CBIO 4: Tissue Invasion and Metastasis Flashcards
1
Q
What are the stages of the metastatic process?
A
- Invasion of adjacent tissue by cells from the primary tumour
- Vascularisation of the primary tumour and intravasation of cancer cells (entering the blood vessel)
- Transport of metastatic cells through the circulatory system.
- Cell arrest at a secondary site and extravasation of cancer cells (escaping the blood vessel)
- Growth of tumour in a secondary organ/site
2
Q
What does it mean clinically when a metastatic tumour is diagnosed?
A
- it indicates a terminal disease
- While our knowledge of tumour biology is increasing, and all-stage survival across all cancer types is increasing, the survival of patients with metastatic disease (stage 4) is not improving greatly.
3
Q
Read the graph below that compares the one year survival rates for breast, colon, lung and ovarian cancers in 2004 – 2007 and 2012.
Which of the following stage 4 cancer types have improved between 2004-2007 and 2012?
A
- Improved: Breast cancer
- Not changed: Lung and ovarian cancer
- According to Public Health England data, the one-year survival rate for stage 4 breast cancer has increased from around 50% in 2004-2007 to around 65% in 2012.
- However, for other cancer types the survival rate remains largely unchanged.
4
Q
What are circulating tumour cells (CTCs)?
A
- these are cancer cells that escape from primary tumours into blood or lymphatic vessels to disseminate to other organs
5
Q
Why has screening for CTCs taken so many years to be developed?
A
- Screening for CTCs is technically challenging.
- CTCs are 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).
6
Q
What is a liquid biopsy?
A
- 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.
7
Q
What other fluids apart from blood can you take a liquid biopsy from?
A
- urine
- saliva
- plerual effusions
- cerebrospinal fluid (CSF)
8
Q
What can liquid biopsy do?
A
- it is a minimally invasive method of monitoring of patients, especially when it comes to monitoring tumour evolution over time
9
Q
Why is screening a liquid biopsy challenging?
A
- ctDNA (circulating tumour DNA) is fragmented and highly under-represented compared with tumour DNA
- The small number of CTCs that can be isolated in a blood sample; approximately 1 cell per 1x109 blood cells
10
Q
In order to detect CTCs via a liquid biopsy, what needs to be done?
A
- the sample needs to be enriched
- in other words, the number of CTCs in the sample needs to be increased.
11
Q
What are the two techniques to increase CTCs?
A
- negative enrichment:
- removing other blood cells based on shape, size or other biophysical properties
- it is focused on other cells other than CTCs
- positive enrichment:
- increase CTC numbers by selecting cells expressing specific markers on cell surfaces (surface markers)
- these surface markers can distinguish epithelial cells from blood cells
- e.g. epithelial cell adhesion molecules (EpCAM)
- the limitation is these markers do not distinguish between a malignant and non-malignant epithelial cell
- so isolated CTCs have to be molecularly characterised
12
Q
What technologies are used to analyse liquid biopsies?
A
- Analysis of liquid biopsies requires highly sensitive assays, which have only recently become available.
- At this time, the only FDA-approved platform for the isolation and enumeration of CTCs in patients with metastatic breast, colorectal, or prostate cancer is the CellSearch platform.
- CellSearch uses positive enrichment based on positive expression of EpCAM and negative expression of CD45 (leukocyte-specific molecule)
13
Q
What needs to happen to cancer cells before the invasion-metastasis cascade?
What triggers this? How?
A
- cancer cells need to be disseminated
- the initial trigger for this is genomic instability
- of which chromosome instability (CIN) is present in most human cancers
- this arises as a result of errors in chromosome segregation during mitosis
14
Q
What is the fuel behind the metastatic process?
What are the after-effects of this?
A
- genomic instability is the fuel behind the metastatic process
- it can create many cell subtypes, called clones, which may have metastatic properties
15
Q
What is monoclonal and polyclonal metastasis?
A
- monoclonal metastasis:
- metastatic tumours forming from one cell
- polyclonal metastasis:
- metastatic tumours forming from multiple cells
- which form it forms from is still under debate
16
Q
Explain this diagram
A
- a cancer mass can contain two clones of cancer cells, clone A and clone B.
- From this clone A and clone B mass, a metastasis can be formed containing both clones (polyclonal), only one clone (monoclonal), or one clone which has undergone further genetic alterations (phenotypically heterogenic monoclonal)
17
Q
What two roles does angiogenesis play in tumour progression?
A
- Intravasation: Entering blood vessels
- Extravasation: Escaping blood vessels
18
Q
What are pro-angiogenic molecules?
Why are they needed?
A
- cancer cells release ‘pro-angiogenic’ molecules which promote the formation of new blood vessels
- cancer cells drive the process of angiogenesis with this
- while the primary tumour expands in size and invades surrounding tissues, it requires nutrients and oxygen, which needs to be delivered via the blood vessels
- these are formed around the tumour mass
19
Q
Give an example of a key pro-angiogenic molecule
Describe what happens to the blood vessels formed from its production
A
- vascular endothelial growth factor (VEGF)
- These new blood vessels have a “leaky” structure and permits the tumour cells to enter the blood stream, a process which you now know is called intravasation.
20
Q
What is the epithelial to mesenchymal transition (EMT)?
A
- A key event for stationary tumour cells to escape the tumour mass so they can migrate and invade
- During EMT, tumour cells gain a mesenchymal phenotype through a transdifferentiation process and gain new properties so they can invade, resist stress and disseminate
- Transdifferentiation is the conversion of one cell type into another cell type without going through a pluripotent cell state.
21
Q
Explain this diagram
A
- EMT completely alters the characteristics and behaviour of tumour cells and as a consequence, the BM is breached and the cells intravasate into blood or lymphatic vessels.
- They travel through the blood and lymphatic vessels until they reach their new destination, escape from the vessels (extravasation) and migrate into a new tissue to expand a metastatic colony there
22
Q
What do EMT-like changes do to nonepithelial tumours?
A
- Accumulating evidence suggests that EMT-like changes also lead to a gain in mesenchymal properties and promote malignancy of nonepithelial tumours such as melanoma, sarcoma and leukaemia.
23
Q
What is a pre-metastatic niche?
A
- The new tissue microenvironment after invasion provides a site that promotes colonisation of the tumour cells.
- It can be altered by bone marrow-derived cells or other circulatory factors prior to tumour cell arrival , forming a pre-metastatic niche
24
Q
Briefly recap the basal lamina
A
- The BM is composed of epithelial, endothelial, and stromal cells to separate the epithelium or endothelium from the stroma and interstitial matrix.
- The 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)
- In contrast, the interstitial matrix contains fibrillar collagens, proteoglycans, and various glycoproteins, as you can see in the figure below
25
Q
How do tumour mass and metastatic cancer cells alter the composition of the ECM?
A
- the alterations arise from enzymes called proteases
- they degrade proteins of the ECM by hydrolysis of peptide bonds.
- key proteases:
- aspartic proteases
- cysteine proteases
- serine proteases
- matrix metalloproteinases (MMPs)
26
Q
Describe cathepsins proteases
Types
Roles
A
- Cathepsin D (an aspartic protease)
- cathepsins B, L and H (cysteine proteases)
- cathepsin A (a serine protease).
Role:
- Turnover and degradation of the ECM
- Activation, processing or degradation of various growth factors, cytokines and chemokines
- Influence cell-cell adhesion molecules
27
Q
Describe Urokinase proteases
A
- a serine proteases
- Key player: urokinase receptor, urokinase-type plasminogen activator receptor (uPAR)
Role:
- 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 (a component of provisional ECM), and through this direct interaction, triggers changes in cell morphology, migration and signalling.
- One consequence is the induction of EMT (more on this later).
28
Q
Describe MMP proteases
A
- MMPs can be divided in six groups:
- collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs.
Role:
- Degradation of collagen and other proteins in the ECM;
- Regulation of cell behaviour
29
Q
Label the diagram
A
30
Q
What is the master cell-cell junction?
How does this relate to metastasis?
A
- the master cell-cell junction is the adherens junction (cadherins), that regulates the function of other cell-cell junctions
- In order to break away from the tumour, cells must overcome the mechanisms that cause them to adhere to their neighbours – such as cadherins
31
Q
How do integrins play a role in cancer metastasis?
A
- 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.
- On the other side of the cell membrane, inside the cell, integrins interact with intracellular ligands and recruit focal adhesion kinase (FAK), which undergoes autophosphorylation, formation of complex with Src and activation of both kinases.
- The FAK/Src complex can now activate a plethora of signalling molecules.
32
Q
What phenotypic changes are observed during the epithelial to mesenchymal transition (EMT)?
What are the key players of this?
A
- tumour cells undergoing EMT gain an invasive phenotype by losing their epithelial characteristics and acquiring mesenchymal (stromal) characteristics (see diagram)
- key players are cadherins and integrins
33
Q
Explain in detail how epithelial cells transition to mesenchymal phenotype via EMT
A
- Epithelial cells express high levels of epithelial markers (e.g. E-cadherin, EpCAM and α6β4 integrins).
- However, as cells undergo EMT, the progressive loss of epithelial markers result in the loss of cell-cell adhesion complexes, remodelling of the cell-ECM interactions and production of enzymes to break down the BM and facilitate invasion.
- Increased expression of mesenchymal markers such as N-cadherin.
- This results in the “cadherin switch” which also alters cell adhesion.
- These cells lose their association with epithelial cells and gain enhanced affinity for mesenchymal cells through N-cadherin interactions.
- Changes in the cytoskeleton such as intermediate filament composition results in increased Vimentin expression enabling cancer cells to become motile.
38
Q
What is epithelial migration?
A
- Epithelial migration is 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.
The different modes of cell migration are illustrated in the diagram
44
Q
What is transcoelomic spread?
A
- In transcoelomic metastasis, cancer cells 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.
46
Q
What is leptomeningeal spread?
A
- In leptomeningeal spread, cancer cells travel by the cerebral spinal fluid
- This is characteristic for cancers occurring in the brain, including non-Hodgkin’s lymphoma.
- Cancer cells can travel by the cerebral spinal fluid (CSF).
- They invade the membrane covering the brain (meninges) to form leptomeningeal metastasis within it, or the spinal cord to invade the dura mater (a thick membrane that surrounds the brain and spinal cord) and the epidural space (space between the two layers of the dura mater).
47
Q
Do you know why lymphatic spread leads to haematogenous spread?
In which part of our body is the connection of the lymph to the blood?
A
- Lymphatics drain into blood through the thoracic duct (left lymphatic duct) or the right lymphatic duct
49
Q
Describe the route of prostate cancer spread
A
- The figure illustrates the spread of prostate cancer cells via venous blood vessels (blue), lymph vessels (green), or nerves (yellow).
- Once in the venous blood, CTCs are carried throughout the body via the blood supply, through arterial systems.
- Once they have reached a suitable secondary site, like bone, cells extravasate from the blood and colonise the metastatic site.
53
Q
What are the four key stages that allow metastatic colonisation to occur?
A
- Priming
- Licensing
- Initiation
- Progression
54
Q
Describe priming
A
- 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).
- Exosomes are small, cell-derived vesicles that promote cell-cell communication.
- They carry cargoes that can either accelerate of suppress metastasis.
- These factors reach the pre-metastatic niche via the circulation and are believed to prepare it for subsequent invasion and thus prepare the formation of an immature pre-metastatic niche.
66
Q
How can in vitro approaches also study the interactions between cancer cells and other cell types?
A
- using co-culture studies
67
Q
Describe microfluidic devices
A
- Microfluidic devices can be also used to study cancer cell interactions with other cells.
- As illustrated below, a microfluidic device contains a set of micro-channels inside a mould (made of various polymers).
- 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.
68
Q
How do you recreate in vivo microenvironment in the microfluidic device?
A
72
Q
What was Caenorhabditis elegans used to study?
Describe the study
A
- to recreate the process of a cell crossing the basement membrane.
- During the developmental stage of C.elegans, a single cell has to breach two underlying basement membranes as part of normal morphogenesis.
- This simple model allowed scientists to recognise the first steps in activating the cell invasion programme.
- Cell cycle is arrested in G1, chromatin is modified, and a set of transcription factors is upregulated.
- In C.elegans one of the transcription factors was found to be FOS-1A, which is the orthologue of proto-oncogene FOS, a protein strongly associated with cancer cell invasion.
