Normal brain tissue reaction after proton irradiation

Protonentherapie ist eine wichtige Behandlungsmodalität in der Radioonkologie. Aufgrund einer vorteilhaften Dosisverteilung im bestrahlten Volumen kann diese Bestrahlungsmethode das tumorumgebende Normalgewebe schützen. Dadurch können Nebenwirkungen in bestimmten Patientenpopulationen, zum Beispiel Kindern oder Patienten mit Gehirntumoren, verringert werden. Trotzdem können nach Protonenbestrahlung von Gehirntumorpatienten Normalgewebsschäden auftreten. Gründe dafür können der notwendige klinische Sicherheitssaum im Normalgewebe, der Einfluss der relativen biologischen Wirksamkeit RBE sowie eine erhöhte Strahlensensitivität bestimmter Gehirnregionen sein. Um diese Aspekte zu beleuchten, werden geeignete präklinische Modelle für die Normalgewebsreaktion im Gehirn nach Protonenbestrahlung benötigt. Darüber hinaus kann eine Risikostratifizierung der Patienten durch die Vorhersage von Nebenwirkungswahrscheinlichkeiten oder der Tumorantwort den Behandlungserfolg erhöhen. Auch hier können präklinische Modelle helfen, um neue prädiktive Biomarker zu finden und um die zugrunde liegenden Mechanismen strahleninduzierter Gehirnschäden besser zu verstehen. Das Ziel dieser Dissertation war die Etablierung und Charakterisierung von adäquaten präklinischen Modellen für die Untersuchung von strahleninduzierten Normalgewebsschäden im Gehirn. Diese Modelle bilden die Grundlage für zukünftige Studien zur Untersuchung von RBE Effekten, der spezifische Strahlensensitivität einzelner Gehirnregionen und neuer Biomarker. Die getesteten Modellsysteme waren in vitro Kulturen von adulten organotypischen Gehirnschnitten, Tumorschnittkultur sowie in vivo Bestrahlung von Gehirnsubvolumina, jeweils mit dem Modellorganismus Maus. Die Etablierung eines Bestrahlungssetups in der experimentellen Protonenanlage und dessen dosimetrische Charakterisierung waren von großer Bedeutung für die Durchführung der biologischen Experimente. Ein weiteres Hauptziel war die Definition klinisch relevanter Endpunkte für frühe und späte Nebenwirkungen. Die Gewebsschnitte wurden durch Messungen des Zellüberlebens und der Entzündungsreaktion, sowie mittels in situ Analyse von Zellmorphologie und DNA Schäden untersucht. Als ergänzendes Modell wurde die Tumorschnittkultur etabliert und ähnliche Endpunkte analysiert. Adulte Gehirnschnitte stellten sich als ungeeignet für präklinische Experimente in der Radioonkologie heraus. Die Messungen von Zelltod und Entzündungswerten zeigten eine starke Zellreaktion auf die Inkulturnahme, aber keine auf die Protonenbestrahlung. In der Histologie wurden gestörte Zellmorphologie, reduzierte Vitalität und eingeschränkte Reparaturfähigkeit von DNA Schäden beobachtet. Daher sollten für strahlenbiologische Experimente andere 3D Zellkulturmodelle in Betracht gezogen werden, wie zum Beispiel Organoide oder durch Tissue Engineering hergestellte Kulturen. Durch die Publikation der Daten leistet diese Dissertation einen wichtigen Beitrag zur aktuellen Forschung, da so künftig die limitierten Ressourcen, die für strahlenbiologische Experimente mit Protonen zur Verfügung stehen, auf relevantere Modelle verwendet werden können. Die Bestrahlung von Gehirnsubvolumina in Mäusen wurde mit dem Ziel etabliert, klinisch vergleichbare Felder zu erreichen. Das gewählte Zielvolumen war der rechte Hippocampus; der Protonenstrahl sollte in der Mitte des Gehirns stoppen. Im Rahmen des Projekts wurde ein Arbeitsablauf für präzise und reproduzierbare Bestrahlung entwickelt. Zur Verifizierung wurde der induzierte DNA Schaden ausgewertet und anschließend mit Monte-Carlos Dosissimulationen korreliert. Die Maushirnbestrahlung lieferte wertvolle Ergebnisse für frühe Zeitpunkte (d.h. innerhalb 24 h nach Bestrahlung). Im Verlauf des Projekts wurde ein Algorithmus erstellt, der schnell und zuverlässig die räumliche Verteilung des DNA Schadens in Relation zur Gesamtzellzahl analysiert. Diese Auswertung zeigte, wie bei der Bestrahlungsplanung vorgesehen, ein Stoppen des Protonenstrahls im Gehirn. Eine anschließende Korrelation der Schadensverteilung mit der applizierten Dosis weist nach, dass das Modell einen wichtigen Beitrag zur Untersuchung des RBE leisten kann. In einer darauf folgenden Studie wurde der Dosis-Zeitverlauf der beobachteten Strahlenreaktion des Normalgewebes genauer beleuchtet. Dafür wurden Untersuchungen des Allgemeinzustands der Versuchstiere, regelmäßige Magnetresonanztomografie (MRI) Messungen über einen Zeitraum von sechs Monaten, sowie abschließende Histologie korreliert. Die Volumenzunahme des Kontrastmittelaustritts, die den Zusammenbruch der Blut-Hirn-Schranke anzeigt, wurde konturiert; aus diesen Daten entstand ein prädiktives Dosis-Volumen Modell. Die Pilotstudie konnte eine dosisabhängige Strahlenreaktion nachweisen, die sich im Zusammenbruch der Blut-Hirn-Schranke, einer Hautreaktion mit vorrübergehender Alopezie, Gewichtsabnahme und zelluläre Veränderung äußerte. Das von den MRI Messungen abgeleitete Modell konnte zuverlässig das Eintreten der Nebenwirkungen, den Krankheitsverlauf, sowie die geschätzte Überlebensdauer der Mäuse vorhersagen. Zusätzlich konnte ein Zusammenhang zwischen den MRI Bildänderungen und den pathologischen Gewebsveränderungen beobachtet werden. Durch die außerordentlich homogene Strahlenreaktion der Tiere können aus den vorliegenden Daten künftig zuverlässig geeignete Dosen für spezifische experimentelle Endpunkte bestimmt werden. Zusammenfassend wurden in dieser Arbeit zwei präklinische Modelle für die Protonengehirnbestrahlung etabliert, nämlich organotypische Gewebsschnitte als 3D Zellkulturmodell sowie in vivo Bestrahlung von Gehirnsubvolumina in Mäusen. Während Zellkulturexperimente die Erwartungen nicht erfüllen konnten, stellen sich die Tierexperimente als hervorragendes Modell für translationale Radioonkologie heraus, welches zusätzlich für andere Strahlenqualitäten eingesetzt werden kann. Darauf basierend können aktuelle und zukünftige Studien die Ursachen von strahleninduzierten Normalgewebsschäden im Gehirn beleuchten, RBE Effekte untersuchen und neue prädiktive Biomarker erforschen.:Contents Abstract i Zusammenfassung v Publications ix List of Figures xiii List of Acronyms and Abbreviations xiv 1 Introduction 3 2 Background 5 2.1 Proton therapy for brain cancer treatment 5 2.1.1 Fundamentals of radiobiology 5 2.1.2 Proton therapy 6 2.1.3 Tumors of the central nervous system 8 2.2 Radiation effects on brain cells 8 2.2.1 Neurons and myelin 9 2.2.2 Blood-brain barrier 9 2.2.3 Astrocytes 10 2.2.4 Microglia 10 2.3 Principles of histology 11 2.3.1 Hematoxylin & eosin staining 12 2.3.2 Immunohistochemistry 13 2.3.3 Bioimage analysis 13 2.4 Techniques in medical imaging 14 2.4.1 Projectional radiography 14 2.4.2 Computed tomography 14 2.4.3 Magnetic resonance imaging 15 2.5 Preclinical models for radiation injury 17 2.5.1 Technical requirements 17 2.5.2 In vitro models 17 2.5.3 Small animal models 18 3 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy 19 3.1 Aim of the study 19 3.2 Conclusion 19 3.3 Author’s contribution 19 3.4 Publication 21 4 High-precision image-guided proton irradiation of mouse brain sub-volumes 41 4.1 Aim of the study 41 4.2 Conclusion 41 4.3 Author’s contribution 41 4.4 Publication 43 5 Late side effects in normal mouse brain tissue after proton irradiation 51 5.1 Aim of the study 51 5.2 Conclusion 51 5.3 Author’s contribution 52 5.4 Publication 53 6 Discussion 71 6.1 Establishment of preclinical models for radiooncology 71 6.1.1 3D cell culture 71 6.1.2 In vivo irradiation of brain subvolumes 73 6.2 Current applications of the mouse model 75 6.2.1 Ongoing data analysis 75 6.2.2 Innovating on-site imaging 76 6.2.3 RBE investigations 77 6.3 Future studies of radiation-induced brain tissue toxicities 79 Acknowledgement XV Supplementary Material XVII 1 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy XVII 2 High-precision image-guided proton irradiation of mouse brain sub-volumes XXVI 3 Late side effects in normal mouse brain tissue after proton irradiation XXXIProton therapy is an important modality in radiation oncology. Due to a favorable dose distribution in the irradiated volume, this treatment allows to spare tumor-surrounding normal tissue. Although this protection can lead to reduced side effects in certain patient populations, such as brain tumor or pediatric patients, normal tissue toxicities can occur to some extend. This could be due to clinical safety margins around the tumor that lead to dose deposition in the normal tissue. The underlying causes might also be related to relative biological effectiveness (RBE) variations or elevated radiosensitivity of certain brain regions. To address these issues, suitable preclinical models for normal brain tissue reaction after proton therapy are needed. In addition, patient stratification to predict the tumor response or the probability of side effects will contribute to increased treatment effectiveness. Preclinical models can improve the process of finding new predictive biomarkers and help to understand underlying mechanisms of radiation-induced brain injury. The aim of this thesis was to establish and characterize suitable preclinical models of brain tissue irradiation effects and set the base for future studies designed to reveal RBE effects, brain region specific radiation sensitivities, and novel biomarkers. The tested model systems were in vitro organotypic brain slice culture (OBSC) and in vivo irradiation of brain subvolumes, both on mouse brain tissue. Setup establishment at the experimental proton beam line and subsequent dosimetry built the foundation for conducting the biological experiments. Additionally, one main goal was defining clinically relevant endpoints for both short- and long-term effects. For OBSC, assays for cell death and inflammation, as well as in situ analysis of cell morphology and DNA damage induction were tested. As comparative model to OBSC, tumor slice culture was established and the results were also used for proton investigation. Adult OBSC turned out as inadequate model for preclinical experiments in radiation oncology. The assays measuring cell death and inflammation indicated a severe reaction during the first days in culture, but no response to irradiation. Histology revealed deficient cell morphology, reduced vitality and impaired DNA damage repair. In conclusion, other 3D cell culture models, such as organoids or tissue engineered constructs, should be considered for radiobiological experiments with protons. By publishing the observations, this thesis contributes to conserving the limited resources of proton radiobiology for more meaningful models. A methodology for irradiation of mouse brain subvolumes was established with a focus on creating fields comparable to clinical practice. The chosen target was the right hippocampus and the goal was to stop the proton beam in the middle of the brain. The project included a workflow for this precise irradiation in a robust and reproducible manner. Evaluation of the induced DNA damage and its correlation to Monte Carlo dose simulations were used for verification. Irradiation of mouse brain subvolumes yielded valuable results for early (i.e. within 24 h after irradiation) time points. An evaluation algorithm was designed for fast and robust analysis of spatial DNA damage distribution in relation to the total cell count. This ratio showed that the beam stopped in the brain tissue, in accordance to the treatment planning. Furthermore, the DNA damage could be reliably correlated with the dose simulation, which proves the value of the presented model for future RBE studies. In a follow-up experiment, the dose-time relationship of induced normal tissue reactions was analysed. For this, scoring of the animals' health status was combined with regular MRI measurements over the course of up to 6 months, and final histopathology. The volume increase of contrast agent leakage - representing breakdown of the blood brain barrier (BBB) - was contoured and the data was used to create a dose-volume response model. This pilot study on long-term radiation effects revealed dose-dependent normal tissue toxicities, including breakdown of the BBB, a skin reaction with temporary alopecia, weight reduction and changes on the cellular level. The model derived from MRI data reliably predicts onset of side effects, volume of brain damage as well as the expected animal survival. In addition, MRI image changes could be correlated to underlying tissue alterations by histopathology. Due to the uniform radiation response of the animals this data set enables to determine endpoint-specific dose values in future experiments. In conclusion, two preclinical models for proton brain irradiation were established, namely OBSC as 3D cell culture model and in vivo irradiation of mouse brain subvolumes. While the former could not yield the anticipated results, the latter emerged as excellent model for translational radiooncology, which can also be applied for experiments with other radiation types. Ongoing and future studies will focus on revealing the causes of normal brain tissue toxicities, studying RBE effects, and investigating new predictive biomarkers.:Contents Abstract i Zusammenfassung v Publications ix List of Figures xiii List of Acronyms and Abbreviations xiv 1 Introduction 3 2 Background 5 2.1 Proton therapy for brain cancer treatment 5 2.1.1 Fundamentals of radiobiology 5 2.1.2 Proton therapy 6 2.1.3 Tumors of the central nervous system 8 2.2 Radiation effects on brain cells 8 2.2.1 Neurons and myelin 9 2.2.2 Blood-brain barrier 9 2.2.3 Astrocytes 10 2.2.4 Microglia 10 2.3 Principles of histology 11 2.3.1 Hematoxylin & eosin staining 12 2.3.2 Immunohistochemistry 13 2.3.3 Bioimage analysis 13 2.4 Techniques in medical imaging 14 2.4.1 Projectional radiography 14 2.4.2 Computed tomography 14 2.4.3 Magnetic resonance imaging 15 2.5 Preclinical models for radiation injury 17 2.5.1 Technical requirements 17 2.5.2 In vitro models 17 2.5.3 Small animal models 18 3 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy 19 3.1 Aim of the study 19 3.2 Conclusion 19 3.3 Author’s contribution 19 3.4 Publication 21 4 High-precision image-guided proton irradiation of mouse brain sub-volumes 41 4.1 Aim of the study 41 4.2 Conclusion 41 4.3 Author’s contribution 41 4.4 Publication 43 5 Late side effects in normal mouse brain tissue after proton irradiation 51 5.1 Aim of the study 51 5.2 Conclusion 51 5.3 Author’s contribution 52 5.4 Publication 53 6 Discussion 71 6.1 Establishment of preclinical models for radiooncology 71 6.1.1 3D cell culture 71 6.1.2 In vivo irradiation of brain subvolumes 73 6.2 Current applications of the mouse model 75 6.2.1 Ongoing data analysis 75 6.2.2 Innovating on-site imaging 76 6.2.3 RBE investigations 77 6.3 Future studies of radiation-induced brain tissue toxicities 79 Acknowledgement XV Supplementary Material XVII 1 Applying Tissue Slice Culture in Cancer Research – Insights from Preclinical Proton Radiotherapy XVII 2 High-precision image-guided proton irradiation of mouse brain sub-volumes XXVI 3 Late side effects in normal mouse brain tissue after proton irradiation XXX

Models for Translational Proton Radiobiology—From Bench to Bedside and Back

The number of proton therapy centers worldwide are increasing steadily, with more than two million cancer patients treated so far. Despite this development, pending questions on proton radiobiology still call for basic and translational preclinical research. Open issues are the on-going discussion on an energy-dependent varying proton RBE (relative biological effectiveness), a better characterization of normal tissue side effects and combination treatments with drugs originally developed for photon therapy. At the same time, novel possibilities arise, such as radioimmunotherapy, and new proton therapy schemata, such as FLASH irradiation and proton mini-beams. The study of those aspects demands for radiobiological models at different stages along the translational chain, allowing the investigation of mechanisms from the molecular level to whole organisms. Focusing on the challenges and specifics of proton research, this review summarizes the different available models, ranging from in vitro systems to animal studies of increasing complexity as well as complementing in silico approaches

Slice2Volume: Fusion of multimodal medical imaging and light microscopy data of irradiation-injured brain tissue in 3D.

The dataset contains comprehensive image data for a total of nine mice, which underwent normal tissue brain irradiation with 90 MeV protons. In particular, the image data comprise cone-bem computed tomographies (CBCT), Monte Carlo beam transport simulations based on those CTs, regular magnetic resonance imaging (MRI) follow-up (≥ 26 weeks), a co-aligned DSURQE mouse brain atlas and scanned whole-brain tissue sections with histochemical and immunofluorescent markers for morphology (H&E), cell nuclei (DAPI), astrocytes (GFAP), microglia (Iba1), the intermediate filament protein Nestin, proliferation (Ki67), neurons (NeuN) and oligodendrocytes (OSP). The volumetric image data (i.e. CBCT, MRI and brain atlas) were co-aligned using the ImageJ plugin Big Warp. The CBCT data was used as spatial reference to allow for mask-based, slice-wise alignment of CBCT and light microscopy image data in 3D with the scriptable registration tool Elastix. We provide the data in raw format and as aligned data sets, as well as their spatial transformations.Chunked zip: The histological data are stored as chunked .zip files (*.zip.001 - *.zip.0XX). In order to unpack the data, download all chunks into the same directory, then unpack

Slice2Volume: Fusion of multimodal medical imaging and light microscopy data of irradiation-injured brain tissue in 3D.

The dataset contains comprehensive image data for a total of nine mice, which underwent normal tissue brain irradiation with 90 MeV protons. In particular, the image data comprise cone-bem computed tomographies (CBCT), Monte Carlo beam transport simulations based on those CTs, regular magnetic resonance imaging (MRI) follow-up (≥ 26 weeks), a co-aligned DSURQE mouse brain atlas and scanned whole-brain tissue sections with histochemical and immunofluorescent markers for morphology (H&E), cell nuclei (DAPI), astrocytes (GFAP), microglia (Iba1), the intermediate filament protein Nestin, proliferation (Ki67), neurons (NeuN) and oligodendrocytes (OSP). The volumetric image data (i.e. CBCT, MRI and brain atlas) were co-aligned using the ImageJ plugin Big Warp. The CBCT data was used as spatial reference to allow for mask-based, slice-wise alignment of CBCT and light microscopy image data in 3D with the scriptable registration tool Elastix. We provide the data in raw format and as aligned data sets, as well as their spatial transformations

DataSheet_1_Combined proton radiography and irradiation for high-precision preclinical studies in small animals.pdf

Background and purposeProton therapy has become a popular treatment modality in the field of radiooncology due to higher spatial dose conformity compared to conventional radiotherapy, which holds the potential to spare normal tissue. Nevertheless, unresolved research questions, such as the much debated relative biological effectiveness (RBE) of protons, call for preclinical research, especially regarding in vivo studies. To mimic clinical workflows, high-precision small animal irradiation setups with image-guidance are needed.Material and methodsA preclinical experimental setup for small animal brain irradiation using proton radiographies was established to perform planning, repositioning, and irradiation of mice. The image quality of proton radiographies was optimized regarding the resolution, contrast-to-noise ratio (CNR), and minimal dose deposition in the animal. Subsequently, proof-of-concept histological analysis was conducted by staining for DNA double-strand breaks that were then correlated to the delivered dose.ResultsThe developed setup and workflow allow precise brain irradiation with a lateral target positioning accuracy ofConclusionProton radiography enables fast and effective high-precision lateral alignment of proton beam and target volume in mouse irradiation experiments with limited dose exposure. In the future, this will enable irradiation of larger animal cohorts as well as fractionated proton irradiation.</p