In Vitro Models in Endometriosis Research
Transcript of In Vitro Models in Endometriosis Research
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In Vitro Models in Endometriosis Research
ESHRE Campus Course, Leuven 2009
In Vitro Models in Endometriosis Research
Patrick Groothuis
Senior Research Scientist
Women’s Health Department
Section Endometriosis and Hormone Dependent Disorders
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General R&D process
Lead FindingLead Finding Lead OptimizationLead Optimization
Discovery Full Developmentand Launch
Target LeadLeadOptimization
Selection phase Phase ISafety/tolerability
Registration
MarketingPhase II
Proof-of-concept1 2 3 4
Early clinicaldevelopment
Phase III
Efficacy
29/7/2009
ResearchResearch DevelopmentDevelopment
Prioritization over projects
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Target discovery
Target identification• Internal Research
• Literature Research - Collaborations• Bioinformatics (external databases)
• Research on physiology / disease (models)
In vivo validation• Tissue distribution
Functional interference• siRNA
Overexpression• Transfection
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• Tissue distribution • Transgenic animals• Knockout animals• Human genetics
• siRNA• Blocking antibodies
• Antisense• Induce mutations
• Transfection• Transduction
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Flow chart LO
• Phase 1a In vitro efficacy in screening assay
• Phase 1b In vitro efficacy in biologically relevant
assay
• Phase 2a PK, In vivo efficacy
49/7/2009
• Phase 2a PK, In vivo efficacy
• Phase 2b In vivo efficacy in therapeutic/predictive
model, PK/PD
• Selection phase PK dog/monkey, bioavailability,
safety, tolerability
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“ Priorities for endometriosis research: recommendations from an international consensus workshop “
• Xth World Congress on Endometriosis, Melbourne, Australia
• Moderator Peter Rogers, Monash University
• Problem:
– Disease with high social and healthcare impact
– No non-invasive diagnostic tools available
– Research is under-funded
– Current therapies are not satisfactory: poor efficacy, high relapse rate
59/7/2009
– Current therapies are not satisfactory: poor efficacy, high relapse rate
– Practical difficulties to investigate this complex disease
– Lack of novel targets and drugs
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“ Priorities for endometriosis research: recommendations from an international consensus workshop “
• Recommendations:
– A better understanding of the role of the eutopic endometrium in the
establishment and continuation of endometriosis is required
– Develop non-invasive diagnostic tools
– Biomarkers are required
– Appropriate in vitro and therapeutic models are needed for studying
different aspects of endometriosis pathophysiology
69/7/2009
What are appropriate in vitro models ?
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Pathogenesis
79/7/2009
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In vitro models to study endometriosis
– Endometrial cells/tissue from women without
endometriosis
– Endometrial cells/tissue from lesions/cysts
– Immortalized cells from lesions/cysts
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How should we employ them ?
Focus on particular biological processes that contribute to the total complexity of the disease, i.e.:
• adhesion
• angiogenesis
• proliferation/apoptosis
• progesterone resistance
• estrogen metabolism
99/7/2009
Create conditions mimicking in vivo situation by combining primary cells/tissues with other cellular/tissue components, i.e.:
• menstrual endometrium
• (Activated) macrophages, lymphocytes
• ECM components
• peritoneum/mesothelium
• chick chorioallantois membrane model (model for peritoneum)
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Primary cell/tissue cultures from endometrium
(from women without endometriosis)
109/7/2009
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Endometrium function in part
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Endometrium function in part
dictated by paracrine, Juxtacrine and matricrine
interactions
(E. Marbaix; Punyadeera et al., 2004 JSBMB)
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100.00
150.00
Pro
lacti
n m
U/L
Primary cultures of endometrial stromal cells are steroid-
hormone responsive
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Regulation of prolactin production by SPRMS in primary endometrial stromal cells
0.00
50.00
0 10-10 10-9 10-8 10-7 10-6
Conc Org 30506 (M)
Pro
lacti
n m
U/L
0
+0.5 nM Org2058
Conc SPRM 1
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Responsiveness of primary stromal cells to steroid
hormones reduces in time
Prolactin production in stromal culture in different passages (n=2)
treated w ith Org 2058 and DHT
3000
4000
5000
pro
lac
tin
(m
U/L
)
P0
P1
P3
P6
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0
1000
2000
control P
pro
lac
tin
(m
U/L
)
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NRs in stroma determine proliferative response
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(Cunha et al., 2004 Arch Histol Cytol)
Recombination and renal capsule transplantation
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Chick chorioallantoic membrane model
This only works when transplanting tissue fragments
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(Nap et al., 2004)
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Control E2 ICI
Short term explant cultures human endometrium are responsive to estrogen
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(Punyadeera et al., 2004 JSBMB)
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Effect of progestins/SPRMs on MMP activation in cultured
explants of pre-menopausal endometrium
3 day incubation
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P+E P+E+APNo hormone
Silver-stained reticulin (collagen) fibers
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Effect of progestins/SPRMs on MMP activation in cultured
explants of post-menopausal endometrium
proMMP9
MMP9
no H P E+P AP P+AP E P E C E+P P E E+P
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Gelatin zymography 8%, day 3
no H P E+P AP P+AP E P E C E+P P E E+P
SPRM 100nM SPRM 1000 nM
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Pathogenesis
199/7/2009
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α6β1α2β1 α5β1
Integrin expression on shed menstrual endometrium
Preferably shed menstrual endometrium should be used
209/7/2009
(Koks et al., 2000 Mol Hun reprod)
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Endometrium
Coculture menstrual endometrium and peritoneum
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(Groothuis et al., 1999; Koks et al., 2000)
Mesothelium
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Coculture menstrual endometrium and ECM
229/7/2009
Adhesion of menstrual endometrium to extracellular matrix is largely mediated by
integrin alpha(6)beta(1) and laminin interaction.
(Koks et al., 200 Mol Hum Reprod)
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Endometrium also adheres to mesothelial surface
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(Witz et al., 2002 Fertil Steril)
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Coculture menstrual endometrium and mesothelial cells
249/7/2009
Binding is mediated by CD44 – hyaluronic acid interaction
(Dechaud et al., 2001 Fertil Steril)
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Neg MMP1 MMP2
MMP3 MMP7 MMP9-2
Shed endometrium contains viable cells that express MMPs and TIMPs
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MMP9-1 TIMP-2 TIMP-1
(Koks et al., 2000 Fertil Steril)
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Chick chorioallantoic membrane model
Antegradely shed menstrual endometrium forms lesions in CAM
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(Nap et al., 2004)
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Endometrium is angiogenic
Chick chorioallantois membrane model
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ENDOMETRIUM
(Maas et al., 2001; Nap et al., 2005)
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Angiostatic therapy inhibits lesion formation in the CAM
Chick chorioallantois membrane model
Intact
BACAM control
CAM + endo
Intact
BACAM control
CAM + endo
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Necrotic
CAM + endo + inh
(Nap et al., 2005)
Necrotic
CAM + endo + inh
(Nap et al., 2005)
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Summary
• Endometrium tissue is suitable to study basic processes involved in the early pathogenesis of endometriosis
• The tissue context is critical, particularly with regard to steroid hormone responsiveness and implantation
299/7/2009
• Endometrium tissue is not likely suitable for the generation of therapeutic models, many endometriosis-related characteristics are lacking
– i.e. changes in cell endometrial physiology, altered immune system
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Primary cell/tissue cultures from lesions/cysts
309/7/2009
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Eutopic stroma
Stromal cell cultures from ovarian cysts
319/7/2009
Eutopic stroma
Ectopic stroma
(Noble et al., 1997 JCEM)
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Bulun hypothesis
• ERβ and SF1 are upregulated in ovarian endometriosis (demethylation)
• COX-2 is induced and consequently PGE2 and aromatase production
329/7/2009
(Bulun, 2009, NEJM)
• Not validated by others; only shown in stromal cells from ovarian cysts
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Explant cultures endometriosis tissue
• One publication: Sharpe-K et al., Fertil Steril 1993
• Proteomics study which led to the discovery of the endometriosis-specific haptoglobin isoform
339/7/2009
(Bulun, 2009, NEJM)
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Human primary xenografts:• Tissue from ovarian cysts and peritoneal endometriosis is transplanted in pockets
made in abdominal wall
• OVX RAG-2γ(c) Knockout mice, E2 and P4 pellets, to support four menstrual cycles
Xenografts with explants of endometriotic tissue
349/7/2009
(Greenberg and Slayden, AJOG 2004)
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Immortalised cells from lesions/cysts
359/7/2009
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Examples
SV40 T-antigen transformed epithelial cells from peritoneal
endometriosis (Akoum et al., 1999 Am J Pathol)
- Nuclear receptor status unknown, many chromosomal aberrations
hTERT-immortalized endometriotic stromal cells (Annunziata et al., 2009 Fertil Steril)
369/7/2009
- No details on lesion type/location, no characterization of cell line
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SV40 T-antigen transformed epithelial and stromal cell
lines from peritoneal endometriosis
379/7/2009
(Banu et al., Fertil Steril, 2008)
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SV40 T-antigen transformed epithelial and stromal cell lines from peritoneal endometriosis
389/7/2009
(Banu et al., Fertil Steril, 2008)
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COX2 overexpression in endometriosis
Neg control Ectopic
399/7/2009
Eutopic patient Eutopic control
Banu et al., 2008
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Prostaglandin receptor overexpression in endometriosis
Ectopic
Eutopic patient
409/7/2009Banu et al., 2008
Eutopic control
Neg control
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Prostaglandins regulate cell survival in endometriosis
419/7/2009
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Conclusions
• The choice of in vitro model for target validation and to demonstrate mechanism of action, depends on the target and the biological mechanism of interest
• There is a need for more studies using explant cultures of endometriotic tissue as well as for more cell lines derived from
429/7/2009
endometriotic tissue as well as for more cell lines derived from endometriotic tissues
• Models should be validated with reference drugs
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439/7/2009
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In vitro assays in LO
• Factors dictating selection assays HO and LO are different
• Reproducibility may be compromised• biological variation
• variation expression target
• Biological relevance
• Biological connection in vitro assays and therapeutic models
449/7/2009
• Biological connection in vitro assays and therapeutic models • tissue context
• Predict clinical efficacy• demand for returning therapeutically relevant data
• check/search for biomarkers