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3R-Project 144-15

Development of in vitro three-dimensional multi-cellular culture models to study the role of heterotypic interactions during colorectal carcinomatous invasive process

Curzio Rüegg, Sarah Cattin

Department of Medicine, Chair of Pathology, University of Fribourg, Switzerland


curzio.ruegg@unifr.ch,, sarah.cattin@unifr.ch

Keywords: colorectal cancer; tumour microenvironment; heterotypic interactions; reduction; replacement

Duration: 2 years Project Completion: 2017

Background and Aim

Colorectal cancer (CRC) is the third most frequent cause of mortality due to cancer in Europe. Local invasion and distant metastasis are the critical steps that negatively impact patient survival. Although cells of the tumour microenvironment (TME) are known to promote invasion and metastasis, the mechanisms underlying these processes are only partially understood. An understanding of how the cells from the TME promote invasion in CRC is crucial for the development of novel anti-invasive therapies.

The study of heterotypic cellular interactions in vivo is difficult, owing to constraints in accessing the tissue and in modulating specific cells or intercellular interactions. In vitro models mimicking the TME would be invaluable in studying the role of individual cells of the TME and their interactions with CRC-cells.

We have developed an in vitro three-dimensional (3D) model to study the interactions between fibroblasts and CRC-cells. This model is now well suited to study the interactions between cancer cells and others, such as endothelial and immune/inflammatory cells, from the TME. In this project, we aim to improve this 3D in vitro culture model by introducing additional cell types of the TME, and by studying their interactions, with a view to improve our understanding of their role in promoting cancer cells invasion as the first step in the metastatic process. Finally, we will validate the in vivo observations, in order to set on a firm footing a robust alternative to study the heterotypic cellular interactions in the TME.

Method and Results

We have already developed a 3D in vitro co-culture model consisting of fibroblasts and CRC-cells. To improve this model, endothelial cells (Ea.hy296) will be embedded together with or without human fibroblasts in a 3D-Matrigel-matrix to generate blood-vessel-like structures. Several CRC-cell lines with different degrees of invasiveness, such as SW620, HCT116 and HT29, will be included in the matrix in the form of spheroids to better mimic a tumorous nodule. All cells will be fluo-labelled with mCherry, Azurite or GFP to track them specifically. Co-cultures will be monitored for up to two weeks using time-lapse fluorescence microscopy, with a view to ascertain how the TME-cells induce CRC-cells invasion and progression. 3D cultures will then be treated with cytokines, growth factors, blocking antibodies or pharmacological inhibitors of signalling to determine the key elements that are implicated in CRC-cells invasion. Monocytes will then be added to complete the model and to study their role in this context.

To recommend the use of these heterotypic models as an in vitro surrogate of the TME in vivo, it is essential to demonstrate that the in vitro effects of several stromal cells on tumoral ones can be reproduced in vivo. To this end, colon-cancer cells will be injected subcutaneously or orthotopically into the caecum of immune compromised mice in the absence or presence of fibroblasts or endothelial cells. Progression of the tumour will be monitored on the basis of the luciferase activity of the CRC-cells. Preliminary results are really promising and show that our 3D in vitro model correctly mimic the in vivo interaction between cells of the tumour microenvironment and cancer cells.

Conclusions and Relevance for 3R

Tumour-host interaction is now emerging to be a critical event in tumoral growth and development. However, a study of the TME is a complex undertaking, since many cells and factors act simultaneously. A differential evaluation of the underlying mechanisms in vivo is thereby rendered extremely difficult.

The development of our in vitro assay recreates a simplified and well-controlled TME. This model could promote progression in this field, reduce costs, and minimize animal use. An improvement in our understanding of how tumoral and host cells interact afford opportunities for elucidating the molecules and mechanisms that are implicated in the invasive process underlying metastasis.

References


  • McAllister, S.S., and Weinberg, R.A. (2014). The tumour-induced systemic environment as a critical regulator of cancer progression and metastasis. Nature cell biology 16, 717-727.

  • Lorusso, G. & Ruegg, C. The tumour microenvironment and its contribution to tumour evolution toward metastasis. Histochemistry and cell biology 130, 1091-1103, doi:10.1007/s00418-008-0530-8 (2008).

  • Lee, G. Y., Kenny, P. A., Lee, E. H. & Bissell, M. J. Three-dimensional culture models of normal and malignant breast epithelial cells. Nature methods 4, 359-365, doi:10.1038/nmeth1015 (2007).

  • Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M. & Nielsen, L. K. Method for generation of homogeneous multicellular tumour spheroids applicable to a wide variety of cell types. Biotechnology and bioengineering 83, 173-180, doi:10.1002/bit.10655 (2003).

  • Knuchel, S., Anderle, P., Werfelli, P., Diamantis, E. & Ruegg, C. Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin alphavbeta5-mediated adhesion. Oncotarget (2015).

Figures

Figure 1
Figure 1: Representation of the 3D-model strategy

Figure 2
Figure 2: Brightfield and fluorescent images of 7-days-old 3D co-culture assay with GFP-expressing SW620 cancer cells and mCherry-expressing Fibroblasts. Magnification: x10.



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