B REAST CANCER CELL LINES GROWN IN A THREE - DIMENSIONAL CULTURE MODEL :

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Thèse présentée en vue de l’obtention du grade de Docteur en Sciences Agronomiques et Ingénierie Biologique

B REAST CANCER CELL LINES GROWN IN A THREE - DIMENSIONAL CULTURE MODEL :

A STEP TOWARDS TISSUE - LIKE PHENOTYPES AS ASSESSED BY FTIR IMAGING

Margarita S MOLINA

École Interfacultaire de Bioingénieurs - Université Libre de Bruxelles Centre de Biologie Structurale et de Bioinformatique

Service de Structure et Fonction des Membranes Biologiques

JURY

Promoteur:

Prof. Erik G OORMAGHTIGH (SFMB, ULB, Belgique)

Président:

Prof. Vincent R AUSSENS (SFMB, ULB, Belgique)

Secrétaire:

Prof. Fabrice H OMBLÉ (SFMB, ULB, Belgique)

Prof. Ganesh S OCKALINGUM (URCA, Reims, France)

Février 2018

Dr. Nathalie P ICOLLET -D' HAHAN (CEA, Grenoble, France)

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A ^BSTRACT

Despite the possible common histopathological features at diagnosis, cancer cells present within breast carcinomas are highly heterogeneous in their molecular signatures. This heterogeneity is responsible for disparate clinical behaviors, treatment responses and long- term outcomes in breast cancer patients. Although the few histopathological markers can partially describe the diversity of cells found in tumor tissue sections, the full molecular characterization of individual cancer cells is currently impossible in routine clinical practice. In this respect, Fourier transform infrared (FTIR) microspectroscopic imaging of histological sections allows obtaining, for each pixel of tissue images, hundreds of independent potential markers, which makes this technique a particularly powerful tool to distinguish cell types and subtypes. As a complement to the conventional clinicopathological evaluation, this spectroscopic approach has the potential to directly reveal molecular descriptors that should allow identifying different clonal lineages found within a single tumor and therefore provide knowledge relevant to diagnosis, prognosis and treatment personalization. Yet, interpretation of infrared (IR) spectra acquired on tissue sections requires a well-established calibration, which is currently missing.

Conventionally, mammary epithelial cells are studied in vitro as adherent two-dimensional

(2D) monolayers, which lead to the alteration of cell-microenvironmental interplay and

consequently to the loss of tissue structure and function. A number of key in vivo-like

interactions may be re-established with the use of three-dimensional (3D) laminin-rich

extracellular matrix (lrECM)-based culture systems. The aim of this thesis is to investigate

by FTIR imaging the influence of the in vitro growth environment (2D culture versus 3D

lrECM culture and 3D monoculture versus 3D co-culture with fibroblasts) on a series of

thirteen well-characterized human breast cancer cell lines and to determine culture

conditions generating spectral phenotypes that are closer to the ones observed in malignant

breast tissues. The reference cell lines cultured in a physiologically relevant basement

membrane model and having undergone formalin fixation, paraffin embedding (FFPE), a

routine treatment used to preserve clinical tissue specimens, could contribute to the

construction of a spectral database. The latter could be ultimately employed as a valuable

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tool to interpret IR spectra of cells present in tumor tissue sections, particularly through the recognition of unique spectral markers.

To achieve the goal, we developed and optimized, in a first step, the preparation of samples

derived from traditional 2D and 3D lrECM cell cultures in order to preserve their

morphological and molecular relevance for FTIR microspectroscopic analysis. We then

highlighted the importance of the influence of the growth environment on the cellular

phenotype by comparing spectra of 2D- and 3D-cultured breast cancer cell lines between

them. A particular focus was placed to establish a correlation between FTIR spectral data

and publicly available microarray-based gene expression patterns of the whole series of

breast cancer cell lines grown in 2D and 3D lrECM cultures. Our results revealed that,

although based on completely different principles, gene expression profiling and FTIR

spectroscopy are similarly sensitive to both the cell line identity and the phenotypes

induced by cell culture conditions. We also identified by FTIR imaging changes in the

chemical content occurring in the microenvironment surrounding cell spheroids grown in

3D lrECM culture model. Finally, we illustrated the impact of the in vivo-like

microenvironment on the IR spectra of breast cancer cell lines grown in 3D lrECM co-

culture with fibroblasts and compared them with spectra of cell lines grown in 3D lrECM

monoculture. Unsupervised statistical data analyses reported that cells grown in 3D co-

cultures produce spectral phenotypes similar to the ones observed in FFPE tumor tissue

sections from breast carcinoma patients. Altogether, our results suggest that FFPE samples

prepared from 3D lrECM cultures of breast cancer cell lines and studied by FTIR

microspectroscopic imaging provide reliable information that could be integrated in the

setting up of a recognition model aiming to identify and interpret specific spectral

signatures of cells present in breast tumor tissue sections.

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T ABLE OF CONTENTS

Abstract ... i

Abbreviations and symbols ... 1

Chapter I. General introduction ... 3

1. B REAST CANCER ... 3

2. M ODELING BREAST CANCER IN A THREE - DIMENSIONAL IN VITRO CULTURE ... 25

3. I NFRARED SPECTROSCOPY ... 33

R EFERENCES ... 41

Chapter II. Aim of the thesis ... 53

Chapter III. Preparing cell culture samples for FTIR microspectroscopic analysis: description of methodology ... 55

1. C ELL CULTURE ... 58

2. S AMPLE PREPARATION FOR FTIR MICROSPECTROSCOPIC ANALYSIS ... 61

3. C ONSTRUCTION OF A SPECTRAL DATABASE ... 66

R EFERENCES ... 69

Chapter IV. Infrared imaging of MDA-MB-231 breast cancer cell line phenotypes in 2D and 3D cultures. ... 71

A BSTRACT ... 72

1. I NTRODUCTION ... 73

2. M ATERIALS AND METHODS ... 76

3. R ESULTS ... 79

4. C ONCLUSION ... 86

R EFERENCES ... 88

Chapter V. Comparison of 2D and 3D cultures of human breast cancer cell lines by FTIR imaging ... 93

A BSTRACT ... 94

1. I NTRODUCTION ... 95

2. M ATERIALS AND METHODS ... 97

3. R ESULTS ... 101

4. D ISCUSSION ... 114

R EFERENCES ... 116

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Chapter VI. Gene expression data and FTIR spectra provide a similar phenotypic

description of breast cancer cell lines in 2D and 3D cultures ... 119

A BSTRACT ... 120

1. I NTRODUCTION ... 121

2. M ATERIALS AND METHODS ... 123

3. R ESULTS ... 128

4. D ISCUSSION ... 136

R EFERENCES ... 140

Chapter VII. FTIR imaging of MCF-7 spheroids and their vicinity in Matrigel-embedded 3D cultures ... 149

A BSTRACT ... 150

1. I NTRODUCTION ... 151

2. M ATERIALS AND METHODS ... 152

3. R ESULTS ... 155

4. D ISCUSSION ... 157

R EFERENCES ... 158

F IGURES ... 161

Chapter VIII. FTIR imaging of the 3D extracellular matrix used to grow spheroids of breast cancer cell lines ... 167

A BSTRACT ... 168

1. I NTRODUCTION ... 169

1. M ATERIALS AND METHODS ... 170

2. R ESULTS ... 173

3. D ISCUSSION ... 180

R EFERENCES ... 183

Chapter IX. nfluence of fibroblasts on the phenotype of breast cancer cell lines grown in 3D culture evidenced by FTIR imaging ... 189