Investigations of the migraine-provoking effect of levcromakalim in patients with migraine with aura.

BACKGROUND/HYPOTHESIS: Experimental provocation studies have yielded important insights in migraine pathophysiology. Levcromakalim has been previously shown to induce migraine-like attacks with and without aura. In this study, we aim to further explore the migraine aura-inducing potential of levcromakalim. METHODS: In a double-blind, randomized, placebo-controlled cross-over study, 27 adult participants with migraine with aura received intravenous infusions of levcromakalim and saline. Headache, aura and associated symptoms were evaluated for 24 hours following administration of the study drug. The primary endpoint was occurrence of migraine-like attacks with or without aura in the 24-hour observation period. RESULTS: Thirteen participants developed migraine-like attacks on the active day only (P = 0.0098), and four participants developed aura on the active day only (P = 0.68). The median time to onset of migraine-like headache was three hours, and the median time to onset of aura was 27.5 minutes. CONCLUSION/INTERPRETATION: Our findings affirm the potent migraine-inducing effect of levcromakalim. We observed a lower induction-rate of migraine aura than previously reported. Further studies are warranted to identify predictors of migraine aura following levcromakalim. CLINICALTRIALS.GOV IDENTIFIER: NCT04905654.

Clinical features of migraine with aura: a REFORM study.

BACKGROUND: About one-third of persons with migraine experience transient neurologic symptoms, referred to as aura. Despite its widespread prevalence, comprehensive clinical descriptions of migraine with aura remain sparse. Therefore, we aimed to provide an in-depth phenotypic analysis of aura symptoms and characteristics in a cross-sectional study of a large sample of adults diagnosed with migraine with aura. METHODS: Data were extracted from the baseline characteristics of participants in the Registry for Migraine (REFORM) study - a single-center, prospective, longitudinal cohort study. Participants were adults diagnosed with migraine aura, reporting ≥ 4 monthly migraine days in the preceding 3 months. Trained personnel conducted in-person semi-structured interviews, capturing details on the nature, duration, localization, and progression of individual aura symptoms. RESULTS: Of the 227 enrolled participants with migraine with aura, the mean age was 41.1 years, with a predominant female representation (n = 205 [90.3%]). Visual aura was present in 215 (94.7%) participants, somatosensory aura in 81 (35.7%), and speech and/or language aura in 31 (13.7%). A single type of aura was observed in 148 (65.2%) participants, whilst 79 (34.8%) reported multiple aura types. Most participants (n = 220 [96.9%]) described their aura symptoms as positive or gradually spreading. Headache in relation to aura was noted by 218 (96.0%) participants, with 177 (80.8%) stating that the onset of aura symptoms preceded the onset of headache. CONCLUSIONS: This study offers a detailed clinical depiction of persons with migraine with aura, who were predominantly enrolled from a tertiary care unit. The findings highlight potential gaps in the available literature on migraine with aura and should bolster clinicians' acumen in diagnosing migraine with aura in clinical settings.

A patterns of care analysis of hyperthermia in combination with radio(chemo)therapy or chemotherapy in European clinical centers.

PURPOSE: The combination of hyperthermia (HT) with radio(chemo)therapy or chemotherapy (CT) is an established treatment strategy for specific indications. Its application in routine clinical practice in Europe depends on regulatory and local conditions. We conducted a survey among European clinical centers to determine current practice of HT. METHODS: A questionnaire with 22 questions was sent to 24 European HT centers. The questions were divided into two main categories. The first category assessed how many patients are treated with HT in combination with radio(chemo)therapy or CT for specific indications per year. The second category addressed which hyperthermia parameters are recorded. Analysis was performed using descriptive methods. RESULTS: The response rate was 71% (17/24) and 16 centers were included in this evaluation. Annually, these 16 centers treat approximately 637 patients using HT in combination with radio(chemo)therapy or CT. On average, 34% (range: 3-100%) of patients are treated in clinical study protocols. Temperature readings and the time interval between HT and radio(chemo)therapy or CT are recorded in 13 (81%) and 9 (56%) centers, respectively. The thermal dose quality parameter "cumulative equivalent minutes at 43 °C" (CEM43°C) is only evaluated in five (31%) centers for each HT session. With regard to treatment sequence, 8 (50%) centers administer HT before radio(chemo)therapy and the other 8 in the reverse order. CONCLUSION: There is a significant heterogeneity among European HT centers as to the indications treated and the recording of thermometric parameters. More evidence from clinical studies is necessary to achieve standardization of HT practice.

Investigations of the subarachnoid space as a potential link between aura and headache in migraine: A case-control MRI study.

BACKGROUND: The connection between migraine aura and headache is poorly understood. Some patients experience migraine aura without headache, and patients with migraine aura with headache commonly experience milder headaches with age. The distance between the cerebral cortex and the overlying dura mater has been hypothesized to influence development of headache following aura. We tested this hypothesis by comparing approximated distances between visual cortical areas and overlying dura mater between female patients with migraine aura without headache and female patients with migraine aura with headache. METHODS: Twelve cases with migraine aura without headache and 45 age-matched controls with migraine aura with headache underwent 3.0 T MRI. We calculated average distances between the occipital lobes, between the calcarine sulci, and between the skull and visual areas V1, V2 and V3a. We also measured volumes of corticospinal fluid between the occipital lobes, between the calcarine sulci, and overlying visual areas V2 and V3a. We investigated the relationship between headache status, distances and corticospinal fluid volumes using conditional logistic regression. RESULTS: Distances between the occipital lobes, calcarine sulci and between the skull and V1, V2 and V3a did not differ between patients with migraine aura with headache and patients with migraine aura without headache. We found no differences in corticospinal fluid volumes between groups. CONCLUSION: We found no indication for a connection between visual migraine aura and headache based on cortico-cortical, cortex-to-skull distances, or corticospinal fluid volumes overlying visual cortical areas. Longitudinal studies with imaging sequences optimized for measuring the cortico-dural distance and a larger sample of patients are needed to further investigate the hypothesis.

Migraine aura-like episodes following sclerotherapy for varicose veins of the lower extremities-A systematic review.

OBJECTIVE: This systematic review provides a summary and evaluation of cases of migraine aura-like episodes elicited by sclerotherapy of veins of the lower extremities and discusses possible underlying mechanisms. BACKGROUND: Sclerotherapy is a commonly used treatment for varicose veins. Symptoms resembling migraine aura have been reported during and following sclerotherapy of the lower extremities, suggesting that sclerotherapy may elicit migraine aura. METHODS: We searched PubMed for articles reporting neurological complications that were transient and fully reversible following sclerotherapy treatment for varicose veins in the lower limbs. There were no restrictions regarding article language or publication date. Only original studies and case reports were included. Two authors independently reviewed included articles in detail. Data were extracted from each article, including details on symptoms, previous migraine history, sclerotherapy method, and the presence of a right-to-left cardiac shunt in patients. We evaluated whether episodes fulfilled modified International Classification of Headache Disorders, 3rd edition, criteria for 1.2 Migraine with aura or 1.5.2 Probable migraine with aura. RESULTS: The search yielded 777 articles, 28 of which were included. Twenty-six articles reported 119 episodes of transient neurological symptoms in 34,500 sclerotherapy sessions. Two additional articles reported six episodes of transient neurological symptoms with no specification of the number of sessions. Of the 125 episodes, 119 involved transient visual disturbances, and eight met the modified criteria for Probable migraine with aura. In most episodes (98%), clinical information was insufficient to determine if the criteria were fulfilled. CONCLUSIONS: Symptoms that are clinically indistinguishable from migraine with aura attacks may occur following sclerotherapy, although this likely is rare. Microembolization through a right-to-left shunt triggering cortical spreading depolarization is a possible mechanism. Our findings are limited by infrequent specific assessments for neurological complications and a low level of detail in the description of symptoms in the available literature. Future prospective studies are needed to determine this phenomenon's incidence and underlying mechanisms.

Clinical wIRA-hyperthermia: heating properties and effectiveness in lower trunk regions and its accordance with ESHO quality criteria for superficial hyperthermia.

PURPOSE: The heating characteristics of water-filtered infrared-A (wIRA) radiation were investigated in vivo in two body regions of healthy humans according to the quality standards of the European Society for Hyperthermic Oncology (ESHO) using an irradiance (infrared-A) of 146 W m-2 as recommended for clinical superficial hyperthermia (HT). METHODS: wIRA was applied to the abdominal wall and lumbar region for 60 min. Skin surface temperature was limited to ≤43 °C. Tissue temperatures were measured invasively at 1-min intervals before, during and after wIRA exposure using five fiber-optical probes at depths of 1-20 mm. RESULTS: Significant differences between body regions occurred during the heating-up phase at depths of 5-15 mm. Thermal steady states were reached at depths ≤5 mm after exposures of 5-6 min, and ≤20 mm after 20 min. On average, the minimum requirements of ESHO were exceeded in both regions by the following factors: ≈3 for the heating rate, ≈2 for the specific absorption rate and ≈1.4 for the temperature rise. Tissue depths with T90 ≥ 40 °C and T50 > 41 °C were ≤10 mm, and ≤20 mm for Tmax ≤ 43 °C. The temperature decay time after termination of irradiation was 1-5 min. Corresponding temperatures were ≤42.2 °C for CEM43 and ≤41.8 °C for CEM43T90, i.e., they are inadequate for direct thermal cell killing. CONCLUSIONS: Thermography-controlled wIRA-HT complies with the ESHO criteria for superficial HT as a radiosensitizer and avoids the risk of thermal skin toxicity.

Krüppel-like factor 7 influences translation and pathways involved in ribosomal biogenesis in breast cancer.

BACKGROUND: Ribosomal biogenesis and ribosomal proteins have attracted attention in the context of tumor biology in recent years. Instead of being mere translational machineries, ribosomes might play an active role in tumor initiation and progression. Despite its importance, regulation of ribosomal biogenesis is still not completely understood. METHODS: Using Gene Set Enrichment Analysis of RNA sequencing and proteomical mass spectrometry data in breast cancer cells expressing Krüppel-like factor 7 (KLF7), we identified processes altered by this transcription factor. In silico analyses of a cohort of breast cancer patients in The Cancer Genome Atlas confirmed our finding. We further verified the role of KLF7 the identified ribosomal processes in in vitro assays of mammary carcinoma cell lines and analyses of breast cancer patients' tissue slices. RESULTS: We identified the transcription factor Krüppel-like factor 7 (KLF7) as a regulator of ribosomal biogenesis and translation in breast cancer cells and tissue. Highly significant overlapping processes related to ribosomal biogenesis were identified in proteomics and transcriptomics data and confirmed in patients' breast cancer RNA Seq data. Further, nucleoli, the sites of ribosomal biogenesis, were morphologically altered and quantitatively increased in KLF7-expressing cells. Pre-rRNA processing was identified as one potential process affected by KLF7. In addition, an increase in global translation independent from proliferation and transcription was observed upon exogenous KLF7 expression in vitro. Importantly, in a cohort of breast cancer patients, KLF7-expression levels correlated with aggressiveness of the intrinsic breast cancer subtype and tumor grading. Moreover, KLF7 correlated with nucleolar characteristics in human breast tumor tissue, indicating a role for KLF7 in ribosomal biogenesis. CONCLUSION: In mammary carcinoma, KLF7 is involved in ribosomal biogenesis. Alterations of ribosomal biogenesis has far reaching quantitative and qualitative implications for the proteome of the cancer cells. This might influence the aggressiveness of cancer cells.

Interleukin-6 as surrogate marker for imaging-based hypoxia dynamics in patients with head-and-neck cancers undergoing definitive chemoradiation-results from a prospective pilot trial.

PURPOSE: Intratumoral hypoxia increases resistance of head-and-neck squamous cell carcinoma (HNSCC) to radiotherapy. [18F]FMISO PET imaging enables noninvasive hypoxia monitoring, though requiring complex logistical efforts. We investigated the role of plasma interleukin-6 (IL-6) as potential surrogate parameter for intratumoral hypoxia in HNSCC using [18F]FMISO PET/CT as reference. METHODS: Within a prospective trial, serial blood samples of 27 HNSCC patients undergoing definitive chemoradiation were collected to analyze plasma IL-6 levels. Intratumoral hypoxia was assessed in treatment weeks 0, 2, and 5 using [18F]FMISO PET/CT imaging. The association between PET-based hypoxia and IL-6 was examined using Pearson's correlation and multiple regression analyses, and the diagnostic power of IL-6 for tumor hypoxia response prediction was determined with receiver-operating characteristic analyses. RESULTS: Mean IL-6 concentrations were 15.1, 19.6, and 31.0 pg/mL at baseline, week 2 and week 5, respectively. Smoking (p=0.050) and reduced performance status (p=0.011) resulted in higher IL-6 levels, whereas tumor (p=0.427) and nodal stages (p=0.334), tumor localization (p=0.439), and HPV status (p=0.294) had no influence. IL-6 levels strongly correlated with the intratumoral hypoxic subvolume during treatment (baseline: r=0.775, p<0.001; week 2: r=0.553, p=0.007; week 5: r=0.734, p<0.001). IL-6 levels in week 2 were higher in patients with absent early tumor hypoxia response (p=0.016) and predicted early hypoxia response (AUC=0.822, p=0.031). Increased IL-6 levels at week 5 resulted in a trend towards reduced progression-free survival (p=0.078) and overall survival (p=0.013). CONCLUSION: Plasma IL-6 is a promising surrogate marker for tumor hypoxia dynamics in HNSCC patients and may facilitate hypoxia-directed personalized radiotherapy concepts. TRIAL REGISTRATION: The prospective trial was registered in the German Clinical Trial Register (DRKS00003830). Registered 20 August 2015.

Improved Oxygenation of Human Skin, Subcutis and Superficial Cancers Upon Mild Hyperthermia Delivered by WIRA-Irradiation.

Clinical trials have shown that mild hyperthermia (HT) serves as an adjunct to cancer treatments such as chemo- and radiotherapy. Recently, a high efficacy of mild HT immediately followed by hypofractionated radiotherapy (RT) in treatment of recurrent breast cancer has been documented if temperatures of 39-43 °C are achieved for 40-60 min. In the present study, temperature and oxygenation profiles were measured in superficial tissues of healthy volunteers exposed to water-filtered infrared-A- (wIRA)- irradiation, to verify that adequate thermal doses together with the improved tumor oxygenation necessary for radiosensitisation are obtained. Experiments were performed using a wIRA-system equipped with two wIRA-radiators, each with a thermography camera for real-time monitoring of the skin surface temperature. Temperatures within the abdominal wall were measured with fibre optic sensors at defined tissue depths (subepidermal, and 1-20 mm inside the tissue). The corresponding tissue pO2 values were assessed with fluorometric microsensors. In selected situations, hyperspectral tissue imaging was used to visualise the oxygenation status of normal skin and superficial tumours in patients. Pre-treatment skin surface temperature was 34.6 °C. Upon wIRA exposure, average skin surface temperatures reached 41.6 °C within 5-12 min. Maximum tissue temperatures of 41.8 °C were found at a tissue depth of 1 mm, with a steady decline in deeper tissue layers (41.6 °C @ 5 mm, 40.8 °C @ 10 mm, 40.6 °C @ 15 mm, and 40.1 °C @ 20 mm). Effective HT levels ≥39 °C were established in tissue depths up to 25 mm. Tissue heating was accompanied by a significant increase in tissue pO2 values [e.g., at a tissue depth of 13 mm mean pO2 rose from 46 mmHg to 81 mmHg (@ T = 40.5 °C). In the post-heating phase (+ 5 min), pO2 was 79 mmHg (@ T = 38 °C) and 15 min post-heat pO2 was 72 mmHg (@ T = 36.8 °C)]. pO2 values remained elevated for 30-60 min post-heat. Non-invasive monitoring of normal skin and of recurrent breast cancers confirmed the improved O2 status by wIRA-HT. In conclusion, wIRA-irradiation enables effective tissue heating (T = 39-43 °C) associated with distinct increases in blood flow and pO2. These adjustments unequivocally meet the requirement for effective radiosensitisation.

Human Mesenchymal Stromal Cells Do Not Cause Radioprotection of Head-and-Neck Squamous Cell Carcinoma.

Radiotherapy of head-and-neck squamous cell carcinoma (HNSCC) can cause considerable normal tissue injuries, and mesenchymal stromal cells (MSCs) have been shown to aid regeneration of irradiation-damaged normal tissues. However, utilization of MSC-based treatments for HNSCC patients undergoing radiotherapy is hampered by concerns regarding potential radioprotective effects. We therefore investigated the influence of MSCs on the radiosensitivity of HNSCCs. Several human papillomavirus (HPV)-negative and HPV-positive HNSCCs were co-cultured with human bone marrow-derived MSCs using two-dimensional and three-dimensional assays. Clonogenic survival, proliferation, and viability of HNSCCs after radiotherapy were assessed depending on MSC co-culture. Flow cytometry analyses were conducted to examine the influence of MSCs on irradiation-induced cell cycle distribution and apoptosis induction in HNSCCs. Immunofluorescence stainings of γH2AX were conducted to determine the levels of residual irradiation-induced DNA double-strand breaks. Levels of connective tissue growth factor (CTGF), a multifunctional pro-tumorigenic cytokine, were analyzed using enzyme-linked immunosorbent assays. Neither direct MSC co-culture nor MSC-conditioned medium exerted radioprotective effects on HNSCCs as determined by clonogenic survival, proliferation, and viability assays. Consistently, three-dimensional microwell arrays revealed no radioprotective effects of MSCs. Irradiation resulted in a G2/M arrest of HNSCCs at 96 h independently of MSC co-culture. HNSCCs' apoptosis rates were increased by irradiation irrespective of MSCs. Numbers of residual γH2AX foci after irradiation with 2 or 8 Gy were comparable between mono- and co-cultures. MSC mono-cultures and HNSCC-MSC co-cultures exhibited comparable CTGF levels. We did not detect radioprotective effects of human MSCs on HNSCCs. Our results suggest that the usage of MSC-based therapies for radiotherapy-related toxicities in HNSCC patients may be safe in the context of absent radioprotection.

FAK inhibition radiosensitizes pancreatic ductal adenocarcinoma cells in vitro.

INTRODUCTION: Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase protein frequently overexpressed in cancer and has been linked to an increase in the stem cell population of tumors, resistance to therapy, and metastatic spread. Pharmacological FAK inhibition in pancreatic cancer has received increased attention over the last few years, either alone or in combination with other therapeutics including chemotherapy and immunotherapy. However, its prognostic value and its role in radioresistance of pancreatic ducal adenocarcinoma (PDAC) is unknown. METHODS AND MATERIALS: Using the TCGA and GTEx databases, we investigated the genetic alterations and mRNA expression levels of PTK2 (the encoding-gene for FAK) in normal pancreatic tissue and pancreatic cancer and its impact on patient survival. Furthermore, we evaluated the expression of FAK and its tyrosine domain Ty-397 in three pancreatic cancer cell lines. We went further and evaluated the role of a commercial FAK tyrosine kinase inhibitor VS-4718 on the viability and radiosensitization of the pancreatic cell lines as well as its effect on the extracellular matrix (ECM) production from the pancreatic stellate cells. Furthermore, we tested the effect of combining radiation with VS-4718 in a three-dimensional (3D) multicellular pancreatic tumor spheroid model. RESULTS: A database analysis revealed a relevant increase in genetic alterations and mRNA expression of the PTK2 in PDAC, which were associated with lower progression-free survival. In vitro, there was only variation in the basal phosphorylation level of FAK in cell lines. VS-4718 radiosensitized pancreatic cell lines only in the presence of ECM-producing pancreatic stellate cells and markedly reduced the ECM production in the stromal cells. Finally, using a 3D multicellular tumor model, the combination of VS-4718 and radiotherapy significantly reduced the growth of tumor aggregates. CONCLUSION: Pharmacological inhibition of FAK in pancreatic cancer could be a novel therapeutic strategy as our results show a radiosensitization effect of VS-4718 in vitro in a multicellular 2D- and in a 3D-model of pancreatic cancer.

Symptomatic migraine: A systematic review to establish a clinically important diagnostic entity.

OBJECTIVE: To determine if a clinical presentation indistinguishable from migraine can occur due to an underlying condition or pathology, that is, "symptomatic migraine." BACKGROUND: It is currently not clear whether migraine truly can be caused by an underlying condition or pathology. Characterization of the etiology and clinical features of possible symptomatic migraine is of significant clinical importance and further may help elucidate the pathophysiology of migraine. METHODS: We devised operational diagnostic criteria for "symptomatic migraine" and "possible symptomatic migraine" requiring strong evidence for a causal relation between underlying cause and migraine symptoms adhering strictly to diagnostic criteria. PubMed was searched for case reports of symptomatic migraine from inception to March 2020. Only articles published in English or German were included. No restrictions were placed on study design. Relevant references in the articles were also included. Papers were systematically reviewed by two independent reviewers for detailed clinical features of migraine as well as the proposed underlying conditions and the effects of treatment of these conditions. RESULTS: Our search retrieved 1726 items. After screening, 109 papers comprising 504 cases were reviewed in detail. Eleven patients with migraine with aura (MWA) fulfilled our working criteria for symptomatic migraine, and 39 patients fulfilled our criteria for possible symptomatic migraine. The most common etiologies of symptomatic migraine were arteriovenous malformations, carotid stenosis, dissection or aneurysm, brain infarctions, meningioma, and various intra-axial tumors. CONCLUSIONS: Symptomatic MWA, indistinguishable from idiopathic MWA, may occur due to cortical lesions or microembolization. We found no clear evidence supporting the existence of symptomatic migraine without aura although we did identify possible cases. Our findings are limited by the available literature, and we suggest that prospective studies are needed.

Tspan8 is expressed in breast cancer and regulates E-cadherin/catenin signalling and metastasis accompanied by increased circulating extracellular vesicles.

Tspan8 exhibits a functional role in many cancer types including pancreatic, colorectal, oesophagus carcinoma, and melanoma. We present a first study on the expression and function of Tspan8 in breast cancer. Tspan8 protein was present in the majority of human primary breast cancer lesions and metastases in the brain, bone, lung, and liver. In a syngeneic rat breast cancer model, Tspan8+ tumours formed multiple liver and spleen metastases, while Tspan8- tumours exhibited a significantly diminished ability to metastasise, indicating a role of Tspan8 in metastases. Addressing the underlying molecular mechanisms, we discovered that Tspan8 can mediate up-regulation of E-cadherin and down-regulation of Twist, p120-catenin, and ß-catenin target genes accompanied by the change of cell phenotype, resembling the mesenchymal-epithelial transition. Furthermore, Tspan8+ cells exhibited enhanced cell-cell adhesion, diminished motility, and decreased sensitivity to irradiation. As a regulator of the content and function of extracellular vesicles (EVs), Tspan8 mediated a several-fold increase in EV number in cell culture and the circulation of tumour-bearing animals. We observed increased protein levels of E-cadherin and p120-catenin in these EVs; furthermore, Tspan8 and p120-catenin were co-immunoprecipitated, indicating that they may interact with each other. Altogether, our findings show the presence of Tspan8 in breast cancer primary lesion and metastases and indicate its role as a regulator of cell behaviour and EV release in breast cancer. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Multipotent mesenchymal stromal cells are sensitive to thermic stress - potential implications for therapeutic hyperthermia.

Purpose: Hyperthermia demonstrated clinical efficacy in multimodal cancer treatment. Multipotent mesenchymal stromal cells (MSCs) as part of the tumor-supporting stroma modulate tumor response and tissue regeneration after hyperthermia. We aimed to investigate the effects of hyperthermia on the survival, stem cell characteristics and heat shock expression of human MSCs.Materials and methods: Human MSCs and normal human dermal fibroblasts (NHDFs) were exposed to temperatures between 37 °C and 44 °C for 60 min, and hyperthermic sensitivity was examined by clonogenicity, proliferation and viability assays. The influence of 42 °C hyperthermia on the MSCs' adhesion potential, migratory capacity, surface marker expression and multi-lineage differentiation capability was investigated. Cell cycle distribution, apoptosis and senescence after 42 °C hyperthermia were determined by flow cytometry and ß-galactosidase staining. Heat shock protein expression was determined by Western Blots.Results: MSCs exhibited decreased clonogenic survival after 40 °C and 42 °C hyperthermia compared to NHDFs, while proliferative activity and viability were comparable after hyperthermia up to 44 °C. MSC adhesion was reduced after 42 °C hyperthermia, while the characteristic surface marker expression and the migratory ability remained unaffected in 42 °C hyperthermia-exposed MSCs. 42 °C hyperthermia diminished the adipogenic differential potential of all tested MSC samples. A pronounced G2/M arrest was found after 42 °C hyperthermia and was associated with increased apoptosis and senescence levels in MSCs. MSCs exhibited slightly lower heat shock protein levels compared to NHDFs.Conclusion: Human MSCs exhibit a thermosensitive phenotype which reduced the multipotent cells' regenerative abilities, resulting in impaired tissue regeneration after hyperthermia treatment or thermal injuries. On the other hand, tumor-associated MSCs may be efficiently targeted by hyperthermia treatment.

Isothiazolinones are still widely used in paints purchased in five European countries: a follow-up study.

BACKGROUND: An increasing incidence of contact allergy to methylisothiazolinone (MI) has been seen, caused, in particular, by cosmetic products and paints. A study from 2015 showed that 93.0% of paints bought in five European countries contained MI. New regulations have been discussed for paints in the EU, which may have influenced this market. OBJECTIVES: To re-evaluate the use and concentrations of MI and four other isothiazolinones in water-based wall paints. METHODS: Water-based white wall paints (n = 60) were purchased in retail stores in five European countries: Denmark, France, Germany, Sweden, and the United Kingdom. The paints were analysed for isothiazolione content by the use of high-performance liquid chromatography coupled to ultraviolet detection, and the results were confirmed with high-performance liquid chromatography-tandem mass spectrometry. RESULTS: MI was identified in 55 (91.7%) of the paints, with concentrations ranging from 1.1 to 142.7 ppm. The other isothiazolinones were identified in 20.0% [methylchloroisothiazolinone (MCI)] to 88.3% [benzisothiazolinone (BIT)] of the paints. BIT concentrations varied significantly between countries, whereas MI and MCI concentrations did not. There were no statistically significant differences in MI, MCI and BIT concentrations between the current study and the 2015 study. CONCLUSIONS: MI and other isothiazolinones are widely used in paints available in Europe. Their use does not seem to be decreasing.

Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedisTM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.

From Localized Mild Hyperthermia to Improved Tumor Oxygenation: Physiological Mechanisms Critically Involved in Oncologic Thermo-Radio-Immunotherapy.

(1) Background: Mild hyperthermia (mHT, 39-42 °C) is a potent cancer treatment modality when delivered in conjunction with radiotherapy. mHT triggers a series of therapeutically relevant biological mechanisms, e.g., it can act as a radiosensitizer by improving tumor oxygenation, the latter generally believed to be the commensurate result of increased blood flow, and it can positively modulate protective anticancer immune responses. However, the extent and kinetics of tumor blood flow (TBF) changes and tumor oxygenation are variable during and after the application of mHT. The interpretation of these spatiotemporal heterogeneities is currently not yet fully clarified. (2) Aim and methods: We have undertaken a systematic literature review and herein provide a comprehensive insight into the potential impact of mHT on the clinical benefits of therapeutic modalities such as radio- and immuno-therapy. (3) Results: mHT-induced increases in TBF are multifactorial and differ both spatially and with time. In the short term, changes are preferentially caused by vasodilation of co-opted vessels and of upstream normal tissue vessels as well as by improved hemorheology. Sustained TBF increases are thought to result from a drastic reduction of interstitial pressure, thus restoring adequate perfusion pressures and/or HIF-1α- and VEGF-mediated activation of angiogenesis. The enhanced oxygenation is not only the result of mHT-increased TBF and, thus, oxygen availability but also of heat-induced higher O2 diffusivities, acidosis- and heat-related enhanced O2 unloading from red blood cells. (4) Conclusions: Enhancement of tumor oxygenation achieved by mHT cannot be fully explained by TBF changes alone. Instead, a series of additional, complexly linked physiological mechanisms are crucial for enhancing tumor oxygenation, almost doubling the initial O2 tensions in tumors.

Protocol of the HISTOTHERM study: assessing the response to hyperthermia and hypofractionated radiotherapy in recurrent breast cancer.

Introduction: Breast cancer is globally the leading cancer in women, and despite the high 5-year survival rate the most frequent cause of cancer related deaths. Surgery, systemic therapy and radiotherapy are the three pillars of curative breast cancer treatment. However, locoregional recurrences frequently occur after initial treatment and are often challenging to treat, amongst others due to high doses of previous radiotherapy treatments. Radiotherapy can be combined with local hyperthermia to sensitize tumor cells to radiation and thereby significantly reduce the required radiation dose. Therefore, the combination treatment of mild local hyperthermia, i.e. locally heating of the tissue to 39-43°C, and re-irradiation with a reduced total dose is a relevant treatment option for previously irradiated patients. The mechanisms of this effect in the course of the therapy are to date not well understood and will be investigated in the HISTOTHERM study. Methods and analyses: Patients with local or (loco)regional recurrent breast cancer with macroscopic tumors are included in the study. Local tumor control is evaluated clinically and histologically during the course of a combination treatment of 60 minutes mild superficial hyperthermia (39 - 43°C) using water-filtered infrared A (wIRA) irradiation, immediately followed by hypofractionated re-irradiation with a total dose of 20-24 Gy, administered in weekly doses of 4 Gy. Tumor and tumor stroma biopsies as well as blood samples will be collected prior to treatment, during therapy (at a dose of 12 Gy) and in the follow-up to monitor therapy response. The treatment represents the standard operating procedure for hyperthermia plus re-irradiation. Various tissue and blood-based markers are analyzed. We aim at pinpointing key mechanisms and markers for therapy response which may help guiding treatment decisions in future. In addition, quality of life in the course of treatment will be assessed and survival data will be evaluated. Registration: The study is registered at the German Clinical Trials Register, Deutsches Register Klinischer Studien (DRKS00029221).

Towards unravelling biological mechanisms behind radiation-induced oral mucositis via mass spectrometry-based proteomics.

Objective: Head and neck cancer (HNC) accounts for almost 890,000 new cases per year. Radiotherapy (RT) is used to treat the majority of these patients. A common side-effect of RT is the onset of oral mucositis, which decreases the quality of life and represents the major dose-limiting factor in RT. To understand the origin of oral mucositis, the biological mechanisms post-ionizing radiation (IR) need to be clarified. Such knowledge is valuable to develop new treatment targets for oral mucositis and markers for the early identification of "at-risk" patients. Methods: Primary keratinocytes from healthy volunteers were biopsied, irradiated in vitro (0 and 6 Gy), and subjected to mass spectrometry-based analyses 96 h after irradiation. Web-based tools were used to predict triggered biological pathways. The results were validated in the OKF6 cell culture model. Immunoblotting and mRNA validation was performed and cytokines present in cell culture media post-IR were quantified. Results: Mass spectrometry-based proteomics identified 5879 proteins in primary keratinocytes and 4597 proteins in OKF6 cells. Amongst them, 212 proteins in primary keratinocytes and 169 proteins in OKF6 cells were differentially abundant 96 h after 6 Gy irradiation compared to sham-irradiated controls. In silico pathway enrichment analysis predicted interferon (IFN) response and DNA strand elongation pathways as mostly affected pathways in both cell systems. Immunoblot validations showed a decrease in minichromosome maintenance (MCM) complex proteins 2-7 and an increase in IFN-associated proteins STAT1 and ISG15. In line with affected IFN signalling, mRNA levels of IFNß and interleukin 6 (IL-6) increased significantly following irradiation and also levels of secreted IL-1ß, IL-6, IP-10, and ISG15 were elevated. Conclusion: This study has investigated biological mechanisms in keratinocytes post-in vitro ionizing radiation. A common radiation signature in keratinocytes was identified. The role of IFN response in keratinocytes along with increased levels of pro-inflammatory cytokines and proteins could hint towards a possible mechanism for oral mucositis.