Radioresistance and Transcriptional Reprograming of Invasive Glioblastoma Cells.

PURPOSE: Infiltrative growth pattern is a hallmark of glioblastoma (GBM). Radiation therapy aims to eradicate microscopic residual GBM cells after surgical removal of the visible tumor bulk. However, in-field recurrences remain the major pattern of therapy failure. We hypothesized that the radiosensitivity of peripheral invasive tumor cells (peri) may differ from the predominantly investigated tumor bulk. METHODS AND MATERIALS: Invasive GBM populations were generated via debulking of the visible tumor core and serial orthotopic transplantation of peri cells, and sustained proinvasive phenotype of peri cells was confirmed in vitro by scratch assay and time lapse imaging. In parallel, invasive GBM cells were selected by transwell assay and from peri cells of patient-derived 3-dimensional spheroid cultures. Transcriptome analysis deciphered a GBM invasion-associated gene signature, and functional involvement of key pathways was validated by pharmacologic inhibition. RESULTS: Compared with the bulk cells, invasive GBM populations acquired a radioresistant phenotype characterized by increased cell survival, reduced cell apoptosis, and enhanced DNA double-strand break repair proficiency. Transcriptome analysis revealed a reprograming of invasive cells toward augmented activation of epidermal growth factor receptor- and nuclear factor-κB-related pathways, whereas metabolic processes were downregulated. An invasive GBM score derived from this transcriptional fingerprint correlated well with patient outcome. Inhibition of epidermal growth factor receptor and nuclear factor-κB signaling resensitized invasive cells to irradiation. Invasive cells were eradicated with similar efficacy by particle therapy with carbon ions. CONCLUSIONS: Our data indicate that invasive tumor cells constitute a phenotypically distinct and highly radioresistant GBM subpopulation with prognostic impact that may be vulnerable to targeted therapy and carbon ions.

Simultaneous targeting of TGF-ß/PD-L1 synergizes with radiotherapy by reprogramming the tumor microenvironment to overcome immune evasion.

Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor ß (TGF-ß). We report that a bifunctional fusion protein that simultaneously inhibits TGF-ß and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ T cells. Intriguingly, targeting of the TGF-ß trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.

Charged Particle and Conventional Radiotherapy: Current Implications as Partner for Immunotherapy.

Radiotherapy (RT) has been shown to interfere with inflammatory signals and to enhance tumor immunogenicity via, e.g., immunogenic cell death, thereby potentially augmenting the therapeutic efficacy of immunotherapy. Conventional RT consists predominantly of high energy photon beams. Hypofractionated RT regimens administered, e.g., by stereotactic body radiation therapy (SBRT), are increasingly investigated in combination with cancer immunotherapy within clinical trials. Despite intensive preclinical studies, the optimal dose per fraction and dose schemes for elaboration of RT induced immunogenic potential remain inconclusive. Compared to the scenario of combined immune checkpoint inhibition (ICI) and RT, multimodal therapies utilizing other immunotherapy principles such as adoptive transfer of immune cells, vaccination strategies, targeted immune-cytokines and agonists are underrepresented in both preclinical and clinical settings. Despite the clinical success of ICI and RT combination, e.g., prolonging overall survival in locally advanced lung cancer, curative outcomes are still not achieved for most cancer entities studied. Charged particle RT (PRT) has gained interest as it may enhance tumor immunogenicity compared to conventional RT due to its unique biological and physical properties. However, whether PRT in combination with immune therapy will elicit superior antitumor effects both locally and systemically needs to be further investigated. In this review, the immunological effects of RT in the tumor microenvironment are summarized to understand their implications for immunotherapy combinations. Attention will be given to the various immunotherapeutic interventions that have been co-administered with RT so far. Furthermore, the theoretical basis and first evidences supporting a favorable immunogenicity profile of PRT will be examined.

Case of suspected theobromine poisoning in dairy cattle.

High-yielding dairy cows collapsed and died displaying signs of a disturbed central nervous system (muscle tremor, convulsion) and a considerably reduced body condition score. An intense diagnostic screening did not allow to confirm a definite diagnosis. Therefore, further analyses including an evaluation of feeds and feeding were initiated. The herd was fed a total mixed ration (TMR) based on corn and grass silage of moderate nutritive value supplemented with various amounts of chocolate chips. In retrospect, the amount of chocolate chips added to the respective TMR batches could not be quantified. These chips were purchased from a company producing bakery and chocolate products for human consumption and added to the silage and therefore to the TMR in order to increase the energy intake of the animals. Because the TMR, which was fed during the time of the incidence, was no longer available, a sample of a later batch of silage was examined. The amount of chocolate chips was quantified (0.44% per fresh matter), and a theobromine analysis was performed in the chocolate chips of the available batch (69.7 mg/100 g fresh matter). Because of the possible link between chocolate intake and observed signs, an immediate cessation of using the chocolate chips was recommended in addition to an optimisation of the TMR, that is an increase of the energy density. Even though the theobromine intake during the time of the incidence is unknown and information about toxicity of theobromine in ruminants is limited, we suspect that the feeding of chocolate in this case caused all signs including the sudden death of the cows. Further reasons are that no differential diagnoses were established and the problems at the farm stopped after removing the chocolate from the TMR.

Assessment of RBE-Weighted Dose Models for Carbon Ion Therapy Toward Modernization of Clinical Practice at HIT: In Vitro, in Vivo, and in Patients.

PURPOSE: Present-day treatment planning in carbon ion therapy is conducted with assumptions for a limited number of tissue types and models for effective dose. Here, we comprehensively assess relative biological effectiveness (RBE) in carbon ion therapy and associated models toward the modernization of current clinical practice in effective dose calculation. METHODS: Using 2 human (A549, H460) and 2 mouse (B16, Renca) tumor cell lines, clonogenic cell survival assay was performed for examination of changes in RBE along the full range of clinical-like spread-out Bragg peak (SOBP) fields. Prediction power of the local effect model (LEM1 and LEM4) and the modified microdosimetric kinetic model (mMKM) was assessed. Experimentation and analysis were carried out in the frame of a multidimensional end point study for clinically relevant ranges of physical dose (D), dose-averaged linear energy transfer (LETd), and base-line photon radio-sensitivity (α/ß)x. Additionally, predictions were compared against previously reported RBE measurements in vivo and surveyed in patient cases. RESULTS: RBE model prediction performance varied among the investigated perspectives, with mMKM prediction exhibiting superior agreement with measurements both in vitro and in vivo across the 3 investigated end points. LEM1 and LEM4 performed their best in the highest LET conditions but yielded overestimations and underestimations in low/midrange LET conditions, respectively, as demonstrated by comparison with measurements. Additionally, the analysis of patient treatment plans revealed substantial variability across the investigated models (±20%-30% uncertainty), largely dependent on the selected model and absolute values for input tissue parameters αx and ßx. CONCLUSION: RBE dependencies in vitro, in vivo, and in silico were investigated with respect to various clinically relevant end points in the context of tumor-specific tissue radio-sensitivity assignment and accurate RBE modeling. Discovered model trends and performances advocate upgrading current treatment planning schemes in carbon ion therapy and call for verification via clinical outcome analysis with large patient cohorts.

Modeling the Effect of Hypoxia and DNA Repair Inhibition on Cell Survival After Photon Irradiation.

Mechanistic approaches to modeling the effects of ionizing radiation on cells are on the rise, promising a better understanding of predictions and higher flexibility concerning conditions to be accounted for. In this work we modified and extended a previously published mechanistic model of cell survival after photon irradiation under hypoxia to account for radiosensitization caused by deficiency or inhibition of DNA damage repair enzymes. The model is shown to be capable of describing the survival data of cells with DNA damage repair deficiency, both under norm- and hypoxia. We find that our parameterization of radiosensitization is invariant under change of oxygen status, indicating that the relevant parameters for both mechanisms can be obtained independently and introduced freely to the model to predict their combined effect.

Metabolisable energy intake and growth of privately owned growing dogs in comparison with official recommendations on the growth curve and energy supply.

The correct assumption of metabolisable energy (ME) requirement is essential for the nutrition consultation and diet formulation. In young dogs, too high energy supply can accelerate growth and thus lead to developmental orthopaedic diseases. The aim of the present study was to collect the data on ME intake and body weight (BW) development in privately owned growing dogs in order to compare these data with the current recommendations. Our hypothesis was that the actual ME intake of healthy young dogs would be lower than the actual recommendation. The data of 493 privately owned puppies (median age at first consultation 21 weeks, the median expected mature BW 30 kg) on ME intake, actual and expected mature BW were collected and compared with recommendations of the Society of Nutrition Physiology (GfE, Meyer and Zentek and NRC). In 243 dogs, there was a follow-up. The actual BW did not deviate systematically from the calculated expected BW (R2  = .929). The ME intake significantly decreased with age (p < .05) and significantly increased with expected mature BW (p < .05). There was no significant interaction between these two parameters (p > .05). Sex had no effect on the ME intake (p > .05). The ME intake of young dogs with a history of skeletal problems or of food allergy did not differ systematically from healthy dogs of similar age and expected mature BW. The ME intake was considerably below NRC recommendations, especially in younger puppies (>8-17 weeks: 78%, >17-26 weeks: 83% of NRC recommendation). A predictive linear equation for ME intake was developed: ME intake (MJ) = (1.063 - 0.565 × [actual BW/expected mature BW]) × actual BW0.75 .

Factorial calculation of calcium and phosphorus requirements of growing dogs.

Calcium and phosphorus requirements for growing dogs can be calculated by different methods. The current standard feeding recommendations are based on experimental data derived from young giant breed puppies. In order to determine the absolute requirement, an extrapolation via metabolisable energy requirement is recommended. Another approach is to calculate the requirement factorially, taking into account the endogenous losses and the amount of calcium and phosphorus retained due to tissue accretion during growth as well as the expected availability of these nutrients. The working hypothesis was that both methods are valid and lead to comparable results in young puppies of a high mature body weight (BW). Yet, deviations for other age and mature BW groups were expected. Thus, the aim of the present study was to compare the results of both methods using exemplary puppies of different age and mature BW groups. The hypotheses could be verified for calcium. The extrapolated requirements overestimate the factorial requirements by up to 59.7% for puppies <60kg mature BW and/or >6 months of age. In case of phosphorus requirement, the deviations between both methods are overall very high in all stages. Taking into account the potentially harmful effects of calcium and phosphorus excess, the feeding recommendations based on the extrapolation should be reconsidered.

Biophysical modeling and experimental validation of relative biological effectiveness (RBE) for 4He ion beam therapy.

BACKGROUND: Helium (4He) ion beam therapy provides favorable biophysical characteristics compared to currently administered particle therapies, i.e., reduced lateral scattering and enhanced biological damage to deep-seated tumors like heavier ions, while simultaneously lessened particle fragmentation in distal healthy tissues as observed with lighter protons. Despite these biophysical advantages, raster-scanning 4He ion therapy remains poorly explored e.g., clinical translational is hampered by the lack of reliable and robust estimation of physical and radiobiological uncertainties. Therefore, prior to the upcoming 4He ion therapy program at the Heidelberg Ion-beam Therapy Center (HIT), we aimed to characterize the biophysical phenomena of 4He ion beams and various aspects of the associated models for clinical integration. METHODS: Characterization of biological effect for 4He ion beams was performed in both homogenous and patient-like treatment scenarios using innovative models for estimation of relative biological effectiveness (RBE) in silico and their experimental validation using clonogenic cell survival as the gold-standard surrogate. Towards translation of RBE models in patients, the first GPU-based treatment planning system (non-commercial) for raster-scanning 4He ion beams was devised in-house (FRoG). RESULTS: Our data indicate clinically relevant uncertainty of ±5-10% across different model simulations, highlighting their distinct biological and computational methodologies. The in vitro surrogate for highly radio-resistant tissues presented large RBE variability and uncertainty within the clinical dose range. CONCLUSIONS: Existing phenomenological and mechanistic/biophysical models were successfully integrated and validated in both Monte Carlo and GPU-accelerated analytical platforms against in vitro experiments, and tested using pristine peaks and clinical fields in highly radio-resistant tissues where models exhibit the greatest RBE uncertainty. Together, these efforts mark an important step towards clinical translation of raster-scanning 4He ion beam therapy to the clinic.

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy.

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (HILIC) column (100 x 2.1 mm, 1.7 µm). Serum samples (250 µL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 to 2000 ng/mL for HGA and from 10 to 1000 ng/mL for MCPA-carnitine. HGA and MCPA-carnitine showed acceptable accuracy and precision (bias -3.0% to 1.1%; RSD 9.2% to 12.4%). The limit of quantification (LOQ) was defined as the lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from -79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples, respectively. Applicability was tested in 10 authentic AM cases. In all specimens, relevant amounts of HGA and MCPA-carnitine were found (570-2000 ng/mL; ~8.5-150 ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA-carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM.

Overcoming hypoxia-induced tumor radioresistance in non-small cell lung cancer by targeting DNA-dependent protein kinase in combination with carbon ion irradiation.

BACKGROUND: Hypoxia-induced radioresistance constitutes a major obstacle for a curative treatment of cancer. The aim of this study was to investigate effects of photon and carbon ion irradiation in combination with inhibitors of DNA-Damage Response (DDR) on tumor cell radiosensitivity under hypoxic conditions. METHODS: Human non-small cell lung cancer (NSCLC) models, A549 and H1437, were irradiated with dose series of photon and carbon ions under hypoxia (1% O2) vs. normoxic conditions (21% O2). Clonogenic survival was studied after dual combinations of radiotherapy with inhibitors of DNA-dependent Protein Kinase (DNAPKi, M3814) and ATM serine/threonine kinase (ATMi). RESULTS: The OER at 30% survival for photon irradiation of A549 cells was 1.4. The maximal oxygen effect measured as survival ratio was 2.34 at 8 Gy photon irradiation of A549 cells. In contrast, no significant oxygen effect was found after carbon ion irradiation. Accordingly, the relative effect of 6 Gy carbon ions was determined as 3.8 under normoxia and. 4.11 under hypoxia. ATM and DNA-PK inhibitors dose dependently sensitized tumor cells for both radiation qualities. For 100 nM DNAPKi the survival ratio at 4 Gy more than doubled from 1.59 under normoxia to 3.3 under hypoxia revealing a strong radiosensitizing effect under hypoxic conditions. In contrast, this ratio only moderately increased after photon irradiation and ATMi under hypoxia. The most effective treatment was combined carbon ion irradiation and DNA damage repair inhibition. CONCLUSIONS: Carbon ions efficiently eradicate hypoxic tumor cells. Both, ATMi and DNAPKi elicit radiosensitizing effects. DNAPKi preferentially sensitizes hypoxic cells to radiotherapy.

Comparative analysis of the effects of a sphingosine kinase inhibitor to temozolomide and radiation treatment on glioblastoma cell lines.

Glioblastoma multiforme (GBM) exhibits high resistance to the standard treatment of temozolomide (TMZ) combined with radiotherapy, due to its remarkable cell heterogeneity. Accordingly, there is a need to target alternative molecules enhancing specific GBM autocrine or paracrine mechanisms and amplifying the effect of standard treatment. Sphingosine 1-phosphate (S1P) is such a lipid target molecule with an important role in cell invasion and proliferation. Sphingosine kinase inhibitors (SKI) prevent S1P formation and induce increased production of reactive oxygen species (ROS), which may potentiate radiation cytotoxicity. We analyzed the effect of SKI singular versus combined treatments with TMZ and radiation on 2 human GBM cell lines characterized by a lack of MGMT expression and low or high expression of the anti-oxidant enzyme, glutathione peroxidase 1 (GPx1). Effects were drug concentration-, cell line-dependent and partly ROS-mediated. Clonogenic survival assay demonstrates that SKI was more effective than TMZ in increasing the sensitivity of U87 cells, which express low GPx1 amount, to a 2 Gy X-ray dose. Addition of both SKI and TMZ drastically decreased U87 cells survival compared with the combination temozolomide/radiation. SKI less effectively than TMZ sensitized LN229 cells to the 2 Gy X-ray dose. Its combination to TMZ in absence of irradiation was as efficient as TMZ combination with X-ray. We provide first evidence for SKI as an alternative or complementary treatment to TMZ, and for efficient combinations of low doses of drugs and X-ray. These may help as novel bi-modal and tri-modal therapies to contend with GBM heterogeneity.