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

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

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ResearchResearch DevelopmentDevelopment

Prioritization over projects

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

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

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• 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

“ 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

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– Current therapies are not satisfactory: poor efficacy, high relapse rate

– Practical difficulties to investigate this complex disease

– Lack of novel targets and drugs

“ 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

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What are appropriate in vitro models ?

Pathogenesis

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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

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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)

Primary cell/tissue cultures from endometrium

(from women without endometriosis)

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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)

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

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

)

NRs in stroma determine proliferative response

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(Cunha et al., 2004 Arch Histol Cytol)

Recombination and renal capsule transplantation

Chick chorioallantoic membrane model

This only works when transplanting tissue fragments

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(Nap et al., 2004)

Control E2 ICI

Short term explant cultures human endometrium are responsive to estrogen

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(Punyadeera et al., 2004 JSBMB)

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

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

Pathogenesis

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α6β1α2β1 α5β1

Integrin expression on shed menstrual endometrium

Preferably shed menstrual endometrium should be used

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(Koks et al., 2000 Mol Hun reprod)

Endometrium

Coculture menstrual endometrium and peritoneum

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(Groothuis et al., 1999; Koks et al., 2000)

Mesothelium

Coculture menstrual endometrium and ECM

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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)

Endometrium also adheres to mesothelial surface

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(Witz et al., 2002 Fertil Steril)

Coculture menstrual endometrium and mesothelial cells

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Binding is mediated by CD44 – hyaluronic acid interaction

(Dechaud et al., 2001 Fertil Steril)

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)

Chick chorioallantoic membrane model

Antegradely shed menstrual endometrium forms lesions in CAM

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(Nap et al., 2004)

Endometrium is angiogenic

Chick chorioallantois membrane model

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ENDOMETRIUM

(Maas et al., 2001; Nap et al., 2005)

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)

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

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• 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

Primary cell/tissue cultures from lesions/cysts

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Eutopic stroma

Stromal cell cultures from ovarian cysts

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Eutopic stroma

Ectopic stroma

(Noble et al., 1997 JCEM)

Bulun hypothesis

• ERβ and SF1 are upregulated in ovarian endometriosis (demethylation)

• COX-2 is induced and consequently PGE2 and aromatase production

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(Bulun, 2009, NEJM)

• Not validated by others; only shown in stromal cells from ovarian cysts

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

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(Bulun, 2009, NEJM)

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

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(Greenberg and Slayden, AJOG 2004)

Immortalised cells from lesions/cysts

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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)

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- No details on lesion type/location, no characterization of cell line

SV40 T-antigen transformed epithelial and stromal cell

lines from peritoneal endometriosis

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(Banu et al., Fertil Steril, 2008)

SV40 T-antigen transformed epithelial and stromal cell lines from peritoneal endometriosis

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(Banu et al., Fertil Steril, 2008)

COX2 overexpression in endometriosis

Neg control Ectopic

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Eutopic patient Eutopic control

Banu et al., 2008

Prostaglandin receptor overexpression in endometriosis

Ectopic

Eutopic patient

409/7/2009Banu et al., 2008

Eutopic control

Neg control

Prostaglandins regulate cell survival in endometriosis

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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

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endometriotic tissue as well as for more cell lines derived from endometriotic tissues

• Models should be validated with reference drugs

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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

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• Biological connection in vitro assays and therapeutic models • tissue context

• Predict clinical efficacy• demand for returning therapeutically relevant data

• check/search for biomarkers