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

Comprehending cellular changes of radiation-induced brain injury is crucial to prevent and treat the pathology. We provide a unique open dataset of proton-irradiated mouse brains consisting of medical imaging, radiation dose simulations, and large-scale microscopy images, all registered into a common coordinate system. This allows dose-dependent analyses on single-cell level.

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

Background and purpose: Proton 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 methods: A 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. Results: The developed setup and workflow allow precise brain irradiation with a lateral target positioning accuracy of<0.26mm for in vivo experiments at minimal imaging dose of<23mGy per mouse. The custom-made software for image registration enables the fast and precise animal positioning at the beam with low observer-variability. DNA damage staining validated the successful positioning and irradiation of the mouse hippocampus. Conclusion: Proton 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.

Sensitization of Patient-Derived Colorectal Cancer Organoids to Photon and Proton Radiation by Targeting DNA Damage Response Mechanisms.

Pathological complete response (pCR) has been correlated with overall survival in several cancer entities including colorectal cancer. Novel total neoadjuvant treatment (TNT) in rectal cancer has achieved pathological complete response in one-third of the patients. To define better treatment options for nonresponding patients, we used patient-derived organoids (PDOs) as avatars of the patient's tumor to apply both photon- and proton-based irradiation as well as single and combined chemo(radio)therapeutic treatments. While response to photon and proton therapy was similar, PDOs revealed heterogeneous responses to irradiation and different chemotherapeutic drugs. Radiotherapeutic response of the PDOs was significantly correlated with their ability to repair irradiation-induced DNA damage. The classical combination of 5-FU and irradiation could not sensitize radioresistant tumor cells. Ataxia-telangiectasia mutated (ATM) kinase was activated upon radiation, and by inhibition of this central sensor of DNA damage, radioresistant PDOs were resensitized. The study underlined the capability of PDOs to define nonresponders to irradiation and could delineate therapeutic approaches for radioresistant patients.

Orthotopic Glioblastoma Models for Evaluation of the Clinical Target Volume Concept.

In times of high-precision radiotherapy, the accurate and precise definition of the primary tumor localization and its microscopic spread is of enormous importance. In glioblastoma, the microscopic tumor extension is uncertain and, therefore, population-based margins for Clinical Target Volume (CTV) definition are clinically used, which could either be too small-leading to increased risk of loco-regional recurrences-or too large, thus, enhancing the probability of normal tissue toxicity. Therefore, the aim of this project is to investigate an individualized definition of the CTV in preclinical glioblastoma models based on specific biological tumor characteristics. The microscopic tumor extensions of two different orthotopic brain tumor models (U87MG_mCherry; G7_mCherry) were evaluated before and during fractionated radiotherapy and correlated with corresponding histological data. Representative tumor slices were analyzed using Matrix-Assisted Laser Desorption/Ionization (MALDI) and stained for putative stem-like cell markers as well as invasion markers. The edges of the tumor are clearly shown by the MALDI segmentation via unsupervised clustering of mass spectra and are consistent with the histologically defined border in H&E staining in both models. MALDI component analysis identified specific peaks as potential markers for normal brain tissue (e.g., 1339 m/z), whereas other peaks demarcated the tumors very well (e.g., 1562 m/z for U87MG_mCherry) irrespective of treatment. MMP14 staining revealed only a few positive cells, mainly in the tumor border, which could reflect the invasive front in both models. The results of this study indicate that MALDI information correlates with microscopic tumor spread in glioblastoma models. Therefore, an individualized CTV definition based on biological tumor characteristics seems possible, whereby the visualization of tumor volume and protein heterogeneity can be potentially used to define radiotherapy-sensitive and resistant areas.

Combined Systemic Drug Treatment with Proton Therapy: Investigations on Patient-Derived Organoids.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

Radiomics-based tumor phenotype determination based on medical imaging and tumor microenvironment in a preclinical setting.

BACKGROUND AND PURPOSE: Radiomics analyses have been shown to predict clinical outcomes of radiotherapy based on medical imaging-derived biomarkers. However, the biological meaning attached to such image features often remains unclear, thus hindering the clinical translation of radiomics analysis. In this manuscript, we describe a preclinical radiomics trial, which attempts to establish correlations between the expression of histological tumor microenvironment (TME)- and magnetic resonance imaging (MRI)-derived image features. MATERIALS & METHODS: A total of 114 mice were transplanted with the radioresistant and radiosensitive head and neck squamous cell carcinoma cell lines SAS and UT-SCC-14, respectively. The models were irradiated with five fractions of protons or photons using different doses. Post-treatment T1-weighted MRI and histopathological evaluation of the TME was conducted to extract quantitative features pertaining to tissue hypoxia and vascularization. We performed radiomics analysis with leave-one-out cross validation to identify the features most strongly associated with the tumor's phenotype. Performance was assessed using the area under the curve (AUCValid) and F1-score. Furthermore, we analyzed correlations between TME- and MRI features using the Spearman correlation coefficient ρ. RESULTS: TME and MRI-derived features showed good performance (AUCValid,TME = 0.72, AUCValid,MRI = 0.85, AUCValid,Combined=0.85) individual tumor phenotype prediction. We found correlation coefficients of ρ=-0.46 between hypoxia-related TME features and texture-related MRI features. Tumor volume was a strong confounder for MRI feature expression. CONCLUSION: We demonstrated a preclinical radiomics implementation and notable correlations between MRI- and TME hypoxia-related features. Developing additional TME features may help to further unravel the underlying biology.

Plasticity within Aldehyde Dehydrogenase-Positive Cells Determines Prostate Cancer Radiosensitivity.

Tumor heterogeneity and cellular plasticity are key determinants of tumor progression, metastatic spread, and therapy response driven by the cancer stem cell (CSC) population. Within the current study, we analyzed irradiation-induced plasticity within the aldehyde dehydrogenase (ALDH)-positive (ALDH+) population in prostate cancer. The radiosensitivity of xenograft tumors derived from ALDH+ and ALDH-negative (ALDH-) cells was determined with local tumor control analyses and demonstrated different dose-response profiles, time to relapse, and focal adhesion signaling. The transcriptional heterogeneity was analyzed in pools of 10 DU145 and PC3 cells with multiplex gene expression analyses and illustrated a higher degree of heterogeneity within the ALDH+ population that even increases upon irradiation in comparison with ALDH- cells. Phenotypic conversion and clonal competition were analyzed with fluorescence protein-labeled cells to distinguish cellular origins in competitive three-dimensional cultures and xenograft tumors. We found that the ALDH+ population outcompetes ALDH- cells and drives tumor growth, in particular upon irradiation. The observed dynamics of the cellular state compositions between ALDH+ and ALDH- cells in vivo before and after tumor irradiation was reproduced by a probabilistic Markov compartment model that incorporates cellular plasticity, clonal competition, and phenotype-specific radiosensitivities. Transcriptional analyses indicate that the cellular conversion from ALDH- into ALDH+ cells within xenograft tumors under therapeutic pressure was partially mediated through induction of the transcriptional repressor SNAI2. In summary, irradiation-induced cellular conversion events are present in xenograft tumors derived from prostate cancer cells and may be responsible for radiotherapy failure. IMPLICATIONS: The increase of ALDH+ cells with stem-like features in prostate xenograft tumors after local irradiation represents a putative cellular escape mechanism inducing tumor radioresistance.

Molecular Response to Combined Molecular- and External Radiotherapy in Head and Neck Squamous Cell Carcinoma (HNSCC).

Combination treatment of molecular targeted and external radiotherapy is a promising strategy and was shown to improve local tumor control in a HNSCC xenograft model. To enhance the therapeutic value of this approach, this study investigated the underlying molecular response. Subcutaneous HNSCC FaDuDD xenografts were treated with single or combination therapy (X-ray: 0, 2, 4 Gy; anti-EGFR antibody (Cetuximab) (un-)labeled with Yttrium-90 (90Y)). Tumors were excised 24 h post respective treatment. Residual DNA double strand breaks (DSB), mRNA expression of DNA damage response related genes, immunoblotting, tumor histology, and immunohistological staining were analyzed. An increase in number and complexity of residual DNA DSB was observed in FaDuDD tumors exposed to the combination treatment of external irradiation and 90Y-Cetuximab relative to controls. The increase was observed in a low oxygenated area, suggesting the expansion of DNA DSB damages. Upregulation of genes encoding p21cip1/waf1 (CDKN1A) and GADD45α (GADD45A) was determined in the combination treatment group, and immunoblotting as well as immunohistochemistry confirmed the upregulation of p21cip1/waf1. The increase in residual γH2AX foci leads to the blockage of cell cycle transition and subsequently to cell death, which could be observed in the upregulation of p21cip1/waf1 expression and an elevated number of cleaved caspase-3 positive cells. Overall, a complex interplay between DNA damage repair and programmed cell death accounts for the potential benefit of the combination therapy using 90Y-Cetuximab and external radiotherapy.

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.

Radiotherapy enhances uptake and efficacy of 90Y-cetuximab: A preclinical trial.

BACKGROUND AND PURPOSE: Systemic molecular radiotherapy utilizes internal irradiation by radionuclide-labeled tumor-targeting agents with the potential to destroy (micro-)metastases. However, doses that are applicable in solid tumors do not reach the levels nessecary for tumor control. Thus, the combination of molecular and external radiotherapy is a promising treatment strategy, as enhanced tumor doses can be delivered with and without minor overlapping toxicities. Here, we combined a 90Y-labeled anti-EGFR antibody (Cetuximab) with clinically relevant fractionated radiotherapy in a preclinical trial using head and neck squamous cell carcinoma xenograft tumors. MATERIALS AND METHODS: To model 90Y-Cetuximab uptake for treatment schedule optimization, FaDu-bearing mice were injected with near-infrared-labeled-Cetuximab at different time points during radiotherapy with differing doses. Cetuximab uptake was longitudinally followed by in vivo-optical imaging. Tumor control probability experiments with fractionated radiotherapy (30 fx, 6 weeks, 8 dose groups/ arm) in combination with 90Y-Cetuximab were performed to test the curative potential. RESULTS: Imaging of near-infrared-labeled-Cetuximab uptake revealed that low to moderate external beam doses can enhance antibody uptake. Using the optimized schedule, combination of molecular and external radiotherapy using 90Y-Cetuximab at a dose that did not result in permanent tumor inactivation in previous experiments, led to substantially increased tumor control compared to radiotherapy alone. CONCLUSION: Our results indicate that combination of radiolabeled therapeutics with clinically relevant fractionated radiotherapy has a remarkable potential to improve curative treatment outcome. Application of some radiation dose prior to injection may improve drug uptake and enable patient stratification and treatment personalization via a corresponding PET-tracer during therapy.

Applying Tissue Slice Culture in Cancer Research-Insights from Preclinical Proton Radiotherapy.

A challenge in cancer research is the definition of reproducible, reliable, and practical models, which reflect the effects of complex treatment modalities and the heterogeneous response of patients. Proton beam radiotherapy (PBRT), relative to conventional photon-based radiotherapy, offers the potential for iso-effective tumor control, while protecting the normal tissue surrounding the tumor. However, the effects of PBRT on the tumor microenvironment and the interplay with newly developed chemo- and immunotherapeutic approaches are still open for investigation. This work evaluated thin-cut tumor slice cultures (TSC) of head and neck cancer and organotypic brain slice cultures (OBSC) of adult mice brain, regarding their relevance for translational radiooncology research. TSC and OBSC were treated with PBRT and investigated for cell survival with a lactate dehydrogenase (LDH) assay, DNA repair via the DNA double strand break marker γH2AX, as well as histology with regards to morphology. Adult OBSC failed to be an appropriate model for radiobiological research questions. However, histological analysis of TSC showed DNA damage and tumor morphological results, comparable to known in vivo and in vitro data, making them a promising model to study novel treatment approaches in patient-derived xenografts or primary tumor material.

High-precision image-guided proton irradiation of mouse brain sub-volumes.

BACKGROUND AND PURPOSE: Proton radiotherapy offers the potential to reduce normal tissue toxicity. However, clinical safety margins, range uncertainties, and varying relative biological effectiveness (RBE) may result in a critical dose in tumor-surrounding normal tissue. To assess potential adverse effects in preclinical studies, image-guided proton mouse brain irradiation and analysis of DNA damage repair was established. MATERIAL AND METHODS: We designed and characterized a setup to shape proton beams with 7 mm range in water and 3 mm in diameter and commissioned a Monte Carlo model for in vivo dose simulation. Cone-beam computed tomography and orthogonal X-ray imaging were used to delineate the right hippocampus and position the mice. The brains of three C3H/HeNRj mice were irradiated with 8 Gy and excised 30 min later. Initial DNA double-strand breaks were visualized by staining brain sections for cell nuclei and γH2AX. Imaged sections were analyzed with an automated and validated processing pipeline to provide a quantitative, spatially resolved radiation damage indicator. RESULTS: The analyzed DNA damage pattern clearly visualized the radiation effect in the mouse brains and could be mapped to the simulated dose distribution. The proton beam passed the right hippocampus and stopped in the central brain region for all evaluated mice. CONCLUSION: We established image-guided proton irradiation of mouse brains. The clinically oriented workflow facilitates (back-) translational studies. Geometric accuracy, detailed Monte Carlo dose simulations, and cell-based assessment enable a biologically and spatially resolved analysis of radiation response and RBE.

Late Side Effects in Normal Mouse Brain Tissue After Proton Irradiation.

Radiation-induced late side effects such as cognitive decline and normal tissue complications can severely affect quality of life and outcome in long-term survivors of brain tumors. Proton therapy offers a favorable depth-dose deposition with the potential to spare tumor-surrounding normal tissue, thus potentially reducing such side effects. In this study, we describe a preclinical model to reveal underlying biological mechanisms caused by precise high-dose proton irradiation of a brain subvolume. We studied the dose- and time-dependent radiation response of mouse brain tissue, using a high-precision image-guided proton irradiation setup for small animals established at the University Proton Therapy Dresden (UPTD). The right hippocampal area of ten C57BL/6 and ten C3H/He mice was irradiated. Both strains contained four groups (nirradiated = 3, ncontrol = 1) treated with increasing doses (0 Gy, 45 Gy, 65 Gy or 85 Gy and 0 Gy, 40 Gy, 60 Gy or 80 Gy, respectively). Follow-up examinations were performed for up to six months, including longitudinal monitoring of general health status and regular contrast-enhanced magnetic resonance imaging (MRI) of mouse brains. These findings were related to comprehensive histological analysis. In all mice of the highest dose group, first symptoms of blood-brain barrier (BBB) damage appeared one week after irradiation, while a dose-dependent delay in onset was observed for lower doses. MRI contrast agent leakage occurred in the irradiated brain areas and was progressive in the higher dose groups. Mouse health status and survival corresponded to the extent of contrast agent leakage. Histological analysis revealed tissue changes such as vessel abnormalities, gliosis, and granule cell dispersion, which also partly affected the non-irradiated contralateral hippocampus in the higher dose groups. All observed effects depended strongly on the prescribed radiation dose and the outcome, i.e. survival, image changes, and tissue alterations, were very consistent within an experimental dose cohort. The derived dose-response model will determine endpoint-specific dose levels for future experiments and may support generating clinical hypotheses on brain toxicity after proton therapy.

Multi-modality bedding platform for combined imaging and irradiation of mice.

Preclinical imaging and irradiation yields valuable insights into clinically relevant research topics. While complementary imaging methods such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) can be combined within single devices, this is technically demanding and cost-intensive. Similarly, bedding and setup solutions are often specific to certain devices and research questions. We present a bedding platform for mice that is compatible with various preclinical imaging modalities (combined PET/MRI, cone beam CT) and irradiation with photons and protons. It consists of a 3D-printed bedding unit (acrylonitrile butadiene styrene, ABS) holding the animal and features an inhalation anesthesia mask, jaw fixation, ear pins, and immobilization for the hind leg. It can be embedded on mounting adaptors for multi-modal imaging and into a transport box (polymethyl methacrylate, PMMA) for experiments outside dedicated animal facilities while maintaining the animal's hygiene status. A vital support unit provides heating, inhalation anesthesia, and a respiration monitor. We dosimetrically evaluated used materials in order to assess their interaction with incident irradiation. Proof-of-concept multi-modal imaging protocols were used on phantoms and mice. The measured attenuation of the bedding unit for 40/60/80/200 kV X-rays was less than 3%. The measured stopping-power-ratio of ABS was 0.951, the combined water-equivalent thickness of bedding unit and transport box was 4.2 mm for proton energies of 150 MeV and 200 MeV. Proof-of-concept imaging showed no loss of image quality. Imaging data of individual mice from different imaging modalities could be aligned rigidly. The presented bed aims to provide a platform for experiments related to both multi-modal imaging and irradiation, thus offering the possibility for image-guided irradiation which relies on precise imaging and positioning. The usage as a self-contained, stand-alone unit outside dedicated animal facilities represents an advantage over setups designed for specific devices.

Pre-clinical imaging for establishment and comparison of orthotopic non-small cell lung carcinoma: in search for models reflecting clinical scenarios.

OBJECTIVE:: Clinically relevant animal models of non-small cell lung carcinoma (NSCLC) are required for the validation of novel treatments. We compared two different orthotopic transplantation techniques as well as imaging modalities to identify suitable mouse models mimicking clinical scenarios. METHODS:: We used three genomically diverse NSCLC cell lines [National Cancer Institute (NCI)-H1703 adenosquamous cell carcinoma, NCI-H23 adenocarcinoma and A549 adenocarcinoma) for implanting tumour cells either as spheroids or cell suspension into lung parenchyma. Bioluminescence imaging (BLI) and contrast-enhanced cone beam CT (CBCT) were performed twice weekly to monitor tumour growth. Tumour histological data and microenvironmental parameters were determined. RESULTS:: Tumour development after spheroid-based transplantation differs probably due to the integrity of spheroids, as H1703 developed single localised nodules, whereas H23 showed diffuse metastatic spread starting early after transplantation. A549 transplantation as cell suspension with the help of a stereotactic system was associated with initial single localised tumour growth and eventual metastatic spread. Imaging techniques were successfully applied to monitor longitudinal tumour growth: BLI revealed highly sensitive qualitative data, whereas CBCT was associated with less sensitive quantitative data. Histology revealed significant model-dependent heterogeneity in proliferation, hypoxia, perfusion and necrosis. CONCLUSION:: Our developed orthotopic NSCLC tumours have similarity with biological growth behaviour comparable to that seen in the clinic and could therefore be used as attractive models to study tumour biology and evaluate new therapeutic strategies. The use of human cancer cell lines facilitates testing of different genomic tumour profiles that may affect treatment outcomes. ADVANCES IN KNOWLEDGE:: The combination of different imaging modalities to identify tumour growth with subsequent use in treatment planning and orthotopic transplantation techniques to develop initially single lesions to ultimate metastases pave the way towards representative pre-clinical NSCLC models for experimental testing of novel therapeutic options in future studies.

Characterization of a switchable chimeric antigen receptor platform in a pre-clinical solid tumor model.

The universal modular chimeric antigen receptor (UniCAR) platform redirects CAR-T cells using a separated, soluble targeting module with a short half-life. This segregation allows precise controllability and flexibility. Herein we show that the UniCAR platform can be used to efficiently target solid cancers in vitro and in vivo using a pre-clinical prostate cancer model which overexpresses prostate stem cell antigen (PSCA). Short-term administration of the targeting module to tumor bearing immunocompromised mice engrafted with human UniCAR-T cells significantly delayed tumor growth and prolonged survival of recipient mice both in a low and high tumor burden model. In addition, we analyzed phenotypic and functional changes of cancer cells and UniCAR-T cells in association with the administration of the targeting module to reveal potential immunoevasive mechanisms. Most notably, UniCAR-T cell activation induced upregulation of immune-inhibitory molecules such as programmed death ligands. In conclusion, this work illustrates that the UniCAR platform mediates potent anti-tumor activity in a relevant in vitro and in vivo solid tumor model.

Development of a genetic sensor that eliminates p53 deficient cells.

The TP53 gene fulfills a central role in protecting cells from genetic insult. Given this crucial role it might be surprising that p53 itself is not essential for cell survival. Indeed, TP53 is the single most mutated gene across different cancer types. Thus, both a theoretical and a question of significant practical applicability arise: can cells be programmed to make TP53 an essential gene? Here we present a genetic p53 sensor, in which the loss of p53 is coupled to the rise of HSV-TK expression. We show that the sensor can distinguish both p53 knockout and cells expressing a common TP53 cancer mutation from otherwise isogenic TP53 wild-type cells. Importantly, the system is sensitive enough to specifically target TP53 loss-of-function cells with the HSV-TK pro-drug Ganciclovir both in vitro and in vivo. Our work opens new ways to programming cell intrinsic transformation protection systems that rely on endogenous components.

Proton radiography for inline treatment planning and positioning verification of small animals.

INTRODUCTION: As proton therapy becomes increasingly well established, there is a need for high-quality clinically relevant in vivo data to gain better insight into the radiobiological effects of proton irradiation on both healthy and tumor tissue. This requires the development of easily applicable setups that allow for efficient, fractionated, image-guided proton irradiation of small animals, the most widely used pre-clinical model. MATERIAL AND METHODS: Here, a method is proposed to perform dual-energy proton radiography for inline positioning verification and treatment planning. Dual-energy proton radiography exploits the differential enhancement of object features in two successively measured two-dimensional (2D) dose distributions at two different proton energies. The two raw images show structures that are dominated by energy absorption (absorption mode) or scattering (scattering mode) of protons in the object, respectively. Data post-processing allowed for the separation of both signal contributions in the respective images. The images were evaluated regarding recognizable object details and feasibility of rigid registration to acquired planar X-ray scans. RESULTS: Robust, automated rigid registration of proton radiography and planar X-ray images in scattering mode could be reliably achieved with the animal bedding unit used as registration landmark. Distinguishable external and internal features of the imaged mouse included the outer body contour, the skull with substructures, the lung, abdominal structures and the hind legs. Image analysis based on the combined information of both imaging modes allowed image enhancement and calculation of 2D water-equivalent path length (WEPL) maps of the object along the beam direction. DISCUSSION: Fractionated irradiation of exposed target volumes (e.g., subcutaneous tumor model or brain) can be realized with the suggested method being used for daily positioning and range determination. Robust registration of X-ray and proton radiography images allows for the irradiation of tumor entities that require conventional computed tomography (CT)-based planning, such as orthotopic lung or brain tumors, similar to conventional patient treatment.

Adhesion- and stress-related adaptation of glioma radiochemoresistance is circumvented by ß1 integrin/JNK co-targeting.

Resistance of cancer stem-like and cancer tumor bulk cells to radiochemotherapy and destructive infiltration of the brain fundamentally influence the treatment efficiency to cure of patients suffering from Glioblastoma (GBM). The interplay of adhesion and stress-related signaling and activation of bypass cascades that counteract therapeutic approaches remain to be identified in GBM cells. We here show that combined inhibition of the adhesion receptor ß1 integrin and the stress-mediator c-Jun N-terminal kinase (JNK) induces radiosensitization and blocks invasion in stem-like and patient-derived GBM cultures as well as in GBM cell lines. In vivo, this treatment approach not only significantly delays tumor growth but also increases median survival of orthotopic, radiochemotherapy-treated GBM mice. Both, in vitro and in vivo, effects seen with ß1 integrin/JNK co-inhibition are superior to the monotherapy. Mechanistically, the in vitro radiosensitization provoked by ß1 integrin/JNK targeting is caused by defective DNA repair associated with chromatin changes, enhanced ATM phosphorylation and prolonged G2/M cell cycle arrest. Our findings identify a ß1 integrin/JNK co-dependent bypass signaling for GBM therapy resistance, which might be therapeutically exploitable.