Periodic Reporting for period 1 - MECHANOIDS (Probing and controlling the three-dimensional organoid mechanobiology)
Okres sprawozdawczy: 2019-09-01 do 2021-08-31
Interaction between cells and their mechanical microenvironment plays a key role in the regulation of development, physiology and disease. Cell behaviour is also regulated by the dimensionality of the microenvironment: 2D cultures display biological traits that generally differ from those of 3D tissues, thus limiting their potential in biomedical research. This limitation has been addressed by the recent development of Organoids, which faithfully preserve a number of distinctive tissue traits. Still, current biophysical tools and conceptual frameworks are not yet suitable to probe and understand the mechanobiology of 3D organoid systems.
This project aimed to quantify and manipulate the mechanobiology of normal gut and colorectal cancer organoids, and other 3D cell tissues. We developed tools to quantify the 3D stresses applied by an organoid embedded in a gel. We also measured the tension, pressure and contractility exerted by cell tissues surrounding a “tumor-like surrogate” gel, key parameters in organ growth and tumour progression.
Pressure and contractility were related with key biological parameters such as geometry, cell adhesion and proliferation. Proteins were fluorescently tagged in the cells that allowed us to track individual cells, as well as monitoring their polarization and adhesion.
The mechanical state of the organoids was also modified through optogenetic means. Proteins were expressed in the cells to control Rho-GTPases upon illumination, thereby steering the local stresses at will. This allowed to modify organoid pressure, contractility and geometry.
As organoids emerge as novel tools in biomedical research, we expect that studying and manipulating their mechanobiology will bring key insight into disease and development processes, and potential new therapeutic targets.
This project aimed to quantify and manipulate the mechanobiology of normal gut and colorectal cancer organoids, and other 3D cell tissues. We developed tools to quantify the 3D stresses applied by an organoid embedded in a gel. We also measured the tension, pressure and contractility exerted by cell tissues surrounding a “tumor-like surrogate” gel, key parameters in organ growth and tumour progression.
Pressure and contractility were related with key biological parameters such as geometry, cell adhesion and proliferation. Proteins were fluorescently tagged in the cells that allowed us to track individual cells, as well as monitoring their polarization and adhesion.
The mechanical state of the organoids was also modified through optogenetic means. Proteins were expressed in the cells to control Rho-GTPases upon illumination, thereby steering the local stresses at will. This allowed to modify organoid pressure, contractility and geometry.
As organoids emerge as novel tools in biomedical research, we expect that studying and manipulating their mechanobiology will bring key insight into disease and development processes, and potential new therapeutic targets.
During this project, the researcher created the computational and analysis tools needed to be able to measure the 3D deformation of very soft gels and to calculate the internal pressure and the contractility of cell clusters from the stresses that it applies on the soft gel. The knowledge acquired during this part was disseminated through a peer-reviewed review paper [https://doi.org/10.1038/s42254-020-0184-6].
The researcher created Colorectal Cancer organoid mutants expressing fluorescent proteins such as actin, CAAX and e-cadherin, and optogenetic contractility constructs through lentiviral transfection. These mutants and the previously mentioned tools were used to study the interplay between structure, shape and stresses.
The tools generated by the researcher were applied to the study of the mechanics of crypts in intestinal organoids, published in a peer-reviewed paper [https://doi.org/10.1038/s41556-021-00699-6]; and to study the interplay between Cancer-Associated Fibroblast contractility and tumor mechanotransduction, disseminated in the form of a preprint [https://doi.org/10.1101/2021.04.05.438443].
The researcher created Colorectal Cancer organoid mutants expressing fluorescent proteins such as actin, CAAX and e-cadherin, and optogenetic contractility constructs through lentiviral transfection. These mutants and the previously mentioned tools were used to study the interplay between structure, shape and stresses.
The tools generated by the researcher were applied to the study of the mechanics of crypts in intestinal organoids, published in a peer-reviewed paper [https://doi.org/10.1038/s41556-021-00699-6]; and to study the interplay between Cancer-Associated Fibroblast contractility and tumor mechanotransduction, disseminated in the form of a preprint [https://doi.org/10.1101/2021.04.05.438443].
During this project, the researcher has created and applied tools to help study mechanobiology in three-dimensions, helping to further the current applicability of 2D mechanobiology techniques to more complex problems that might be relevant for development and disease.