modelling cancer

in silico

sequence cancer genomes for somatic mutations
limitation - can’t detect characteristic of mutations
e.g. driverML predicts moonlighting genes or genes that are more slowly mutated

disadvantages of tissue culture

cell lines derived from patients may not be good representation of genetics
engineered cells may not be correct type

alternatives to cell culture

3D organoids - self organising 3D structures derived from SCs or tumour cells e.g. CRC organoids
3D bioprinting

identifying if a gene product is an oncogene

transgenic mice - tissue specific promoter
modelling translocation events

oncogenic transgenic mice

is ALK an oncogenic RTK
transgenic mice in different founder lines developed lymphoma but one line was slower
issue due to random integration of oncogene

modelling translocation events - MLL fusions

Cre-lox - Cre is directed to LoxP sites and excises floxed gene
study - MLL translocations in leukaemia
MLL-AF9 only caused cancer under Lmo2 promoter = causes cancer in myeloid cells
MLL-Enl only causes cancer under Lck promoter = causes cancer in T cells

modelling translocation events - EML4-ALK

CRISPR/cas9 system
gRNA guides break points of inversion
cas9 inverts

studying oncogene addiction

conditional inducible transgenic mice with oncogene
can switch on and off to see regression
combination with TSG inhibition and repletion

KRAS in lung cancer

rtTA, add dox and tTA binds operator element leading to KRAS expression
lineage specific promoter
cross with inkA/Ark null mouse = no TSG
re supply TSG and cancer still progresses = oncogene addiction

is a gene product a TSG

TSG restoration in transgeinc mice
loxP-stop-loxP-p53 X Cre-ER
add tamoxifen = stop removed = p53 expressed
tumour regresses but doesn’t disappear

questions about cancer stem cells

what are cells of origin
does cancer arise from a CSC
how do oncogenes drive cancer cells from committed progenitors
how frequent are CSCs

finding cancer originating cells

method 1 - take HSC, CLM and CMP from a mouse and fuse to OG and transfer into irradiated mouse, monitor tumour growth and phenotype compared to parent cell population
method 2 - start with developed tumour and sort cells according to ontogeny, transfer to irradiated mouse and see which cells propagate tumours, dilutions and further transplants to identify CSCs, phenotype by FACS

identity CSCs in leukaemia

Bcr-Abl can only be oncogenic in stem-like cells
MOZ-TIF2 is oncogenic in committed progenitors
identity of CSCs is oncogene dependent

how oncogenes drive cancer development from committed progenitors

MLL-AF9 GFP transgenic mice
label cells with lineage Abs and FACS sorting
found GMP cells that were transgene positive
L-GMP cells expressed self renewal program
oncogene conferred tissue specific stem cell properties in a committed progenitor

how frequent are CSCs

zebrafish expressing 3 different colours of fluoro-myc under rag2 promoter= T-ALL development
each colour marks a clone or subpopulation
transplanted cells into new ish and saw which cells could regenerate a whole tumour
many different clones could regenerate tumours
shows CSCs are frequent

leukaemia CSCs in NSG mice

SCID leukaemia initiating cells - CD34+CD38-
can differentiate
cancer initiating cell in leukaemia is a tissue specific stem cell

modelling OIS

KRAS expressing mice develop adenomas - positive for beta galactosidase staining, p16, p15 and SHEF

miRNA oncogenes

transduce myc mouse fetal liver cells with miR-17-19b and inject into irradiated myc mouse
more aggressive disease

using mouse models to test for drug efficacy

avastin VEGFRi
lead to tumour regression in nude mice
not translated well to human studies

models for cancer drug studies

GEMMs
patient xengrafts
humanised xenografts
humanised mice with human HSCs