GLADIATOR by EU Research

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. 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 28

The main building blocks of an externally controllable molecular communications platform as proposed by GLADIATOR.

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. 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

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

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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. 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).

Ultrasound transducers for communication and power transfer (left), implantable sensor (middle) and electronics (right).

Induced Monitoring and Reprogramming NSCs: The Sensors and The Transducers 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.

Glioblastoma Multiforme (GBM) as growing bio-nanomachine networks (left) and diagram of the simulations of the molecular diffusion through the brain extracellular space (right).