Contact microscopy with synchrotron radiation

Soft X-ray contact microscopy with synchrotron radiation offers the biologist, and especially the microscopist, a way to morphologically study specimens that could not be imaged by conventional TEM, STEM, or SEM methods (i.e., hydrated samples, samples easily damaged by an electron beam, electron-dense samples, thick specimens, unstained, low-contrast specimens) at spatial resolutions approaching those of the TEM, with the additional possibility to obtain compositional (elemental) information about the sample as well. Although flash X-ray sources offer faster exposure times, synchrotron radiation provides a highly collimated, intense radiation that can be tuned to select specific discrete ranges of X-ray wavelengths or specific individual wavelengths that optimize imaging or microanalysis of a specific sample. This paper presents an overview of the applications of X-ray contact microscopy to biological research and some current research results using monochromatic synchrotron radiation to image biological samples.     

Medical physics aspects of the synchrotron radiation therapies: Microbeam radiation therapy (MRT) and synchrotron stereotactic radiotherapy (SSRT)

Stereotactic Synchrotron Radiotherapy (SSRT) and Microbeam Radiation Therapy (MRT) are both novel approaches to treat brain tumor and potentially other tumors using synchrotron radiation. Although the techniques differ by their principles, SSRT and MRT share certain common aspects with the possibility of combining their advantages in the future. For MRT, the technique uses highly collimated, quasi-parallel arrays of X-ray microbeams between 50 and 600 keV. Important features of highly brilliant Synchrotron sources are a very small beam divergence and an extremely high dose rate. The minimal beam divergence allows the insertion of so called Multi Slit Collimators (MSC) to produce spatially fractionated beams of typically ∼25–75 micron-wide microplanar beams separated by wider (100–400 microns center-to-center(ctc)) spaces with a very sharp penumbra. Peak entrance doses of several hundreds of Gy are extremely well tolerated by normal tissues and at the same time provide a higher therapeutic index for various tumor models in rodents. The hypothesis of a selective radio-vulnerability of the tumor vasculature versus normal blood vessels by MRT was recently more solidified.SSRT (Synchrotron Stereotactic Radiotherapy) is based on a local drug uptake of high-Z elements in tumors followed by stereotactic irradiation with 80 keV photons to enhance the dose deposition only within the tumor. With SSRT already in its clinical trial stage at the ESRF, most medical physics problems are already solved and the implemented solutions are briefly described, while the medical physics aspects in MRT will be discussed in more detail in this paper.     

Ablation of breast cancer stem cells with radiation

Tumor radioresistance leads to recurrence after radiation therapy. The radioresistant phenotype has been hypothesized to reside in the cancer stem cell (CSC) component of breast and other tumors and is considered to be an inherent property of CSC. In this study, we assessed the radiation resistance of breast CSCs using early passaged, patient-derived xenografts from two separate patients. We found a patient-derived tumor in which the CSC population was rapidly depleted 2 weeks after treatment with radiation, based on CD44(+) CD24(-) lin(-) phenotype and aldehyde dehydrogenase 1 immunofluorescence, suggesting sensitivity to radiotherapy. The reduction in CSCs according to phenotypic markers was accompanied by a decrease in functional CSC activity measured by tumor sphere frequency and the ability to form tumors in mice. In contrast, another patient tumor sample displayed enrichment of CSC after irradiation, signifying radioresistance, in agreement with others. CSC response to radiation did not correlate with the level of reactive oxygen species in CSC versus non-CSC. These findings demonstrate that not all breast tumor CSCs are radioresistant and suggest a mechanism for the observed variability in breast cancer local recurrence.     

Imaging performance of phase-contrast breast computed tomography with synchrotron radiation and a CdTe photon-counting detector

PurposeWithin the SYRMA-CT collaboration based at the ELETTRA synchrotron radiation (SR) facility the authors investigated the imaging performance of the phase-contrast computed tomography (CT) system dedicated to monochromatic in vivo 3D imaging of the female breast, for breast cancer diagnosis.MethodsTest objects were imaged at 38 keV using monochromatic SR and a high-resolution CdTe photon-counting detector. Signal and noise performance were evaluated using modulation transfer function (MTF) and noise power spectrum. The analysis was performed on the images obtained with the application of a phase retrieval algorithm as well as on those obtained without phase retrieval. The contrast to noise ratio (CNR) and the capability of detecting test microcalcification clusters and soft masses were investigated.ResultsFor a voxel size of (60 μm)³, images without phase retrieval showed higher spatial resolution (6.7 mm⁻¹ at 10% MTF) than corresponding images with phase retrieval (2.5 mm⁻¹). Phase retrieval produced a reduction of the noise level and an increase of the CNR by more than one order of magnitude, compared to raw phase-contrast images. Microcalcifications with a diameter down to 130 μm could be detected in both types of images.ConclusionsThe investigation on test objects indicates that breast CT with a monochromatic SR source is technically feasible in terms of spatial resolution, image noise and contrast, for in vivo 3D imaging with a dose comparable to that of two-view mammography. Images obtained with the phase retrieval algorithm showed the best performance in the trade-off between spatial resolution and image noise.     

Incidence of radiation toxicity in cervical cancer and endometrial cancer patients treated with radiotherapy alone versus adjuvant radiotherapy

AimThe study was made to evaluate early and late toxicity in a diversified group of patients receiving definitive or adjuvant radiotherapy in terms of clinical diagnosis and treatment methods.BackgroundRadiotherapy is a standard way of treatment in cervical and endometrial cancer patients, both as definitive and adjuvant therapy. But every radiation treatment may be involved with toxicity.Materials and methodsA detailed analysis was performed of 263 patients with gynaecological cancer treated with definitive (90 patients with cervical cancer received radiochemotherapy or radiotherapy exclusively) and adjuvant radiotherapy (38 with cervical and 135 with endometrial cancer).ResultsAcute reactions were found in 51.3% and late reactions were found in 14.8% of patients. It was stated that early (p < 0.007) and late (p < 0.003) post radiation reaction appear more frequently in women treated with definitive than adjuvant radiotherapy. The analysis of the whole group revealed higher rate of toxicity, both early and late, in the gastrointestinal tract than in the urinary system (p < 0.004). Comparing the subgroups, it was found that intestinal reactions occurred more frequently in the definitive radiotherapy group than in the adjuvant one.The occurrence of side effects was associated with the prolongation of total irradiation time due to necessary interruptions of radiotherapy. The comparison of the subgroups showed that interruptions occurred more frequently in patients receiving definitive rather than adjuvant radiotherapy (17.7–2.9%).ConclusionsDefinitive radiotherapy compared with adjuvant treatment may by associated with higher percentage of side effects caused by dose of therapy and correlation with chemotherapy.     

Towards discovery-driven translational research in breast cancer

Discovery-driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number of novel and powerful technologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diagnosed, treated and monitored in the future. Here we present a brief report on long-term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and targets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform 'omic' data sets collected from tissue samples obtained from individual patients. The ultimate goal of this initiative is to coalesce knowledge-based complementary procedures into a systems biology approach to fight breast cancer.     

In vivo K-edge imaging with synchrotron radiation.

We present in this paper two imaging techniques using contrast agents assessed with in vivo experiments. Both methods are based on the same physical principle, and were implemented at the European Synchrotron Radiation Facility medical beamline. The first one is intravenous coronary angiography using synchrotron radiation X-rays. This imaging technique has been planned for human studies in the near future. We describe the first experiments that were carried out with pigs at the ESRF. The second imaging mode is computed tomography using synchrotron radiation on rats bearing brain tumors. Owing to synchrotron radiation physical properties, these new imaging methods provide additional information compared to conventional techniques. After infusion of the contrast agent, it is possible to derive from the images the concentration of the contrast agent in the tumor area for the computed tomography and in any visible vessel for the angiography method.     

Triple-negative breast cancer and radiation therapy

BackgroundThis study aimed to review specific indications of radiation therapy for triple-negative breast cancer (TNBC), and to introduce the hypothesis of TNBC as an independent predictor for postmastectomy radiation therapy (PMRT).Materials and methodsTwo reviewers independently searched two electronic databases (Pubmed and Embase), with the inclusion dates of January 2000 to December 2021, for the following terms: “mastectomy” or “breast conserving surgery” or “lumpectomy”, and “radiation” or “radiotherapy”, and “triple negative” and “recurrence”. All evidence was explored by two reviewers, then organized into a narrative review considering grades of recommendation.ResultsPatients with TNBC are candidates for breast conserving surgery (grade of recommendation B). Postoperative whole-breast irradiation must be offered following breast conserving surgery (grade of recommendation A). Do not omit postoperative radiation therapy in older patients with TNBC (grade of recommendation B). Do not use partial-breast irradiation in patients with TNBC (grade of recommendation B). Postmastectomy radiation therapy should be offered for women with T3–T4 or node-positive TNBC, for any number of positive nodes (grade of recommendation A). Radiation therapy following mastectomy might also benefit patients with T1–T2 node-negative TNBC (grade of recommendation B). For patients treated with neoadjuvant systemic therapy, radiation therapy indication is based on pretreatment features. Retrospective studies suggest that residual TNBC is sensitive to radiation therapy to optimize locoregional control (grade of recommendation C).ConclusionsPostoperative radiation therapy should be offered for most patients with TNBC. Upcoming studies, preferably prospective randomized trials, should evaluate the indications of radiation therapy, especially in the context of novel systemic treatments.     

Enhancing radiotherapy effect in breast cancer with nanoparticles: A review

Amongst all efforts for improving oncological management outcomes, nanoparticles enhanced radiation for breast cancer patient's treatment is a novel approach that has grown interest for research in the last decade. Multiple preclinical data has been published, from all around the globe; however, clinical evidence is still insufficient for implementing the method in routine practice and in disease specific management. Gold nanoparticles (AuNP), which may be among the most studed materials, account for the majority of available data; however, some new materials have also been used in preclinical settings.Without any safety data available at the moment to support an active use, dosimetric in vitro and in vivo information seems to be consistent with a very promising and hopeful panorama for clinical applications. This review evaluates existing dosimetric data in breast cancer tissue, and a probable future impact in treatment choices and patient outcomes, as further investigation is required in a clinical setting.     

Anomalous X-ray diffraction with soft X-ray synchrotron radiation.

Anomalous diffraction with soft X-ray synchrotron radiation opens new possibilities in protein crystallography and materials science. Low-Z elements like silicon, phosphorus, sulfur and chlorine become accessible as new labels in structural studies. Some of the heavy elements like uranium exhibit an unusually strong dispersion at their M(V) absorption edge (lambdaMV = 3.497 A, E(MV) = 3545 eV) and so does thorium. Two different test experiments are reported here showing the feasibility of anomalous X-ray diffraction at long wavelengths with a protein containing uranium and with a salt containing chlorine atoms. With 110 electrons the anomalous scattering amplitude of uranium exceeds by a factor of 4 the resonance scattering of other strong anomalous scatterers like that of the lanthanides at their L(III) edge. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the multi-wavelength anomalous diffraction (MAD) method. The anomalous dispersion of an uranium derivative of asparaginyl-tRNA synthetase (hexagonal unit cell; a = 123.4 A, c = 124.4 A) has been measured for the first time at 4 wavelengths near the M(V) edge using the beamline ID1 of ESRF (Grenoble, France). The present set up allowed to measure only 30% of the possible reflections at a resolution of 4 A, mainly because of the low sensitivity of the CCD detector. In the second experiment, the dispersion of the intensity of 5 X-ray diffraction peaks from pentakismethylammonium undecachlorodibismuthate (PMACB, orthorhombic unit cell; a = 13.003 A, b = 14.038 A, c = 15.450 A) has been measured at 30 wavelengths near the K absorption edge of chlorine (lambdaK = 4.397 A, EK= 2819.6 eV). All reflections within the resolution range from 6.4 A to 3.4 A expected in the 20 degree scan were observed. The chemical state varies between different chlorine atoms of PMACB, and so does the dispersion of different Bragg peaks near the K-edge of chlorine. The results reflect the performance of the beamline ID1 of ESRF at wavelengths beyond 3 A at the end of 1998. A gain by a factor 100 for diffraction experiments with 4.4 A photons was achieved in Autumn 1999 when two focusing mirrors had been added to the X-ray optics. Further progress is expected from area detectors more sensitive to soft X-rays. Both CCD detectors and image plates would provide a gain of two orders of measured intensity. Image plates would have the additional advantage that they can be bent cylindrically and thus cover a larger solid angle in reciprocal space. In many cases, samples need to be cooled: closed and open systems are presented. A comparison with the state of art of soft X-ray diffraction, as it had been reached at HASYLAB (Hamburg, Germany), and as it is developing at the Brookhaven National Laboratory (USA), is given.     

Visualizing rhizosphere chemistry of legumes with mid-infrared synchrotron radiation

A bright synchrotron light source operated by the Lawrence Berkeley National Laboratory served as an external source for infrared (IR) microscopy of plant root microcosms. Mid-IR light from synchrotrons is 2-3 orders of magnitude brighter than conventional sources, providing contrast based on the chemical information in the reflected signal at a spatial resolution near the diffraction-limit of 3-10 microm. In an experiment using plant root microcosms fitted with zinc selenide IR-transmissive windows (50 mm x 20 mm x 1 mm), we describe chemical differences and similarities within the root zone of mung bean (Vigna radiata L.), grown with or without phosphorus, and revealed by reflectance spectromicroscopy. Comparative root and root-exudate profiles are described in sand/silt culture over the wavelength range of 2.5 to 16 pm (4.000 to 650 cm(-1) ) in the mid-IR. the spectral region most useful for the analytical identification of small organic molecules. Root epidermal tissue of plants grown with low phosphorus showed a greater lipid contribution and less lignin than nutrient-sufficient plants. In the zone 200 microm from the root axis, control plants were enriched with simple sugars and monomeric lignin precursors. In low-phosphorus plants, the rhizosphere possessed IR signatures from protein and sugars. Individual soil minerals could be easily discriminated from biological material. Synchrotron IR spectromicroscopy, therefore, complements existing root imaging techniques.     

Prospects for microcomputerized-tomography using synchrotron radiation

A microversion of a computerized tomograph (CT) is described, in which the object is subjected to a successive series of translations with rotation by a small angle in between. The spatial resolution is determined by collimators and translation step lengths and is today, with clinical X-ray tube, of the order of 100 μm. The use of synchrotron radiation instead of X-ray tubes offers the advantages of much higher fluence rates, which can be used to diminish the exposure times from days to minutes or to increase the spatial resolution from 100 μm to about 1 μm. The possibility to receive monoenergetic photons of selectable energy makes it possible to avoid spectral hardening image artifacts, as well as to optimize the information sampling with regard to average absorbed dose or exposure time. Selectable photon energies are valuable also for tomochemistry applications.     

Biological trace-element measurements using synchrotron radiation

The feasibility of performing X-ray fluorescence trace-element determinations at concentrations substantially below the ppm level for biological materials is demonstrated. Conditions for achieving optimum sensitivity were ascertained. Results achieved for five standard reference materials were, in most cases, in excellent agreement with listed values. Minimum detectable limits of 20 ppb were measured for most elements.     

Nitroblue tetrazolium test in patients with breast cancer undergoing mastectomy and radiotherapy

The reduction of nitrotetrazolium blue with stimulated and unstimulated granulocytes was performed in 40 patients with breast cancer. Blood was collected from peripheral vein before surgical intervention, during surgery from vein draining of tumor, two weeks after surgery and three months after surgery when radiotherapy was finished. In parallel experiments the NBT test was made in the peripheral blood of 20 healthy individuals. We have observed decreased reduction of nitrotetrazolium blue in stimulated granulocytes, which may indicate that granulocytes from these patients are unable for efficient stimulation. No evident effect concerning the advancement of disease and applied therapy could be found. The importance of determining the NBT test in cancer patients is briefly discussed.     

Mathematical modelling of radiotherapy strategies for early breast cancer

Targeted intraoperative radiotherapy (Targit) is a new concept of partial breast irradiation where single fraction radiotherapy is delivered directly to the tumour bed. Apart from logistic advantages, this strategy minimizes the risk of missing the tumour bed and avoids delay between surgery and radiotherapy. It is presently being compared with the standard fractionated external beam radiotherapy (EBRT) in randomized trials. In this paper we present a mathematical model for the growth and invasion of a solid tumour into a domain of tissue (in this case breast tissue), and then a model for surgery and radiation treatment of this tumour. We use the established linear-quadratic (LQ) model to compute the survival probabilities for both tumour cells and irradiated breast tissue and then simulate the effects of conventional EBRT and Targit. True local recurrence of the tumour could arise either from stray tumour cells, or the tumour bed that harbours morphologically normal cells having a predisposition to genetic changes, such as a loss of heterozygosity (LOH) in genes that are crucial for tumourigenesis, e.g. tumour suppressor genes (TSGs). Our mathematical model predicts that the single high dose of radiotherapy delivered by Targit would result in eliminating all these sources of recurrence, whereas the fractionated EBRT would eliminate stray tumour cells, but allow (by virtue of its very schedule) the cells with LOH in TSGs or cell-cycle checkpoint genes to pass on low-dose radiation-induced DNA damage and consequently mutations that may favour the development of a new tumour. The mathematical model presented here is an initial attempt to model a biologically complex phenomenon that has until now received little attention in the literature and provides a 'proof of principle' that it is possible to produce clinically testable hypotheses on the effects of different approaches of radiotherapy for breast cancer.     

A six-gene-based signature for breast cancer radiotherapy sensitivity estimation

Breast cancer (BRCA) represents the most common malignancy among women worldwide with high mortality. Radiotherapy is a prevalent therapeutic for BRCA that with heterogeneous effectiveness among patients. Here, we proposed to develop a gene expression-based signature for BRCA radiotherapy sensitivity estimation. Gene expression profiles of BRCA samples from the Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) were obtained and used as training and independent testing dataset, respectively. Differential expression genes (DEGs) in BRCA samples compared with their paracancerous samples in the training set were identified by using the edgeR Bioconductor package. Univariate Cox regression analysis and LASSO Cox regression method were applied to screen optimal genes for constructing a radiotherapy sensitivity estimation signature. Nomogram combining independent prognostic factors was used to predict 1-, 3-, and 5-year OS of radiation-treated BRCA patients. Relative proportions of tumor infiltrating immune cells (TIICs) calculated by CIBERSORT and mRNA levels of key immune checkpoint receptors was adopted to explore the relation between the signature and tumor immune response. As a result, 603 DEGs were obtained in BRCA tumor samples, six of which were retained and used to construct the radiotherapy sensitivity prediction model. The signature was proved to be robust in both training and testing sets. In addition, the signature was closely related to the immune microenvironment of BRCA in the context of TIICs and immune checkpoint receptors’ mRNA levels. In conclusion, the present study obtained a radiotherapy sensitivity estimation signature for BRCA, which should shed new light in clinical and experimental research.