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Several studies show that the combination of high-Z nanoparticles and external radiotherapy leads to an increased radiation effect in tumoral cells without an increase of the patient dose. However, it is not yet clear how the sequence of physical, chemical, and biological mechanisms contributes to the observed synergic effect. The objective of this Ph.D. research project is to develop simulation tools that allow the analysis and interpretation of radiobiology studies with multifunctional nanoparticles (NPs). The work will be developed within the scope of the project “In-beam Time-of-Flight (TOF) Positron Emission Tomography (PET) for proton radiation therapy” (TOP-PET), in particular the tasks involving the evaluation of multifunctional gold NPs (AuNP) as radiosensitizers in the treatment of glioblastomas multiforme (GBM). The student will develop realistic simulations of the irradiation of monolayer (2D) and spheroid (3D) human GBM cell cultures and xenograft animal models, taking into consideration different concentrations and cellular and subcellular distributions of the AuNPs. The simulations will be implemented based on the Geant4 toolkit and in particular with the extension Geant4-DNA that includes models of the physical and chemical processes induced by radiation at the DNA scale. These must describe the laboratory experimental conditions of irradiation with X-rays and Co-60 sources and with proton beams taking into account the cell lines morphology and 2D and 3D cell culture scenarios. The construction of the computational cell models will be developed based on confocal microscopy images of the biological samples. At a later stage, the simulations will be extended to computational models of mice bearing subcutaneous GBM xenografts. The student will also investigate feasible ways of simulating irradiations of biological systems with different levels of oxygenation (e.g. normoxia vs hypoxia). Based on the simulations, the dose distributions at the subcellular scale will be obtained, as well as the temporal distribution of the reactive oxygen species (ROS) induced by the different irradiation conditions, AuNPs distribution, and concentrations. The microdosimetric distributions in cells and tissues will be used to predict cell survival fractions, and single and double-strand breaks of the DNA, using standard mathematical models of the biological effects of radiation, like the local effect model (LEM), among others. The results obtained in the simulations will be compared with the biological in vitro and in vivo experimental results, which will include evaluation of cell viability and survival, as well as cell apoptosis and DNA damage (gamma-H2AX) studies. Moreover, the simulated ROS yields will be also compared with the experimentally determined values. The Ph.D. research activities will be developed at LIP in close collaboration with C2TN-IST and ICNAS groups involved in the tasks of AuNPs synthesis, irradiation, and biological studies related to the TOP-PET project.

Rationale Radiotherapy has always been a trade-off between tumor control and organ toxicity, where healthy organs are in many cases very sensitive to radiation. However, after recent developments in FLASH radiotherapy, this paradigm has been challenged due to a significant reduction in the toxicity of healthy organs [1]. “FLASH” is based on very high dose-rate irradiation (dose rate ≥50 Gy/s), short beamon times (≤100 ms) and large single doses (≥10 Gy) per beam [1]. At the moment, it is still unknown how to apply clinically FLASH to treat patients. Recent clinical implementation of FLASH has focused on delivering a single beam with FLASH to treat cancers on the skin or in the extremities (hands and legs). Single beam doses above 10Gy to 12 Gy is not common in standard radiotherapy, because of the very high risk of radiation toxicity. At the moment it is still unknown how to perform FLASH with multi-beams. PhD Project Goals (G) G1: To study the FLASH effect using Monte Carlo tools such GEANT4, TOPAS, gMicroMC and TRAX-CHEM. The Monte Carlo tools will allow the evaluation of the chemical reaction of proton radiolysis. The PhD project will investigate how the chemical products of this reaction can produce FLASH protection in cancer cells and not in healthy cells. G2: To investigate how to implement multi-beam FLASH in proton radiotherapy using the treatment planning software MATRAD. G3: To investigate how FLASH can be used in combination with other delivery methods such 3D conformal radiotherapy (3D-CRT), intensity modulated radiotherapy (IMRT) and mini- or micro-beams, to maximize the theapeutic benefit. REFERENCES 1. Wilson et al (2020) “Ultra-high dose rate (FLASH) Radiotherapy: Silver bullet or fool’s gold?” Front. Oncol. 9:1563 doi:10.3389/fonc.2019.01563

This PhD research project will focus on the design and preclinical evaluation of a new generation of gold nanoseeds, which will be studied as nanoplatforms for the drug delivery of Pt(IV) prodrugs and as radiosensitizers in radiation therapy of cancer. The overall goal is to contribute for the development of innovative strategies for a more efficient treatment of unresectable solid tumor cancers with reduction of adverse side effects. The proposed research relies on our previous encouraging results with gold nanoparticles (AuNPs) carrying bombesin (BBN) peptides targeted at the gastrin releasing peptide receptor (GRPr) overexpressed in different cancer cells, namely prostate cancer (PCa) cells. We have found that these BBN-containing AuNPs undergo a specific and high uptake in PCa cells and radiosensitize these cancerous cells following exposure to high energy gamma photons (F. Silva et al., Bioconjugate Chemistry 2016, 27 (4), 1153-1164; F. Silva et al., Materials 2020, 13, 513). Herein, we propose to study the radiosensitizing properties of related AuNPs in proton therapy of prostate cancer, while comparing with traditional photon radiation. The originally designed AuNPs will be synthesized using different macrocyclic chelators suitable to coordinate 64Cu. This PET radionuclide will allow an image-guided approach of the intended radiation therapy involving the external irradiation with photon or proton beams of PCa cells or tissues accumulating the AuNPs. Some of the AuNPs will be also derivatized with Pt(IV) prodrugs. The presence of two different high-Z elements (Au and Pt) is expected to induce strongest radiosensitizing effects by intensifying the generation of low-energy electrons (e.g. photoelectrons or Auger electrons). Moreover, synergisms are anticipated between chemotherapeutic and radiotherapeutic effects in the case of the Pt-containing AuNPs, as they carry Pt(IV) prodrugs suffering intracellular glutathione-mediated activation with release of cisplatin or related Pt(II) anticancer metallodrugs. The PhD student will be involved in the different main steps of the devised multidisciplinary research plan, which are listed below. i) Synthesis and characterization of the AuNPs and their radiolabeling with Cu-64; ii) Determination of cellular uptake/subcellular distribution (Au and Pt content) and cytotoxicity of the AuNPs in PCa cell lines; iii) Cell irradiation experiments: irradiation of PCa cells with photon and proton beams (in the presence or absence of AuNPs); assessment of radiobiological effects and mechanisms of cell death; iv) PET imaging studies in PCa xenografts using the AuNPs labeled with 64Cu; v) Animal irradiation experiments: proton and photon irradiation of nude mice bearing PCa xenografts (treated or not with AuNPs); assessment of in vivo antitumoral effects and mechanisms of action. The PhD research work will be developed mainly at C2TN/IST within the framework of the ongoing projects TOP-PET (LISBOA-01-0247-FEDER-045904) and NANOGLIO (PTDC/MED-QUI/29649/2017), leaded in the latter case by the supervisor. The irradiation experiments with photons will be carried out at C2TN. The irradiation experiments with protons will be initially conducted at ICNAS with low energy protons using a 18 MeV cyclotron. As proposed in the TOF-PET project, irradiation experiments with high energy protons will be also performed at MD Anderson for the most promising AuNPs.

Organic scintillators are elementary constituents of ionising radiation detectors and a very popular choice with respect to alternative scintillating materials due to their low cost, low weight, and malleability. For large-scale applications, such as dosimetry for nuclear medical imaging and radiotherapy facilities, cost is a leading aspect to address, as wel as long-term durability in what concerns resistance to radiation and natural ageing. Promising novelties for scintillator materials were recently introduced regarding new low-cost dosimeters based on PET (Polyethylene Terephthalate) and PEN (Polyethylene Naphthalate) but dedicated investigation is still lacking. This proposal consists of R&D on the production and characterisation of new scintillator plates based on PET/PEN for a future application in large dosimetry systems, consti tuting a collaboration between the Institute for Polymers and Composites (IPC) of the University of Minho and LIP's Laboratory of Optics and Scintillating Materials (LOMaC). PET/PEN blends will be produced by melt mixing techniques and characterized. The most performing blend composition ratios will be selected and prototype scintillator plates will be manifactured by compression and injection moulding. The scintillator samples will be characterised in terms of light yield, attenuation length and resistance to ionising radiation at LOMaC.

The fields of Artificial Intelligence (AI) and mathematical programming are increasingly intertwined. Optimization problems lie at the heart of most AI approaches. Regarding optimization events in proton therapy, relative motion between the tumor and the scanning proton beam results in a degradation of the dose distribution, called the interplay effect. Proton therapy is much more susceptible to the interplay effect than traditional radiation therapy with photons due to the quirkiness of the interaction from the beam of particles with the medium. Treatment planning traditionally relies on dosimetric analysis and has become, among other tasks, a key element in the particle therapy process. Regarding patient safety and the success of therapy, its accurate and stable functioning is an issue of the highest importance. This critical task involves the procedures in which radiation oncologists, radiation therapists, medical physicists, and medical dosimetrists create proton treatment plans. Proton therapy is much more susceptible to the interplay effect than traditional radiation therapy with photons due to the characteristics of the interaction with the beam of particles carried with the medium. This study investigates the relationship between beam scanning parameters and the interplay effect, with the goal of finding parameters that minimize the interplay effect. Therefore, the Ph.D. student will be charged with the design of an AI model that can successfully take information from past clinical data (anatomical, dosimetric, medical history, type of tumor, etc) found in the literature to produce outputs that can help the treatment planning team with making more informed decisions and therefore improve treatment outcomes towards a more personalized medical care.

Proton therapy is a promising radiation treatment modality that uses proton beams to treat cancer. Current treatment planning systems rely on an X-ray computed tomography (CT) image of the patient's anatomy to design the treatment plan. While Dual Energy Computer Tomography - DECT shows promising results for converting linear attenuation coefficients into proton stop (ion) power, proton CT (ion) ignores the conversion entirely by taking images of the stopping power directly. Proton/ion CT depends on measurements of the beam energy downstream of the patient, with optional particle-by-particle tracking technology for enhanced resolution. Besides high stopping power accuracy, proton/ion CT offers the prospect of frequent image guidance at the isocenter, enabling both image-guided and adaptive particle therapy. The modality is particularly appealing due to reportedly better contrast to noise ratio at equivalent dose when compared to X-ray CT imaging. The Ph.D. student will be charged with the design of an optimized system regarding the improvement of calculation of the precision of the calibration curve between Hounsfield Unit and Relative Stopping Power, to improve image reconstruction from parameter acquisition and image post-processing, and finally to present a proposal for the implementation of a system based on Proton Tomography feasible and appropriate to the reality of the radioactive installation to be built in Portugal.

Intensity-modulated proton therapy (IMPT) is one of the most important recent developments in radiation oncology. It enables precise conformation of the radiation dose to the target volume, while minimizing radiation to closely surrounding organs. It has the potential to significantly reduce long-term morbidity and improve local control. However, IMPT can deliver radiation to healthy organs along the path of proton beam, which can potentially yield increased radiation toxicity. Recent developments in FLASH radiotherapy has allowed for the delivery of radiation beams at very high dose rates (dose rate ≥50 Gy/s), that ultimately protect healthy organs from radiation effects [1]. At the moment, it is still unknown how to apply clinically FLASH to treat patients. Single beam dose of 10 Gy is not common in standard radiotherapy, because of the very high risk of radiation toxicity. In addition, the true mechanism behind FLASH is still unknown. Combined IMPT and FLASH could potentially improve the therapeutic ratio by allowing improved tumor coverage with IMPT and reducing radiation side effects with FLASH. The main objective of the PhD project is to investigate how to combine IMRT with FLASH for use in a clinical environment, in a collaboration between IST (Portugal) and DKFZ German Cancer Research Center (Germany). PhD Project Goals: Goal #1: To investigate how to combine IMPT with FLASH within a proton therapy center using the treatment planning software MATRAD. The clinical implementation of IMPT-FLASH involves solving an optimization problem that incorporates 1) beam angle optimization, where some angles will be IMPT and others FLASH, 2) dose constraints that will limit the radiation dose to healthy organs, and 3) tumor dose objectives to achieve tumor control,; Goal #2: To study the IMPT-FLASH for various treatment sites such as lung, head and neck and prostate using Monte Carlo tools such as TOPAS. To evaluate dosimetrically using Monte Carlo tools the combined IMPT-FLASH plans for the treatment regions mentioned before with the focus on assessing the “biological gain” of using FLASH radiation beams. [1] Wilson et al. (2020) “Ultra-high dose rate (FLASH) Radiotherapy: Silver bullet or fool’s gold?” Front. Oncol. 9:1563 doi:10.3389/fonc.2019.01563

Background The targeting accuracy of proton therapy (PT) for moving soft-tissue tumours is expected to greatly improve by real-time magnetic resonance imaging (MRI) guidance. The integration of MRI and PT at the treatment isocenter would offer the opportunity of combining the unparalleled soft-tissue contrast and real-time imaging capabilities of MRI with the most conformal dose distribution and best dose steering capability provided by modern PT. However, hybrid systems for MR-integrated PT (MRiPT) have not been realized so far due to a number of hitherto open technological challenges. In recent years, various research groups have started addressing these challenges and exploring the technical feasibility and clinical potential of MRiPT. The aim of this contribution is to review the different aspects of MRiPT, to report on the status quo and to identify important future research topics. Four aspects currently under study in the field of MR-guided proton therapy are (1) modelling and experimental investigations of electromagnetic interactions between the MRI and PT systems, (2) integration of MRiPT workflows in clinical facilities, (3) proton dose calculation algorithms in magnetic fields, and (4) MRI-only based proton treatment planning approaches. PhD Project Goals (G) G1: To investigate how magnetic field alter, through Lorentz force, the proton beam transport within patients. To develop methods for improving dose calculation, optimization and delivery for proton beams within a magnetic field produce by an MRI machine; G2: To study online proton treatment planning based on MRI images. MR images do not contain electron density information that can be converted into SPR or water-equivalent path length information. The project focuses on developing methods to convert MRI information into synthetic CT information. For real-time MR image-guided dose delivery the conversion not only needs to be accurate but also very fast. REFERENCES 1. Hoffmann et al (2020) “MR-guided proton therapy: review and a preview” Radiation Oncol. 15:129 doi: 10.1186/s13014-020-01571-x 2. Spadea et al (2019) “Deep convolution neural networks (DCNN) multi-plane approach to synthetic CT generation MR images – application in brain proton therapy” Int. Journ. Of Rad. Oncol. Biol. Physics 105 (3) 495-503

Radiation therapy requires absorbed dose verification to guarantee accurate delivery of a carefully planned and optimized treatment scheme. In modern radiation therapy, the solution of an "optimal" plan is usually found by mathematically solving an inverse problem, where the physician prescribes the minimum required dose to the tumor and maximum allowed doses to healthy tissues (organs at risk) and a mathematical algorithm finds a solution in the fluence vector space. Each individual of many pencil beams is delivered with the required fluence such that the desired prescribed dose distribution is obtained. This method works not only well for intensity-modulated photon radiotherapy (IMRT) but also for intensity-modulated proton therapy (IMPT). The pre-calculated dose distribution still needs to be validated before treatment with a patient-specific QA procedure, which requires a 3D ionization chamber matrix. For charged particle therapy using protons or ions, verification of the absorbed dose is not sufficient. We need to plan and verify the microscopic, or better, the nanoscopic dose distribution. On very small (nanometer) spatial scales, the energy imparted to a given microscopic volume, e.g., a DNA segment of 1-2 helical turns, is a stochastic quantity. Since ionization is considered the most consequential form of energy deposition in radiobiology, ionization cluster size distributions are fundamental for nanodosimetry. Certain quantities, such as the cumulative frequency of more than j ionizations (Fj), where j could be 2, 3, 4, etc., can characterize the biological effect and serve as upper and lower bounds in a constrained dose optimization problem. We are proposing to replace the problem-loaden concept of relative biological effectiveness (RBE) with a nanodosimetric concept of ionization detail (ID), a physical quantity that can be calculated, optimized, and measured using a compact gas-based nanodosimeter, just like macroscopic dose during treatment planning is calculated and then verified. Nanodosimetry started many years ago, initially with large, mostly stationary nanodosimeters built in several laboratories in Europe and at Loma Linda University (LLU) in the U.S. In recent years, investigators at LLU have built prototypes of a more compact nanodosimeter that needs to be further developed and optimized but could eventually be used to do point measurements of nanodosimetric distributions in a matrix-like 3D nanodosimeter. The PhD activities will be developed at C2TN and at LLU in the U.S. to test the next-generation prototype of a compact nanodosimeter. For the planned experiments, we will utilize at a modern proton therapy center (Northwestern Medicine Proton Therapy Center in Warrenville, Illinois). The project investigators will jointly direct the research at C2TN and LLU. They will meet weekly on Zoom. This proposal encompasses two main components: i) nanodosimetry, using state-of-the-art computational tools to assess quantities of relevance for the assessment of the damage induced by ionizing radiation at the DNA level and to study of biophysical repair mechanisms, and ii) radiobiology, using laboratory assays to quantify the biological effectiveness of different types of ionizing radiation including protons, following cell irradiations The LLU supervisors (Professor Reinhard Schulte) is a leading expert in nanodosimetry. The supervisors at C2TN (Ana Belchior and Octávia Monteiro Gil) feature robust and mature expertise in radiobiology, biological dosimetry, and cytogenetics, as well as a sustained level of international collaborations. Over the years, they have participated in projects and networks of excellence and have published in the scientific domains of relevance. The balance and synergies between the supervisors' expertise will be an added value to attain the proposal's objectives. Access to state-of-the-art irradiation facilities, laboratories, and equipment will be available at the institutions mentioned above.

Rationale: Radiotherapy (RT) is commonly associated to the treatment of cancer. However, low-dose radiotherapy is routinely used to remove toxic protein aggregates in chronic degenerative diseases affecting peripheral organs (e.g. eye, larynx, bladder) (1, 2). These toxic protein crystals and fibrils are similar to those found in senile plaques in Alzheimer´s disease (AD) or in Lewy bodies in Parkinson´s disease (PD), among many other neurodegenerative disorders (3). Mouse models of AD treated with low-dose RT showed an impressive reduction in the accumulation of toxic protein aggregates and a parallel improvement in cognitive performance (4). However, the molecular mechanisms for these beneficial effects of RT, as well as its possible side effects, remain unknown. Proton therapy has several advantages over classic electron/photon-based RT for cancer because of its specific properties (5). High energy proton beams present a characteristic Linear Energy Transfer (LET) profile, with low doses deposited along a well-defined linear entry track, until their velocity is diminished resulting in a much larger LET in the region better known as the Bragg Peak. No energy will be deposited beyond the Bragg peak. These properties allow for precise targeting of tumors at specific depths, with simultaneous reduction of toxicity along their path through healthy tissues surrounding the tumorous region. The fact that neurodegenerative disorders such as AD or PD start in deep parts of the brain make proton therapy a good candidate to treat these disorders. However, no study has been carried out to evaluate its potential and possible benefits versus classic RT. Objectives: In this fundamental and multidisciplinary project in the frontiers between physics and biology, we aim at laying the groundwork for the potential application of proton therapy to AD, PD and other neurodegenerative disorders. To achieve this general goal, we will fulfil the following specific objectives: 1) Implement a simulation tool based on the GEANT4-DNA (6) and TOPAS-nBio (7) engines to study the intrinsic characteristics of the effect of traditional and proton RT on toxic protein amyloid structures; and 2) Characterize and compare the effects of traditional versus proton RT on the formation of toxic amyloid aggregates in vitro and in living human cells Research Plan and Methods: This project has two major components: one in nuclear physics (supervised by Daniel Galaviz) and another in biology of neurodegeneration (supervised by Federico Herrera). Both supervisors are experts in their fields. The candidate will be trained in the use of particle transport simulation tools, with the goal of benchmarking the expected effect of different types of RT on toxic protein aggregates related to neurodegenerative disorders. These include the Amyloid beta peptide and tau protein (AD), alpha-synuclein (PD), huntingtin (Huntington´s disease) or GFAP (Alexander´s disease). The implementation of the GEANT4-DNA and TOPAS simulation toolkits will be considered simultaneously to the benchmark of available nuclear data for the different electromagnetic and nuclear probes under study, in order to target both global and local RT effects. In parallel, the student will analyze the formation of toxic protein amyloid structures from purified proteins in vitro, after irradiation with different sources. These experiments will benchmark the performed simulations. The formation of protein aggregates with or without irradiation will be monitored in real-time by spectroscopy methods and Thioflavin staining, but also imaged at selected time points by atomic force microscopy. Irradiated protein aggregates will be characterized by their dynamics and toxicity on living human cells, as well as by their solubility properties in different detergents and experimental conditions. Possible changes in the sequence of amino acids (if the nuclei of atoms are affected by proton RT, for example) will be determined by mass spectrometry or Nuclear Magnetic Resonance. Once the effects of different RT types have been characterized, further structural studies will be applied to selected experimental conditions. NMR or X-ray crystallography will be the primary methods of choice, as they are readily available in Portugal, but the use of foreign cryo-electron microscopy facilities may be also considered necessary. The PhD programme contemplates a 6-month stay in a foreign host institution. We are already considering therapeutic-grade proton beams in Europe, (https://www.ptcog.ch/index.php/facilities-in-operation), and several state-of-the-art structural biology and bioimaging facilities in these and other European cities (https://www.embl.de/services/cryo-em-platform/). The final host institution will depend on the development and the specific needs of the project. In the framework of the present PhD-program, we expect to provide experimental evidence in favor (or against) the potential use of RT for neurodegenerative disorders, with special focus on proton therapy. While the tasks are well within the range of expertise of the supervisors, this project has several high-risk, high profit goals, and requires a student with a multidisciplinary vocation. The student will learn advanced nuclear physics analysis and simulation tools, but also a wide spectrum of biochemistry methods, in a combination of computational analysis and wet laboratory with recombinant proteins and living cells. 1. Copperman, T. S., Truong, M. T., Berk, J. L., and Sobel, R. K. (2019) "External beam radiation for localized periocular amyloidosis: a case series". Orbit (London) 2. Ceyzériat, K., Tournier, B. B., Millet, P., Frisoni, G. B., Garibotto, V., and Zilli, T. (2020) "Low-Dose Radiation Therapy: A New Treatment Strategy for Alzheimer’s Disease?" J. Alzheimer’s Dis. 3. Knowles, T. P. J., Vendruscolo, M., and Dobson, C. M. (2014) "The amyloid state and its association with protein misfolding diseases". Nat. Rev. Mol. Cell Biol. 4. Marples, B., McGee, M., Callan, S., Bowen, S. E., Thibodeau, B. J., Michael, D. B., Wilson, G. D., Maddens, M. E., Fontanesi, J., and Martinez, A. A. (2016) "Cranial irradiation significantly reduces beta amyloid plaques in the brain and improves cognition in a murine model of Alzheimer’s Disease (AD)". Radiother. Oncol. 5. Paganetti, H. (2017) "Proton beam therapy". In Proton beam therapy 6. Incerti, Sebastien; Douglass, Michael; Penfold, Scott; Guatelli, Susanna; and Bezak, Eva (2016) "Review of Geant4-DNA applications for micro and nanoscale simulations". Faculty of Engineering and Information Sciences - Papers: Part A. 6265. 7. McNamara, Aimee L et al. “Geometrical structures for radiation biology research as implemented in the TOPAS-nBio toolkit.” (2018) Physics in medicine and biology vol. 63,17 175018

Unexpected late effects of proton therapy (PT) have been observed over the last few years as the number of patients treated with PT has increased. These side effects could be related to the uncertainties in the relative high biological effectiveness (RBE) of protons stopping in organs at risk (OAR). RBE values across the spread-out Bragg peak (SOBP) of protons range can generally vary from 0.9 – 1.7 [1]. The use of a constant RBE value of 1.1, which is still the accepted clinical practice, disregards experimental evidence that the RBE depends on many factors, including: i) dose per fraction (higher for smaller dose fractions), ii) Tissue type (higher for late-response tissue, in particular in the brain), iii) LET/energy (increasing up to a maximum around 50-70 keV/µm, then falling) and iv) biological endpoint (higher for late-response endpoints, including brainstem necrosis and blindness). DNA damage generated by particle tracks with higher LET are thought to be more complex and more difficult to repair. This effect will increase the magnitude of RBE in critical healthy tissues when protons are stopped in them. Moreover, the effect of fractionation is not included in current RBE considerations but certainly plays a role. There is evidence that RBE is higher with fractionation, further amplifying the high-LET effect of stopping protons. Bearing this, the main goal of the thesis is to go beyond the state-of-the-art and prove that the use of a constant RBE value of 1.1 disregards experimental evidence that the RBE depends on many factors (describe above). To achieve this, we propose to systematically study the frequency of larger ionization clusters in small target volumes of DNA size experimentally with low-pressure gas-based nanodosimetry and with Monte Carlo track structure simulations (Geant4-DNA, or TOPAS-nBio) and correlate them with radiobiological studies. Accordingly, this proposal encompasses a stepwise multidisciplinary approach combining the following scientific objectives: i) measurement of particle track characteristics to establish nanodosimetric parameters of the clinical proton beams for different energies, using Monte Carlo simulations will be performed. We expect that propane-based ionization cross-sections will become available soon, ii) point measurements of nanodosimetric distributions in a matrix-like 3D nanodosimeter to validated the MC simulations in the Northwestern Medicine Chicago Proton Center, iii) quantification of the early and late biological effects, using 3D cell models, and iv) correlation of prospectively collected clinical proton data of brain and head and neck patients with grade 3+ side effects with the ionization cluster size distributions for the plan. We hypothesize that the maximum number of large ionization clusters for the same not-RBE-weighted dose is significantly higher in patients with side effects in age- and gender-matched patients. For the planned experiments, we will utilize at a modern proton therapy center (Northwestern Medicine Proton Therapy Center in Warrenville, Illinois). The project investigators will jointly direct the research at C2TN and LLU. They will meet weekly on Zoom. The LLU supervisor (Professor Reinhard Schulte) is a leading expert in nanodosimetry. The supervisors at C2TN (Ana Belchior and Octávia Monteiro Gil) feature robust and mature expertise in radiobiology, biological dosimetry, and cytogenetics, as well as a sustained level of international collaborations. Over the years, they have participated in projects and networks of excellence and have published in the scientific domains of relevance. The balance and synergies between the supervisors' expertise will be an added value to attain the proposal's objectives. Access to state-of-the-art irradiation facilities, laboratories, and equipment will be available at the institutions mentioned above. REFERENCES [1] Paganetti H. Relative biological effectiveness (RBE) values for proton beam therapy. Variations asa function of biological endpoint, dose, and linear energy transfer. Phys Med Biol 2014;59:R419-72.

This project aims to develop an exosome-based theranostic nanoplatform for non-invasive early detection and treatment of lung metastasis, using tumor-derived exosomes (EXs) as natural delivery vehicles of radionuclides with diagnostic (64Cu) or therapeutic (177Lu) purposes. This represents a new functionality for EXs integrating advanced molecular imaging technology in early diagnosis and treatment of metastatic tumors and with increased clinical translation potential. The intrinsic advantages of EXs as endogenous nanocarriers featuring excellent biocompatibility and low immunogenicity, and the easy functionalization make them exquisite nanoplatforms for radionuclide-based theranostic for lung metastasis. EXs derived from metastatic cell lines will be used as core platforms for linking with cyclotron produced radionuclides. Copper-64 (64Cu), a longer-lived positron-emitter (t1/2=12.7h), will be conjugated at the surface of EVs following a protocol already established by our group. This targeting ability for metastatic lesions will be extended for radionuclide therapy by functionalizing EXs with 177Lu, which delivers cytotoxic radiation with a maximal tissue penetration of 2mm and a shorter emission range of 1.6mm, which makes it ideal for irradiation of smaller lesions. The therapeutic efficacy of 177Lu-labelled EXs will be tested in mice bearing lung metastasis. We have already established a set of animals’ models modeling lung metastatic disease suitable for this study and have available a broad set of small animal imaging modalities, among which stand out the microPET scanner based on resistive plate chambers with submillimeter spatial resolution (0.3mm) able to detect a small cluster of cells within the range size of the micrometastases. The PhD student will be involved in the different main steps of the devised multidisciplinary research plan, which are listed below: 1. Optimize the process of radiolabeling of EXs with 64Cu or 177Lu 2. Pharmacokinetics and in vivo biodistribution studies of [64Cu]EXs by micro-PET . 3. Exploit the diagnostic value of PET with [64Cu]EXs for lung metastasis 4. Demonstrate the therapeutic efficacy of 177Lu-labelled EXs against lung metastasis 5. Radiobiological effects of 177Lu in metastatic lesions