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15 Novembre 2019 Dormoy Valérian, PhD, HDR MCU, chaire Inserm Modèles in vivo Intraductal xenograft models in breast cancer research Centre Paul Strauss, Strasbourg Institut National de la Santé et de la Recherche Médicale Pathologies Pulmonaires et Plasticité Cellulaire Unité 1250 WORKSHOP Cancéropôle Est Systèmes modèles précliniques en cancérologie

WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

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Page 1: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

15 Novembre 2019

Dormoy Valérian, PhD, HDR

MCU, chaire Inserm

Modèles in vivo

Intraductal xenograft

models in breast cancer

research

Centre Paul

Strauss,

Strasbourg

Institut National de la Santé

et de la Recherche Médicale

Pathologies Pulmonaires

et Plasticité Cellulaire

Unité 1250

WORKSHOP Cancéropôle Est

Systèmes modèles précliniques en

cancérologie

Page 2: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Introduction Methods Validation Applications Conclusions

Environmental context

Role of Epithelial-Mesenchymal Activating Transcription Factors in invasion and

metastasis of ER+ Breast Cancer

Role of ER/PR Receptor and Androgen Receptor in the Human Breast

Epithelium in vivo

The role of the hormone receptors/androgen receptor in breast cancer

Page 3: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

From basal state to cancer

Many therapeutic

options but

invasion remains

the principal

challenge

Introduction Methods Validation Applications Conclusions

Scientific background

Page 4: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

“mo

use

tum

or”

“tu

mo

r

en

viro

nm

en

t”

Introduction Methods Validation Applications Conclusions

Experimental models

Page 5: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Orthotopic

(Intraductal,ID) injection

Subcutaneous

injection

Orthotopic

(fat pad,FP) injection

● Only one type of BC: DCIS recapitulate

pathologic histology (ER/PR/HER2/Ki67)

● Only H&E

● No follow-up of tumor cells establishment and

growth

● Not enough material to initiate pre-clinical

studies

● Hormones supplementation :

+ estradiol / + progesterone

Improvement of the xenograft

Introduction Methods Validation Applications Conclusions

Towards novel animal models in BC?

Page 6: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Krause et al., JOVE, 2013

Introduction Methods Validation Applications Conclusions

Mouse intraductal model (MIND)

Page 7: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Our aim: characterization and validation of the establishment of MIND to study

human breast cancers

Overview:

Introduction Methods Validation Applications Conclusions

Experimental approach

Page 8: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Introduction Methods Validation Applications Conclusions

Cancer cell lines & microenvironment + validation of

MIND-PDXs

Page 9: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Introduction Methods Validation Applications Conclusions

Cancer cell lines & microenvironment

All cell lines grow intraductally

(except MDAMB231)

PCA and PAM50 to classify 48 breast

cancer cell lines: MCF-MIND clustered

with luminal

MIND

recapitulate

tumor

progression

from DCIS to

invasion

Page 10: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

1st Gen MIND

Tumor dissociation

+ single cells

Infection

GFP-Luc

ID injection

2nd Gen

ANALYSIS

WM

H&E

Alu

IHC markers

X Gen

PRE-CLINICAL

STUDIES

IVIS

HR+

Her2+

TNBC

Our aim: characterize and validate the establishment of Mice IntraDuctally

(MIND) injected to study human breast cancers

Introduction Methods Validation Applications Conclusions

PDXs-MIND

Page 11: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

● Case n° T4, M#3/4L

Radiance <1E+05 2.7E+05 8.6E+05 4.8E+06 1E+07 1.7E+07 2E+07

Weeks after injection 1-3 3 6 9 12 15 18

In vivo Imaging System (IVIS) measurements

Radiance <1E+05 3,90E+05 8,10E+06 2,60E+07 7,70E+07 9,00E+07 1,50E+09

Weeks after injection 1 2 3 4 5 6 12

● Case n° T10, M#5/3R

No visible/palpable tumors

Introduction Methods Validation Applications Conclusions

In vivo monitoring of cancer cells

Page 12: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Distribution of the human cells in the mammary glands

1. Fluorescence of GFP-positive cells

Case n°M

2

Case n°T

10

Case n°T

5

2mm

Case n°T

9

Human primary cells are present

in the mammary ducts and

expend over time

Introduction Methods Validation Applications Conclusions

What are we monitoring in vivo?

Page 13: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Case n°T4 Case n°T5 Case n°T7 Case n°M1

2. Wholemount Carmine

2mm

The pattern of localization and the expansion of the human primary cells vary

from one PDX to another

Introduction Methods Validation Applications Conclusions

What are we monitoring in vivo?

Distribution of the human cells in the mammary glands

Page 14: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

3. Alu-ISH

Case n°T

2

H&E MID Alu-ISH MID

Case n°T

1C

ase n°T

10

50µm2mm

Introduction Methods Validation Applications Conclusions

What are we monitoring in vivo?

Distribution of the human cells in the mammary glands

Page 15: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

50µm

Recapitulation of the histopathological features

● 3 types:

(1) morphology

(size, mitosis…)

(2) type (ID,

IDC…)

(3) histological

markers (ER, PR,

Ki67, Her2…)

Case n°T2

Case n°T10

H&E patient H&E MID

H&E patient H&E MID

Morphology and types

are recapitulated by the

MIND

Introduction Methods Validation Applications Conclusions

What are we monitoring in vivo?

Page 16: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Ca

se

T6

9C

ase

T7

3C

ase

T7

6C

ase

T7

8

Alu-ISH ER PR Ki67

Introduction Methods Validation Applications Conclusions

MIND conserved HR/proliferative signature

Recapitulation of the histopathological features

ER/PR status are conserved & proliferative status is similar

Page 17: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Growth curves for each patients: tumors

Luminal B Luminal A TNBC

All human primary tumor specimen established in MIND PDXs

Introduction Methods Validation Applications Conclusions

Input acquired with in vivo follow-up

Page 18: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Growth curves for each patients: mammoplasty

Human primary cells established but do NOT form tumors

Introduction Methods Validation Applications Conclusions

Input acquired with in vivo follow-up

Page 19: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Growth curves for each patients: BRCA mutants

Possible investigation of the roles of mutations in the MIND PDXs

Introduction Methods Validation Applications Conclusions

Input acquired with in vivo follow-up

Page 20: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

1st GenTumor dissociation

+ single cellsInfection

GFP-Luc ID injection

FP

ID

ID+

2nd Gen

Characterization

& Treatments

Introduction Methods Validation Applications Conclusions

2nd generations for pre-clinical studies

Page 21: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Treatment of MIND ER-/Her2-

Doxorubicine 3mg/kg/day i.Tu (50µg/mouse/week i.p.)

Cyclophosphamide 10mg/kg in drinking water

Time after ID injection (in weeks)

1 2 3 4 5 6 7 8 9 10Fo

ld i

ncre

ase i

n r

ad

ian

ce (

fro

m w

1)

1

10

100

1000

10000

individual CTL mean CTL individual TMTmeanTMT

Introduction Methods Validation Applications Conclusions

MIND-therapies to test patient’s response?

Page 22: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Time after ID injection (in days)

0 20 40 60 80

Fo

ld i

ncre

ase i

n r

ad

ian

ce (

fro

m d

ay0)

0,001

0,01

0,1

1

10

100

Treatment of MIND ER+

Faslodex (Fulvestrant)

25mg/mouse/week s.c.

individual CTL mean CTL individual TMTmeanTMT

Introduction Methods Validation Applications Conclusions

MIND-therapies to test patient’s response?

Page 23: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Modelisation of cancer cell establishment: insertion? growth? differentiation?

Factors necessary/sufficient for cancer cell establishment: adhesion proteins?

hormone signaling?

Factors necessary/sufficient for cancer cell invasion/migration

Mammary duct

Cancer cell

establishment

& proliferation

Hyperplasia &

invasion

Cancer cell

injection &

adhesion

> Understand & characterize MIND model

> Identify new targets/markers for pre-clinical & clinical studies

Introduction Methods Validation Applications Conclusions

Understanding cancer cells establishment, growth

and invasion

Page 24: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

3 approaches:

1. RNAseq analysis:

MCF7 at the time of injection vs ID for

1/2/3/5 days

>identification of targets to prevent

establishment of the cancer cells

3. 3D culture of mammary epithelial cells and microinjection of cancer cells to

follow in vivo establishment

>assay for drug testing & identify molecular actors in cancer cell

establishment, growth and invasion

Introduction Methods Validation Applications Conclusions

Understanding cancer cells establishment, growth

and invasion

2. In vivo follow-up of cancer cell establishment

ID injections of MCF7 cells in NSG-GFP mice and 2-photons live imaging

Page 25: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

4L 5L1mm

5L4R

Day 14

Day 1

ID injectionIVIS

MCF7-GFP-Luc

Monitoring Sacrifice Analysis

> Insertion of the cancer cells and proliferation

Introduction Methods Validation Applications Conclusions

Characterizing tumor patterns in MIND

Page 26: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

In less than 3 days, human cancer cells replace endogenous epithelium

Introduction Methods Validation Applications Conclusions

Characterizing tumor patterns in MIND

Page 27: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Montage on Z-Project

●DAY 5

Z-project MAX intensity Movie z-stack (2fps)

●Vibratome

sections: 250 µm

● LSM700

● Z-Stack 50µm

● 40X Oil

GFP

DNA

K8

Ki67

Introduction Methods Validation Applications Conclusions

Characterizing tumor patterns in MIND

Page 28: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Characterization of 4 different patterns in pairs primary tumor/PDX: flat (T),

lobular (LOB), in situ (IS), invasive (INV)

ER PgRHEHEHE

HE ER

ERKi67HE

ERHE HE

FLAT

LOBULAR

IN SITU

INVASIVE

Introduction Methods Validation Applications Conclusions

Characterizing tumor patterns of HR+ tumors in MIND

F F FF

F

F

FF

F

Page 29: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

ID injection

MCF7-GFP-Luc

Ex vivo culture

Leica SP5 (2-photons)

Collection of MG

imaging

Live imaging of ID injected cells

> Characterize ID models

> System to analyze cancer cell

establishment/growth/migration

Introduction Methods Validation Applications Conclusions

Identifying cellular events during the first steps of

cancerogenesis

Page 30: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Introduction Methods Validation Applications Conclusions

Identifying cellular events during the first steps of

cancerogenesis

Page 31: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

1. FACSorting and cell culture

2. Microinjection 3. Live imaging

MG

collection

MG

dissociation

FACS

3D cell culture

Injection of

1/2/4/X cells

Collection of

I-spheroides

and LSM

3D culture of mammary epithelial cells and microinjection of cancer cells to follow

in vivo establishment

Collection of spheres

Introduction Methods Validation Applications Conclusions

Identifying cellular events during the first steps of

cancerogenesis

T0-72h

Page 32: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

> Microinjection of mammary epithelial

spheroids with breast cancer cells

Introduction Methods Validation Applications Conclusions

Identifying cellular events during the first steps of

cancerogenesis

Page 33: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

MIND-PDXs for drug testing?

MIND-PDXs to understand cellular and molecular events of

cancerogenesis?

MIND-PDXs recapitulate facefully histopathological features

MIND established with cell lines to manipulate environment

& experimental conditions

MIND-PDX in breast cancer research

Introduction Methods Validation Applications Conclusions

Page 34: WORKSHOP Cancéropôle Est · 1/2/4/X cells Collection of I-spheroides and LSM 3D culture of mammary epithelial cells and microinjection of cancer cells to follow in vivo establishment

Thank you

for your

attention

Institut National de la Santé

et de la Recherche Médicale

Pathologies Pulmonaires

et Plasticité Cellulaire

Unité 1250