutrients and oxygen?
angiogenesis
How is angiogenesis promoted?
molecules released by cancer cells called pro-angiogenic molecules. E.g: vascular endothelial growth factor (VEGF)
How does VEGF work?
VEGF production encourages blood vessels to grow towards tumour, allowing tumour cells to enter blood stream
How do the proliferating tumour cells gain movement ability?
To escape the tumour mass, cells must also overcome the mechanisms holding them to their neighbouring cells or to the surrounding matrix. For this, they must break down the tissue architecture
What is the EMT process?
Epithelial to mesenchymal transition: special enzymes are produced, adhesion molecules are broken down, and cells acquire new shapes. By acquiring a new shape or phenotype, cells become more motile - losing cell polarity and cell adhesion, to gain invasive and migratory property.
What is breached causing intravasation?
The basement membrane (BM)
What forms a premetastatic niche?
Bone-marrow derived cells or other circulating factors prior to tumour cell arrival
What do the initial stages of cancer invasion into surrounding tissues require?
The degradation and remodelling of the basement membrane.
What produces the BM and what is it’s function?
- BM is produced jointly by epithelial, endothelial, and stromal cells
- To separate the epithelium or endothelium from the stroma and interstitial matrix
What is BM and what is it composed of?
BM is a specialised type of extracellular matrix (ECM) as it is more compact, less porous and has a distinctive composition (consisting of type IV collagen, laminins, fibronectin and linker proteins including nidogen and entactin)
What does interstitial matrix contain?
fibrillar collagens ( I and III)
proteoglycans
various glycoproteins
(hyaluronan and fibronectin)
What is the ECM divided into?
The BM and interstitual matrix
What alters the composition of the ECM?
progressing tumour mass and metastatic cancer cells
How is the EM altered by tumour mass and metastatic cancer cells?
ECM alteration occurs via its degradation through enzymes called proteases that degrade proteins by hydrolysis of peptide bonds.
What are the key proteases that degrade the ECM?
- aspartic proteases
- cysteine proteases
- serine proteases
- matrix metalloproteinases (MMPs)
What types of proteases are the following?
- Cathepsin D
- Cathespin B,L and H
- Cathespin A
Cathepsin D = aspartic proteases
Cathespin B,L and H = cysteine proteases
Cathespin A =serine proteases
What is the role of cathepsins?
- turnover and degradation of the ECM;
- activation, processing or degradation of various growth factors, cytokines and chemokines;
- influence cell-cell adhesion molecules
What does uPAR stand for and what is it?
urokinase receptor, urokinase-type plasminogen activator receptor
What does uPAR do?
- regulation of urokinase proteolytic activity and ECM components degradation;
- regulation of cell adhesion, migration, proliferation and survival by interactions with other transmembrane receptors, like integrins.
- uPAR binds also to vitronectin (component of provisional ECM), and through this direct interaction triggers changes in cell morphology, migration and signalling, for example by inducing epithelial-mesenchymal transition (EMT).
What does MMPs stand for?
matrix metalloproteinases
What are the six groups MMPs can be divided into?
collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs.
What is the role of MMPs?
degradation of collagen and other proteins in ECM;
regulation of cell behaviour
What type of junction regulates the function of other cell-cell junctions, and holds epithelial cells together?
The adherens junction (cadherins)
How do integrins act?
1) Integrins interact with specific ECM ligands, sensing the outside-in signals and becoming active. Through this, they can sense the ECM alternations and trigger cells to undergo responsive changes.
2) inside the cell, integrins interact with intracellular ligands and recruit focal adhesion kinase (FAK), which undergo autophosphorylation, formation of complex with Src and activation of both kinases.
What does FAK/Src complex do following activation?
activates a plethora of signalling molecules
What are two key players in the process of EMT?
Alterations in cadherins and integrins
What are the key changes in cells during EMT?
- lack of cell-cell adhesion (reduction in epithelial cadherin)
- dysmorphic shape.
These morphological changes facilitate the invasiveness and motility of cancer cells.
Upon arrival at the new site, what happens to the cancer cells?
They undergo the reverse process of mesenchymal to epithelial transition (MET) to form a new carcinoma. During MET cancer cells proliferate and re-express their epithelial characteristics.
What protein plays a role in producing forward cell protrusions?
filamentous (F)-actin
What is movement of cancer cells called? What is it facilitated by?
mesenchymal migration, representing cell migration on a basement membrane
- forward cell protrusions
How do round or ellipsoid cells migrate?
Amoeboid migration: non-actin protrusions known as blebs can be also present. These are produced through hydrostatic pressure and cytoplasmic flow
Describe epithelial migration
Migration of a group of cells that are connected through cell-cell adhesions. These groups of cells can move as clusters, sheets, strands or fluid-like masses. This process is also termed bulk migration
What are the two types of individual cell migration?
- Mesenchymal
- Amoeboid
What are he four types o multicellular migration?
- Cluster
- Solid strand
- Strand (lumen)
- Strand (protrusion)
What is a common method of metastasis for carcinomas?
lymphatic spread, but this is not common for sarcomas
Name three other routes of metastasis?
transcoelomic,
perineural,
leptomeningeal
What is transcoelomic spead?
spread through the surfaces of body cavities. For example, ovarian cancer can spread into the peritoneal cavity, and in cases of lung cancer, the cancer can spread into the pleural cavity.
What is the space between the visceral peritoneum and the peritoneum surrounding the abdominal wall called?
the peritoneal cavity
What is perineural spread?
Cancer cells spread into the layers of nerves. This type of spread can be found in the head and neck, prostate and colorectal cancers, that are highly innervated
What is leptomeningeal spread?
Cancer cells can travel by the cerebral spinal fluid (CSF). They can invade the membrane covering the brain (meninges) to form lepromeningeal metastasis within it, or the spinal cord to invade the dura mater and epidural space
What is a method of preventing cancer from reaching other parts of the body via lymph nodes?
Tumour-draining lymph nodes are clinically removed
why does lymphatic spread lead to haematogenous spread? In which part of our body is the connection of the lymph to the blood?
Lymphatics drain into blood through the thoracic duct (left lymphatic duct) or the right lymphatic duct.
What determines where cancer cells travel to?
Blood flow characteristics and the structure of the vascular system can regulate the patterns of metastatic dissemination.
In ovarian cancer where do metastatic cells can grow?
peritoneal cavity
in the ascites fluid
by attaching to the surface of peritoneal organs.
Which tumour phenotypes tend to metastasise regardless of vascular anatomy or rate of blood flow?
melanomas: their metastases form largely in the lungs and also in pulmonary and ovarian tissue
What are the four stages to ensure metastatic colonisation succeeds?
1) Priming
2) Licensing
3) Initiation
4) Progression
What happens during priming?
Highly proliferative cancer cells at the primary site become hypoxic (low oxygen levels) and inflammatory. They release different systemic mediators including cytokines, ECM remodelling enzymes and extracellular vesicles (exosomes)
What are exosomes? How do they promote the formation of an immature pre-metastatic niche?
Small, cell-derived vesicles that promote cell-cell communication. They can transport suppressive or metastasis accelerating cargoes. These factors can reach the pre-mastic niche via the circulation and are believed to prepare it for subsequent invasion (formation of an immature pre-metastastic niche).
How can the ECM be remodelled?
by recruiting supporting cells, like bone marrow-derived cells and immune cells - host stroma remodel the local microenvironment and provide metastasis promoting cytokines and chemokines. A mature pre-metastatic niche is formed.
After breaking down ECM of the colonised site, what do the cancer cells do?
New ECM components are produced, like matrix protein tenascin C produced by breast cancer cells in lungs.
During initiation, what signals do cancer cells send to stromal cells (e.g.fibroblasts)? What does this do?
transforming growth factor (TGF)-β
- deposit more matrix proteins, e.g. perIostin, a binding partner of tenascin C, or fibronectin.
What cross-linking enzymes do cancer cells secrete? What does this do?
LOX and PLOD2, to stiffen the ECM and increase integrin/focal adhesion signalling to favour metastasis
Only which integrins are preferentially expressed in the new cancer site?
Only those integrins that facilitate tissue invasion are preferentially expressed. Micrometastases are formed
What is a macrometastasis?
pre-metastatic niche can host more cancer cells and promote metastatic tumour to grow and expand
What does dormancy mean?
When the metastatic process pauses. The dormant tumour cells can reside in new tissues or organs for many years, only to restart at some point to form a metastatic mass
What are disseminated tumour cells?
CTCs that infiltrate distant organs and survive there: hey can be present there for many years without developing metastasis. The metastatic process can be delayed by years due to the dormant phase of metastatic cells
Clinically what does the dormant phase mean?
Dormant phase is described as the time between removal of the primary tumour and a relapse in a patient who was clinically disease free
What are the two theories about how dormancy arises?
1) The DTCs are in a state of proliferative quiescence.
2) Micrometastases become dormant due to insufficient vascularisation or through immune defences.
What are the areas of interest about dormancy which may lead to possible therapies?
- Identification of the microenvironments that promote dormancy;
- Relationships between stem cell pathways and dormancy;
- The molecules/triggers behind reactivation of dormant cells.
How is cell migration studied?
- Scratch assay (also known as wound-healing assay)
- Boyden chambers
- Micropatterning and pre-forming rings or paths
- Microfluidic devices
How do microfluidic devices work?
- Contains a set of micro-channels inside a mould: The micro-channels are connected together to be able to control fluid flow, but also to pump nutrients or drugs. Using microfluidic devices, we can represent the blood flow, while also being able to co-culture different cell types
What model was used to study cell migration and cytokine signalling?
the social amoeba Dictyostelium discoideum
To study how cells cross the basement membrane, what model was used?
nematode worm Caenorhabditis elegans
uring the developmental stage of C.elegans, a single cell has to breach two underlying basement membranes as part of normal morphogenesis: how does it do this?
Cell cycle is arrested in G1, chromatin is modified, and a set of transcription factors is upregulated
What was one of the transcription factors found in C.elegans?
FOS-1A, which is the orthologue of proto-oncogene FOS, a protein strongly associated with cancer cell invasion
What is an Orthologue?
Genes in different species that evolved from a common ancestral gene
What is an orthotopic injection?
cancer cells are injected in the original site of the tumour - to represent primary tumour
What are xenograft models?
tissue from one species being grafted into another species
How do you study the circulation of cancer cells in vivo?
Cancer cells can be injected into the blood vessels of mice. This is called an intravenous injection and can help in studying metastatic colonisation.
What is a subcutaneous injection?
Tumour cells originating from one area injected into the skin far away from the original site of injection
What is an intravenous injection?
Tumour cells injected into a vein and through circulation reach the organ the cancer cells were derived from, where they form a metastasis
Define Allograft
Transplant of human tumour cells in immunocompromised mice
Define orthotopic
Same primary site transplant
Define PDX
Patient derived xenograft
What are the advantages and disadvantages
Ad: Can see the route of metastasis (track with radioactive glucose GfP - faster metabolism), personalised medicine, representative of tumour in patient
Neg: Can be cellular rejected, wrong size of tumour, not necessarily predictive of therapeutics
What are the 3Rs
Replacement
Reduction
Refinement
What is shRNA
Short hairpin RNA
same as siRNA
