Periodic Reporting for period 1 - MagDock (Advanced 3D in vitro models based on magnetically-driven docking of modular microscaffolds)
Reporting period: 2023-01-01 to 2024-06-30
Glioblastoma (GBM) represents one of the most malignant brain tumors., developing from complex biochemical interactions between malignant and non-malignant cells, with the latter playing a relevant role since the early phases of cancer progression. These cell-cell interactions result into a three-dimensional (3D) microenvironment, hardly obtainable in vitro due to its high complexity.
3D models mimicking in vivo cellular behavior provide relevant information on tumor development and response to novel pharmacological, physical, and immunological therapies. However, currently available techniques do not enable 3D co-culture system assembly and structuring on demand, and thus they do not allow a strikingly precise spatiotemporal control of the interactions among different cell types. To date, a generation of devices supporting activities of biomedical research laboratories and pharmaceutical companies, by enabling a straightforward assembly of different cell types into a biomimetic 3D co-culture system, is indeed missing. In this regard, the development of 3D co-culture systems recapitulating the GBM microenvironment would represent a valuable opportunity to investigate the unclear origins and causes of this tumor. Most importantly, such multicellular model mimicking the complexity of the GBM niche would enable performing high-throughput tests with innovative anticancer drugs, as well as with other therapies (e.g. antiangiogenic treatments) involving non-malignant cells (e.g. neuronal/glial progenitors, astrocytes, microglia, and endothelial cells) that support tumor growth.
Two-photon lithography (TPL) represents an innovative technology, which allows fast prototyping of 3D structures with nanometer resolution for many applications. This microfabrication approach is exploited to reproduce different 3D microenvironments, featured by several physio-pathologic conditions. In MagDock, we proposed a 3D multi-culture system on magnetic scaffold self-assembly, faithfully mimicking GBM microenvironment complexity (Figure 1). Specifically, modular magnetic structures bear GBM cells, neuronal cells, and endothelial cells. Tumor core will be obtained by culturing GBM cells inside a magnetic scaffold shaped like a great dodecahedron. Healthy cells are instead cultured on tetrahedron-shaped magnetic scaffolds, that assemble around the tumor core by interfacing to its complementary indentations and thanks to magnetic interactions. Finally, vascular-mimicking tubular structures, serving as scaffolds for endothelial cells, assemble on the external part of the 3D multicellular system to mimic the presence of the blood-brain barrier.
This proof-of-concept prototype has been exploited to perform tests on a representative chemotherapy drug, allowing the evaluation of anticancer effects on malignant cells and side-effects on non-malignant cells associated with the tumor.
3D models mimicking in vivo cellular behavior provide relevant information on tumor development and response to novel pharmacological, physical, and immunological therapies. However, currently available techniques do not enable 3D co-culture system assembly and structuring on demand, and thus they do not allow a strikingly precise spatiotemporal control of the interactions among different cell types. To date, a generation of devices supporting activities of biomedical research laboratories and pharmaceutical companies, by enabling a straightforward assembly of different cell types into a biomimetic 3D co-culture system, is indeed missing. In this regard, the development of 3D co-culture systems recapitulating the GBM microenvironment would represent a valuable opportunity to investigate the unclear origins and causes of this tumor. Most importantly, such multicellular model mimicking the complexity of the GBM niche would enable performing high-throughput tests with innovative anticancer drugs, as well as with other therapies (e.g. antiangiogenic treatments) involving non-malignant cells (e.g. neuronal/glial progenitors, astrocytes, microglia, and endothelial cells) that support tumor growth.
Two-photon lithography (TPL) represents an innovative technology, which allows fast prototyping of 3D structures with nanometer resolution for many applications. This microfabrication approach is exploited to reproduce different 3D microenvironments, featured by several physio-pathologic conditions. In MagDock, we proposed a 3D multi-culture system on magnetic scaffold self-assembly, faithfully mimicking GBM microenvironment complexity (Figure 1). Specifically, modular magnetic structures bear GBM cells, neuronal cells, and endothelial cells. Tumor core will be obtained by culturing GBM cells inside a magnetic scaffold shaped like a great dodecahedron. Healthy cells are instead cultured on tetrahedron-shaped magnetic scaffolds, that assemble around the tumor core by interfacing to its complementary indentations and thanks to magnetic interactions. Finally, vascular-mimicking tubular structures, serving as scaffolds for endothelial cells, assemble on the external part of the 3D multicellular system to mimic the presence of the blood-brain barrier.
This proof-of-concept prototype has been exploited to perform tests on a representative chemotherapy drug, allowing the evaluation of anticancer effects on malignant cells and side-effects on non-malignant cells associated with the tumor.
The microscaffolds used for the tumor core are represented by a great dodecahedron (GD) characterized by twenty small tetrahedron-shaped indentations, which represent the main docking stations of the assembly system. The microscaffolds used for culturing the healthy cell types are instead small tetrahedrons (Ts). Structures have been provided with magnetic features through the coating with a Ti-Ni-Ti triple-magnetic layer; the presence of an external magnet induces the movement of the 3D structures, enhancing the probability of their contact and assembly.
After demonstrating the expression of typical molecular markers of cultured cells, the assembly of the multicellular system mimicking GBM foci interfaced to non-malignant cells of the peritumoral niche was carried out by combining a GFP-U87 GBM cells-bearing GD with Ts seeded with hCMEC/D3 endothelial cells and hNSCs-derived neurons. The analyses revealed as the GBM cells are interfaced with the non-malignant cell types; iInterestingly, it is possible to observe a small group of GFP-U87 GBM cells that are disconnected from the main colony in the GD, and interfaced with the hCMEC/D3 cells: a clear hint of GBM cell migration phenomena.
The multicellular GBM niche model was used in combination with a real-scale 3D BBB system, realized in the framework of a previous ERC PoC (BBBhybrid, 832045).
The nutlin-3a (nut-3a) anticancer drug was selected as testing molecule, since previous evidence indicated selective anticancer effects toward GBM cells. The viability of malignant cells (GFP-U87 GBM cells) in GDs and of non-malignant cells (hCMEC/D3 brain endothelial cells and hNSCs-derived neurons) in Ts has been assessed in terms of cells positive for ethidium homodimer-1 (ethD-1, Figure 2a). Results showed a significantly higher % of ethD-1+ malignant cells (36.7 ± 3.6%) versus non-malignant cells (13.5 ± 1.5%; Figure 2b). Immunofluorescence analyses were then performed to investigate whether the increased cell death was specifically induced by nut-3a through the activation of the p53 apoptotic pathway (Figure 2c). Our findings report a significantly higher % of p53+ malignant cells (37.8 ± 13.4%) versus non-malignant cells (5.8 ± 2.9%, Figure 2d), suggesting that the higher % of GBM dead cells is associated with a higher expression of p53, which is known to be pharmacologically activated by nut-3a.
Overall, the described developed prototype allowed the screening of multiple features of a promising anticancer drug, nutlin-3a, that are fundamental for the treatment of brain cancer, such as the BBB crossing capabilities, apoptotic efficacy against GBM cells, and side effects on non-malignant brain cells.
After demonstrating the expression of typical molecular markers of cultured cells, the assembly of the multicellular system mimicking GBM foci interfaced to non-malignant cells of the peritumoral niche was carried out by combining a GFP-U87 GBM cells-bearing GD with Ts seeded with hCMEC/D3 endothelial cells and hNSCs-derived neurons. The analyses revealed as the GBM cells are interfaced with the non-malignant cell types; iInterestingly, it is possible to observe a small group of GFP-U87 GBM cells that are disconnected from the main colony in the GD, and interfaced with the hCMEC/D3 cells: a clear hint of GBM cell migration phenomena.
The multicellular GBM niche model was used in combination with a real-scale 3D BBB system, realized in the framework of a previous ERC PoC (BBBhybrid, 832045).
The nutlin-3a (nut-3a) anticancer drug was selected as testing molecule, since previous evidence indicated selective anticancer effects toward GBM cells. The viability of malignant cells (GFP-U87 GBM cells) in GDs and of non-malignant cells (hCMEC/D3 brain endothelial cells and hNSCs-derived neurons) in Ts has been assessed in terms of cells positive for ethidium homodimer-1 (ethD-1, Figure 2a). Results showed a significantly higher % of ethD-1+ malignant cells (36.7 ± 3.6%) versus non-malignant cells (13.5 ± 1.5%; Figure 2b). Immunofluorescence analyses were then performed to investigate whether the increased cell death was specifically induced by nut-3a through the activation of the p53 apoptotic pathway (Figure 2c). Our findings report a significantly higher % of p53+ malignant cells (37.8 ± 13.4%) versus non-malignant cells (5.8 ± 2.9%, Figure 2d), suggesting that the higher % of GBM dead cells is associated with a higher expression of p53, which is known to be pharmacologically activated by nut-3a.
Overall, the described developed prototype allowed the screening of multiple features of a promising anticancer drug, nutlin-3a, that are fundamental for the treatment of brain cancer, such as the BBB crossing capabilities, apoptotic efficacy against GBM cells, and side effects on non-malignant brain cells.
We have been able to generate non-degradable 3D prismatic-shaped magnetic scaffolds colonizable by cells. Furthermore, the shape-coding of the scaffolds allowed us to detect migration phenomena from one structure to another one, identified by confocal imaging of their assembly. Alternative assembly approaches reported in the literature, such as those ones exploiting magnetic iron oxide-loaded hydrogels, conversely do not allow for a real 3D assembly and shape-coding. Eventually, our approach differentiates from other magnetic-based co-culture methods that involve the cellular uptake of magnetic nanoparticles or beads, that are known to interfere with biological processes through different biochemical pathways, thus affecting the experimental outcome.
Our findings show as our brain-on-a-chip system represents an excellent biomimetic platform for testing drug delivery through biological membranes and the functional selectivity of the anticancer effects. The development of a 3D co-culture system recapitulating the GBM microenvironment, in principle, can be exploited not only for performing predictive tests on anticancer drugs, but it may also result into a versatile platform to test other therapeutic approaches (e.g. antiangiogenic treatments) acting on non-malignant cells (e.g. neuronal/glial progenitors, astrocytes, microglia, and endothelial cells) that support the tumor growth.
The prototype developed in MagDock will be part of a soon-to-be-launched start-up (provisionally named NABIS -NAnostructured on-demand 3D-printed BIostructureS-). Commercialization strategies aiming at technology valorization and ensuring fast-track access to the market have been implemented in the NABIS business plan elaborated at conclusion of the project.
Our findings show as our brain-on-a-chip system represents an excellent biomimetic platform for testing drug delivery through biological membranes and the functional selectivity of the anticancer effects. The development of a 3D co-culture system recapitulating the GBM microenvironment, in principle, can be exploited not only for performing predictive tests on anticancer drugs, but it may also result into a versatile platform to test other therapeutic approaches (e.g. antiangiogenic treatments) acting on non-malignant cells (e.g. neuronal/glial progenitors, astrocytes, microglia, and endothelial cells) that support the tumor growth.
The prototype developed in MagDock will be part of a soon-to-be-launched start-up (provisionally named NABIS -NAnostructured on-demand 3D-printed BIostructureS-). Commercialization strategies aiming at technology valorization and ensuring fast-track access to the market have been implemented in the NABIS business plan elaborated at conclusion of the project.