Periodic Reporting for period 3 - GLADIATOR (Next-generation theranostics of brain pathologies with autonomous externally controllable nanonetworks: a trans-disciplinary approach with bio-nanodevice interfaces) Reporting period: 2021-01-01 to 2021-12-31 Summary of the context and overall objectives of the project Brain pathologies are highly complex. Despite recent progress, their prognosis is grim, defining a high societal challenge. Bridging life sciences, bio-nanotechnology, engineering and ICT, GLADIATOR promises a vanguard and comprehensive theranostic (therapeutic+diagnostic) solution for brain malignancies. GLADIATOR will provide, for the first time, a working prototype of a complete, autonomous and clinically applicable, nanonetwork-based, molecular communications system based on the conceptual framework of Externally Controllable Molecular Communications (ECMC). Using Glioblastoma Multiforme (GBM) tumors, the most detrimental of brain pathologies, as a proof-of-concept case, GLADIATOR will implement a platform of cell-based and electronic components, consisting of1. Implantable autologous cell organoids, consisting of engineered induced neural stem cells (iNSCs), which will release specifically designed exosomal vesicles, acting as bio-nano-machines, delivering reprogramming (therapeutic) miRNAs and building nano-networks. Interfering with the underlying biological environment, they will provide a revolutionary intervention both killing the tumor cells but also reducing their aggressiveness and recurrence. 2. A hybrid bio-electronic interface, consisting of coupled external and implantable devices, which will enable communication channels with fluorescent bio-nano-machines, released by the modified cancer cells, via micro-optoelectronic sensors. 3. A wireless ECMC network integrating the cellular, sub-cellular and electronic components. This system will autonomously monitor the spatiotemporal tumor evolution and recurrence and generate, on demand, appropriate reprogramming interventions, by increasing iNSC renewal and multiplication via external radiofrequency stimulation. GLADIATOR establishes the feasibility and innovation potential towards a far-reaching transformation in the investigation and management of complex malignancies and potentially other major central nervous system pathologies. It also promotes the emerging supra-disciplines of “bio-nano-machine diagnostics” and a profound shift towards “nano-network therapeutics” which lay the grounds for future autonomous, closed-loop, externally controllable, micro- or nano- scale devices for disease management. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first two years of the project, the consortium partners have worked towards the biological and technological goals of GLADIATOR:1. The University of Oulu (UOULU) has constructed a library of gene constructs and subsequently genetically engineered glioblastoma cells to produce and release exosomes (EX), delivering sensing signatures, as the first set of optically active bio-nanomachines. They have also showed that radiofrequency exposure induces genetically-modified fluorophore expression in iNSC. To further expand the understanding of these RF effects, EPOS and the University of Cyprus (UCY) are proceeding with a multi-parametric study to further elucidate the effect and provide a system recapitulating the in vivo situation. 2. For the generation of organoids, EPOS-Iasis, Ltd (EPOS) has investigated the direct induction of neural stem cells (iNSC) from readily available human fibroblasts from the Fraunhofer Institute for Biomedical Technology (FRAU) biobank. At the same time, FRAU has provided the consortium with iNSC derived from readily available pluripotent stem cells (iPSCs), and expanded in organoid form. A new technology for the cryopreservation and re-expansion of such organoid structures, has been established for the first time. EPOS is also investigating hybrid scaffolds, combining the high surface area, morphology and porosity of electrospun fibers with the three-dimensional structure and swellable properties of hydrogel matrices in a single amalgamated 3-D structure, better mimics the mechanical properties and functions of the brain ECM.3. To facilitate the understanding and control of the molecular communications network, the Waterford Institute of Technology (WIT), the Norwegian University of Science and Technology (NTNU) and the Osaka University (OU) groups have been developing theoretical molecular communication models to investigate the actions of the EX transmitters, the EX diffusion and binding to the receivers, and is finally integrating all models to create an end-to-end channel capacity and delay model for molecular communication via EXs.4. Parts of the electronic implantable and wearable components of the project are being developed by UCY and FRAU. The partners have performed thorough modelling and optimization of the hardware. Subsequently, they begun the construction of the optoelectronics for the implantable hybrid sensing devices and the ultrasound-based communication and power transfer hardware for the external wearable devices (patches). The design and experimentation for the wearable, met-material, antenna for RF excitation of the implantable cells is in progress. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The GLADIATOR consortium is already producing original research results beyond the state of the art, demonstrating1. Formation of the cellular components of the proposed platform with a complete characterization of transdifferentiated cells and protocol optimization, comparing the iNSC derived from iPSC and assessment of transfection efficacy in transdifferentiated iNSCs vs iPSC derived. 2. Experiments to validate the effects of RF exposure on cells, and to study RF effects on the expression of fluorescent proteins under the control of various promoters. Increases in proliferation as well as protein production have already been demonstrated.3. A sustainable three-dimensional (3D) scaffold comprised of biocompatible hydrogel and of electrospun nanofibers, as the technological embodiment of 3D GBM tumor constructs and the proposed organoids. 4. The building blocks of a theoretical communication model, which include exosomal release distribution and binding, in an expanded molecular communications modelling platform. 5. The initial designs for novel technologies for ultrasound-based transcranial power transfer and passive communication schemes, which are currently being tested experimentally, as well as considering various designs of metasurfaces for transcranial RF transmission.When the proposed platform is completed and becomes clinically available, it is expected to have a significant societal impact. The advances in cancer management, enabled by the innovations in GLADIATOR, will improve patient prognosis and prolong survival by minimizing recurrences and reducing drug toxicity. Improved health, extended life expectancy and productivity, reduced sick-leaves, shorter hospitalizations, reduced return visits, less personnel and caregiver involvement will also have a positive effect on the already overstrained Health Care Systems. The ground-breaking biological and nanotechnology-based innovations of GLADIATOR, are also expected to have significant economic impact since they can enter into significant market segments. For example, the microelectronic medical implants and micro-sensors markets were valued at $35B in 2016 and expected to grow to $56B by 2021. The Point of Care (PoC) diagnosis market was valued at $10.3B in 2016 growing to $33.7B by 2025. GLADIATOR can lead to high-gain innovations that can have a long-lasting positive impact on Europe’s science and industry, with benefits proportional to the associated high-risks of the project.