Workshop on Advanced Radiotherapies and Diagnostic in Oncology

12 mar 2026, 09:00 → 13 mar 2026, 19:00 Europe/Rome

Auditorium (Edificio A) (CNR - Area della Ricerca di Pisa)

Cancer care remains one of the most complex challenges for modern healthcare systems. While radiotherapy continues to play a central role in the treatment of more than half of all cancer patients, increasing clinical complexity and expanding biological knowledge are driving the need for new therapeutic paradigms. Advanced radiation delivery techniques, innovative diagnostic tools, novel radiopharmaceuticals and radiotracers are essential to further improve cancer treatment precision, reduce toxicity, and enable truly personalized oncological care. Among advanced radiation delivery techniques, the possibility of exploiting Ultra High Dose Rate and the FLASH effect, has attracted considerable interest opening new perspectives for the treatment of radio-resistant tumors, potentially shortening treatment times, and improving the overall sustainability of healthcare systems. Other modalities are also emerging, like the mini and micro-beam approach that could provide standalone-alone effective solutions or could be combined with the sparing effect of UHDR beams and FLASH.

Within this evolving landscape, the Spoke 1 – Advanced Radiotherapies and Diagnostics in Oncology of the Tuscany Health Ecosystem has fostered a broad, multidisciplinary collaboration among Tuscan research centers, hospitals, and technology developers to close the gap between lab and clinics, also enabling leaps towards technology readiness and industry engagement.

The workshop “Advanced Radiotherapies and Diagnostics in Oncology” is conceived as a platform to explore future developments sparked by this extensive regional collaboration. By bringing together physicists, biologists, engineers, clinicians, and industry partners, the event highlights the value of integrated research models in advancing and translating emerging oncological technologies.

The central aim of the meeting is to engage national and international experts in a forward-looking discussion on new research directions, emerging technologies, and opportunities for international collaboration beyond the Italian PNRR framework. The workshop seeks to identify shared priorities and potential joint initiatives capable of accelerating innovation and clinical impact at both European and global levels.

Hosted in Pisa at the CNR Research Area, the event marks the conclusion of the PNRR-funded activities of the Spoke 1 project, while simultaneously serving as a starting point for new scientific trajectories. By strengthening the link between advanced research and clinical practice through innovation, the workshop underscores Tuscany’s ambition to position itself as a reference hub for next-generation radiotherapy and oncological diagnostics, open to international partnerships and long-term collaborative growth. 

A visit will be organized during the workshop to the Intense Laser Irradiation Laboratory of the National Institute of Optics that operates inside the CNR campus in Pisa.

The workshop topics include:

• Innovative Approaches to Oncologic Radiotherapy: Radiation Sources
• Radiobiological Effects of Ionizing Radiation: Mechanisms, Modelling, and Experimental Validation
• High dose rate and FLASH Radiotherapy: from Preclinical to Clinical
• Advanced Diagnostics of Radiation Deposition and Conformality
• Tumor-Targeted Radionuclides, Radiotracers and Radiopharmaceuticals

 

For further information about the "THE-Spoke1" project activities and participants check the pages of the previous events:

• 3° INCONTRO SULL’ECOSISTEMA TOSCANO PER L’INNOVAZIONE - SPOKE 1
• 4° INCONTRO SULL’ECOSISTEMA TOSCANO PER L’INNOVAZIONE - SPOKE 1

 

The event is kindly sponsored by:

CNR-Informativa Eventi.pdf

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giovedì 12 marzo

09:30 → 10:00 Registration

10:00 → 12:30 KEYNOTES SESSION 1

10:00 Innovating Radiotherapy: The Tuscany Health Ecosystem and Beyond

The Tuscany Health Ecosystem is fostering a coordinated innovation pipeline in radiotherapy, spanning fundamental research to high–technology readiness level (TRL) deployment. Core activities include advanced radiation source development including disruptive laser-driven sources, tailored for precision and ultra-high dose rate applications, dedicated studies on FLASH effect to elucidate radiobiological mechanisms, optimized dosimetry, and translation studies into clinically viable solutions.
Preclinical research programs integrate physics, biology, and medical sciences to validate novel treatment paradigms and accelerate translational pathways. Complementing these efforts, the development of innovative radiopharmaceuticals expands the therapeutic landscape toward targeted and combined modality approaches. To ensure rapid impact, cascade calls involving clinical, industrial and complementary research capabilities are supporting high-TRL technology maturation, clinical validation, and industrial uptake.
An overview of the project will be given with attention on the ongoing effort towards an integrated strategy to further strengthen the scope, by the promotion of newly established spin-offs and public–private partnerships, cooperation with similar international initiatives in the European research landscape, promoting Tuscany as a competitive hub for radiotherapy innovation at the European and global level.

Speaker: Leonida Antonio GIZZI (Istituto Nazionale di Ottica, Pisa)

Workshop_Pisa_THE_2026_Gizzi.pptx

10:30 Radiation Oncology: Clinical Aims and Technological Horizons

Radiation oncology has benefited from the digital era by improving its performance in terms of irradiation precision and selectivity. This has enabled dose escalation to regions of interest (ROIs) and dose reduction to organs at risk (OARs), supported by the availability of Volumetric Modulated Arc Therapy (VMAT). The introduction of proton beams, carbon ions (hadrons), and boron neutron capture therapy (BNCT) has further expanded the therapeutic armamentarium, although these technologies remain challenging to disseminate widely.
A major contributor to improved clinical outcomes has been the concomitant use of cytotoxic antineoplastic agents (cisplatin, 5-fluorouracil, mitomycin, gemcitabine), which enhance DNA damage. More recently, monoclonal antibody therapies and immunotherapeutic agents have raised new questions regarding their integration with radiotherapy. High doses per fraction, exceeding 8 Gy, have been shown to elicit immunological effects and to induce cytotoxic activity at sites distant from the irradiated volume (the abscopal effect).
Accordingly, treatment strategies initially evolved through modification of target volumes and the achievement of very high doses; subsequently, attention shifted to the type of radiation employed, leveraging higher linear energy transfer (LET) and biological effective dose (BED).
More recently, research has focused on the biological effects of dose-rate modulation, driven by growing evidence that very high dose-rate irradiation can selectively damage tumor cells (the FLASH effect). The development of very high-energy electron (VHEE) beams enables replication of the FLASH effect at depths comparable to those achieved with megavoltage (MV) photon dose deposition.
Finally, radiation oncology will need to engage with emerging pharmacological evidence and the field of network medicine, marked by the decline of the “one disease–one target–one drug” paradigm. This model is being replaced by the recognition that each disease is characterized by “disease modules” composed of multiple targets shared across different diseases, which may respond to the effects of multiple drugs.

Speaker: Dr. Maurizio PORTALURI (ASST Papa Giovanni XXIII Hospital, Bergamo)

2_cnr_ino_slide formato sfondo blu_PORTALURI_2026.pdf

2_cnr_ino_slide formato sfondo blu_PORTALURI_2026.pptx

11:00 Laser-Driven Particle Acceleration in Oncology: Technological Progress, Radiobiological Exploration, and Translational Perspectives

Laser-driven particle acceleration represents an emerging and potentially transformative technology in radiation oncology. At the Extreme Light Infrastructure (ELI), substantial advances over the past two years have led to improved beam stability, higher repetition rates, enhanced spectral control, and more reproducible delivery conditions. These developments have enabled, for the first time, controlled and repeatable irradiation experiments in preclinical biological models. The distinctive physical characteristics of laser based neutrons and laser-accelerated protons and electrons - including ultrashort pulse durations and ultra-high instantaneous dose rates a nd extreme small beam size - create irradiation conditions that differ fundamentally from those produced by conventional cyclotron- or LINAC-based systems. Recent studies have explored biological endpoints such as DNA double-strand break induction (γH2AX), apoptosis, clonogenic survival, and organism-level responses in both in vitro and in vivo systems. However, important challenges remain. In particular, absolute dosimetry under ultrashort-pulse, high-fluence conditions is not yet fully established, and uncertainties in dose quantification, spectral distribution, and shot-to-shot variability currently limit definitive biological interpretation. While comparative investigations with conventionally accelerated beams are ongoing, these technical constraints preclude firm conclusions regarding relative biological effectiveness or potential dose-rate–dependent modulation effects. Nevertheless, the recent improvements in accelerator performance have made meaningful radiobiological experimentation feasible and have generated a growing body of preliminary data. Rather than demonstrating superiority over conventional modalities at this stage, laser-driven particle beams should be regarded as a promising experimental platform for probing new irradiation regimes and testing hypotheses that extend beyond current accelerator capabilities. Further progress in dosimetric standardization, beam monitoring, and reproducibility will enable to translate these experimental advances into robust comparative radiobiology and, ultimately, to assess clinical relevance. The field is thus transitioning from technological proof-of-concept toward structured biological validation, marking an important step in the development of next-generation radiotherapy concepts.

Speaker: Prof. Katalin HIDEGHETY (Department of Oncotherapy, University of Szeged)

AdvancedRT_WS_pisa2026.pptx

11:30 Preclinical experiments with high dose rate laser plasma electron accelerators at ELI Beamlines

Laser plasma electron accelerators can produce very high energy electron beams up to GeV level in <100 m2 size experimental rooms. This is possible since plasma can sustain 1000x stronger accelerating gradients than conventional radio-frequency linear accelerators. At ELI Beamlines user facility the L3-HAPLS laser has been used in the ELBA beamline to generate electron beams up to 500 MeV at 3.3 Hz repetition rate. These electron beams are available to users for preclinical experiments. The peak dose rate of these beams has been estimated by Monte Carlo simulations with the code FLUKA. Dosimetry has been performed with Gafchromic films.

Speaker: Dr. Gabriele Maria GRITTANI (ELI Beamlines)

1130_Grittani PISA 2026.pdf

12:00 Radioprotection in Space, and Molecular Mechanisms Underpinning Proteinogenic Radiation Injuries

Exposure to ionizing radiation presents one risk factor in human space flight. To protect astronauts on future long duration missions better, we have to address three scientific frontiers, namely (i) elucidation of the molecular damage mechanisms, (ii) identification of tissue specific responses and (iii) rationalizing our understanding of radioprotectant molecules. Similarly, predictive markers for genotype specific responses still lack for ideal future classification. By assessing classification into radiosensitivity (acute response) or radiosusceptibility (cancer onset likelihood) or radiodegeneration (aging effects) across over 11,000 in-vitro and in-vivo studies, we present an ontology of key terms and one example of a mechanistic damage cascade that hinges on chemical changes in proteins. This we can call “molecular radiation injuries”. A brief overview of all possible such molecular radiation injuries, with focus on oxidation-induced protein alterations, is given.

Speaker: Alexandra WALDHERR

12:30 → 12:45 Workshop Group Photo (All Partecipants)

12:45 → 14:00 Lunch break

14:00 → 16:00 KEYNOTES SESSION 2

14:00 Advances in Radiotherapy: From Established Standards to Emerging Physical and Clinical Frontiers

Radiotherapy has undergone profound technological and conceptual evolution over the past decades, becoming increasingly precise, image-guided, and patient-centric. This presentation provides a comprehensive overview of the current state of the art in radiotherapy from a clinical-physics perspective, highlighting how established standards are continuously refined through advances in medical physics, imaging integration, and delivery technologies. Finally, we will briefly touch upon how emerging radiation sources may support the implementation of novel irradiation paradigms

Speaker: Dr. Damiano DEL SARTO (Casa di Cura Privata San Rossore)

slide_delsarto_cnr_marzo.pptx

14:30 SOD Inhibition as Modulator of Mediated Radioprotection in Conventional and UHDR Irradiation

Ionizing radiation triggers a sequence of physical, physico-chemical and chemical events from
femtoseconds to milliseconds before entering a biological response window that extends from
seconds to days and years. Within this cascade, superoxide dismutases (SODs) in living cells critically regulate the conversion of superoxide to hydrogen peroxide (H2O2), thereby shaping downstream redox signaling and damage responses. We investigated whether transient pharmacological inhibition of SOD1 prior to irradiation modulated radioprotection under conventional (CONV) and ultra-high dose rate (UHDR) modalities both in vitro and in a in vivo model. Short-term SOD1 inhibition did not impair viability in healthy (HaCaT, 16HBE) and cancer (SAS, SCC25) cell lines, and allowed for enzymatic recovery after drug removal. In vitro irradiation studies using electrons (CONVand UHDR) showed a FLASH-like survival benefit in healthy cells under UHDR, while cancer cells exhibited no protection. Importantly, SOD1 inhibition induced protection in healthy cells also under CONV (electrons, X-Rays) irradiation mode, while cancer cells exhibited no protection. Additionally, in a CAM in vivo model, SOD1 inhibition enhanced embryo survival after both dose rates, without reducing tumor control. These data support SOD1 inhibition as a strategy to modulate early H₂O₂ dynamics, potentially influencing how initial chemical events propagate into subsequent biological responses, and extending FLASH-like radioprotection beyond UHDR.

Speaker: Dr. Francesca PAGLIARI (DKFZ)

Pisa_Pagliari_2026_confidential_new.pptx

Pisa_Pagliari_2026_general.pdf

15:00 From ultrafast laser–plasma radiation sources to clinical impact: radiobiology and cancer research at the Extreme Light Infrastructure

Ultrahigh-intensity laser systems now enable the generation of particle beams with instantaneous dose rates reaching 10⁷–10¹² Gy/s and pulse durations down to the femtosecond regime. The Extreme Light Infrastructure (ELI) is emerging as a unique open-access platform where such laser-driven protons, ions, electrons, and neutrons can be systematically investigated for radiobiology and medical applications.
These sources provide unprecedented experimental conditions to probe radiation–matter interactions at ultrafast timescales and extreme dose rates, offering potential insights into radiochemical dynamics, DNA damage formation, and FLASH-like biological effects. However, realizing their therapeutic relevance requires rigorous dosimetry, standardized irradiation protocols, and reproducible biological models across facilities.
We present a coordinated roadmap for advancing ultrafast laser-driven radiation biology at ELI [1]. The strategy integrates cross-facility harmonization, dedicated instrumentation development, and structured collaboration between laser physicists, radiation biologists, medical physicists, and clinicians. Preparatory experiments at users’ home laboratories and systematic benchmarking are identified as essential steps toward clinical validation.
By consolidating infrastructure, methodology, and interdisciplinary collaboration, ELI aims to provide the scientific foundation necessary to evaluate and potentially translate ultrafast laser-driven radiation into next-generation therapeutic modalities.

Speaker: Dr. Daniele MARGARONE (ELI Beamlines)

1500_Margarone_Radiotherapy-workshop_Pisa.pptx

15:30 High Intensity X-ray Microbeams for the Treatment of Cancer with a Compact High-Power X-ray Tube

Speaker: Dr. Liam DAY (Technical University of Munich)

2026-03-12_ilil_workshop.pptx

16:00 → 16:30 Coffee break

16:30 → 18:30 KEYNOTES SESSION 3

16:30 FLASH Radiotherapy Induces Unique Early Immuno-Transcriptomic Responses Despite Iso-Efficacy with Conventional Dose Rates in a Murine Model of Triple-Negative Breast Cancer

While radiotherapy is a key component in the management of more than 80% of breast cancer,
the dose required to achieve a high probability of tumour control can overlap with the rates of
radiation-induced normal tissue side effects, hindering effective treatment. FLASH-RT, delivered
at ultra-high dose rates, was shown to generate comparable anti-tumour effects compared to
conventional dose rate RT (CONV-RT), but spared healthy tissues from side effects. This novel
technique could offer the potential of dose escalation to treat aggressive tumour subtypes such
as triple negative breast cancer (TNBC).
In the present study, we investigated TNBC responses to FLASH-RT across gene transcription,
immune infiltration, and anti-tumour efficacy. Using subcutaneous and orthotopic 4T1 or
orthotopic MDA-MB-231 murine TNBC models, female mice received single (14 or 20 Gy) or
fractionated (5×5.2 or 10×3 Gy) doses of either CONV-RT (0.2 Gy/s) or FLASH-RT (7.7×10⁶ Gy/s).
Tumour growth was monitored in all models, and 4T1 tumour samples were collected 24 hours,
5 days, and 2 weeks post-irradiation for bulk RNA sequencing, flow cytometry, and histology.
All regimens demonstrated iso-efficacy between FLASH and CONV-RT in delaying tumour growth
and prolonging survival. RNA sequencing revealed that, 24 hours post-irradiation, FLASH-RT
upregulated genes associated with lymphoid cell differentiation, activation, recruitment, and
anti-viral/innate immune responses compared to CONV-RT. However, histology and flow
cytometry analysis showed no differences in immune cell infiltration between modalities. We
hypothesize that FLASH-RT’s enhanced expression of immunoregulatory genes observed 24h
post-irradiation may underlie this discrepancy.
In summary, while FLASH- and CONV-RT are equally effective in delaying tumour growth and
modulating the immune response in murine TNBC, transcriptomic analysis uncovered distinct
immunomodulatory profiles, suggesting nuanced mechanisms that warrant further investigation.

Speaker: Adrien ARRIGO (University of Antwerp (BE))

17:00 Dosimetry and radiobiology of laser-driven ion beams

Laser-driven ion acceleration via ultra-intense laser–matter interaction has rapidly progressed in recent years, demonstrating the capability to generate high-current, short-pulse ion beams with unique temporal and spectral characteristics. These beams, inherently different from those produced by conventional accelerators, open new perspectives for multidisciplinary applications, particularly in radiation therapy research. However, their broad energy spectra, shot-to-shot fluctuations, ultra-high dose rates, and pulsed time structure pose significant challenges for beam transport, dosimetry, and radiobiological assessment.
At ELI Beamlines, the laser-driven ion beamline ELIMAIA has been developed to provide controlled and selectable proton beams for user applications. Within this framework, the ELIMED initiative, a collaboration with Istituto Nazionale di Fisica Nucleare, focuses on the realization of a dedicated beam transport and dosimetric section tailored to medical and radiobiological studies.
This presentation will first review the present status of laser-driven ion acceleration and beam transport solutions, highlighting the technological strategies adopted to handle spectral selection, beam shaping, and stability. Particular emphasis will be devoted to the development and commissioning of the ELIMED dosimetric chain, designed to operate under high instantaneous dose-rate conditions. The system integrates online and absolute dosimeters, including secondary emission monitors, multi-gap ionization chambers, radiochromic films, and a dedicated Faraday cup, enabling accurate shot-by-shot dose monitoring.
Results obtained during the first commissioning campaigns with a ~20 MeV laser-driven proton beam will be discussed, demonstrating dose measurements in the range of tens of mGy per shot with uncertainties below 10%. The reliability of the dosimetric system enabled the first radiobiological irradiations at ELI Beamlines, where cell cultures were exposed to intense pulsed proton beams. Initial radiobiological investigations, aimed at assessing cellular response under ultra-high dose-rate conditions, will be presented and discussed in the context of both conventional and emerging FLASH radiotherapy research.
The upcoming perspectived in Italian laser driven beamlines in the field of the realisation of new multidisciplinary beamlines, will be also discussed icluding the INO-CNR (Pisa, IT) and the ucoming INFN I-LUCE facility (Catania, IT).

Speaker: Dr. G. A. Pablo CIRRONE (INFN)

CirronePisaWorkshop2026.pptx

17:30 Differential biological response of cell cultures to single-pulse femtosecond and picosecond radiation at mean dose-rates between 10^10 and 10^13 Gy/s

We have recently developed novel irradiation platforms capable of delivering multi-Gy doses in a single pulse with durations ranging from 20 picosecond down to 150 femtoseconds (mean dose-rates up to 10^13 Gy/s). Irradiation of a series of normal and tumoral cell lines, in both 2D and 3D geometries, consistently indicate a significantly different response when compared to conventional and FLASH-like dose-rates, which promise significant benefits for radiotherapeutic applications. In this talk, we will review our current results in the area and discuss future steps towards in-vivo studies.

Speaker: Hannah MAGUIRE (Queen's University Belfast)

Hannah_Maguire_CNR.pptx

18:00 kHz laser-driven electron beams for pre-clinical applications

The extremely high electric fields sustained by plasma make the Laser Wakefield Acceleration
(LWFA) the most compact technique to generate very highly relativistic electron beams in the
MeV-to-GeV regime. However, the limited repetition rate and low efficiency of this technology
has, to date, prevented people from unleashing its full potential as a unique source for basic
research, biomedical applications and high flux sources of secondary radiations as hard X-rays. In recent years a new direction emerged showing the possibility to accelerate electron beams at 1 kHz repetition rate. We previously reported the acceleration of collimated, quasi-monoenergetic electron beams with record energies driven by a 1 kHz repetition rate, < 3 TW power-scalable OPCPA laser system. In this talk, I will show the actual status of the ALFA beamline for the acceleration of kHz beams that is nowadays open to users worldwide, offering on-demand in-air irradiations with high average dose rate (up to Gy/sec). The in-air beam profiling, pointing and dose rate characterization will be presented, highlighting the importance of the stability and the precision of delivering the electron beams, requested for medical applications such as radiotherapy treatments.

Speaker: Carlo Maria LAZZARINI (ELI Beamlines Facility)

Lazzarini_Pisa2026_kHz laser-driven electron beams for pre-clinical applications.pdf

venerdì 13 marzo

08:50 → 09:00 Welcome and Opening Remarks

09:00 → 09:30 SESSION 1: INNOVATIVE APPROACHES TO ONCOLOGIC RADIOTHERAPY: RADIATION SOURCES

09:00 WP1-Low-Energy UHDR Electron Beams at CPFR: Advancing Toward Clinical Use

Speaker: Prof. Maria Giuseppina BISOGNI (INFN-UNIPI)

WP1 bisogni.pdf

09:15 WP1-Laser-Plasma Accelerated VHEE Source: Beam Characterization and Dosimetric Validation for Advanced Radiotherapy

Within the Tuscany Health Ecosystem, Work Package 1 of Spoke 1 was devoted to the development of innovative radiation sources for oncologic radiotherapy, with a specific focus on the production of laser-plasma accelerated Very High Energy Electron (VHEE) beams.
Laser-Plasma Acceleration (LPA) enables intrinsically compact accelerator architectures by exploiting extremely high accelerating gradients. These sources deliver ultra-short pulses characterized by extremely high instantaneous dose rates. Through repetition-rate optimization, they have the potential to achieve clinically relevant average dose rates, thereby supporting compatibility with the FLASH regime and opening new perspectives for the treatment of deep-seated tumors.
The activity was centered on comprehensive beam characterization, including charge measurement, energy spectrum reconstruction, and depth-dose distribution assessment. Dosimetric validation was carried out through dedicated experimental campaigns and complemented by Monte Carlo simulations to ensure accurate dose reconstruction.
The fully characterized VHEE beams were employed in in vitro radiobiology studies to investigate their biological effectiveness and to assess their therapeutic potential in comparison with conventional X-rays. This integrated approach bridged source development, beam diagnostics, and preclinical validation, paving the way toward the future clinical translation of laser-driven VHEE radiotherapy.

Speaker: Simona PICCININI (INO-CNR)

Piccinini_5°-THE.pptx

09:30 → 10:00 HIGHLIGHT SESSION

09:30 WP2-Radiobiological Evaluation of Laser-Plasma-Accelerated Very High-Energy Electrons (VHEEs)

This research investigates the radiobiological response of human peripheral blood leukocytes (PBLs) exposed to laser-driven very high-energy electrons (VHEEs) delivered at an instantaneous ultra-high dose rate exceeding 10¹² Gy/s. PBLs serve as a valuable in vitro model for studying radiation-induced effects due to their high radiosensitivity, largely non-proliferative state in vivo, and widespread use in radiobiological assessments. Their well-characterized response to ionizing radiation provides critical insights into DNA damage and repair mechanisms, as well as overall cellular stress responses in normal human cells, making them an essential model for both mechanistic and translational research. PBLs are particularly suitable for examining radiation-induced chromosomal damage because they are inherently synchronized in the G0 phase of the cell cycle, ensuring uniform radiosensitivity. The micronucleus (MN) assay is a sensitive cytogenetic method used to detect and quantify chromosomal damage by measuring the formation of small extranuclear bodies—micronuclei—arising from acentric chromosome fragments or whole chromosomes excluded during cell division, making it a robust marker of radiation exposure.
Moreover, telomere length has emerged as a novel and sensitive endpoint of cellular radiation response. Telomeres, the protective caps at the ends of chromosomes, are essential for maintaining genome stability, and their excessive shortening can lead to genomic instability. Since mitochondrial DNA lacks protective histones and has limited repair capacity compared to nuclear DNA, it is particularly vulnerable to reactive oxygen species generated by ionizing radiation. mtDNA copy number, which reflects mitochondrial content per cell, serves as an indirect indicator of mitochondrial health and radiation-induced cellular damage.
Ionizing radiation affects not only directly irradiated cells but also neighboring non-irradiated cells through the bystander effect. This phenomenon includes increased apoptosis, micronucleus formation, DNA strand breaks, altered regulatory protein expression, and reduced clonogenic survival. These responses are mediated by intercellular communication via gap junctions and soluble factors released into the extracellular environment and may have important clinical implications, particularly for tissues outside the primary radiation field during radiotherapy.
In this study, we analyzed the biological effects of VHEE bunches on both nuclear and mitochondrial DNA, considering direct and bystander responses. Our findings demonstrate that laser-plasma-accelerated VHEEs elicit distinct radiobiological responses, with evidence of micronuclei formation and telomere shortening, yet induce less chromosomal damage and telomere reduction compared to conventional X-rays at equivalent doses. These results provide an important reference for evaluating the radiobiological properties and therapeutic potential of VHEEs in preclinical studies, particularly in the context of their ultra-high instantaneous dose rates.

Speaker: Andrea BORGHINI (IFC CNR)

THE_ Spoke 1_Borghini 13.3.2026.pdf

09:45 Cascade Call - LPA – STAR: Laser-Plasma Acceleration – Stabilization for Radiotherapy

Laser-plasma accelerators (LPAs) can generate high-quality electron beams in compact, fully optical setups, with promising applications in radiotherapy. However, beam stability is limited by laser pointing fluctuations at various timescales, caused by thermal drifts, air currents, and mechanical vibrations. These instabilities affect the reproducibility of electron generation and must be mitigated to meet clinical standards, which require dose uncertainties below 5–7% and spatial accuracy in the millimeter range.??We propose a compact, vacuum-compatible system for active beam stabilization based on diagnostics performed on a replica of the main laser beam. Installed near the laser-plasma interaction point, the system enables real-time measurement of laser position and direction at focus using cameras and Position-Sensitive Detectors (PSDs). Fast-processing electronics drive feedback to the final steering mirrors, suppressing high-frequency jitter. This approach aims to enhance LPA stability and support its transition toward medical applications.

Speaker: Guido GIORGI (Vacuum FAB)

10:00 → 10:30 SESSION 2: SIMULATIONS, MOLECULAR MECHANISMS VALIDATION AND RADIOBIOLOGICAL EFFECT MODELLING

10:00 WP2-Integrating modeling and data analysis to understand and quantify the FLASH effect

As part of the activities of THE WP2, we have focused our efforts on understanding the mechanisms underlying the FLASH effect, i.e., the sparing of healthy tissue resulting from Ultra High Dose Rate (UHDR) irradiation, possibly with pulsed radiation and/or spatial fractionation (micro-mini-beam), and on quantifying it for clinical translation. To this end, we have developed a strategy that combines data organization and analysis with multiscale modeling and implemented this strategy in a single platform, to be used as a reference for data collection and processing and for calculating dose modification factors that correct treatment planning for the FLASH effect.

Data collection was realized on a customized database for outcomes from experiments conducted at the ElectronFlash linac of Centro Pisano Flash RadioTherapy (CPFR) . The platform, based on XNAT technology and built with the support of the cascade call FLASH_IT and with the collaboration of all the experimental groups participating in Spoke1, maps diverse experimental workflows into a unified data scheme, where beam parameters and biological variables are linked together for easy search and selection. A significant effort was dedicated to standardizing heterogeneous data , addressing ambiguities in physical definitions and biological endpoints for both in vivo and in vitro experiments . Besides irradiation parameters (dose rate, spatial and temporal structure of the beam, …), the platform includes structural and environmental variables of tissues and cells (strain, age, type of tissue, cell line/cycle, physiological/pathological state…), experimental outputs (clonogenic assays, toxicity scores, etc.), and files of diverse structure and formats with additional information (e.g., microscopy or MRI images, Excel or text files).

The integration of these data with modeling occurs in two directions: data are used to optimize models, while simulations deliver additional data to fill the gaps of experiments . More specifically, we performed extensive atomistic Monte Carlo, and Reactive Molecular dynamics , to assess the role of oxygen in the early phases of radical creation and to simulate the damage on DNA , on time and size scales difficult to access experimentally. These data are used together with experimental ones for the parameterization of low-resolution models with a higher empirical level, to simulate the evolution of damage on larger space and time scales , for more direct comparison with experiments. The combination of experimental and simulation data shed light on the mechanisms of FLASH effect, which, aside non-linear tracks interaction effects enhanced by oxygen, call into play a main role of differential diffusion and evolution of several agents, not only ROS and radicals but also scavengers, metabolic messengers and other molecules, which are currently under study, to clarify further mechanistic details.

In parallel, we developed a minimalistic empirical model specifically aimed at distilling the information (both experimental and simulation) and targeting directly the final effect, possibly with the aid of machine learning based algorithms . The model is built with the following ingredients: (i) minimal number of parameters (ii) two stages describing the local (sub-cellular) and the cell level damage (possibly extendable to tissue level) (iii) inclusion of stochastic-thresholds mechanisms and diffusivity-evolution of damaging agents. Our model reproduces the FLASH sparing effect as the dose rate is increased and incorporates the time dynamics and spatial fractionation of the beam. The simulations performed with the model indicate that the FLASH effect appears directly dependent on diffusivity, an intrinsic property of the cell/tissue type/status, while the temporal dynamics of delivery and space fractionation (beam characteristics) seem to modulate it. A main advantage of this model is the low number of parameters, which allows its optimization using a small number of data and can therefore be used, once optimized for different tissues/cell types, to evaluate the dose modifying factor to correct treatment planning systems for FLASH effect.

In conclusions, we designed and implemented a platform for data collection on FLASH effect from various origins (experimental and simulation) and in a variety of formats, expandable and customizable, which allow searching sorting and selecting data for further analysis. The platform is being populated with data and will remain beyond the THE project, and will be made available to the community. We performed simulations studies revealing, aside a possible amplification of non linear local irradiaton effect due to oxygen, a main role of diffusivity of damaging agents in the FLASH effect. These aspects are currently under analysis with further simulations. In the meanwhile, we addressed the calculation of the dose modifying factor with a minimalistic empirical stochastic model that includes the main determinants (mainly diffusivity / evolution of damaging agents) and is able to include the beam time pulsation and space fractionation. Once parameterized for different tissues, this model can efficiently and directly evaluate the dose modifying factor for the treatment planning.

Speaker: Camilla SCAPICCHIO (INFN sezione di Pisa)

Scapicchio_WP2_dataplatform.pptx

10:15 Integrating modeling and data analysis to understand and quantify the FLASH effect

Speakers: Camilla SCAPICCHIO (INFN sezione di Pisa) , Valentina TOZZINI (CNR-NANO)

Tozzini.pdf

Tozzini.pptx

10:30 → 11:00 Coffee break

11:00 → 11:30 SESSION 3: RADIOBIOLOGICAL EFFECTS OF IONIZING RADIATION: IN VITRO (CELL COLTURE) AND IN VIVO

11:00 High-Speed Radiation, Low Neural Damage: the visual system as a window on FLASH radiotherapy

Vision is the most dominant human sense and the visual system represents a sensitive marker of biological and physical insults. Here, we exploited this system to compare the effects of ultra-high dose rate radiotherapy (FLASH) and conventional dose rate irradiation (CONV) in healthy C57BL/6 mice and in a preclinical models of ocular melanoma and glioblastoma. Irradiations were performed with a new-generation linear accelerator (LINAC) capable of alternating between FLASH and CONV modalities, enabling a direct comparison of regimens.
We analyzed two critical levels of the visual pathway: the retina, where photo-transduction and radiation toxicity often initiate, and the primary visual cortex, where visual signals are processed. In healthy animals, FLASH markedly reduced retinal inflammation, with lower microglial activation and decreased expression of molecular stress markers, and modulated cortical responses in a dose-dependent manner. Both in melanoma and glioblastoma model, FLASH and CONV showed comparable efficacy in reducing tumor size, yet FLASH better preserved the integrity of non-tumoral tissues. Importantly, both retina and cortex displayed dose-dependent sensitivity to irradiation, underscoring the visual system as a highly responsive readout for treatment-induced effects.
These findings demonstrate that FLASH radiotherapy can mitigate radiation-induced toxicity in the visual system without compromising tumor control. By preserving retinal and cortical integrity while maintaining antitumor efficacy, FLASH emerges as a promising therapeutic approach for ocular melanoma and potentially other conditions where vision preservation is critical.

Speaker: Gabriele SANSEVERO (IN CNR)

Sansevero_THE_Finale (4).pptx

11:15 FLASH Radiotherapy selectively protects healthy glia while enhancing glioma cytotoxicity through redox–mitochondrial modulation

FLASH radiotherapy (FLASH-RT) delivers radiation at ultra-high dose rates (UHDRs), achieving strong antitumor efficacy while minimizing normal tissue toxicity. However, the cellular and metabolic mechanisms underlying this ‘FLASH effect’ remain unclear. Here, we compared the impact of FLASH versus conventional (CONV) irradiation (8 Gy in vitro) in glioma GL261 cells and healthy primary glial cultures. Multiparameters, including cytotoxicity, oxidative stress, mitochondrial function, and bioenergetics were evaluated through real-time population-based assays, flow cytometry, Seahorse analysis, and gene expression profiling. In healthy glial cells, FLASH-RT markedly attenuated ROS accumulation, preserved mitochondrial membrane potential and respiration, and maintained viability, while CONV-RT induced sustained oxidative stress and mitochondrial dysfunction. FLASH-treated glial cells showed transcriptional signatures of cell cycle arrest and senescence rather than apoptosis, indicating activation of repair and survival pathways. In contrast, FLASH-RT in tumor cells enhanced ROS production, mitochondrial depolarization, and metabolic impairment, leading to pronounced cell death and early activation of autophagy-related genes. Both modalities reduced glioma cell viability by 72 h, but only FLASH preserved mitochondrial integrity in non-tumoral cells. Pharmacological inhibition of PARP, calpains, and necroptosis further revealed that CONV-RT–induced metabolic collapse is primarily PARP- and necroptosis-dependent, whereas FLASH-RT triggers a more distributed and modulable stress response, with glial cells showing minimal inhibitor sensitivity and GL261 cells exhibiting multimodal vulnerability. These findings demonstrate, for the first time, how FLASH-RT reprograms a cell-type-specific metabolism that spares healthy cells while promoting glioma cell death, providing a mechanistic basis for its therapeutic advantage and supporting its further development as a next-generation radiotherapy approach.

Speaker: Beatrice D'ORSI (CNR-IN)

THE meeting_march 2026_DORSI.pptx

11:30 → 12:00 SESSION 4: FLASH RADIOTHERAPY: PRECLINICAL

11:30 WP4-The Sparing Effect of Electron FLASH Radiotherapy on Murine Skin: Molecular Mechanisms and Systemic Responses

Background and Aim: The molecular mechanisms underlying the FLASH effect remain largely unknown. We previously demonstrated that electron FLASH radiotherapy (FLASH-RT) achieves tumor control comparable to conventional radiotherapy (CONV-RT) in a murine melanoma model, while significantly reducing damage to surrounding skin. Here, we investigated the molecular and histological correlates of this sparing effect, extending our analysis to adjacent tissues and systemic parameters.
Methods: Healthy mice received 35 Gy to the posterior leg using either CONV or FLASH modes via a low-energy LINAC equipped with a triode-gun Electron-Flash system. Skin, white adipose tissue (WAT), and muscle from the irradiated area were collected at sacrifice and analyzed by RNA sequencing, optical microscopy, and transmission electron microscopy. Mice were monitored for 20 weeks post-treatment to assess circulating metabolic and inflammatory markers.
Results: CONV-RT and FLASH-RT altered the expression of 2,461 and 93 genes, respectively, in the skin. Pathway analysis of upregulated genes revealed no enriched terms specific to FLASH-RT, whereas 81 enriched pathways were identified in the CONV-RT group, mainly related to inflammation, fibrosis, and cell death. In WAT, transcriptomic signatures indicated reduced functionality following CONV-RT but not FLASH-RT. In muscle, key protein synthesis pathways (mTOR, Akt, S6) were altered only in CONV-treated mice. These molecular findings were corroborated by histological analyses. At the systemic level, CONV-RT mice showed decreased levels of the satiety hormone leptin and increased levels of the appetite stimulator ghrelin. FLASH-RT mice showed no such alterations.
Conclusions: Our findings highlight a marked difference in tissue and systemic responses between CONV-RT and FLASH-RT. FLASH-RT minimizes inflammatory and metabolic disruptions in normal tissues, supporting its therapeutic potential for reducing radiotoxicity without compromising tumor control.

Aknowledgements: FPS, PNRR-THE, MIRO

Speaker: Giulia FURINI (CNR-IFC)

FURINI_Session4.pptx

11:45 WP4-Preserving Tissue Integrity: Differential Impact of FLASH and Conventional Radiotherapy on White Adipose Tissue

Background: Subcutaneous white adipose tissue (scWAT) is a major energy reservoir and an active endocrine organ. Owing to its widespread distribution, scWAT is inevitably exposed during conventional (CONV) radiotherapy (RT). Although RT remains a cornerstone of cancer treatment, its therapeutic efficacy is limited by toxicity to surrounding healthy tissues. Ultra-high dose rate (FLASH) RT has recently emerged as a promising modality capable of preserving tumor control while markedly reducing normal tissue damage, the so-called FLASH effect. Clinical evidence indicates that individuals exposed to RT during childhood frequently develop lipodystrophy and metabolic dysfunction later in life; however, the underlying mechanisms remain poorly understood. Notably, the effects of FLASH RT on WAT have not yet been investigated.
Aim: To examine and compare the effects of FLASH and CONV RT on adipocytes and subcutaneous white adipose tissue.
Materials and Methods: In vitro studies were performed using the human SGBS preadipocyte/adipocyte cell line, while in vivo experiments were conducted in male C57BL/6 mice. Confluent undifferentiated SGBS cells were irradiated and subsequently induced to differentiate or irradiated at terminal differentiation. Irradiations were delivered using a linear accelerator capable of administering both CONV and FLASH RT at doses of 4, 8, and 16 Gy. Triglyceride accumulation and adipocyte function were assessed by Oil Red O staining and gene expression analyses.
For in vivo studies, mice received a single 35 Gy irradiation to the proximal hind limb and were sacrificed 70 days post-irradiation. scWAT from the irradiated region was analyzed using histological approaches (H&E and Sirius Red staining, immunohistochemistry), light and transmission electron microscopy, and bulk RNA sequencing.
Results: RT impaired adipogenic differentiation in a dose-dependent manner, with a clear sparing effect of FLASH irradiation at 4–8 Gy. Mature adipocytes displayed relative radioresistance, with a significant protective effect of FLASH observed at 8 Gy. In vivo, both irradiation regimens induced a reduction in scWAT mass despite comparable total body weights, with fat loss being more pronounced following CONV-RT. Transcriptomic profiling of irradiated scWAT revealed robust activation of inflammatory and neurodegenerative pathways after CONV-RT, whereas FLASH-RT induced minimal to no transcriptional alterations. Consistently, histological and ultrastructural analyses showed marked cellular damage in CONV-RT treated mice, including increased vacuolization, lipid spill-over, and reduced PLIN1 immunoreactivity. These alterations were markedly attenuated or absent following FLASH-RT.
Conclusions: These findings demonstrate that WAT homeostasis is highly sensitive to CONV-RT, whereas FLASH-RT more effectively preserves its structure and function. This differential response has important implications for the long-term metabolic health of cancer survivors. Moreover, we provide novel mechanistic insights into RT-induced WAT dysfunction and identify new trajectories for understanding and potentially mitigating RT-related metabolic sequelae.

Speaker: Gaia SCABIA (CNR Istituto di Fisiologia Clinica)

GaiaScabia_THE workshop_DEF2_12 marzo 2026.pptx

12:00 → 12:30 HIGHLIGHT SESSION

12:00 WP1-Monte Carlo Simulation–Driven Optimization of eFLASH Radiotherapy for Uveal Melanoma

Uveal melanoma represents the most common primary intraocular malignancy in adults.
Current treatment strategies include eye-preserving stereotactic radiotherapy (SRT) and
enucleation in advanced-stage disease. In this study, we investigate the feasibility of lowenergy
electron ultra-high dose-rate radiotherapy (eFLASH-RT) as a novel treatment
modality for localized uveal melanoma. The FLASH effect, characterized by reduced toxicity
to normal tissues while maintaining tumor control, may offer substantial advantages in terms
of clinical outcome and ocular preservation.
To this end, a comprehensive Monte Carlo (MC) simulation study was implemented to model
the ElectronFlash (EF) linear accelerator of Centro Pisano Flash RadioTherapy (CPFR) and
to generate patient-specific FLASH-RT treatment plans using both real and synthetic
electron spectra up to 30 MeV. The potential radiobiological sparing effect of FLASH-RT
plans was also considered, introducing the Dose Modifying Factor. Then, a quantitative and
reproducible scoring system, integrating physical and radiobiological metrics derived from
dose–volume histograms, was developed to enable objective comparison among clinically
delivered SRT treatments on real patients and simulated FLASH-RT treatment plans.
Comparative analysis between simulated FLASH-RT plans and clinically delivered SRT
treatments showed comparable target coverage, with PTV homogeneity achieved by
FLASH-RT comparable to that of SRT. However, the results indicate that a FLASH sparing
factor of approximately 45% would be required to achieve a meaningful reduction in normal
tissue exposure.
Overall, these results demonstrate the technical and dosimetric feasibility of electron
FLASH-RT as a potential organ-preserving strategy for uveal melanoma. Moreover, this
study provides a generalizable and reproducible methodology for the evaluation of FLASHRT
treatment plans, including a scoring framework that allows objective comparison between
conventional and FLASH modalities in terms of RBE. Nonetheless, extensive preclinical
studies are necessary to validate the magnitude and consistency of the FLASH effect in
ocular tissues prior to clinical translation.

Speaker: Camilla SCAPICCHIO (INFN sezione di Pisa)

Scapicchio_WP1_highlights.pdf

12:15 WP4-Comparative Effects of FLASH and Conventional Radiotherapy on Corneal Collagen Organization

Whole brain radiotherapy (WBRT) remains an essential treatment for brain metastases and primary intracranial malignancies, but conventional radiotherapy (CONV-RT) incidentally exposes ocular structures to radiation, potentially leading to vision-threatening complications. FLASH radiotherapy (FLASH-RT) delivers ultra-high dose rates (≥40 Gy/s) in milliseconds, compared to CONV-RT where the dose delivery is ≤1 Gy/min over several minutes. This study aims to evaluate comparative effects of FLASH-RT versus CONV-RT on corneal collagen morphology using second-harmonic generation (SHG) imaging in a murine WBRT model. C57BL/6J wild type mice were assigned to control, FLASH-RT, and CONV-RT groups and evaluated acutely (4 days) and chronically (40 days) post irradiation at 20, 15 and 10 Gy, respectively. SHG microscopy quantified collagen organization using forward-to-backward (F/B) signal ratios and assessed corneal thickness as markers of radiation-induced damage. In CONV-RT we found significantly decreased F/B ratios compared to controls (p < 0.05), indicating substantial collagen disorganization, and increased corneal thickness, due edema and inflammation. Conversely, FLASH-RT maintained F/B ratios comparable to controls and prevented excessive thickening of the cornea, demonstrating preserved extracellular matrix integrity across both timepoints. FLASH-RT preserves corneal collagen architecture compared to CONV-RT during whole brain irradiation, demonstrating significant potential for minimizing ocular toxicity in WBRT. These findings support clinical investigation of FLASH-RT as a tissue-sparing modality for brain cancer treatment

Speaker: Riccardo CICCHI (Istituto Nazionale di Ottica (CNR-INO))

12:30 → 14:00 Lunch break

14:00 → 14:30 SESSION 5: FLASH RADIOTHERAPY - FROM PRECLINICAL TO CLINICAL

14:00 WP5-Beyond the THE Project: What We Learned and the Clinical Consequences for Trial Design and Patient Selection

The THE Project (Spoke 1, WP1.5) provided a structured translational framework for the
clinical implementation of low-energy electron FLASH radiotherapy, integrating dosimetric
protocol development, treatment planning adaptation, device upgrade, and preclinical
validation. Beyond technical feasibility, the project generated key insights into trial design
and patient selection for first-in-human FLASH applications.
A central lesson was that technological optimization—beam monitoring, applicator
refinement, collimation systems, and positioning solutions—is a prerequisite but not
sufficient condition for clinical translation. Early-phase trials must prioritize
reproducibility, strict dosimetric control, and minimization of technical variability to ensure
safety and scientific credibility.
Patient selection emerged as equally critical. Early FLASH trials require highly restricted
and homogeneous populations, limited confounding variables, and simple, measurable
endpoints. T1 non-melanoma skin cancers were selected as an ideal model due to their
accessibility, geometric definition, and suitability for precise high-dose irradiation with low
energy electrons. The skin provides a controlled in vivo environment to assess toxicity, local
control, and oxygen-dependent radiobiological mechanisms central to the FLASH effect.
Accordingly, the primary endpoint focused on skin toxicity up to six months, with
hierarchical evaluation of local control.
Overall, the THE Project underscores that the credibility of FLASH radiotherapy will depend
on rigorous early trial design, conservative indication selection, and multidisciplinary
integration, ensuring safety, reproducibility, and robust clinical translation.

Speaker: Fabiola PAIAR (Unipi)

Paiar.pdf

14:30 → 15:00 SESSION 6: IN SITU ADVANCED DIAGNOSTICS OF RADIATION DEPOSITION AND CONFORMALITY

14:30 WP6-Plastic Scintillator-Based Dosimetry for FLASH Radiotherapy

Ultra-high dose rates delivered in FLASH radiotherapy introduce significant challenges for conventional dosimetry, potentially affecting detector response and reliability. In a scenario where comprehensive and fully established dosimetric guidelines are still evolving, plastic scintillators represent a promising alternative due to their fast temporal response, water equivalence, and versatility in detector design. Implemented as scintillating fibres, sheets, or three-dimensional volumes, these detectors enable dose measurements and the investigation of spatial dose distributions under FLASH irradiation conditions. This contribution presents the main characteristics of a set of plastic scintillator-based dosimeters and reports experimental measurements performed at the CPFR facility, highlighting their potential for high-dose-rate beam characterisation.

Speaker: Eleonora RAVERA (UniPi)

THE_03_26_Ravera.pdf

14:45 WP6-Toward Preclinical Validation of Laser-Driven VHEE Beams: Advancing Dose Delivery and Conformality

Speaker: Luca LABATE (CNR - Istituto Nazionale di Ottica)

20260313_workshopTHE.pptx

15:00 → 15:30 HIGHLIGHT SESSION

15:00 Real-Time Dose Monitoring via Non-Destructive Charge Measurement of Laser-Driven Electrons for Medical Applications

Laser-accelerated electron beams in the Very High-Energy Electron (VHEE) range are receiving increasing interest for biomedical applications. Furthermore, compact accelerator systems for the exciting field of FLASH radiotherapy may be made precisely accessible by laser-driven VHEE beams. Nevertheless, radiobiology experiments carried out using laser-driven beams require the real-time knowledge of the dose delivered to the sample. For this reason, we have developed an online dose monitoring procedure, using an Integrating Current Transformer (ICT) coupled to a suitable collimator, that allows the estimation of the delivered dose on a shot-to-shot basis under well-defined assumptions. Such a dose measuring method through electron charge was validated through cross-calibration with standard offline dosimetry using RadioChromic Films (RCFs).

Speaker: David GREGOCKI (Consiglio Nazionale delle Ricerche - Istituto Nazionale di Ottica)

Workshop_26_Gregocki.pptx

15:15 Cascade Call-JET Target for Laser Electron Acceleration (JET-LEA)

The generation of high-power, ultrashort pulsed electron beams is a key characteristic of
modern radiotherapy techniques based on the FLASH effect.
One of the most promising techniques in this field is Laser-Linacs (LL), which is based on the
Laser Wakefield Acceleration (LWFA) process. In this process, a high-power, ultra-short laser
pulse is focused into a gas-jet target to generate a plasma wave that can accelerate electrons
injected into the plasma at the correct phase.
Recent studies and simulations have shown that using a gas jet target with a structured
chemical gradient can improve control over the depth of electron injection into the plasma
and improve the energy spectrum of the resulting electron beam.
In this context, the JET-LEA (JEt Target for Laser Electron Acceleration) project was started with
the objective of designing and characterising a continuous recirculating gas jet target.
The JET-LEA project and its main results will be presented during the workshop.

Speaker: Dr. Raffaele BUOMPANE (Università degli Studi della Campania "Luigi Vanvitelli")

Workshop_Pisa_BaC_JET-LEA.pdf

15:30 → 16:00 Coffee break

16:00 → 16:30 SESSION 7: SYNTHESIS AND PRODUCTION OF TUMOR-TARGETED RADIONUCLIDES, RADIOTRACERS AND RADIOPHARMACEUTICALS FOR EXPERIMENTAL STUDIES

16:00 Preclinical in vitro evaluation of monomeric radiotracers to support the development of new heterodimeric theragnostic agents selective for the tumor microenvironment

Background: Cancer theranostics integrates molecular imaging and targeted radionuclide therapy using matched radiopharmaceutical pairs with identical molecular scaffolds labeled with diagnostic radionuclides such as fluorine-18 or gallium-68, and therapeutic emitters such as lutetium-177 [1]. Recent advances have led to the clinical translation of selective radiotracers exploiting differential protein overexpression between malignant and normal tissues [2]. However, monospecific agents often show limited efficacy due to intratumoral heterogeneity and dynamic tumor microenvironment interactions [3]. To overcome these constraints, heterodimeric radiotracers capable of simultaneously targeting two molecular determinants have been developed, improving specificity, uptake, sensitivity, and retention [1], [4], [5]. Among emerging targets, Fibroblast activation protein and Carbonic anhydrase IX are particularly promising tumor microenvironment associated targets for diagnostic and therapeutic applications [6], [7]. Despite their translational potential, preclinical evaluation of dual-targeted radiotracers remains challenging due to the limited availability of in vitro models that endogenously co-express both proteins at levels and patterns comparable to those observed in human tumors. Additionally, in vitro characterization of monomeric counterparts is critical to establishing a robust benchmark dataset while simultaneously highlighting critical biological and methodological challenges to be addressed in the design and evaluation of heterodimeric radiotracers.
Methods: We systematically assessed FAP and CAIX expression across a panel of cell lines to identify a biologically relevant dual-positive in vitro model. Monomeric radiotracers [68Ga]Ga-DOTA-FAPI-04 and [68Ga]Ga -DOTA-CAI were characterized for cellular uptake, specificity, and internalization. Binding assays, molecular dynamics simulations, and enzymatic inhibition studies were performed for CAIX-selective compound to elucidate the molecular basis of its interaction with carbonic anhydrase isoforms.
Results: U87MG cells were identified as the most suitable dual-positive model, exhibiting endogenous co-expression of FAP and CAIX. [68Ga]Ga-DOTA-FAPI-04 displayed specific uptake and predominant internalization in FAP-positive cells correlating with FAP expression. In contrast, in vitro studies of [⁶⁸Ga]Ga-DOTA-CAI, including uptake, blocking, and saturation assays, did not demonstrate CAIX-specific or saturable binding. Molecular dynamics simulations predicted stable interactions with CA II, IX, and XII and indicated that gallium complexation does not affect the sulfonamide-zinc anchoring mechanism. Enzyme inhibition assays confirmed nanomolar potency against tumor-associated CA IX and XII, showing preserved intrinsic affinity. In contrast, direct radiotracer binding assays showed limited association with tumor-associated isoforms, which was attributed to aggregation of recombinant CA IX and XII under the assay conditions.
Conclusions: This study identified a biologically relevant co-expressing cellular model and characterized monomeric FAPI- and CAI-based radiotracers, establishing a reference dataset for heterodimer evaluation. Future work will focus on further validation and optimization of CAIX-targeted tracer

Speaker: Costanza FABBRI (Scuola Superiore Sant'Anna - Istituto di Fisiologia clinica CNR Pisa)

Costanza Fabbri_meeting THE_2026.pdf

Costanza Fabbri_meeting THE_2026.pptx

16:30 → 17:00 SESSION 8: SYNTHESIS AND PRODUCTION OF TUMOR-TARGETED RADIONUCLIDES, RADIOTRACERS AND RADIOPHARMACEUTICALS FOR CLINICAL USE

16:30 WP8-Profeti Project

Glioblastoma represents the most frequent malignant primary brain tumor, with a median overall survival of approximately 15 months and a 5-year survival rate close to 5%. In this setting, positron emission tomography (PET) using O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) has demonstrated significant value for diagnosis, treatment planning, and therapy monitoring.
Despite the strong clinical evidence, the limited national availability of [18F]FET has historically restricted its widespread adoption in routine practice in Italy.
The PROFETI project was conceived to overcome this barrier by establishing a coordinated
network integrating research institutions, industrial partners, and local healthcare stakeholders. The initiative aimed to enable multi-site GMP production and create the conditions for sustainable distribution of the radiopharmaceutical.
Building on the technology transfer activities initiated in Pisa, PROFETI supported the
implementation of [18F]FET manufacturing capabilities in additional Italian sites, including Rome, Udine, and Milan, through a structured pathway based on:
• definition of functional and performance requirements;
• validation of analytical methods and comprehensive personnel training;
• process validation and harmonization across sites;
• preparation for regulatory submission to the Italian Medicines Agency (AIFA);
• development of a collaborative framework for continuous quality improvement and shared
problem solving.
The creation of an Italian network of GMP-authorized centers represents a major advancement in the national exploitation of public healthcare investments, ensuring broader and more equitable access to advanced molecular imaging.
This coordinated effort has the potential to significantly impact the management of more than 6,500 patients diagnosed with glioma every year in Italy, enabling standardized, high-quality diagnostic support and fostering future clinical and research applications.

Speaker: Dr. Anna NOTARO (Curium Pharma)

Notaro 130326.pptx

16:45 WP8-Tuscany Health Ecosystem (THE)

Within the Tuscany Health Ecosystem, Spoke 1 – WP8 is dedicated to the synthesis and production of tumor-targeted radionuclides, radiotracers and radiopharmaceuticals intended for clinical use.
The overarching objective is to ensure the availability of medicinal products authorized for
administration in humans, supporting diagnosis and follow-up of patients affected by glioma and other oncological and neurological diseases.
The work package focused on three major translational pathways: technology transfer and GMP authorization of Fluoroethyl-L-tyrosine, technology transfer of Fluorodopa, and process validation activities for FAPI-74.
For [18F]FET, the transfer technology transfer was successfully finalized in February and GMP
authorization was released from Agenzia Italiana del Farmaco (AIFA) in April 2024, enabling
routine clinical availability of the tracer in Italy.
Technology transfer for [18F]FDOPA was completed in September 2024, followed by national
regulatory authorization released in August 2025, further strengthening the infrastructure for
molecular imaging in neurology, particularly in parkinsonian syndromes.
In parallel, the WP achieved production of three consecutive validation batches of [18F]FAPI-74 under controlled conditions. This radiopharmaceutical will be manufactured under AIFA
authorization within prospective clinical investigations, including the DETECT Study, aimed at
evaluating [18F]FAPI PET in hepatocellular carcinoma, and the TARG Study, focused on targeting the tumor microenvironment in gliomas.
Overall, WP8 represents a concrete example of rapid translation from research to clinical
application, reinforcing regional and national capacity to deliver innovative, GMP-compliant
radiopharmaceuticals and to support multicenter clinical research.

Speaker: Michela POLI (CNR-IFC)

Poli 130326.pptx

17:00 → 17:30 FINAL REMARKS