GLADIATOR: Transforming Glioblastoma Care
We spoke to Professor Costas Pitris about GLADIATOR, a project that aims to revolutionise brain pathology diagnosis and treatment by using Molecular Communications systems. This theranostic solution, focused on glioblastoma, integrates autonomous molecular nanonetworks of engineered cells and innovative bio-electronics for personalised interventions, marking a paradigm shift in oncology research.
Brain tumours are characterized by a highly complex and heterogenous nature that varies significantly among patients and within different regions of the tumour itself. Glioblastoma multiforme is an aggressively fast-growing brain tumour with a bleak prognosis and a high likelihood of recurrence. By bridging life sciences, bio-nanotechnology, engineering, and information and communication technology (ICT) the interdisciplinary EU FET-Open project GLADIATOR aims to develop a theranostic (therapuetic+diagnostic) solution for the early detection and eradication of brain malignancies, such as glioblastoma multiforme. The project aims to provide clinicians with continuous, long-term, in vivo monitoring of cancer recurrence or metastasis by developing an implantable personalized and multifunctional platform.
presence of cancer and externally initiating the therapy.” explains Prof. Pitris.
The brain tumour reprogramming and monitoring platform consists of autologous, engineered, induced neural stem cells (iNSCs) that release or detect specialized exosomes (EXs) which act as natural nanoparticles or bionanomachines. The interaction with these “communication channels” within the biological environment represents a breakthrough theranostic intervention. “To make this clearer, imagine the cells of a brain tumour transmitting messages through vesicles. The vesicles are then detected by the neural stem cells which are genetically engineered to recognize the signal. The signal is measured by an implantable optoelectronic sensor which then transmits a message to an external unit. The external unit analyses this message and through a radio frequency (RF) antenna, activates the engineered therapeutic cells. These cells are genetically engineered to transmit messages in the form of reprogramming exosomes. These vesicles carry specific sequences of noncoding microRNA, targeting various tumour pathways. The reprogramming vesicles, when activated by the external RF signal, kill the tumour cells. In essence, we’ve developed a closed-loop system for continuous monitoring and treatment. The initial goal is to detect the presence or recurrence of cancer in cases when sensors are implanted into individuals who have undergone surgery and chemotherapy for brain tumours.” says Prof. Pitris.
Induced Monitoring and Reprogramming NSCs: The Sensors and The Transducers
Molecular Communication is a discipline inspired by ICT but in a biological environment. The project GLADIATOR uses molecular communication principles to understand the underlying cellular and sub-cellular processes which are modelled as interactive bio-nanomachines. The consortium plans to manage these processes by externally controlling diagnostic and therapeutic interventions. Externally controlled molecular communications enable the interrogation of implanted diagnostic cells to extract
GLADIATOR is creating the first working prototype of a clinically applicable, nanonetwork-based, Molecular Communications platform based on the conceptual framework of Externally Controllable Molecular Communications. This platform has the potential to significantly transform the management of brain malignancies by providing an autonomous system that integrates both diagnostic capabilities and reprogramming, i.e. therapeutic, interventions. Genetically engineered cells can sense the presence of cancer and offer reprogramming interventions that can halt the disease’s progression. This introduces a promising novel avenue for effective therapy.
information on the status of the disease (diagnostic) and manipulate the therapeutic cells to stop the disease’s progression (therapeutic). “The idea is to use molecular communications, specifically externally controllable molecular communications, to affect the way cells behave in the body. Molecular communication is the equivalent of telecommunication, but here, the transmitter and the receiver are actually cells within the body, and they communicate through molecules instead of electromagnetic waves. The idea behind GLADIATOR is that we could use such communication method to affect the behaviour of rationally engineered cells to fight cancer. The concept involves receiving molecular messages indicating the
The building blocks of the proposed externally controllable molecular communications platform are the sensor, detector, reader, controller, actuator, and transducer. The sensor and transducer are the cellular components of GLADIATOR. The in vitro and in vivo developments during GLADIATOR culminated in the formation of the sensor and transducer cells, namely the monitoring induced Neural Stem Cells (M-iNSCs) and the reprogramming induced Neural Stem Cells (R-iNSCs).
The M-iNSCs were engineered to convert the signals from the cancer cells, i.e. the reporting exosomes, into a readable fluorescent signal. The first step in creating the monitoring cells was obtaining high-quality induced neural stem cells (iNSCs), which were generated from human induced pluripotent stem cells. Non-labeled iNSCs were initially produced by Fraunhofer Institute of Biomedical Technology (FRAU) and sent to partners University of OULU (UOULU) and innovative SME EPOS Iasis R&D, Ltd (EPOS). Subsequently, GFP-tagged iNSCs were successfully produced from the parental stem cell line. Once the iNSCs were developed, FRAU focused on investigating cryopreservation protocols for the longterm storage of iNSC organoids, an essential component for their exploitation. Despite cryo-induced injury mechanisms, successful recovery was observed for all samples. The next goal was to achieve scalable and controllable organoid formation. Towards that end FRAU demonstrated suitable, automated, options with no biological disadvantages. EPOS contributed by using advanced nanobiotechnology and bioengineering to create biomimetic organoids with structural stability. They developed hybrid scaffolds by combining hydrogels and electrospun nanofibers, treated with brain extracellular matrix polymers. This innovation led to a
modular organoid that can be implanted into the brain. Currently, EPOS is working on a capsule for delivering clinical-grade organoids in vivo, making progress toward pre-clinical testing and final proof of concept.
In lab tests, the growth of the sensor cells (M-iNSCs) was observed in the presence of glioblastoma-derived cells. A special medium was used to grow the cells and their behaviour was observed with a time-lapse video under a fluorescent confocal microscope. The study revealed that fluorescent extracellular vesicles released by both sensor and glioblastoma cells can be tracked in vitro. The researchers at UOULU used an affinity-based chip platform to study the presence of certain molecules on the surface of the exosomes released by the glioblastoma cells. They revealed that these molecules were present in a portion of the exosomes captured on special chips. Additionally, they tested whether glioblastoma cells could release fluorescently labelled extracellular vesicles that attach to the iNSCs. The results showed that these vesicles were indeed present, both floating freely and attached to iNSCs.
The reprogramming induced neural stem cells (R-iNSCs) release a re-programming (therapeutic) agent following RF induction. Two variations of these cells were developed: one producing a toxic protein targeting glioblastoma cells, and the other expressing a therapeutic molecule (miRNA34a) under the control of specific gene promoters.
UOULU designed genetic constructs to monitor cell behaviour, extracellular vesicle production, and protein generation. Various RF exposure setups were tested to find the conditions that initiate the therapeutic response without harming the cells. EPOS aimed to overcome limitations in therapeutic reprogramming. They explored strategies like identifying RF-responsive promoters and developing an optimal reporting system for in-vivo monitoring.
Detector: Implantable Hybrid Optoelectronic Device
The implantable hybrid sensor is a device that combines light-emitting and lightdetecting components with cells that can detect specific biological markers. It excites the fluorescence of the M-iNSCs, detects the signal, and transmits it to an external central unit. The device includes various parts like micro light sources (μ-LEDs), micro light detectors (μ-PDs), and other components to process and transmit the signals. In this context, FRAU developed an innovative approach of powering the implant and receiving the signal data with a trans-cranial ultrasound-based system. In the initial stages, prototypes of these devices were created and tested in a collaboration between the University of Cyprus (UCY), FRAU and EPOS. They were designed to emit light at a specific wavelength to excite cell fluorescence and then detect the emitted signals from these cells. These prototypes were carefully tested in different in vitro conditions to ensure they worked effectively. The next step involved integrating biological structures to these sensors, i.e. scaffold membranes with M-iNSCs. These membranes act as a supportive environment for cell adhesion and growth. The team experimented with different types of membranes and found that 3D membranes worked better than 2D ones, especially when exposed to ultraviolet light. This step is crucial as it prepares the sensor for interaction with living cells. The researchers then explored methods of making the sensor smaller and more flexible. They designed miniature sensors that could be implanted in living organisms with devices that were more adaptable and suitable for real-world applications. To prove that these sensors could work inside a living system, they conducted experiments where they implanted the sensors and monitored them over time in mice. The goal was to see how the fluorescence signals changed, indicating potential tumour growth. The results showed that the sensors could effectively monitor changes within the living organism.
Reader: External wearable patch that provides power and communication
The patch is used for trans-cranial power transfer to and communication with an implantable device. It uses ultrasound-based methods and has gone through various stages of development. The patch comprises two main modes of operation: power transfer to the implant (continuous wave) and retrieving information transfer signals (pulse echo). The continuous wave power transfer mode generates continuous wave signals with adjustable voltage
Actuator: External Radiofrequency Induction System
The external radiofrequency system induces a therapeutic response from the reprogramming cells (R-iNSCs). The UOULY, Norwegian University of Science and Technology (NTNU), EPOS, and UCY teams first conducted radiofrequency experiments on cells using antennas designed for use in the incubation chambers. The EPOS and UCY teams performed experiments in mice using
“The initial goal is to detect the presence or recurrence of cancer in cases where sensors are implanted into individuals who have undergone surgery and chemotherapy for brain tumours.”
that provide the power to operate the implant. The pulse-echo mode generates burst signals for information retrieval with various adjustable parameters. The researchers used simulationbased models to design ultrasound transducers for the hybrid sensor and the external patch. The size of the transducer was chosen based on energy requirements. Power transfer experiments were conducted to see how well the ultrasound transfers power. The impact of factors like frequency, distance, and voltage were investigated. The FRAU and UCY research teams designed and fabricated the patch and implantable electronics.
a ceramic patch antenna with circular polarization in a well-designed exposure chamber. A wearable antenna system was also designed to deliver the radiofrequency signal to the cells. The UCY research team designed an antenna for the head that needs to be small, light, flexible, and emit signals in a specific frequency range. They used metamaterials to make the antenna smaller while maintaining effectiveness. Wearable antennas were designed for human, and mouse applications, considering the specific requirements in each case.
Central Unit: Closing the loop
The consortium is currently working on developing a controller, that will close the loop in the external control of the molecular communications platform. To deliver the proposed platform, NTNU, the Waterford Institute of Technology (WIT) and Osaka University (OU) formulated an end-toend simulator to explore the molecular communication processes and develop models that will be critical in creating a complete and effective theranostic platform.
In Vivo ExperimentsThe Orthotopic Glioblastoma Multiforme Tumour Model in Mice
The researchers at EPOS used orthotopic implantation to introduce tumour cells with high oncogenic potential into mice. This method provides a unique tool for the development of in vivo models mimicking human tumour evolution and clinical characteristics. The tumour cells used were U87-MG cells. EPOS studied the tumorigenicity of these cells, and how blood vessel formation correlates with tumour growth, animal behaviour, and overall neurological function. These findings helped to determine at which point in time during the experiment to intervene effectively. The orthotopic implantation process involves anesthetizing the mice, placing them in a specialized apparatus, and injecting tumour cells into the mice’s brains. A control injection with a harmless substance is done for comparison.
In the in vivo studies, three groups of mice were involved: Group 1(Control) had no induced neural stem cells (iNSCs) and no radiofrequency (RF) exposure, Group 2 had iNSCs without RF exposure, and Group 3 had iNSCs with RF
exposure. The Milabs Optical Module was used for imaging the mice before radiofrequency exposure and every 48 hours until they expired. An RF exposure chamber was designed, where the mouse head was exposed to ~900 MHz RF for 30 minutes one-week post-implantation and once more a day later. Evaluating the response to RF, researchers found no notable difference in tumour size, as measured using fluorescence, between the control and non-RF-exposed groups. However there were statistically significant differences in total tumour fluorescence intensity and survival when mice implanted with the iNSCs were exposed to RF. Mice without RF exposure expired by day 19, while those with RF exposure had a slightly longer, by 2.5 times, survival.
The significant achievements of the GLADIATOR project have been made possible with the contribution of leading scientific institutions including the University of Oulu (UOULU), Oulu, Finland, EPOS-lasis, Ltd (EPOS), Nicosia, Cyprus, the Fraunhofer Institute for Biomedical Technology (FRAU), Sulzbach, Germany, the Waterford Institute of Technology (WIT), Waterford, Ireland, the Norwegian University of Science and Technology (NTNU), Trondheim, Norway, Osaka University (OU), Osaka, Japan, and the University of Cyprus (UCY), Nicosia, Cyprus.
Once completed and clinically available, the proposed platform is expected to have a substantial societal impact by enhancing cancer management, improving patient prognosis, minimizing cancer recurrences, reducing drug toxicity, and contributing to overall health system efficiency. In the future, the results of this project could extend life expectancy, enhance productivity, and alleviate the burden on healthcare systems.
GLADIATOR
A paradigm shift in Oncology Research via externally controllable molecular communications for “bio-nanomachine diagnostics”
Project Objectives
The GLADIATOR multidisciplinary consortium proposes a vanguard and comprehensive theranostic (therapeutic+diagnostic) solution for the management of brain pathologies based on an externally controllable molecular communication platform with rationally engineered cells and implantable and external hardware to monitor and treat brain tumours.
Project Funding
This project has received funding from the European Union’s Horizon 2020 programme under Grant Agreement No. 828837.
Project Partners
Key Players: University of Cyprus • University of Oulu • Fraunhofer Institute for Biomedical Engineering • Waterford Institute of Technology • Norges teknisknaturvitenskapelige universitet • Osaka University • EPOS-Iasis, R&D https://www.fet-gladiator.eu/index.php/ about/key-players
Contact Details
Project Coordinator, Professor Contantinos Pitris
Department of Electrical and Computer Engineering
School of Engineering
University of Cyprus 75 Kallipoleos street P.O.Box 20537
CY-1678 Nicosia
Cyprus
T: +357 22 892297
E: cpitris@ucy.ac.cy
W: https://www.fet-gladiator.eu
W: http://www.eng.ucy.ac.cy/cpitris/
Professor Contantinos Pitris
Professor Contantinos Pitris is a Professor at the KIOS Research and Innovation Center of Excellence at the Department of Electrical and Computer Engineering of the University of Cyprus. His research interests include Lasers, Biomedical Optics, Optical Imaging, Optical Coherence Tomography, Fluorescence and Raman Spectroscopy, Medical Instrumentation, Biomedical Signals and Systems, Medical Diagnostics.
