Factors released by low and high-LET irradiated fibroblasts modulate migration and invasiveness of pancreatic cancer cells.

Introduction: Radiotherapy represents a major treatment option for patients with pancreatic cancer, however, its benefits remain limited also due to the ability of cancer cells to migrate to the surrounding tissues. Low-LET ionizing radiation is well known to promote tumor cell migration and invasion, nevertheless, little data provided by studies using high-LET radiation has led to ambiguous findings. What is hypothesized to be fundamental in the modulation of migration of tumor cells exposed to ionizing radiation is the influence of the microenvironment. Therefore, the properties of cells that populate the tumor stroma cannot be ignored when studying the influence of radiation on the migratory and invasive capacity of cancer cells. This is especially important in the case of pancreatic malignancies that are characterized by an abundance of stromal cells, including cancer-associated fibroblasts, which are known to orchestrate the cross-talk with tumor cells. Aim: The current study aims to investigate whether the presence of factors released by irradiated fibroblasts affects the migratory and invasive capacity of pancreatic cancer cells exposed to different doses of photons or C-ions. Materials and methods: AsPC-1 and AG01522 cells were irradiated with the same dose of photons or C-ions at room temperature. Through Boyden chamber assay, we tested whether factors secreted by irradiated fibroblasts may influence tumor cell migration, while the invasiveness of AsPC-1 cells was assessed using matrigel precoated inserts in which medium collected from non-irradiated (0 Gy), photon and C-ion irradiated fibroblasts, was added. Data were analyzed by Student t-test using GraphPad software. The mean ± s.d. was determined with a significance level of p<0.05. Results: In the presence of conditioned medium collected from 1 Gy and 2 Gy photon irradiated fibroblasts, the number of migrated tumor cells increased (P<0.0360, P<0.0001) but decreased at 4 Gy dose (P<0.002). There was a trend of reduction in migration (P<0.0460, P<0.038, P<0.0024, P<0.0002), as well as a decrease in invasiveness (P<0.0525, P<0.0035, P<0.0868, P<0.0310) after exposure to 0.5 Gy, 1 Gy, 2 Gy and 4 Gy of C-ions. Conclusions: The presence of irradiated fibroblasts affected the invasiveness capability of pancreatic cancer cells, probably by the reciprocal release of soluble factors whose production is differently modulated after high or low-LET radiation. Understanding the effects of irradiation on the metastatic potential of pancreatic cancer cells is of utmost importance for improving the outcome and tailoring the therapeutic approach. This challenging scenario requires a continuous and multidisciplinary approach that involves clinicians together with researcher experts in oncological and radiation treatment. In the last years, including preclinical experiences in a multidisciplinary approach has proved to be a winning strategy in clinical oncological research.

Assessment of the Exposure to Gradient Magnetic Fields Generated by MRI Tomographs: Measurement Method, Verification of Limits and Clearance Areas through a Web-Based Platform.

This work is the result of a campaign of measures of exposure levels to magnetic field gradients (GMF) generated by magnetic resonance imaging (MRI) tomographs, to which both healthcare staff and any persons accompanying patients who remain inside the magnet room are exposed while performing a diagnostic Investigation. The study was conducted on three MRI tomographs with a static magnetic induction field up to 1.5 T installed in two hospitals of Lombardy. The study aims to characterize electromagnetic emissions within the magnet room and the definition of a measurement method suitable for assessing the level of exposure of healthcare personnel and any persons accompanying patients. The measurements performed concerned the determination of the weighted peak index for magnetic induction, due to the diagnostic GMF, relating to the action levels for the workers and the reference levels for the general population, in force in the European Union. Thanks to the defined experimental setup, the use of two different measuring instruments, and the software resources of the WEBNIR platform, it was possible to identify, for both categories of exposed persons, the "clearance" space, i.e., the distance from the magnet of the tomograph that guarantees health protection concerning the exposure to GMF, according to the indications of the standards in force. The method used showed that the exposure levels to GMF are substantially safe for professionally exposed workers who do not carry specific risks. For workers particularly sensitive to the specific risk, as well as to individuals part of the population, it is however advisable to maintain a distance from the magnet of about one meter to prevent sensorial neuromuscular stimulation effects.

Patient dose in angiographic interventional procedures: A multicentre study in Italy.

PURPOSE: The Council Directive 2013/59/EURATOM considers interventional radiology to be a special practice involving high doses of radiation and requiring strict monitoring to ensure the best quality assurance programs. This work reports the early experience of managing dose data from patients undergoing angiography in a multicentre study. MATERIALS AND METHODS: The study was based on a survey of about 15,200 sample procedures performed in 21 Italian hospitals centres involved on a voluntary basis. The survey concerned the collection of data related to different interventional radiology procedures: interventional cardiology, radiology, neuroradiology, vascular surgery, urology, endoscopy and pain therapy from a C-Arm and fixed units. The analysis included 11 types of procedures and for each procedure, air-kerma, kerma-area product and fluoroscopy time were collected. RESULTS: The duration and dose values of fluoroscopic exposure for each procedure is strongly dependent on individual clinical circumstances including the complexity of the procedure; the observed distribution of patient doses was very wide, even for a specified protocol. The median values of the parameters were compared with the diagnostic reference levels (DRL) proposed for some procedures in Italy (ISTISAN) or internationally. This work proposes local DRL values for three procedures. CONCLUSION: This first data collection serves to take stock of the situation on patient's dosimetry in several sectors and is the starting point for obtaining and updating DRL recalling that these levels are dependent on experience and technology available.

Morphological Analysis of Amoeboid-Mesenchymal Transition Plasticity After Low and High LET Radiation on Migrating and Invading Pancreatic Cancer Cells.

BACKGROUND/AIM: Cell migration and invasion are fundamental components of tumor cell metastasis that represent the biggest threat to the survival and quality of life of cancer patients. There is clear evidence that ionizing radiation can differently modulate migration and invasiveness of cancer cells depending on the cell lines, the doses and the radiation types investigated. This suggests that motile cells are able to adopt different migration strategies according to their molecular characteristics and external signals. MATERIALS AND METHODS: In this study, a morphological analysis was performed on pancreatic cancer Aspc-1 cells to evaluate the amoeboid-mesenchymal mobility transition in several experimental conditions considering the role played by factors released by normal and tumor cells, in basal conditions and after low and high Linear Energy Transfer (LET) irradiation. RESULTS AND CONCLUSION: The migratory behavior of Aspc-1 cells is modulated by factors released by normal fibroblasts and tumor cells, and this is in turn modulated by both the radiation dose and the radiation quality.

[Safety of use assessment in a radio-frequency medical device]. / Valutazione della sicurezza d'impiego di un'apparecchiatura elettromedicale ad emissione di radiofrequenza.

The authors assessed the operating safety physical parameters of a bipolar radiofrequency device for aesthetic purposes. According to both Italian and EU guidelines, the authors considered: magnetic field environmental emission levels, electricity induced in the opertator's limbs, operator's exposure and radiofrequency specific absorbance rate (SAR) in treated tissues. Measurements were carried out with isotropic sensors and an inductive current indicator. Results pointed out excellent safety levels regarding environment, operators and patients as well, although such radiofrequency equipment cannot be used on patients with pacemakers, neurostimulators and other vital function controlling devices.

Effects of single or combined treatments with radiation and chemotherapy on survival and danger signals expression in glioblastoma cell lines.

The success of chemo- and radiotherapy in glioblastoma multiforme, the most common and lethal primary brain tumour, could rely on the induction of immunogenic tumour cell death and on the induction of anticancer immune response. In this study we investigated cell survival to single treatments or combination of X-rays and temozolomide in glioblastoma cell lines (T98G and U251MG) and we attempted to identify danger signals (HMGB1 and HSP70) released by dying cells in the microenvironment that could activate antitumour immunity contributing to the therapeutic efficacy of conventional treatments. Our data suggest that HSP70 translocates from cytoplasm to extracellular environment after an increase in radiation dose and HMGB1 translocates from the nucleus to the cytoplasm and subsequently is released into the extracellular space, confirming a role of these proteins as signals released after radiation-induced damage in glioblastoma cells. We also could state that TMZ had limited effectiveness in activating HMGB1 and HSP70 signalling and, instead, an adjuvant effect was observed in some combined treatments, depending on schedule, cell line, and timing. A big challenge in tumour therapy is, therefore, to identify the most beneficial combination and chronology of multiple treatment options to contribute to the improvement of the therapeutic outcome.

Late oral mucosa alterations after radiotherapy for head and neck cancer assessed by exfoliative cytology.

AIM: Late oral mucosa changes after radiotherapy for head and neck cancer have been poorly studied. This study aimed to determine long-term effects of radiotherapy on oral mucosa using exfoliative oral cytology. PATIENTS AND METHODS: Fifty patients with cancer were enrolled, five of whom in order to validate microscopic analysis. Smears were collected at programmed visit; a score was used to rank possible cytological alterations. Presence of inflammation was also microscopically described and compared to blood count tests. RESULTS: Epithelial cells revealed a peculiar 'folding' phenotype, not related to chemotherapy, total dose, or to the effective dose delivered to mucosa. Inflammation described was related to the score for 'folding' cells; moreover, score decreased in the presence of a higher lymphocyte count, while it was not altered by neutrophil count. CONCLUSION: We suggest application of exfoliative cytology to study radiation injury and the variability of individual response of oral mucosa to radiation.

Mouse embryonic stem cells irradiated with γ-rays differentiate into cardiomyocytes but with altered contractile properties.

Embryonic stem cells (ESCs) for their derivation from the inner cell mass of a blastocyst represent a valuable in vitro model to investigate the effects of ionizing radiation on early embryonic cellular response. Following irradiation, both human and mouse ESCs (mESCs) maintain their pluripotent status and the capacity to differentiate into embryoid bodies and to form teratomas. Although informative of the maintenance of a pluripotent status, these studies never investigated the capability of irradiated ESCs to form specific differentiated phenotypes. Here, for the first time, 5Gy-irradiated mESCs were differentiated into cardiomyocytes, thus allowing the analysis of the long-term effects of ionizing radiations on the differentiation potential of a pluripotent stem cell population. On treated mESCs, 96h after irradiation, a genome-wide expression analysis was first performed in order to determine whether the treatment influenced gene expression of the surviving mESCs. Microarrays analysis showed that only 186 genes were differentially expressed in treated mESCs compared to control cells; a quarter of these genes were involved in cellular differentiation, with three main gene networks emerging, including cardiogenesis. Based on these results, we differentiated irradiated mESCs into cardiomyocytes. On day 5, 8 and 12 of differentiation, treated cells showed a significant alteration (qRT-PCR) of the expression of marker genes (Gata-4, Nkx-2.5, Tnnc1 and Alpk3) when compared to control cells. At day 15 of differentiation, although the organization of sarcomeric α-actinin and troponin T proteins appeared similar in cardiomyocytes differentiated from either mock or treated cells, the video evaluation of the kinematics and dynamics of the beating cardiac syncytium evidenced altered contractile properties of cardiomyocytes derived from irradiated mESCs. This alteration correlated with significant reduction of Connexin 43 foci. Our results indicate that mESCs populations that survive an ionizing irradiation treatment are capable to differentiate into cardiomyocytes, but they have altered contractile properties.

New frontiers for astrocytic tumours.

Glioblastoma multiforme, the most common type of primary brain tumour, remains an unsolved clinical problem. A great deal of work has been done in an effort to understand the biology and genetics of glioblastoma multiforme, but clinically effective treatments remain elusive. It is well known that malignant gliomas develop resistance to chemo- and radiotherapy. In this review we evaluated the literature data regarding therapeutic progress for the treatment of astrocytic tumours, focusing our attention on new frontiers for glioblastoma. The research studies performed in in vitro and in vivo models show that the application of hyperthermia using magnetic nanoparticles is safe and could be a promising tool in the treatment of glioblastoma patients. Our efforts are focused towards new fields of research, for example nanomedicine and the study of the uptake and cytotoxic effects of magnetic nanoparticles. The improvement of the quality of life of patients, by increasing their survival rate is the best result to be pursued, since these tumours are considered as ineradicable.

Mouse embryonic stem cells that survive γ-rays exposure maintain pluripotent differentiation potential and genome stability.

This study aimed to investigate the cell cycle, apoptosis, cytogenetics and differentiation capacity of mouse embryonic stem cells (mESCs) that survived a single dose of 2 or 5 Gy γ-rays during a period of up to 96 h of culture. After 2 Gy irradiation and 24 h culture, compared to control, a significant majority of cells was blocked at the G2/M phase and a massive apoptosis was recorded. Between 48 and 72 h post-irradiation, the parameters used to describe the cell cycle and apoptosis returned similar to those of control samples. When mESCs were irradiated with 5 Gy, a small fraction of cells, even after 96 h of culture, still presented clear evidences of a G2/M block and apoptosis. The cytogenetic analysis performed at 96 h showed that the structural stability of the aberrations did not change significantly when comparing control and 2 or 5 Gy-treated populations. However, the chromosomal damage observed in the progeny of the survived cells after 5 Gy exposure is significantly higher than that observed in control samples, although it is mostly of the stable and transmissible type. Ninety-six hours after irradiation, the survived mESCs maintained their undifferentiated status and capability to differentiate into the three germ layers. Overall, these results indicate a commitment of mESCs to maintain pluripotency and genome stability.

Cell death forms and HSP70 expression in U87 cells after ionizing radiation and/or chemotherapy.

BACKGROUND: Several types of cell death can induce the activation of the immune system by releasing factors of damage. AIM: To identify different cell death modalities using U87 glioblastoma cell line after radio-chemotherapy treatments by analyzing the expression of HSP70 after γ-ray irradiation and temozolomide treatment. MATERIALS AND METHODS: U87 cell line was irradiated with 2, 4, 8, 10 and 20 Gy, treated with 200 µM of temozolomide, or subjected to combined treatments of these. RESULTS: Forty-eight hours after treatment, apoptosis was more prevalent than necrosis, especially after γ-radiation with a somewhat dose-dependent trend. Temozolomide did not seem to induce significant modifications of the pattern of cell death and protein expression. Temozolomide-induced cell death was lower or equal to that obtained with high dose radiotherapy. A low synergism between the two treatments was observed. CONCLUSION: This cell line showed a marked radioresistance to conventional clinical doses, showing attempts to repair the induced damage, while temozolomide treatment showed no cytotoxic effects.

In vitro analysis of ultrasound second generation contrast agent diluted in saline solution.

PURPOSE: To evaluate the changes in response of a second-generation sonographic contrast agent diluted in saline solution at different concentrations when different scanning techniques, and saline solution temperatures and pH were used. MATERIALS AND METHODS: A series of tests was devised to analyse the behaviour of the sonographic contrast agent at different concentrations, temperatures, pH and scanning techniques. Latex balloons were used as phantoms. These were filled with 0.9% Sodium Chloride solution mixed with a suspension of sulphur hexafluoride microbubbles stabilised with phospholipids (SonoVue ) and scanned with the harmonic imaging technique at low acoustic pressure. For each image obtained, we calculated the mean grey-scale level values and the standard deviations of grey-scale level were calculated within a ROI on the US images obtained; the resulting data were used to create echogenicity curves of UCA the echo-enhancer over time at the different conditions tested. RESULTS: We noted that SonoVue maintains adequate backscatter properties even at low concentration (0.15%) in N/S solution. The amount of echogenicity can be considered adequate for the average duration of an ultrasound examination. These properties are not affected by the scanning technique used on the phantom containing the UCA contrast agent/NaCl solution. The pH of the saline solution does not affect the survival of the microbubbles. Temperature becomes a significant value beyondover 33-37 degrees C; this condition favours rupture of the microbubbles with subsequent reduction of the echogenicity after 10-15 min, and complete disappearance after 30 min. US beam attenuation related to the concentration of the microbubbles in the saline solution volume has a non-linear behaviour; at low amplification levels, attenuation becomes more significant when using SonoVue concentrations higher than 0.4%. Unexpected greater improvement in US beam transmission was experienced for each of the three SonoVue concentrations tested, and especially for 0.2% and 0.4%, compared to saline solution alone. CONCLUSIONS: Our results show that a second-generation contrast agent may be used even diluted at low concentration in saline solution. This characteristic opens up new diagnostic perspectives possibilities for the use of contrast-enhanced harmonic imaging; in particular, its can be suggested used in hollow organs and functional studies. These in vitro results require by confirmation by clinical applications which are under evaluation and experimentation.