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working towards world class cervical
screening systems for Irish women
John J O’Leary MD, PhD, MSc, MA, FRCPath, FFPathRCPI, FTCD
[Trinity College Dublin]
HRB and IMDA Seminar: From clinical research to medical technologies
Molecular Pathology research group [TCD]
• Member lab for HRB PhD student /scholars programme
• Member of the Biomedical Diagnostics Institute [BDI], Dublin City University
– [SFI funded programme]
• Current group size = 38
– 14 Post docs
– 21 PhD students
– 2 Technicians
– 1 Lab administrator
• KPIs
– 16 PhDs completed since 1999
– 6 MScs completed since 1999
– Publications in Nature, Nature Medicine, Nature Immunology, The Lancet,
PLoS One, Cancer Research, Molec Cancer etc.
– 3 patents filed
– Funding: EU 5th, 6th and 7th FP, Health Research Board Ireland, Irish Cancer Society,
Emer Casey Foundation, The Baggot Street Hospital Trust, The Friends of the Coombe Hospital, SFI
– 12 strategic industrial partners
– Grant income in past 4 years: 19 million euros
PREG consortium(Proteomics Research
Enhancing Gestation)
Research consortia based in our laboratories
ASCMicroPlat
Area 1: Automated cytoscreening
Area 2: Development of a virtual slide based EQA
system
Area 3: Attitudinal and psychological survey of the
Irish population in relation to attitudes to:
- cervical screening
- HPV testing
- HPV vaccination
- Psychosocial impact of HPV testing and
HPV results
Area 4: Education outreach programme
Area 5: HPV DNA and RNA testing in:
- women with BNA/CIN1 in the colposcopic
setting
- women post LLETZ treatment
- women >30 years
Area 6: HPV DNA and RNA testing in HIV disease
Area 7: Stratifying vaccine responses
Area 8: Cervical cancer biochip development
www.cerviva.ie
Virtual slide web-based EQA system
Research efforts in cervical cancer
Basic science
Industrial
collaboration
Translational
Medicine
Cervical cancer:
basic science and
translational medicine
Kelly JG, Clin Chim Acta.2010 Aug 5;411(15-16):1027-33.
Sheedy FJ,. Nature Immunol. 2010 Feb;11(2):141-7..
Baier T, Lab Chip. 2009 Dec 7;9(23):3399-405.
Martin CM, Methods Mol Biol. 2009;511:333-59.
Martin CM,. Mol Diagn Ther.2007;11(5):277-90.
Martin CM, Expert Rev Mol Diagn. 2006 Mar;6(2):217-29.
Membrane traffic protein (MF00267)
Molecular function unclassified (MF00208)
Select regulatory molecule (MF00093)
Transcription factor (MF00036)
Hydrolase (MF00141)
Extracellular matrix (MF00178)
Receptor (MF00001)
Miscellaneous function (MF00197)
Cell adhesion molecule (MF00040)
Nucleic acid binding (MF00042)
Signalling molecule (MF00016)
Gene dysregulation in cervical cancer
Upregulated genes
Geminin
mcm 2,3,4,5,7,10
p16
HOX C6
HOX C10
Topoisomerase IIa
Survivin
Prostaglandin E synthase
Serine/threonine kinase 6
Cyclins A2, B2, E1, E2, F,
E2F
cdc 6, 14, 28
hTERT
cdcA11(nuf2)
SMAD 3, 4
p16 a marker of pre-cancer and cancer
p16(INK4a)
p16(INK4a)
Mechanism of interaction between
p16 and HPV E7
Cell cycle dysregulation in
cervical cancer
Trends in Mol Biol
MCM 3
MCM5 Geminin
Selective targeting of:
a). E6/E7 HPV genes
b). E7 oncoproteins[the gene gun approach]
Downstream
Analysis
RN
A T
arg
eti
ng
Pro
tein
Targ
eti
ng
Model system
siRNA design
Evaluation of optimal reagents for transfection
Optimisation of experimental conditions
Silencing of HPV16 oncogenes
Whole genome expression profiling
Cellular Characterisation
Dose response curves -sulindac
Dose Response curves –sulindac derivatives
Evaluation of COX expression & activity
Evaluation of E6 and E7 expression
E7E6
HPV E6/E7 silencing using siRNA
RNA analysis
Concomitant silencing of HPV 16 E7 and E6
0
20
40
60
80
100
120
140
160
180
200
Untreated Mock Negative E7#1 E7#2 E7#3 E7#4 E7#5
mR
NA
(%
co
ntr
ol)
E7
E6/E6*I
E6
Protein analysis
Post knockdown
• Shift in phosphorylation state of pRb
• Accumulation of p53 and p21
E7#1
E7#2
E7#3
E7#4
Ne
gati
ve
Mo
ck
Un
tre
ate
d
GAPDH
ppRb
pRb
GAPDH
p21
E7#1
E7#2
E7#3
E7#4
Ne
gati
ve
Mo
ck
Un
tre
ate
d
GAPDH
p53
E7#1
E7#2
E7#3
E7#4
Ne
gati
ve
Mo
ck
Un
tre
ate
d
Effect of E7 siRNA
MCM2
MCM3
MCM4
MCM5
MCM6
MCM7
MCM8
MCM10
CDC6
Cdt1
MCM5
MCM3
Apoptotic profile
Cell cycle profile
0
10
20
30
40
50
60
70
80
90
100
Untreated Mock Negative E7#1 E7#2 E7#3 E7#4
Num
ber
of
cells
(%)
Live Early Apoptosis Late Apoptosis
0
10
20
30
40
50
60
70
80
90
100
Unt Mock Negative E7#1 E7#2 E7#3 E7#4
Num
ber
of
cells
(%)
G1 S G2/M
** ****
**
**
**
**
**
* *
*p<0.05, **p<0.01
Altered cell cycle not apoptotic profile
Silencing of E6/E7 induces senescence
• Cells permanently and irreversibly lose the ability to undergo cell division.
• Characteristics:
1. G1 arrest
2. Enlarged morphology
3. Flattened morphology
4. Loss of cell contact
5. Increased granularity
6. Auto-fluorescence
7. Expression of SA-β-gal
Negative E7#1
Sulindac: molecular pharmacological targeting
of HPV transforming proteins
NSAID
Non-selective inhibitor
of COX-1/2
NSAIDs have been
shown to reduce the
risk of many cancers
Sulindac induces apoptosis
in cervical cancer cells
0
10
20
30
40
50
60
70
80
90
100
Unt DMSO 57.5μM 115μM 230μM 400μM
Num
ber
of
cells
(%)
Live
Early Apotosis
Late Apoptosis
0
10
20
30
40
50
60
70
80
90
100
Unt DMSO 57.5μM 115μM 230μM 400μM
Num
ber
of
cells
(%)
Live
Early Apotosis
Late Apoptosis
*
***
***
**
**
**
**
**
**
**
**
24hrs post sulindac treatment
48hrs post sulindac treatment
*p<0.05, **p<0.01, ***p<0.001
Effect of sulindac on HPV18 E6 & E7
Unt DMSO 57.5 M 115 M 230 M 400 M
0.1
1
10E6
E7
0.5
2
Rela
tive Q
uantity
Untr
eate
d
DM
SO
57.5μM
115uM
230μM
400μM
HPV18 E7
GAPDH
Untr
eate
d
DM
SO
57.5μM
115uM
230μM
400μM
GAPDH
p53Representative
of HPV18 E6
expression
RNA expression Protein expression
Sulindac treatment results
in degradation of E7
‘The HPV-cancer stem cell paradigm
in cervical cancer’
p63 expression in cervical pre-cancer
lesions
Member of p53 family
- 6 isoforms due to 2 promoters – TA & ∆N, differential splicing = α, β, γ.
- ∆Np63α – maintains basal, progenitor cell population.
- Epithelial cells predominantly express ∆Np63α isotype.
Cervical cancer:
industrial collaborations[towards the creation of devices]
“Automated Cancer Screening Based on
Real-Time PCR”
“Platform for Advanced Single cell
manipulation and Analysis”
Figure 1| Single-cell-manipulator (SCM) system for printing single-cells. (a) SCM main component consists of (1) dispenser chip mounted to the aluminium case
that hosts the piezo-stack actuator, (2) Target for single-cell printing (e.g 96 well plate) mounted on motorized linear stage, (3) external illumination (cold lamp),
(4) Objective of CCD camera for image recognition and cell detection and (5) Reservoir. (b) Enlarged view of dispenser chip assembly (1) using transparent
PMMA for mechanical fixture (2) and fluidic connection. (c) Image from CCD camera focused on the nozzle showing HeLa cells approaching the nozzle orifice.
The red square marks the ROI or sensing region from where the motion detection algorithm works to detect single-cells. Scale bar, 100 μm. (d) Dispenser chip
fabricated from Silicon/glass. The on-demand dispensing mechanism consists of 3 principle steps: (i) the priming stage where liquid fills the fluid channel by
capillary force, (ii) the dispensing stage, which is achieved by fast displacement of the silicon membrane by the piezo-stack actuator and (iii) the liquid refill stage
during which the actuator retracts to its initial position. The driving parameters to control the droplet formations are the maximum actuator displacement (in μm)
and the actuator extension velocity (in μm/ms).
Nature Methods 2010: submitted
Biomedical Diagnostics Institute 2
Metastasis: important biological mechanisms
The Metastatic ’Highway’
Circulating tumour cells:
- differentiated tumour cells
- cancer stem cells [CSCs]
- live cells
- cells undergoing apoptosis
- cells undergoing anoikis
- senescent cells
- dead cells
- CTCs can be:
cloaked with platelets, natural opsonins [IgG] and auto-antibodies
CTC platelet ‘cloaking’
ex-vivo evidence
In Vitro Adhesion Assay -Platelet Adhesion to Ovarian Cell Lines
HIO
E-8
059
M
A27
80
A27
80ci
s
Bla
nk
Fibrinog
en
-50
0
50
100
150
******
******
*** P<0.001
Cell Line
Pla
tele
t A
dh
esio
n(N
orm
ali
se
d A
ga
ins
t A
dh
es
ion
to
Fib
rin
og
en
)
Non-shear
Shear
59M
59M
Deadly allies: the fatal interplay between platelets and
metastasizing cancer cells Luise Erpenbeck and Michael P. Schön
Blood First Edition Paper, pre-published online March 1, 2010; DOI 10.1182/blood-2009-10-247296
In-vivo evidence of the effect of
platelets on cancer cell metastatic potential
Oc-1 + PLTs CD42a + AV.006Oc-1 + PLTs CD42a + AV.006
0 200 400 600 800 1000FSC-H
0 200 400 600 800 1000FSC-H
Oc-1 + PLTs + EDT A.008Oc-1 + PLTs + EDT A.008
0 200 400 600 800 1000FSC-H
0 200 400 600 800 1000FSC-H
Oc-1 + PLTs + Reopro.009Oc-1 + PLTs + Reopro.009
0 200 400 600 800 1000FSC-H
0 200 400 600 800 1000FSC-H
Oc-1 + PLTs + RGDS.010Oc-1 + PLTs + RGDS.010
0 200 400 600 800 1000FSC-H
0 200 400 600 800 1000FSC-H
(1) Platelets + OC-1 cells (2) Platelets treated with EDTA + OC-1 cells
(3) Platelets treated with Reopro + OC-1 cells (4) Platelets treated with RGDS peptide + OC-1 cells
Data is expressed as % CD42a positive OC-1 cells following incubation with platelets (resting, EDTA, Reopro,
or RGDS peptide treated). PE fluorescence (y-axis), Forward scatter (x-axis)
37 % CD42a
positive
0.56 % CD42a
positive
0.38 % CD42a
positive
0 % CD42a
positive
Platelet Cloaked Cells
Un-Cloaked Cells
Platelet
Exposure
Platelet
Releasate
Exposure
Alteration of gene
expression
Anti-apoptotic Signalling
Induction of proteases
Alteration of gene expression
Alteration of cell cycle genes
Alteration of metabolic genes
Anti-apoptotic, anti-autophagy,
pro-angiogenic signalling
Pro-proliferative signalling
Anti-apoptotic, anti-autophagy,
pro-angiogenic signalling
Alteration of gene expression
SK-OV 3
59M
59M
A2780cis Su
rviv
al
in t
he v
ascu
latu
re a
nd
su
ccessfu
l m
eta
sta
sis
Platelet cloaking of cancer cells directly
inhibits apoptosis and autophagy
Circulation tumour cell detection chip
“OncoChip”
2nd generation sequencing
characterisation of CTCs
The BIODISCOVERY TRANSLATIONAL
approach
Thanks to the following scientists:
Orla SheilsCara MartinDermot KennyKarl EganPaul SmythDolores CahillSharon O’TooleLoretto PilkingtonBrian MacCraithLuke LeeClaus PoulsenMichael GallagherCynthia HeffronKatharine AstburyLouise KehoeCathy SpillaneJamie McInerneyBrian SheppardAmanda CotterMarek RadomskiCatriona LoganNiamh O’SullivanSimone GuentherRichard HenfreySteve PictonNiall Swann
Sue CahillKaren DenningCathy AllenJinghuan LiSinead AherneStephen FinnRichard FlavinEsther O’ReganMartina RingNoel BolgerHelen KeeganAmanda MurphyCaroline HughesAlex LaoisMark LawlerCiara BarrettEdgar MocanuAnna RadomskiSalah ElbaruniJon SherlockBill WatsonLucy NorrisAstrid PotratzChris StreckRay SamahaNoreen GleesonTom D’Arcy
Acknowledgements
Royal City of Dublin Trust
