Periodic Reporting for period 4 - 4DBIOSERS (Four-Dimensional Monitoring of Tumour Growth by Surface Enhanced Raman Scattering)
Reporting period: 2023-04-01 to 2024-09-30
The main objective of 4DbioSERS was the fabrication of three-dimensional scaffolding to guide the growth of artificial tumors in controlled environments, to monitor the evolution of tumor cells in real time and to record the release of tumor metabolites and other indicators of cell activity under different conditions. This was a project geared towards the study of cancer, melanoma and breast cancer in particular, seeking to better understand the growth and dynamics of tumors, while avoiding the need for animal experimentation. 4DbioSERS was funded by the European Research Council (ERC), as part of the prestigious ERC Advanced Grants scheme, awarded to high-risk and high-gain projects.
We proposed the use of micrometric scaffolds, to be fabricated by means of modern 3D bioprinting technology, in which we would incorporate metal nanoparticles for their use as nanosensors. Mixtures of tumor cells, other types of human cells and extracellular components can be cultured inside such scaffolds, aiming to reproduce the structure and behavior of real tumors as faithfully as possible. The nanosensors would then allow us to detect biomarkers related to tumor evolution under different conditions. We could thus alter temperature or pH (either globally or locally), but also add anticancer drugs, so that more effective treatments can be subsequently designed. By accurately labeling specific cell types, we could watch their behavior inside the artificial tumor, including the potential segregation of certain types of cells toward a specific location leading to tumor heterogeneity, or eventually watching metastatic cancer cells leave the tumor core. Detection of biomarkers and monitoring of cells was based on nanotechnology. Our nanosensors acted as substrates for a spectroscopic technique called surface-enhanced Raman scattering (SERS), which is capable of analyzing a broad variety of substances with very fine spatial resolution, even at extremely low concentrations. SERS detection requires the use of metal (gold) nanoparticles for amplification of characteristic molecular signals, thereby becoming sensors and even highly specific labels for bioimaging. The implementation of such tumor replicas should ultimately enable drug testing on patient samples (organoids) and contribute to the expected transition into personalized medicine.
We proposed the use of micrometric scaffolds, to be fabricated by means of modern 3D bioprinting technology, in which we would incorporate metal nanoparticles for their use as nanosensors. Mixtures of tumor cells, other types of human cells and extracellular components can be cultured inside such scaffolds, aiming to reproduce the structure and behavior of real tumors as faithfully as possible. The nanosensors would then allow us to detect biomarkers related to tumor evolution under different conditions. We could thus alter temperature or pH (either globally or locally), but also add anticancer drugs, so that more effective treatments can be subsequently designed. By accurately labeling specific cell types, we could watch their behavior inside the artificial tumor, including the potential segregation of certain types of cells toward a specific location leading to tumor heterogeneity, or eventually watching metastatic cancer cells leave the tumor core. Detection of biomarkers and monitoring of cells was based on nanotechnology. Our nanosensors acted as substrates for a spectroscopic technique called surface-enhanced Raman scattering (SERS), which is capable of analyzing a broad variety of substances with very fine spatial resolution, even at extremely low concentrations. SERS detection requires the use of metal (gold) nanoparticles for amplification of characteristic molecular signals, thereby becoming sensors and even highly specific labels for bioimaging. The implementation of such tumor replicas should ultimately enable drug testing on patient samples (organoids) and contribute to the expected transition into personalized medicine.
We have initially optimized the production of SERS-encoded nanoparticles, employing chemical methods that allowed to achieve a wide variety of codes, complemented with antibodies for selective labeling of different cell types. 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 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 succeeded to formulate polymer hydrogel inks for 3D printing, leading to scaffolds where cells can be cultured in 3D. By employing SERS codes and 3D confocal microscopy, we demonstrated the possibility to monitor the initial stages of cancer cell aggregation into tumor-like aggregates, which provides information about parameters that can promote or inhibit this crucial event. On the other hand, working on purposely designed breast and skin cancer models, we were also able to watch in real time the dynamics of cancer cells leaving the tumor core and promoting metastatic events.
3D printed scaffolds could also be loaded with nanosensors, which 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. This technology was also used to evaluate the mechanism employed by certain types of tumors to escape the action of the immune system. By monitoring the presence of selected metabolites in the tumor microenvironment, it was found that healthy fibroblasts near the tumor can be recruited to provide essential biomolecules that allow cancer cell proliferation, while reprogramming nearby immune cells, so they do not act against the tumor. This result shows the power of SERS to explore biomolecular processes involved in cancer development.
We also demonstrated the possibility to print more realistic tumor models, comprising a dense tumor core with cancer cells, surrounded by a stromal compartment where fibroblasts and endothelial cells were present, to reproduce the real tumor microenvironment. With this realistic system, we additionally studied the effect of drugs, including the diffusion through the medium and how it correlates with the death of tumor cells.
The design of these models has been protected by an European patent application, aiming at the development of a platform for anticancer drug testing with no need for animal experimentation. With the support of an ERC proof-of-concept grant, the production of tumor models by 3D bioprinting has been automatized and we are in the process of validating high-throughput production, so it can be offered as a testing platform for pharma companies.
3D printed scaffolds could also be loaded with nanosensors, which 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. This technology was also used to evaluate the mechanism employed by certain types of tumors to escape the action of the immune system. By monitoring the presence of selected metabolites in the tumor microenvironment, it was found that healthy fibroblasts near the tumor can be recruited to provide essential biomolecules that allow cancer cell proliferation, while reprogramming nearby immune cells, so they do not act against the tumor. This result shows the power of SERS to explore biomolecular processes involved in cancer development.
We also demonstrated the possibility to print more realistic tumor models, comprising a dense tumor core with cancer cells, surrounded by a stromal compartment where fibroblasts and endothelial cells were present, to reproduce the real tumor microenvironment. With this realistic system, we additionally studied the effect of drugs, including the diffusion through the medium and how it correlates with the death of tumor cells.
The design of these models has been protected by an European patent application, aiming at the development of a platform for anticancer drug testing with no need for animal experimentation. With the support of an ERC proof-of-concept grant, the production of tumor models by 3D bioprinting has been automatized and we are in the process of validating high-throughput production, so it can be offered as a testing platform for pharma companies.
A new technology has been implemented for the production of realistic tumor models, containing nanosensors that allow continuous monitoring of relevant biomolecules in situ, during cancer development. The technology is based on 3D bioprinting combined with SERS detection of molecular analytes and bioimaging. By using nanoparticle-based substrates, the Raman scattering signals of targeted biomolecules can be largely amplified and monitored over time to reveal the behavior of cancer cells. This processes have allowed us to discover unknown relationships between cancer cells and the neighboring immune system, thereby offering solutions to cancers with poor prognosis. High-resolution imaging of tumors in 3D offers the possibility to monitor cancer evolution, as demonstrated for breast cancer and melanoma, where the onset of metastasis has been observed in real time.