High-Resolution Microscopic Characterization of Tunneling Nanotubes in Living U87 MG and LN229 Glioblastoma Cells.

Tunneling nanotubes (TNTs) are fine, nanometer-sized membrane connections between distant cells that provide an efficient communication tool for cellular organization. TNTs are thought to play a critical role in cellular behavior, particularly in cancer cells. The treatment of aggressive cancers such as glioblastoma remains challenging due to their high potential for developing therapy resistance, high infiltration rates, uncontrolled cell growth, and other aggressive features. A better understanding of the cellular organization via cellular communication through TNTs could help to find new therapeutic approaches. In this study, we investigate the properties of TNTs in two glioblastoma cell lines, U87 MG and LN229, including measurements of their diameter by high-resolution live-cell stimulated emission depletion (STED) microscopy and an analysis of their length, morphology, lifetime, and formation by live-cell confocal microscopy. In addition, we discuss how these fine compounds can ideally be studied microscopically. In particular, we show which membrane-labeling method is suitable for studying TNTs in glioblastoma cells and demonstrate that live-cell studies should be preferred to explore the role of TNTs in cellular behavior. Our observations on TNT formation in glioblastoma cells suggest that TNTs could be involved in cell migration and serve as guidance.

Chromatin Organization after High-LET Irradiation Revealed by Super-Resolution STED Microscopy.

Ion-radiation-induced DNA double-strand breaks can lead to severe cellular damage ranging from mutations up to direct cell death. The interplay between the chromatin surrounding the damage and the proteins responsible for damage recognition and repair determines the efficiency and outcome of DNA repair. The chromatin is organized in three major functional compartments throughout the interphase: the chromatin territories, the interchromatin compartment, and the perichromatin lying in between. In this study, we perform correlation analysis using super-resolution STED images of chromatin; splicing factor SC35, as an interchromatin marker; and the DNA repair factors 53BP1, Rad51, and γH2AX in carbon-ion-irradiated human HeLa cells. Chromatin and interchromatin overlap only in protruding chromatin branches, which is the same for the correlation between chromatin and 53BP1. In contrast, between interchromatin and 53BP1, a gap of (270 ± 40) nm is visible. Rad51 shows overlap with decondensed euchromatic regions located at the borders of condensed heterochromatin with further correlation with γH2AX. We conclude that the DNA damage is repaired in decondensed DNA loops in the perichromatin, located in the periphery of the DNA-dense chromatin compartments containing the heterochromatin. Proteins like γH2AX and 53BP1 serve as supporters of the chromatin structure.

Proton-FLASH: effects of ultra-high dose rate irradiation on an in-vivo mouse ear model.

FLASH-radiotherapy may provide significant sparing of healthy tissue through ultra-high dose rates in protons, electrons, and x-rays while maintaining the tumor control. Key factors for the FLASH effect might be oxygen depletion, the immune system, and the irradiated blood volume, but none could be fully confirmed yet. Therefore, further investigations are necessary. We investigated the protective (tissue sparing) effect of FLASH in proton treatment using an in-vivo mouse ear model. The right ears of Balb/c mice were irradiated with 20 MeV protons at the ion microprobe SNAKE in Garching near Munich by using three dose rates (Conv = 0.06 Gy/s, Flash9 = 9.3 Gy/s and Flash930 = 930 Gy/s) at a total dose of 23 Gy or 33 Gy. The ear thickness, desquamation, and erythema combined in an inflammation score were measured for 180 days. The cytokines TGF-ß1, TNF-α, IL1α, and IL1ß were analyzed in the blood sampled in the first 4 weeks and at termination day. No differences in inflammation reactions were visible in the 23 Gy group for the different dose rates. In the 33 Gy group, the ear swelling and the inflammation score for Flash9 was reduced by (57 ± 12) % and (67 ± 17) % and for Flash930 by (40 ± 13) % and (50 ± 17) % compared to the Conv dose rate. No changes in the cytokines in the blood could be measured. However, an estimation of the irradiated blood volume demonstrates, that 100-times more blood is irradiated when using Conv compared to using Flash9 or Flash930. This indicates that blood might play a role in the underlying mechanisms in the protective effect of FLASH.

Single-Cell Radiation Response Scoring with the Deep Learning Algorithm CeCILE 2.0.

External stressors, such as ionizing radiation, have massive effects on life, survival, and the ability of mammalian cells to divide. Different types of radiation have different effects. In order to understand these in detail and the underlying mechanisms, it is essential to study the radiation response of each cell. This allows abnormalities to be characterized and laws to be derived. Tracking individual cells over several generations of division generates large amounts of data that can no longer be meaningfully analyzed by hand. In this study, we present a deep-learning-based algorithm, CeCILE (Cell classification and in vitro lifecycle evaluation) 2.0, that can localize, classify, and track cells in live cell phase-contrast videos. This allows conclusions to be drawn about the viability of the cells, the cell cycle, cell survival, and the influence of X-ray radiation on these. Furthermore, radiation-specific abnormalities during division could be characterized. In summary, CeCILE 2.0 is a powerful tool to characterize and quantify the cellular response to external stressors such as radiation and to put individual responses into a larger context. To the authors knowledge, this is the first algorithm with a fully integrated workflow that is able to do comprehensive single-cell and cell composite analysis, allowing them to draw conclusions on cellular radiation response.

Planar Proton Minibeam Irradiation Elicits Spatially Confined DNA Damage in a Human Epidermis Model.

Purpose: High doses of ionizing radiation in radiotherapy can elicit undesirable side effects to the skin. Proton minibeam radiotherapy (pMBRT) may circumvent such limitations due to tissue-sparing effects observed at the macro scale. Here, we mapped DNA damage dynamics in a 3D tissue context at the sub-cellular level. Methods: Epidermis models were irradiated with planar proton minibeams of 66 µm, 408 µm and 920 µm widths and inter-beam-distances of 2.5 mm at an average dose of 2 Gy using the scanning-ion-microscope SNAKE in Garching, GER. γ-H2AX + 53BP1 and cleaved-caspase-3 immunostaining revealed dsDNA damage and cell death, respectively, in time courses from 0.5 to 72 h after irradiation. Results: Focused 66 µm pMBRT induced sharply localized severe DNA damage (pan-γ-H2AX) in cells at the dose peaks, while damage in the dose valleys was similar to sham control. pMBRT with 408 µm and 920 µm minibeams induced DSB foci in all cells. At 72 h after irradiation, DNA damage had reached sham levels, indicating successful DNA repair. Increased frequencies of active-caspase-3 and pan-γ-H2AX-positive cells revealed incipient cell death at late time points. Conclusions: The spatially confined distribution of DNA damage appears to underlie the tissue-sparing effect after focused pMBRT. Thus, pMBRT may be the method of choice in radiotherapy to reduce side effects to the skin.

CeCILE - An Artificial Intelligence Based Cell-Detection for the Evaluation of Radiation Effects in Eucaryotic Cells.

The fundamental basis in the development of novel radiotherapy methods is in-vitro cellular studies. To assess different endpoints of cellular reactions to irradiation like proliferation, cell cycle arrest, and cell death, several assays are used in radiobiological research as standard methods. For example, colony forming assay investigates cell survival and Caspase3/7-Sytox assay cell death. The major limitation of these assays is the analysis at a fixed timepoint after irradiation. Thus, not much is known about the reactions before or after the assay is performed. Additionally, these assays need special treatments, which influence cell behavior and health. In this study, a completely new method is proposed to tackle these challenges: A deep-learning algorithm called CeCILE (Cell Classification and In-vitro Lifecycle Evaluation), which is used to detect and analyze cells on videos obtained from phase-contrast microscopy. With this method, we can observe and analyze the behavior and the health conditions of single cells over several days after treatment, up to a sample size of 100 cells per image frame. To train CeCILE, we built a dataset by labeling cells on microscopic images and assign class labels to each cell, which define the cell states in the cell cycle. After successful training of CeCILE, we irradiated CHO-K1 cells with 4 Gy protons, imaged them for 2 days by a microscope equipped with a live-cell-imaging set-up, and analyzed the videos by CeCILE and by hand. From analysis, we gained information about cell numbers, cell divisions, and cell deaths over time. We could show that similar results were achieved in the first proof of principle compared with colony forming and Caspase3/7-Sytox assays in this experiment. Therefore, CeCILE has the potential to assess the same endpoints as state-of-the-art assays but gives extra information about the evolution of cell numbers, cell state, and cell cycle. Additionally, CeCILE will be extended to track individual cells and their descendants throughout the whole video to follow the behavior of each cell and the progeny after irradiation. This tracking method is capable to put radiobiologic research to the next level to obtain a better understanding of the cellular reactions to radiation.

Normal Tissue Response of Combined Temporal and Spatial Fractionation in Proton Minibeam Radiation Therapy.

PURPOSE: Proton minibeam radiation therapy, a spatial fractionation concept, widens the therapeutic window. By reducing normal tissue toxicities, it allows a temporally fractionated regime with high daily doses. However, an array shift between daily fractions can affect the tissue-sparing effect by decreasing the total peak-to-valley dose ratio. Therefore, combining temporal fractions with spatial fractionation raises questions about the impact of daily applied dose modulations, reirradiation accuracies, and total dose modulations. METHODS AND MATERIALS: Healthy mouse ear pinnae were irradiated with 4 daily fractions of 30 Gy mean dose, applying proton pencil minibeams (pMB) of Gaussian σ = 222 µm in 3 different schemes: a 16 pMB array with a center-to-center distance of 1.8 mm irradiated the same position in all sessions (FS1) or was shifted by 0.9 mm to never hit the previously irradiated tissue in each session (FS2), or a 64 pMB array with a center-to-center distance of 0.9 mm irradiated the same position in all sessions (FS3), resulting in the same total dose distribution as FS2. Reirradiation positioning and its accuracy were obtained from image guidance using the unique vessel structure of ears. Acute toxicities (swelling, erythema, and desquamation) were evaluated for 153 days after the first fraction. Late toxicities (fibrous tissue, inflammation) were analyzed on day 153. RESULTS: Reirradiation of highly dose-modulated arrays at a positioning accuracy of 110 ± 52 µm induced the least severe acute and late toxicities. A shift of the same array in FS2 led to significantly inducted acute toxicities, a higher otitis score, and a slight increase in fibrous tissue. FS3 led to the strongest increase in acute and late toxicities. CONCLUSIONS: The highest normal-tissue sparing is achieved after accurate reirradiation of a highly dose modulated pMB array, although high positioning accuracies are challenging in a clinical environment. Nevertheless, the same integral dose applied in highly dose-modulated fractions is superior to low daily dose-modulated fractions.