Periodic Reporting for period 2 - 4DBIOSERS (Four-Dimensional Monitoring of Tumour Growth by Surface Enhanced Raman Scattering)
Reporting period: 2020-04-01 to 2021-09-30
Building three-dimensional scaffolding to guide the growth of tumors in controlled environments, to monitor the evolution of each of their cells in real time and to record the release of tumor metabolites and other indicators of cell activity under different conditions. That is the ultimate aim of the 4DbioSERS project, on which the research group led by Prof. Luis Liz-Marzán is working at CIC biomaGUNE. It is a project geared towards the study of cancer, melanoma and breast cancer in particular, and seeks to better understand the growth and dynamics of tumors, avoiding the need for animal experiments. 4DbioSERS is a five-year project funded by the European Research Council (ERC) as part of the prestigious ERC Advanced Grants call awarded to high-risk and high-gain projects.
We are devising micrometric scaffolds, which can be fabricated by means of the most modern 3D printing technology and incorporate built-in metal nanoparticles that can be used as sensors. Mixtures of tumor cells, other types of cells and extracellular components are cultured inside the scaffolds to reproduce real tumors as faithfully as possible, so that the nanosensors will allow us to detect biomarkers related to tumor evolution under different conditions. We can thus alter the temperature or pH (either globally or locally), but also add drugs or create relevant ambient conditions, so that more effective treatments can be subsequently designed. By specifically labeling some of the cells, we can watch their behavior inside the artificial tumor, including the potential segregation of certain types of cells toward a specific location leading to tumor heterogeneity. Upon completion of the study, we might even be able to watch metastatic events.
Detection of biomarkers and monitoring of cells is carried out by SERS (surface-enhanced Raman spectroscopy), a technique capable of analyzing a broad variety of substances with very fine spatial resolution, even at extremely low concentrations. SERS uses gold or silver nanoparticles as sensors and also as labels, as well as a laser that enables highly selective identification of the molecules close to these nanoparticles.
We are devising micrometric scaffolds, which can be fabricated by means of the most modern 3D printing technology and incorporate built-in metal nanoparticles that can be used as sensors. Mixtures of tumor cells, other types of cells and extracellular components are cultured inside the scaffolds to reproduce real tumors as faithfully as possible, so that the nanosensors will allow us to detect biomarkers related to tumor evolution under different conditions. We can thus alter the temperature or pH (either globally or locally), but also add drugs or create relevant ambient conditions, so that more effective treatments can be subsequently designed. By specifically labeling some of the cells, we can watch their behavior inside the artificial tumor, including the potential segregation of certain types of cells toward a specific location leading to tumor heterogeneity. Upon completion of the study, we might even be able to watch metastatic events.
Detection of biomarkers and monitoring of cells is carried out by SERS (surface-enhanced Raman spectroscopy), a technique capable of analyzing a broad variety of substances with very fine spatial resolution, even at extremely low concentrations. SERS uses gold or silver nanoparticles as sensors and also as labels, as well as a laser that enables highly selective identification of the molecules close to these nanoparticles.
During this first half of the project, we have achieved highly promising results in the direction of the initially established objectives. We have optimized the production of SERS-encoded nanoparticles, with various chemical methods that lead to a wide variety of codes and potential binding of antibodies and other targeting biomolecules. Such encoded particles were used to obtain the 3D reconstruction of biological multilayer constructs comprising different types of cells, with a sufficient resolution to differentiate between each layer of cells over relatively long periods of time. The advantage of the encoded particles used in this system is that they do not degrade over time, unlike fluorescent molecules routinely used for detections of this type. Apart from the acquisition of three-dimensional cellular maps, we have developed a data analysis tool that allows us to count the number of encoded particles per cell, so that we should also be able to monitor events such as cell division. This achievement is a first demonstration of the study of dynamic evolution in cellular systems.
We additionally managed to formulate polymer hydrogel inks for 3D printing, leading to scaffolds where cells can be cultured in 3D, and incorporated nanosensors can provide a map of metabolic processes taking place. The evolution of different metabolite biomarkers has been monitored in real time, which vary during the evolution of cancer cells, in particular in the presence of drugs or other substances. Specially designed substrates were devised to detect concentrations that are small enough to be significant in these tumor cultures. We can also decide where and when a measurement is to be made, so that the state of the biological system can be mapped in space and time. We can thus watch how the tumor cells that are developing in the system itself evolve over time and distinguish their behavior under various conditions. Specifically, we have managed to observe the evolution of two metabolites simultaneously, one increasing its concentration while the other declines, which confirms that we are seeing in real time the metabolic process caused by enzymes that are expressed in these tumor cells. We were also able to watch a drug diffuse through a 3D cell culture system and how this affects the destruction of tumor cells. This is a very important evidence because it enables us to avail ourselves of practically remote detection, owing to the fact that our detector is not in direct contact with the cells, but simply studies the medium surrounding them. This is a significant step forward with respect to the ultimate objective.
We additionally managed to formulate polymer hydrogel inks for 3D printing, leading to scaffolds where cells can be cultured in 3D, and incorporated nanosensors can provide a map of metabolic processes taking place. The evolution of different metabolite biomarkers has been monitored in real time, which vary during the evolution of cancer cells, in particular in the presence of drugs or other substances. Specially designed substrates were devised to detect concentrations that are small enough to be significant in these tumor cultures. We can also decide where and when a measurement is to be made, so that the state of the biological system can be mapped in space and time. We can thus watch how the tumor cells that are developing in the system itself evolve over time and distinguish their behavior under various conditions. Specifically, we have managed to observe the evolution of two metabolites simultaneously, one increasing its concentration while the other declines, which confirms that we are seeing in real time the metabolic process caused by enzymes that are expressed in these tumor cells. We were also able to watch a drug diffuse through a 3D cell culture system and how this affects the destruction of tumor cells. This is a very important evidence because it enables us to avail ourselves of practically remote detection, owing to the fact that our detector is not in direct contact with the cells, but simply studies the medium surrounding them. This is a significant step forward with respect to the ultimate objective.
A new technology has been implemented for the continuous monitoring of relevant molecules in media under development. The technology is based on SERS detection of molecular analytes, by using a regularly structured nanoparticle-based substrate, which is able of largely amplifying the Raman scattering signals of adsorbed molecules. In order to prevent contamination and provide a faithful representation of the system at the selected time, a measurement window is opened by irradiation with a focused laser beam, so that the rest of the substrate remains shielded and ca be used at a later stage. This technology has been protected through a European patent application and can be applied not only for monitoring of cellular systems but also for other applications such as environmental contamination.