Modelling and Observing Cancer Flashcards
(19 cards)
What are the difficulties associated with modelling neural invovlement in cancer in mice?
Mouse models (e.g. Kras/p53 mutants) show different manifestation
* No neuritis (nerve inflammation) seen - will effect immune response which is known to be important
* Epineural rather than neural association (unless cancer cells directly injected in, which is artificial)
* Little neural re-modelling seen
What are the different types of models for cancer (list a few):
Mutagen models (in vitro)
* Ames test (models of mutagenicity): grow cells on a plate missing a growth nutrient (e.g. histidine). A high number of colonies suggests a high mutation rate.
Cell models
* Mortal (Hayflick limit) or immortal (e.g. HeLa cells). These can be transfected with oncogenes
* Environmentally derived mutants (chemical, radiation or viral)
* Cell lines (with specific mutation profile)
Animal models:
* Own derived cell environmentally induced models
* Own derived transgenic cells (human gene inserted) GEMMs
* Endogenous genes in self cells which have mutated (e.g. knock-in or out)
* Transplantation models
What are Wilner’s three validating criteria?
- Face validity: are symptoms/clinical signs similar in the model and humans?
- Construct validity (underlying mechanism): are cytological/biochemical changes comparable (e.g. same gene mutations)?
- Predictive validity (treatment/outcomes): are same drugs/compounds effective in model and humans?
Why are mice good models for cancer research?
- Practically good (cheap, ethically viable,, short reproduction cycle)
- Similar genetics to humans (carry same genes as mutated in cancer)
- Allow many different forms of research – cellular, xenograft, injection of human cells
- Genetic manipulation possible (transgenic mice for selective KOs, optogenetics or chemogenetics
What are the limitations of mouse models? (4 broad reasons)
- Practically: ethically protected species
- Physiology is different from humans (e.g. neural involvement, AOM response, metastasis from lung cancer rare)
- May not respond/tolerate same drugs
- Side effects may not be easy to quantify (e.g. depression; nausea)
Given an example of the differences between an environmentally induced tumour in mouse model and naturally occuring one:
Colorectal cancer model (Li et al 2022):
* Environmentally induced: using AOM injections (alkylating DNA) to mimic natural colorectal tumour.
* AOM mutations mainly in β-catenin; natural mainly APC and K-ras (40-80%) with some β-catenin
* Metastasis from AOM treatment rare; common in humans
* Represents adenoma to adenocarcinoma sequence well but not metastasis.
Lung cancer models:
* Exposure to benzopyrenes or smoke at high dose for short time to induce lung tumours in mice.
* Mutation profile different (new major drive mutations as opposed to huge mutation population)
Detail an example of a transgenic mouse model and how it was used to investigate treatment.
Transgenic model of pancreatic cancer:
* E.g. RIP1-Tag2 model (from 1985)
* Large SV40 T antigen (Tag2) is oncogenic by binding to p53 and rb1 (causing inactivation)
* This is inserted uner control of rat insulin promotor (RIP1) meaning expression only occurs in β-cells of pancreas
* Insulinomas form due to DNA replication and extensive angiogenesis.
Provided a good model to test VEGF2 inhibitor Sunitinib
Detail how Endogenous GEMMs can be used. Give examples.
I.e. changing own mouse cells to observe cancer
Endogeneous cells
* Germ-line knock-in or out (mutations present in all cells from birth)
* Conditional activation (cre-recombinase with lox-stop-lox (conditional knock-in) or flox (conditional knock-out) - better
* Activate in specific cell types/areas using viral injection
* E.g. autochthonous lung cancer (Ad5-SPC-Cre)
Give an example where mouse models have been highly predictive for a cancer treatment success:
Targeting a tumour by inhibiting angiogenesis - Sunitinib
* Transgenic mouse model expressing SV40 large T antigen (Tag) in the rat insulin promotor (RIP) (i.e. RIP1-Tag2 model). Contains expression to β-cells in pancreas (forming insulinomas).
* Tag binds to p53 and Rb suppressors (effectively inactivating them) causing extensive DNA replication and angiogenesis
* Sunitinib is a VEGF inhibitor which was trialled and effective in this model
* Is also effective for specific tumours in humans
High predictability in this case due to the human gene being inserted (therefore similar pathways activated) and a highly specific inhibitor.
What are the different sorts of transplantation models used? What are the benefits/limitations of transplant models?
- Heterotopic (non-primary tumour site e.g. subcutaneous)
- Orthotopic (primary tumour site)
- Patient derived xenografts in humanised or immunodeficient mice to prevent infection (solid or circulating tumour cells)
Benefits:
* Time and cost-effective with easy-monitoring ability.
* May be more translatable (since mutation profile the same which is important for testing mutation targeting therapies)
Limitations
* Some tumour types are yet to be succesfully grown e.g. xenograft of prostate cancer
* Heterogeneity difficult to represent
* Micro-environment to cancer interaction may not be representative
* Immune system interaction may not be representative.
What are the benefits/limitations of humanised mice models?
Mice with human genes or transplanted human immune system cells.
Benefits:
* Reduces change of rejection in transplantation models
* May represent the immune system-cancer interaction known to be significant
* Allows testing of immune target
Limitations:
* Difficult and longer time to form
* Does not represent complete immune system
What can be included when forming a model to allow for easy observation?
- Include a luminescence gene when genetically modifying cells. E.g. luciferase reporter.
- E.g. aT1-luc2 breast cancer model (Zhang et al)
Allows for better quantification of cancer size, density and metastasis
Give an example of conflicting results coming from different models of the same cancer:
Renz et al: SNS is tumourigenic in PDAC - surgical sympathectomy or adrenergic beta blockade significantly prolonged survival.
Guillot et al: SNS is protective in PDAC by limiting number of CD163+ cells: 6-OHDA sympathectomy progresses tumour.
Potential causes of difference:
* Renz using GEMM model, Guillot using transplant model
* Difference in SNS inhibition used. 6-OHDA leads to ROS release (may lead to increased mutation and angiogenesis which progresses tumour)
What are the advantages and limitation of cell lines for cancer research (e.g. lung cancer)?
Advantages:
* Cheap
* Easy to scale
* Genetically modifiable
* Well characterised (e.g. many specific mutations)
Limitations:
* Not representative: no tumour microenvironment
* No immune system
* Little tumour heterogenity seen (known to be important)
What are the advantages and limitation of GEMMs for cancer research (e.g. lung cancer)?
Advantages:
* Can closely resemble human disease (e.g. metastatic pattern of development in neuroendocrine SCLC)
* Good for investigating the role of individual genes
* Relatively easy to GM
* Good for initial drugs testing & toxicity
Limitations:
* Preneoplastic stages do not mimic human cancers well
* Lack of long term exposure results in lower tumour burdens than seen in human cases (may reduce heterogeneity)
* Mouse physiology is not identical
What are passenger mutations? How might they be important?
Mutations which are present but not directly causing cancer progression (as opposed to ‘driver mutations’).
These are seen to an extreme degree in human cancers caused by long term exposure (e.g. melanoma; lung cancer)
Important for two main reasons:
* Add to heterogeneity which is known to contribute to tumour survival and metastasis (Cheung et al…).
* They may be currently labelled as passenger mutations but later discovered to actually play a role in tumour development (i.e. models which do not include them may be poorly representative e.g. GEMM models with few specific KOs).
What are CTX models? Where have they been useful?
Circulating tumour cell derived xenografts (CTXs)
Have been successful for the modelling of SCLC which was previously very difficult to transplant (due to difficulty in obtaining tissue).
How can invasiveness be measured and quantified?
- Time lapse microscopy
- Traction force microscopy
- Boyden chamber
How does a Boyden chamber work?
- Load cell suspension into plate well insert with semi-permeable membrane below
- Invading cells migrate and break through semi-permeable membrane
- Detatch invading cells in buffer and quantify
