In vivo methods of studying cancer cell death

Question 1

studying tumor progression; mouse

Answer 1

mouse traditionally primary model
- mouse mutant strains prone to develop a particular cancer
- use of immunocompromised mouse strains to perform xenotransplants (transplanting human tumor cells)

Question 2

studying tumor progression; zebrafish

Answer 2

alternative with additional advantages
- cheaper
- allows imaging in vivo
- high throughput screen for therapeutic approaches

Question 3

zebrafish melanoma transgenic models

Answer 3

• 50% advanced melanomas harbor a mutation in the BRAF gene
• BRAF ^V600E is most common mutation
• BRAF is a kinase and the V600E mutation = formaiton of a constitutive active form
• transgenic zebrafish was generated that expresses BRAF^V600E in melanocytes
• transgenic fish = malignant melanoma

Question 4

how to visualise these cancerous cells

Answer 4

• GFP ; labels all neural crest progenitors

Question 5

what do metastatic melanoma cells present?

Answer 5

features of neural crest progenitors and re-express crestin

Question 5

using crestin to visualise tumors

Answer 5

combining the crestin marker with BRAF model = visualisation of growing tumors

Question 6

using zebrafish for drug discovery

Answer 6

the models that develop tumors in embryonic/larval stages are useful for high throughput drug discovery screens

Question 7

Tg(mitfa:V12HRas)

Answer 7

transgenic line expressing oncogenic Ras in the melanocytes from moment of specificaiton
- these fish develop too many melanocytes from embryonic stages; larvae are hyperpigmented
- as they grow, adults develop melanoma

Question 8

process of drug discovery screens on in vivo models

Answer 8

• identify cancer relevant ‘proxy’ phenotype in the embryo
• screen chemicals against phenotype in 96-well format
• SAR, chemoinformatics and oulldowns to indentify target
• preclinical evaluation in models and human clinical trials

Question 9

using neural crest formation to find treatment

Answer 9

• metastatic melanoma cells present features of neural crest progenitors
• if drugs can suppress neural crest formation are found, they may also work suppressing melanoma progresison
• combination of drugs worked preventing progression; in melanoma cell cultures, in mouse xenografts
• leflunomide has been taken into phase I/II trials