Periodic Reporting for period 2 - VISCOMATRIX (Dynamic regulation of tissue response by matrix viscoelasticity)
Periodo di rendicontazione: 2022-12-01 al 2024-05-31
The mechanical properties of the extracellular matrix (ECM) have emerged as critical regulators of tissue behaviour in a myriad of diverse processes that expand from morphogenesis to cancer. From the mechanical properties of the ECM, elasticity has attracted most efforts as the main regulator of tissue function. The elasticity of the ECM is generally characterized by its stiffness, which is the stress (force per unit area) needed to induce a given strain (deformation). Increased stiffness is a hallmark of most cancers as cells acquire a malignant phenotype when seeded above their physiological stiffness, tumours are generally stiffer than surrounding tissues8 and tumour stiffness is a property used for medical diagnosis simply by tissue palpation or by imaging techniques. Beyond cancer, elasticity controls processes during development (i.e. differentiation, proliferation, or morphogenesis) and wound healing. Research in this space operates under the assumption that tissues are purely elastic. Purely elastic solid materials immediately deform when they are submitted to a force, maintain a constant deformation as long as the force is held constant and immediately return to their original shape when the force deforming them is removed. However, tissues are not merely elastic but both elastic and viscous (viscoelastic). Contrary to elastic materials, viscous liquid materials’ strain is not instantaneous, and they continuously deform with time under force in an irreversible manner. Biological tissues exhibit a first instantaneous solid elastic response followed by a time-dependent liquid viscous behaviour where forces are dissipated, and stresses are relaxed. The viscoelastic response affects all tissues regardless of their stiffness (i.e. muscle, breast, bone, brain, adipose tissue, liver). The viscoelastic nature of the ECM is starting to emerge as a pivotal determinant of cellular responses. We hypothesize that the viscoelastic properties of the ECM provides a novel fundamental mechanosensitive pathway that control tissue function and works with or overrules elastic responses. The objective of VISCOMATRIX is to understand which are the mechanisms that cells use to sense the properties of 3D viscoelastic ECMs and how these regulate tissue homeostasis and malignant transformation.
VISCOMATRIX is focused on a highly relevant system which is the mammary gland and related mammary tumours. This choice is based on: (i) First, breast tumour impacts approximately 1 in 8 women. ii) Second, breast mammary gland is a viscoelastic tissue. (iii) Third, even if the role of viscoelasticity is unknown, it is well known that mammary gland and tumour development is associated with strong changes in mechanical properties. Our preliminary results show that viscoelasticity significantly affects tissue function. Therefore, as all tissues are viscoelastic, the biomedical impact of the importance of viscoelasticity is expected to be dramatic and impact fields ranging from regenerative medicine to cancer, and fibrosis. In the bioengineering filed, we believe that our findings will affect biomaterial development as well as the development of new imaging techniques to determine the viscoelastic properties of tissues. As to cancer, clinical trials targeting matrix metalloproteinases or integrins have been so far disappointing. The inclusion of viscoelasticity in the equation may provide crucial information to progress disease treatment and develop drugs.
VISCOMATRIX is focused on a highly relevant system which is the mammary gland and related mammary tumours. This choice is based on: (i) First, breast tumour impacts approximately 1 in 8 women. ii) Second, breast mammary gland is a viscoelastic tissue. (iii) Third, even if the role of viscoelasticity is unknown, it is well known that mammary gland and tumour development is associated with strong changes in mechanical properties. Our preliminary results show that viscoelasticity significantly affects tissue function. Therefore, as all tissues are viscoelastic, the biomedical impact of the importance of viscoelasticity is expected to be dramatic and impact fields ranging from regenerative medicine to cancer, and fibrosis. In the bioengineering filed, we believe that our findings will affect biomaterial development as well as the development of new imaging techniques to determine the viscoelastic properties of tissues. As to cancer, clinical trials targeting matrix metalloproteinases or integrins have been so far disappointing. The inclusion of viscoelasticity in the equation may provide crucial information to progress disease treatment and develop drugs.
During these 30 months, we have been able to achieve some of the main milestones of VISCOMATRIX.
During these first years, we were focused on the development of the hydrogels that we have are currently using and will be used throughout the project. Apart from hydrogels, we have been developing the methodology to be able to perform all the aims of the project. We have invested a significant amount of time to the development of a methodology that spans from single cell measurements to in vivo characterization. This methodology will provide broad applicability and a very powerful set of tools for many different applications. We have done most of the second objective and a significant amount of the first objective. As to the main results achieved so far:
Alginate hydrogels: We have been able to obtain alginate that we have irradiated to generate alginate hydrogels with different molecular weights. Different molecular weights allow us to develop alginate hydrogels with different viscoelasticities and stiffness. We have then mechanically characterized these hydrogels using two main techniques: rheology and Atomic Force Microscopy. With these hydrogels we have been able to start the experiments with epithelial cells as described in VISCOMATRIX. Additionally, we are developing materials with some small regions with different properties as described in 3.2 in VISCOMATRIX.
Role of viscoelasticity in breast cells: As hypothesised in VISCOMATRIX, we have observed that MCF10A cells encapsulated in viscoelastic hydrogels are able to undergo EMT and invade the matrix. However, when these cells are seeded in a more elastic matrix spherical symmetry is maintained and cells are unable to invade. Additionally, cell proliferation is enhanced in viscoelastic matrices. Interestingly, stiffness enhances the influence of viscoelasticity. Even though cells are unable to invade in stiff elastic ECMs, cells invade significantly more in stiff viscoelastic ECMs compared to soft viscoelastic ECMs. In accordance with these results, we have done a RNAseq experiment that shows that most cancer pathways are activated in stiff viscoelastic ECMs. This process seems to be regulated by FAK, Arp2/3 complex and Rac1. Taking into account our bulk sequencing results, we are going to perform single cell sequencing experiments in 2024. Furthermore, we have developed a method to quantitatively measure tissue dynamics and force transmission in live experiments. We observe that spheroids migrate significantly faster in viscoelastic hydrogels and exert larger stress transmission.
We have also been able to set up the AFM and microscope to measure both the ECM composition and the mechanical properties of the tissue.
During these first years, we were focused on the development of the hydrogels that we have are currently using and will be used throughout the project. Apart from hydrogels, we have been developing the methodology to be able to perform all the aims of the project. We have invested a significant amount of time to the development of a methodology that spans from single cell measurements to in vivo characterization. This methodology will provide broad applicability and a very powerful set of tools for many different applications. We have done most of the second objective and a significant amount of the first objective. As to the main results achieved so far:
Alginate hydrogels: We have been able to obtain alginate that we have irradiated to generate alginate hydrogels with different molecular weights. Different molecular weights allow us to develop alginate hydrogels with different viscoelasticities and stiffness. We have then mechanically characterized these hydrogels using two main techniques: rheology and Atomic Force Microscopy. With these hydrogels we have been able to start the experiments with epithelial cells as described in VISCOMATRIX. Additionally, we are developing materials with some small regions with different properties as described in 3.2 in VISCOMATRIX.
Role of viscoelasticity in breast cells: As hypothesised in VISCOMATRIX, we have observed that MCF10A cells encapsulated in viscoelastic hydrogels are able to undergo EMT and invade the matrix. However, when these cells are seeded in a more elastic matrix spherical symmetry is maintained and cells are unable to invade. Additionally, cell proliferation is enhanced in viscoelastic matrices. Interestingly, stiffness enhances the influence of viscoelasticity. Even though cells are unable to invade in stiff elastic ECMs, cells invade significantly more in stiff viscoelastic ECMs compared to soft viscoelastic ECMs. In accordance with these results, we have done a RNAseq experiment that shows that most cancer pathways are activated in stiff viscoelastic ECMs. This process seems to be regulated by FAK, Arp2/3 complex and Rac1. Taking into account our bulk sequencing results, we are going to perform single cell sequencing experiments in 2024. Furthermore, we have developed a method to quantitatively measure tissue dynamics and force transmission in live experiments. We observe that spheroids migrate significantly faster in viscoelastic hydrogels and exert larger stress transmission.
We have also been able to set up the AFM and microscope to measure both the ECM composition and the mechanical properties of the tissue.
VISCOMATRIX is a highly ambitious project that tries to resolve the fundamental role that viscoelasticity plays in cell and tissues response. Due to the importance of the question and the lack of previous knowledge, the project requires the development of a new broad methodology to unveil the role of viscoelasticity from molecular mechanisms to in vivo experiments in the context of breast cancer. We expect to elucidate novel mechanisms that could provide critical information to treat and understand breast cancer progression. Besides, even though the project is focused on breast cancer, more than 80% of tumours are derived from epithelial cells. Thus, findings from VISCOMATRIX will likely be applied to other tumours.