Blanc SVSE 5 - Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

3D polyelectrolyte scaffolds for prostatic cell culture : a tool to study the influence of the microenvironment on cancerous phenotype and to identify new biomarkers – BROCOLI

BROCOLI - 3D polyelectrolyte scaffolds for prostatic cell culture

a tool to study the influence of the microenvironment<br />on cancerous phenotype and to identify new<br />biomarkers

2.3. OBJECTIVES, ORIGINALITY AND/OR NOVELTY OF THE PROPOSAL

The objective of this project is to develop a 3D-microculture format that provides a ductal scaffold of polyelectrolyte microtubes to enable prostatic cells to grow into a normal glandular acinar structure. 3D culture systems for prostatic cells currently utilise a matrix of Matrigel or collagen to provide a substrate into which the cells are seeded and then spontaneously develop acinar structures. After developing such acinar structures, the prostatic cells secrete ions, enzymes and hormones into the core of the acinus. However, it is difficult to analyse the secretions from such acini, since they are individual clusters within the 3D matrix and there is no linking collecting ductal structure from which the acinar secretions can be collected. In that case the 3D matrix has to be dissected and the acini removed for analysis.<br />The novelty of this project is to develop a ductal scaffold to support the growth of the prostatic acinar cells, which will allow measurement of the secretory responses of prostate cells in real time.<br />Polyelectrolyte (PE) appears to be the ideal candidate, as films made from these charged polymers are non-cytotoxic and relatively cheap. We have already used PE to make micro-size capsules and tubes and thin films which have supported successfully both lipid and cell adherence and growth. Furthermore, the PE films are viscoelastic, compressible and other physical parameters are easily controlled.

The specific aims of this project are:

1. to construct a bio-chip utilizing polyelectrolyte 3-dimensional ductal micro-structures to provide a controlled biomimetic micro-environment for the growth of exocrine gland cells.

2. to characterise the influence of the micro-environment (i.e. physical substrate and extracellular fluid composition) on the growth of cancerous exocrine gland cells,

3. to characterise the influence of the micro-environment (i.e. physical substrate and extracellular fluid composition) on the physiological function (i.e. secretions) of cancerous exocrine gland cells,

4. to utilize the 3-dimensional bio-chip to assay the role of drugs to ameliorate the physiological function and phenotype of cancerous exocrine gland cells

To achieve those aims the scientific program is organised into 3 phases:

Phase 1: 3-dimensional polyelectrolyte ductal microstructures to provide controlled micro-environment for growth of exocrine gland cells (aim 1)

Phase 2: micro-environment influence on growth and physiological function of exocrine gland cells (aims 2, 3)

Phase 3: assay drugs to ameliorate phenotype and physiological function of exocrine gland cells (aim 4)

Our expected results are in 3 main areas:

1. utlising polyelectrolytes to engineer a 3D cell culture scaffold system to study normal and cancerous prostatic cell lines and to quantify the secretory activity of cells. Our 3D format will provide a means to collect and analyse fluids from the lumen and also to trigger or measure mechanical deformation in the lumen following fluids movement. Therefore, the originality of this project is also to use the microfluidics for analysing the dynamic cell response, rather than previously reported utilisation of microfluidics simply as a tool for constructing the 3D cell culture,

2. that relevant 3D polyelectrolyte micro-environment would provide new insights in the mechanisms controlling epithelial polarity and lumen formation which are to date poorly understood. By modulating chemical, physical and mechanical properties of PE, we can affect the cell polarity dynamics on both sides of epithelial cells: from the external culture medium (allow for dispensing of important reagents and drugs or siRNA) but also from the internal side of the lumen. This is a very original aspect since the important morphological event of polarized fluid movement has been demonstrated in lumen space formation,

3. we propose to (i) create the proof-of-concept of 3D PE scaffolds for epithelial morphogenesis, and (ii) demonstrate that PE are functional in providing the structural and biochemical signals necessary for 3D morphogenesis of epithelial cell organisation. An expected outcome is to provide a protocol for cell culture in a new 3D microenvironment using microspheres and microtubes of PE.

The results and outcomes from this project BROCOLI will provide underlying fundamental research to promote the development of biotechnologies and bioengineering for improved diagnostic and/or in vitro analysis. In these terms, BROCOLI perfectly matches the ITMO (“Institut Thématique Multi-Organisme”) goals and strategies mainly relied on the discovery of new biomarkers and their clinical validation. Moreover, we believe that our multidisciplinary BROCOLI project based on the application of 3D cell culture and polyelectrolytes to biomarkers discovery, suits well to the SNRI (“Stratégie Nationale de la Recherche et de l’Innovation”). One of the SNRI priorities relies indeed on the need for a personalized medicine through the development of better diagnostic tools. BROCOLI proposes a new lab-on-chip analysis tool that answers this need.

We have submitted the following manuscript for publication:

Nathalie Picollet-D’hahan N, Sophie Gerbaud S, Kermarrec F, Alcaraz J-P, Guyon L, Sulpice E, Cinquin P, Gidrol X, Martin DK. (2012). Polyelectrolyte nanofilms modulate attachment, growth and morphology of cancerous prostate cells. Biomaterials (a soumis)

The core of this project is to characterize a novel 3D culture system using polyelectrolyte (PE) scaffolds to construct in vitro 3-dimensional (3D) culture models of prostate tissue and to discover ways to control the microenvironment of cell growth so as to understand (i) the excocrine gland morphogenesis into cell differentiation, and (ii) acini formation and to understand the breakdown of tissue architecture and disorganisation of acini during disease and cancer. The targeted cell-type is prostatic cells in direct connection to the research expertise of TIMC-IMAG and CEA-BGE. The novelty in our approach is to create a 3D branched luminal structure with PE (named BROCOLI) onto which acinar cells will grow. We have already successfully created ion-transporting 3D PE microtubes that can support ion-transporting lipid bilayer membranes and the growth of cells. In parallel, the CEA-Biomics team has obtained preliminary results on 2D and 3D cell culture of normal and tumorigenic prostatic cells with the study of kinases in the regulation of the balance between proliferation, differentiation and cell death in prostatic cells. Therefore, the BROCOLI project combines the partners’ expertise in exocrine gland physiology and bioengineering (UJF), prostatic cell phenotyping and carcinogenesis (CEA-biomics), lab-on-chip and microtechnologies (LETI) and clinical diagnosis of prostate cancer (UJF). We will apply the fundamental knowledge from this project to designing 3D systems of lumen structures that will mimic the exocrine gland lumen structure. The outcomes from this ANR proposal will provide a paradigm-shift in the concept of in vitro 3D cell growth by providing a luminal scaffold microstructure for 3D growth of exocrine tissues. Using this system we will be able to investigate the influence of the microenvironment, and provide significant advances in the 3D tissue engineering of exocrine structures for enhanced understanding of normal and diseased organ development. We aim to demonstrate that 3D PE scaffolds are functional in providing the structural and biochemical signals necessary for 3D morphogenesis of epithelial cell organisation. By coupling 3D tubular PE scaffolds with microfluidics, we also aim to develop a lab-on-chip device for 3D cell culture and quantitative analysis of secretory activity of prostatic cells in order to identify new biomarkers for cancer diagnostic. Those outcomes for this project have application to developing novel lab-on-chip systems for cancer diagnosis and for tissue engineering of exocrine glands. Further, information should lead to the development of novel therapies to effectively prevent and/or treat prostate cancer.

Project coordinator

Monsieur Donald MARTIN (UNIVERSITE GRENOBLE I [Joseph Fourier]) – don.martin@univ-grenoble-alpes.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CEA-LETI COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES - CENTRE DE GRENOBLE
CEA/DSV/iRTSV/BGE/BioMics COMMISSARIAT A L'ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES - CENTRE DE GRENOBLE
UJF UNIVERSITE GRENOBLE I [Joseph Fourier]

Help of the ANR 484,399 euros
Beginning and duration of the scientific project: - 36 Months

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