The extracellular matrix (ECM) is present in all solid tissues and is a master regulator of cell behavior and phenotype. The ECM in each tissue is characterized by its biochemical and biophysical properties, which are modified in many different types of disease, especially cancer. Hyaluronic acid (HA), as a biopolymer, has emerged as a remarkable ECM component by its viscoelastic properties, its hygroscopic capacities and the diversity of cell processes it controls in health and disease. BIOMIMESYS®, a patented HA-based hydroscaffold, is a highly reproducible and physiological matrix that is suitable for long-term 3D cell culture. Using BIOMIMESYS® Oncology - made of HA biofunctionalized with undenaturated collagen I - we investigated the influence of (i) matrix composition (with/without laminin) and (ii) stiffness on the growth and expression of CD44 (a receptor of HA) in spheroids made of breast cancer cell lines (MCF7, MDA-MB-231) and normal cells (MCF10A) along time. Analyses were carried out using high content analysis/screening (HCA/HCS, high throughput confocal microscopy). As a whole, the results showed that the cell growth was lowered in the presence of laminin but promoted with increased stiffness. Except for MCF10A cells, the expression of CD44 had the tendency to increase over time, while the stiffness and the presence of laminin had no significant impact on its expression. Given that BIOMIMESYS® is a highly defined and easily tunable matrix, this physiological 3D cell culture system allows to study the impact of biochemical and biophysical properties of the matrix. By better mimicking the cancer microenvironment - including the ECM - the new generation of 3D cell culture systems might help to discover new effective anti-cancer therapies. In this frame, our aim is to study the response of cancer cells in BIOMIMESYS® to a large set of anti-tumor molecules, using our HCS platform. Keywords: 3D cell culture, hyaluronic acid, breast cancer, stiffness, high content screening

1. Impact of the composition and stiffness of a 3D hyaluronic acid-based
hydroscaffold on breast cancer cell growth and CD44 expression
Abstract
The extracellular matrix (ECM) is present in all solid tissues and is a master regulator of cell behavior and phenotype. The ECM in each tissue is characterized by its
biochemical and biophysical properties, which are modified in many different types of disease, especially cancer. Hyaluronic acid (HA), as a biopolymer, has emerged as a
remarkable ECM component by its viscoelastic properties, its hygroscopic capacities and the diversity of cell processes it controls in health and disease.
BIOMIMESYS®, a patented HA-based hydroscaffold, is a highly reproducible and physiological matrix that is suitable for long-term 3D cell culture. Using BIOMIMESYS®
Oncology - made of HA biofunctionalized with undenaturated collagen I - we investigated the influence of (i) matrix composition (with/without laminin) and (ii) stiffness
on the growth and expression of CD44 (a receptor of HA) in spheroids made of breast cancer cell lines (MCF7, MDA-MB-231) and normal cells (MCF10A) along time.
Analyses were carried out using high content analysis/screening (HCA/HCS, high throughput confocal microscopy).
As a whole, the results showed that the cell growth was lowered in the presence of laminin but promoted with increased stiffness. Except for MCF10A cells, the
expression of CD44 had the tendency to increase over time, while the stiffness and the presence of laminin had no significant impact on its expression. Given that
BIOMIMESYS® is a highly defined and easily tunable matrix, this physiological 3D cell culture system allows to study the impact of biochemical and biophysical properties
of the matrix.
By better mimicking the cancer microenvironment - including the ECM - the new generation of 3D cell culture systems might help to discover new effective anti-cancer
therapies. In this frame, our aim is to study the response of cancer cells in BIOMIMESYS® to a large set of anti-tumor molecules, using our HCS platform.
Keywords: 3D cell culture, hyaluronic acid, breast cancer, stiffness, high content screening
Methods Results
Conclusions and perspectives
Marie Lesaffre1, Noémie Carlier1, Nathalie Maubon1, Zied Souguir1, Elodie Vandenhaute1
1HCS Pharma, Biocentre Fleming, 250 rue Salvador Allende, 59120 Loos, FRANCE
• Cell culture in hydroscaffold
96-well plate with the
lyophilised hydroscaffolds
Scaffold visualized
by SEM
• Cell lines used
• Timeline
MCF10A
“normal-like" breast
epithelial cell line
MCF7
luminal A breast cancer cell line
MDA-MB-231
triple negative breast cancer
cell line
• What is high content analysis/screening?
Source: https://www.nottingham.ac.uk/life-sciences/facilities/slim/cell-signalling-imaging/high-content-screening/index.aspx
• Effect of matrix stiffness on cancer cell growth
• Effect of laminin on cell growth and CD44 expression
These preliminary results show that BIOMIMESYS® is a HA-based relevant matrix to study the impact of the ECM on
cancer cell growth and expression of proteins in long-lasting in vitro studies (one month, which would be difficult - if
not impossible - in 2D cultures). Compatible with all classical downstream analysis methods, the format of
BIOMIMESYS® (96-well, and now 384-well format) can also be beneficial in the frame of phenotypic screening in a
search for new antitumor therapies.
As such, our aim is to use BIOMIMESYS® as a tool for finding efficient molecules against each cancer type, by better
mimicking the structure, the composition and the physico-chemical properties of the pathological ECM.
Calcein green
+ Laminin
MDA-MB-231
Day7 - Laminin (control)
CD44
MDA-MB-231
Day14
The growth of spheroids
seems to be lowered in the
presence of laminin in
BIOMIMESYS®.
 The expression of CD44
varies over time, but the
presence of laminin in
BIOMIMESYS® seems not to
impact its expression…
What about CD44 variants?
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
M D A -M B -2 3 1
T im e (d a y s )
Spheroidarea(µm
2
)
B IO M IM E S Y S ® O n c o lo g y so ft
B IO M IM E S Y S ® O n c o lo g y rig id
7 14 21 28
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
M D A -M B -2 3 1
T im e (d a y s )
Spheroidarea(µm
2
)
B IO M IM E S Y S ® O n c o lo g y so ft
B IO M IM E S Y S ® O n c o lo g y rig id
7 14 21 28
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
M C F 1 0 A
T im e (d a y s )
Spheroidarea(µm
2
)
B IO M IM E S Y S ® O n c o lo g y so ft
B IO M IM E S Y S ® O n c o lo g y rig id
7 14 21 28
~ 1 kPa
~ 10 kPa
as measured by rheology
 The matrix stiffness can be modulated in BIOMIMESYS®, which will allow
us to study in more detail the impact of this parameter in
‘microenvironmentally-relevant’ conditions.