Lentiviral shRNA screen to test the
validity of a gene signature of breast
cancer stem cells using high
throughput mammosphere assays
Jenny Chang Lab
by Bhuvanesh Dave PhD
Tumorigenic Cells: Are we missing the target?
Jones et al. JNCI 96:583, 2004
Tumorigenic Cells: Are we missing the target?
Jones et al. JNCI 96:583, 2004
Tumorigenic Cells: Are we missing the target?
Jones et al. JNCI 96:583, 2004
A Model For “Cancer Stem Cells” In
Treatment Resistance and Disease
Recurrence
Chemotherapy Increases the
Proportion of CD44+/CD24low/- Cells
and Mammosphere-Initiating Cells
A
B
CD44+/CD2430
Mammosphere Formation
Efficiency
130
n=31
120
n=31
110
25
No. of MS/10,000 cells
100
CD44+/CD24-
20
15
10
90
80
70
60
50
40
30
5
20
10
0
0
Initial
Week 3
Observed
Week 12
Predictive
Initial
Week 3
Observed
Week 12
Predictive
Li et al (2008) JNCI 100:672-9
The Proportion of CD44+/CD24- Cells
Correlates With Mammosphere Initiation
Potential
Mammospheres Are Enriched For
Mammosphere-initiating Cells Compared
To The Tumor of Origin
Gene Expression Signature of Chemoresistant
Mammosphere-initiating Cells
The “Signature” Is Preferentially
Manifest in Claudin-Low Tumors
Design of GIPZ shRNAmir Library
Choosing a Cell Line
• In order to confirm our patient derived gene
signature we looked for Claudin Low like cell
lines
• Based on Joe gray’s dataset we narrowed it
down to MDA-MB231, SUM-159PT & HS578T
• We chose SUM159PT because it possessed the
CD44/24 population, which had been shown to
have increased tumorigenic potential.
Lentiviral Screen using open bioystems library and
our gene signature
We started with 493 genes and 1120 shRNA’s for this Lentiviral screen.
Screening for CSC drug targets using shRNA for gene signature
Phase I
Library
Cherry
Picking
of clones
Purification
of DNA
Transfection
Infection
Day 1 transfect
Mammosp
heres
Day 3 collect
Day 4 collect
Visualize
and count
Phase II
Trypsinize
mammospheres
HBSS + serum
Spin and
Resuspend in
MEGM+
PKH26 dye
on Day4
Visualize
and count
SUM159PT cells-Mammospheres plated 081408 and photographed 082008
96 Well
20x
24 Well
6 Well
20x
10x
Testing Controls
• Testing of Controls to Determine the Set of
Controls to be used in the Screen
- Infectivity
- Impact on Mammosphere formation.
% MSFE
0.2
0
Experiments
• Conduct the screen using SUM159PT cells with the aforementioned
controls and genes derived from our signature
• Add some of the Hedgehog and Notch sigaling pathway regulators
to that gene list in order to test for things that have been the usual
suspects in the cancer stem field.
• We added 40 genes from the ALDH1 derived signature from Dr.
Max Wicha’s Group.
• Confirm reduction of gene expression of the control genes by real
time PCR which will be eventually followed by western blots.
Results
• We conducted this screen in 14x96 well
plates each time (8 times)
• We collected information on MS formation
information for primary and secondary MS
using Gelcount from Oxford Optronix
• We have currently analyzed the primary
MS data and we got 151 unique shRNAs
where MS number changes significantly
p=0.05
Z Score
Results of Primary MS formation screen depicted as
a Z-score of all the genes tested in the screen
Candidate Regulators p<0.05
128 shRNA’s representing 108 unique genes showed
significant alteration in the MS formation Efficiency in SUM159
Increasing MSFE
Decreasing MSFE
BT549 p<0.05
exp1
exp2
exp3
exp4
exp5
exp6
exp7
Candidate Regulators p<0.05
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BT549
Increasing MSFE
Decreasing MSFE
BT549
Future Directions
• Do real time PCR of all the targets.
• Liposomal delivery of shRNA in
cells/animals using nanoparticles
• Target two tumor lines based on five target
gene identified from the combination of
these two cell lines.