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Modulating the behaviour of cancer myofibroblasts to control tumour desmoplasia

Periodic Reporting for period 1 - MYO-DESMOPLASIA (Modulating the behaviour of cancer myofibroblasts to control tumour desmoplasia)

Reporting period: 2015-06-01 to 2017-05-31

The tumor micro-environment consists of stromal cells (including fibroblasts and myofibroblasts, also known as cancer associated fibroblasts (CAFs)), the extracellular matrix (ECM) and myriad soluble factors in the extracellular milieu whose importance in cancer progression and metastasis is indisputable. In many types of tumors (e.g. pancreatic and breast cancers), the complex interplay among tumor micro-environment components leads to remodeling and overproduction of the tumor ECM, resulting in a desmoplastic reaction. Desmoplasia is a cancer-specific type of fibrosis, characterized by the presence of CAFs and overproduction of ECM proteins, such as collagen type I. Desmoplasia stiffens the tumor tissue, and as a result, it increases the compressive mechanical forces in the interior of the tumor. Among the wide range of physical alterations that occur during cancer and the many TME constituents that are present, the CAFs, the transforming growth factor beta (TGFβ) and the matrix stiffness stand out as key players, responsible for tumor desmoplasia. One pathway through which extensive ECM synthesis and remodeling occur is the activation of TGFβ along with the mechanical forces exerted on fibroblasts from the ECM and other structural components of the tumor. Specifically, it is known that TGFβ activation and stiffening of the ECM contribute to the conversion of fibroblasts to contractile CAFs. Subsequently, CAFs increase the synthesis of ECM proteins, such as collagens, while TGFβ down-regulates the expression of matrix-depleting metalloproteinases. Furthermore, TGFβ regulates the production of matrix-modifying enzymes, which increase the degree of collagen crosslinking. This mechanism results in desmoplasia and further stiffening of the matrix. Matrix stiffening, in turn, will cause an increase in TGFβ expression and will further fibroblasts conversion. Therefore, it appears to be a positive feedback loop, which gives rise to continuous activation of TGFβ and formation of CAFs that exacerbate tumor desmoplasia. However, the underlying mechanisms, leading to the desmoplastic reaction of solid tumors are not yet fully understood. Elucidation of the role of matrix stiffness and TGFβ in controlling tumor desmoplasia can lead to new approaches for treating cancer. Specifically, it has been shown that targeting tumor desmoplasia can improve the systemic delivery of drugs and hinder metastasis. The specific research objectives of the project were: Research Objective 1: Development of a collagen based ECM model, with pre-determined topography and tunable stiffness. Research Objective 2: Characterization of the mechanical properties and behaviour of fibroblasts and myofibroblasts cultured in the ECM models as a function of TGFβ. Research Objective 3: gene expression analysis of fibroblasts and CAFs in the matrix models to identify genes responsible for ECM production as a function of TGFβ and correlate to cell mechanical properties.
"In the MYO-DESMPOPLASIA project the fellow tested the hypothesis that the increase in ECM stiffness and TGFβ activation have additive effects on tumor desmoplasia. To explore this hypothesis, a combination of cutting-edge techniques were employed. Specifically, two and three dimensional (2D and 3D) collagen ECM models, with tunable stiffness were developed. Subsequently, pancreatic normal fibroblasts (FBs) and CAFs were cultured in the ECM models. Cells nanomechanical behavior and their morphodynamic alterations were investigated with Atomic Force Microscopy (AFM) and light/fluorescence microscopy under the presence or absence of TGFβ and under the influence of different stiffness. Also, cell spheroids were formed and cultured in 3D collagen gels with different stiffness in order to assess cells’ mobility/invasion under the influence of different collagen stiffness and the presence of TGFβ. Finally, the effects of TGFβ and matrix stiffness in the expression pattern of specific genes were investigated using real-time PCR.Our results showed that CAFs present specific characteristic of the myofibroblasts phetotype, such as elongation and a-smooth muscle actin expression, while we demonstrated that CAFs have more lamellipodia and are softer compared to normal FBs. Also, it was revealed that TGFβ and collagen stiffness significantly affect CAFs basic morphodynamic characteristics, such as cell elongation, cell spreading, number of lamellipodia and stress fiber orientation, while this was not the case for normal FBs. For both FBs and CAFs it was found that TGFβ increase their stiffness in term of Young’s modulus values. Moreover, a significant correlation was revealed between cell spreading and RAC expression in both cell lines. Also, RhoA and ROCK expression was altered in CAFs after TGFβ treatment, which in combination with RAC expression is correlated to the increase in the number of lemellipodia, stress fiber orientation, cells’ mobility and stiffness. Although more research is needed to elucidate the exact involvement of TGFβ and ECM stiffness in desmoplasia, these findings provide new insights that need to be taken into consideration for understanding of desmoplasia or even for the development of novel therapeutic approaches for treating cancer having TGFβ as a target molecule.Exploitation and disseminationConferences/forums and public engagement activities-Stylianou, A, Stylianopoulos, T. (2016) ""Investigation of the effect of Tumor Growth Factor-β on Pancreatic Normal and Cancer Associated Fibroblasts"", Cyprus Researchers' Night 2016, 30 September, Nicosia, Cyprus (poster).-Stylianou, A, Gkretsi, V. and Stylianopoulos, T. (2016) ""Effects of Tumor Growth Factor β on Pancreatic Cancer Associated Fibroblasts and Fibroblasts"", EUROAFMForum 2016, June, 22-24, 2016, Geneva, Switzerland (podium)Journal Paper Publications (Peer-reviewed) -Stylianou, A. (2017) ""Atomic Force Microscopy for Collagen-Based Nanobiomaterial"", Journal of Nanomaterials, art. id. 9234627-Kontomaris, S.V and Stylianou, A. (2017) ""Atomic Force Microscopy for University Students, Application in Biomaterials"" European Journal of Physics, 38 (3) -Stylianou, A. and Stylianopoulos, T. (2016) ""Atomic Force Microscopy Probing of Cancer Cells and Tumor Microenvironment Components"" BioNanoScience,6 (1), 33-46 -Gkretsi, V., Stylianou, A., Papageorgios, P., Polydorou, C. and Stylianopoulos, T. (2015) ""Remodeling components of the tumor microenvironment to enhance cancer therapy"" Frontiers in Oncology, 5 (214) -Two research articles containing all the work performed during the period of the project is currently under preparation-A book chapter is under publication: Stylianou, A., Gkretsi, V, Patrickios, C. and Stylianopoulos, T. ""Chapter 29: Exploring the nano-surface of fibrotic tissues with AFM"" In: Fibrosis: Methods and Protocols (Rittié L, ed), Springer."
The MYO-DESMOPLASIA project contributed to the understanding of significant parts of the underlying mechanisms of tumor desmoplasia and the additive effect of ECM stiffness and TGFβ activation. The thorough investigation of the tumor desmoplastic reaction, which is present in many cancers (e.g. pancreatic & breast cancers) it might also contribute to the development of novel markers for early diagnosis of these cancers or the control of tumor metastasis.
TGF-b and matrix stiffness contribute to the conversion of fibroblasts to contractile myofibroblasts