Lecture 7 Flashcards
How can cancer cells be visualised in 2D?
Cell culture assays
- Advantages
- Easy to perform
- Repeatable
- Well-defined parameters
- Disadvantages
- Monoculture
- 2D environment may not be applicable
How can you model invasion in 3D in vitro?
Matrigel invasion assay
- Cells must digest an ECM to reach and travel through a filter to reach a chemoattractant
- Cells can be stained to visualise which ones have travelled through the filter
How can you model migration in 3D in vitro?
Transwell migration assay
- As per matrigel assay but without the ECM for digestion
Which adhesion molecule has been shown to be important using matrigel invasion assays?
N-cadherin
What is a commonly used cancer cell line?
MDA-MB 231
What is a concern with using cells derived from effusions?
The cells have lost their requirement for stromal support and hence their growth in vitro is probably not a good indicator of their growth in vivo
How can you study invasion in vivo?
Intravital imaging
Cover glass placed into skin of animal
Imaging through this ‘window’ shows invasion by the tumour
Expensive and difficult to quantify
Why are blood vessles inefficient-hostile environments for cancer cells?
- Cells may lose ability to adhere (anoikis)
- Undergo MET due to lack of stromal support
- Destruction by hydrodynamic shear forces
How might circulating tumour cells (CTCs) adapt to thie hostile vessel environment?
Recruit platelets to form microthombi - ensheathing the CTCs and protecting them
How has it been shown that CTCs use microthrombi?
- P-selectin, adhesion moelcule needed for thombus formation, knockout mice cannot form microthrombi
- Nf-E2 knockout mouse, no platelets, will have fewer metastases when cancer cells injected into bloodstream
How can cancer cells be removed form circulation?
- CellSearch - targets EpCAM using an antibody
- However, not all cancer cells express EpCAM and some non-cancer cells do express it
- Microfluic devices
- Remove cells based on size - cancer cells larger than most other circulating cells
What can CTCs be used to predict?
Why is it difficult?
Survival rate : fewer CTCs = better survival rate
Hard to remove and in very small numbers
What is a new area of research for find CTCs in blood?
Liquid biopsy
Looks for other factors associated with CTCs - e.g. circulating DNA shed from cancer cells
Where else can tumours cells intravasate into?
The lymphatic system - which drains into the left subclavian vein
How can CTCs be detected in the lymphatics?
What is this commonly used for?
Immunohistochemistry (IHC)
Breast cancer prognosis
Why is lymphatic dispersion called a surogate marker?
Cannot be targeted for therapy
What was the first theory to explain the apparent preference for the site of metasis for different primary tumours?
Seed and Soil hypothesis by Stephen Paget in 1889
Tumour cells (seed) simply have an affinity for a specific tissue (soil)
What was the major issue with Paget’s hypothesis?
If tumour cells are drawn to specific tissue then a breast cancer would commonly travel to the non-malignant breast. However, these contralateral metastases are rare.
Outline the work on targeted metastases of melanoma cells
Injecting B16 melanoma cells into mice lead to tumours developing at the lungs and fragments of lung/ovarian tissue added into smooth muscle. But not at fragments of renal tissue or the injection site. Demonstrated a definite affinitiy to certain areas.
Outline the work on targeted metastases of breast cancer cells
- Breast cancer cells express CXCR4 so are more likely to arrest in tissues which express CXCL12
- Certain cancer cells with elevated IL-4, CXCR4, CTGF and osteopontin have higher bone metastatic potential
Outline the first pass organ theory
James Ewing in 1929
The cancer cells are direcetd by blood flow and hence will colonize at the nexe available organ they pass
Issue with that all cancer cells would first go through heart and lungs and therefore get stuck in thin lung capillary beds.
Issue possible overcome due to arterial-venous shunts which would allow transit to any tissue via arterial circulation
On what two parameters does ability to metastasise depend?
- Frequency that cells are trapped
- Adaptation to microenvironment
What is metastatic latency?
Cancer cells can lay dormant at secondary site for decades - possibly due to adaptation to the new site
What are micrometastases?
<2mm metastatic lesions
What are the possible fates of micrometastases?
- Death
- Quiescence
- Cannot undergo angiogenesis so is kept small
- Resume proliferation
What is the rate-limiting step of metastasis?
Colonisation
What may cause reactivation of colonized cells?
- Injury
- Immune supression
- Mutation
What three types of in vivo xenograph model?
- Subcutaneous
- Microenvironment no accuarte unless using skin cancer cells
- Orthotopic
- Injected into site of origin of tumour
- Experimental metastasis
- Inject into blood and metastasise to site
How can bioluminescent imaging be used?
Cancer cells have biolumenescence and amount of luminescence is relative to number of cells
So can model tumour growth over time
However, the mice are immune deficient so the model is not perfect
What genetic approach is now being used?
Cre expressing cells with floced genes of interest in specific tissue causes tumour to develop in that tissue
What is the pre-metastatic niche?
Signals from primary tumour ‘prepare’ distant sites to support secondary tumour formation
What is the proposed mechanism for the formation of the pre-metastatic niche?
VEGF receptor and bone marrow-derived cells colonize potential sites and
- Upregulate integrins and cytokines
- Accelerate angiogenesis
How are the bone marrow derived cells recruited to the secondary site?
Structural changes induced by LOX from primary tumour
LOX levels are higher in tumours than normal cells, due to hypoxia in tumours (further accelerated by anti-angiogenesis drugs)
Higher LOX levels in tumours associated with more metastasis
Why are secondary breast cancer tumours in the brain thought to being seen now?
Before people would die due to metastases in the bone before the brain metastases could develop, but now bone tumours are better treated.
How do breast cancer metastases in the brain compete?
Proliferative advantage by displaying GABAergic properties and catabolizing GABA to NADH as an energy source
What may be a new target for breast cancer cells?
Sodium channels - which promote migration of breast cancer cells via a neuronal signalling mechanism
e.g. epilepsy drugs
