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.

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.

FLASHlab@PITZ: New R&D platform with unique capabilities for electron FLASH and VHEE radiation therapy and radiation biology under preparation at PITZ.

At the Photo Injector Test facility at DESY in Zeuthen (PITZ), an R&D platform for electron FLASH and very high energy electron radiation therapy and radiation biology is being prepared (FLASHlab@PITZ). The beam parameters available at PITZ are worldwide unique. They are based on experiences from 20 + years of developing high brightness beam sources and an ultra-intensive THz light source demonstrator for ps scale electron bunches with up to 5 nC bunch charge at MHz repetition rate in bunch trains of up to 1 ms length, currently 22 MeV (upgrade to 250 MeV planned). Individual bunches can provide peak dose rates up to 1014 Gy/s, and 10 Gy can be delivered within picoseconds. Upon demand, each bunch of the bunch train can be guided to a different transverse location, so that either a "painting" with micro beams (comparable to pencil beam scanning in proton therapy) or a cumulative increase of absorbed dose, using a wide beam distribution, can be realized at the tumor. Full tumor treatment can hence be completed within 1 ms, mitigating organ movement issues. With extremely flexible beam manipulation capabilities, FLASHlab@PITZ will cover the current parameter range of successfully demonstrated FLASH effects and extend the parameter range towards yet unexploited short treatment times and high dose rates. A summary of the plans for FLASHlab@PITZ and the status of its realization will be presented.

Longitudinally Heterogeneous Tumor Dose Optimizes Proton Broadbeam, Interlaced Minibeam, and FLASH Therapy.

The prerequisite of any radiation therapy modality (X-ray, electron, proton, and heavy ion) is meant to meet at least a minimum prescribed dose at any location in the tumor for the best tumor control. In addition, there is also an upper dose limit within the tumor according to the International Commission on Radiation Units (ICRU) recommendations in order to spare healthy tissue as well as possible. However, healthy tissue may profit from the lower side effects when waving this upper dose limit and allowing a larger heterogeneous dose deposition in the tumor, but maintaining the prescribed minimum dose level, particularly in proton minibeam therapy. Methods: Three different longitudinally heterogeneous proton irradiation modes and a standard spread-out Bragg peak (SOBP) irradiation mode are simulated for their depth-dose curves under the constraint of maintaining a minimum prescribed dose anywhere in the tumor region. Symmetric dose distributions of two opposing directions are overlaid in a 25 cm-thick water phantom containing a 5 cm-thick tumor region. Interlaced planar minibeam dose distributions are compared to those of a broadbeam using the same longitudinal dose profiles. Results and Conclusion: All longitudinally heterogeneous proton irradiation modes show a dose reduction in the healthy tissue compared to the common SOBP mode in the case of broad proton beams. The proton minibeam cases show eventually a much larger mean cell survival and thus a further reduced equivalent uniform dose (EUD) in the healthy tissue than any broadbeam case. In fact, the irradiation mode using only one proton energy from each side shows better sparing capabilities in the healthy tissue than the common spread-out Bragg peak irradiation mode with the option of a better dose fall-off at the tumor edges and an easier technical realization, particularly in view of proton minibeam irradiation at ultra-high dose rates larger than ~10 Gy/s (so-called FLASH irradiation modes).

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.

Dosimetry of heavy ion exposure to human cells using nanoscopic imaging of double strand break repair protein clusters.

The human body is constantly exposed to ionizing radiation of different qualities. Especially the exposure to high-LET (linear energy transfer) particles increases due to new tumor therapy methods using e.g. carbon ions. Furthermore, upon radiation accidents, a mixture of radiation of different quality is adding up to human radiation exposure. Finally, long-term space missions such as the mission to mars pose great challenges to the dose assessment an astronaut was exposed to. Currently, DSB counting using γH2AX foci is used as an exact dosimetric measure for individuals. Due to the size of the γH2AX IRIF of ~ 0.6 µm, it is only possible to count DSB when they are separated by this distance. For high-LET particle exposure, the distance of the DSB is too small to be separated and the dose will be underestimated. In this study, we developed a method where it is possible to count DSB which are separated by a distance of ~ 140 nm. We counted the number of ionizing radiation-induced pDNA-PKcs (DNA-PKcs phosphorylated at T2609) foci (size = 140 nm ± 20 nm) in human HeLa cells using STED super-resolution microscopy that has an intrinsic resolution of 100 nm. Irradiation was performed at the ion microprobe SNAKE using high-LET 20 MeV lithium (LET = 116 keV/µm) and 27 MeV carbon ions (LET = 500 keV/µm). pDNA-PKcs foci label all DSB as proven by counterstaining with 53BP1 after low-LET γ-irradiation where separation of individual DSB is in most cases larger than the 53BP1 gross size of about 0.6 µm. Lithium ions produce (1.5 ± 0.1) IRIF/µm track length, for carbon ions (2.2 ± 0.2) IRIF/µm are counted. These values are enhanced by a factor of 2-3 compared to conventional foci counting of high-LET tracks. Comparison of the measurements to PARTRAC simulation data proof the consistency of results. We used these data to develop a measure for dosimetry of high-LET or mixed particle radiation exposure directly in the biological sample. We show that proper dosimetry for radiation up to a LET of 240 keV/µm is possible.

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.

Focused Ion Microbeam Irradiation Induces Clustering of DNA Double-Strand Breaks in Heterochromatin Visualized by Nanoscale-Resolution Electron Microscopy.

BACKGROUND: Charged-particle radiotherapy is an emerging treatment modality for radioresistant tumors. The enhanced effectiveness of high-energy particles (such as heavy ions) has been related to the spatial clustering of DNA lesions due to highly localized energy deposition. Here, DNA damage patterns induced by single and multiple carbon ions were analyzed in the nuclear chromatin environment by different high-resolution microscopy approaches. MATERIAL AND METHODS: Using the heavy-ion microbeam SNAKE, fibroblast monolayers were irradiated with defined numbers of carbon ions (1/10/100 ions per pulse, ipp) focused to micrometer-sized stripes or spots. Radiation-induced lesions were visualized as DNA damage foci (γH2AX, 53BP1) by conventional fluorescence and stimulated emission depletion (STED) microscopy. At micro- and nanoscale level, DNA double-strand breaks (DSBs) were visualized within their chromatin context by labeling the Ku heterodimer. Single and clustered pKu70-labeled DSBs were quantified in euchromatic and heterochromatic regions at 0.1 h, 5 h and 24 h post-IR by transmission electron microscopy (TEM). RESULTS: Increasing numbers of carbon ions per beam spot enhanced spatial clustering of DNA lesions and increased damage complexity with two or more DSBs in close proximity. This effect was detectable in euchromatin, but was much more pronounced in heterochromatin. Analyzing the dynamics of damage processing, our findings indicate that euchromatic DSBs were processed efficiently and repaired in a timely manner. In heterochromatin, by contrast, the number of clustered DSBs continuously increased further over the first hours following IR exposure, indicating the challenging task for the cell to process highly clustered DSBs appropriately. CONCLUSION: Increasing numbers of carbon ions applied to sub-nuclear chromatin regions enhanced the spatial clustering of DSBs and increased damage complexity, this being more pronounced in heterochromatic regions. Inefficient processing of clustered DSBs may explain the enhanced therapeutic efficacy of particle-based radiotherapy in cancer treatment.

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.

Influence of α-Particle Radiation on Intercellular Communication Networks of Tunneling Nanotubes in U87 Glioblastoma Cells.

Cellular communication plays a crucial role in the coordination and organization of cancer cells. Especially processes such as uncontrolled cell growth, invasion, and therapy resistance (development), which are features of very malignant tumors like glioblastomas, are supported by an efficient cell-to-cell communication in the tumor environment. One powerful way for cells to communicate are tunneling nanotubes (TNTs). These tiny membrane tunnels interconnect cells over long distances and serve as highways for information exchange between distant cells. Here, we study the response of cellular communication via TNTs in U87 glioblastoma cells to homogeneous irradiation with α-particles as a stress factor. We describe the development of TNT networks in certain time steps after irradiation using confocal live-cell imaging and suggest an evaluation method to characterize these communication networks. Our results show that irradiated cells establish their network faster and have more cell-to-cell connections with high TNT content than sham-irradiated controls within the first 24 h. These findings suggest that there is an additional trigger upon radiation damage which results in fast and intensive network formation by TNTs as a radiation damage response mechanism.

Perspectives of cellular communication through tunneling nanotubes in cancer cells and the connection to radiation effects.

Direct cell-to-cell communication is crucial for the survival of cells in stressful situations such as during or after radiation exposure. This communication can lead to non-targeted effects, where non-treated or non-infected cells show effects induced by signal transduction from non-healthy cells or vice versa. In the last 15 years, tunneling nanotubes (TNTs) were identified as membrane connections between cells which facilitate the transfer of several cargoes and signals. TNTs were identified in various cell types and serve as promoter of treatment resistance e.g. in chemotherapy treatment of cancer. Here, we discuss our current understanding of how to differentiate tunneling nanotubes from other direct cellular connections and their role in the stress reaction of cellular networks. We also provide a perspective on how the capability of cells to form such networks is related to the ability to surpass stress and how this can be used to study radioresistance of cancer cells.

Proton pencil minibeam irradiation of an in-vivo mouse ear model spares healthy tissue dependent on beam size.

Proton radiotherapy using minibeams of sub-millimeter dimensions reduces side effects in comparison to conventional proton therapy due to spatial fractionation. Since the proton minibeams widen with depth, the homogeneous irradiation of a tumor can be ensured by adjusting the beam distances to tumor size and depth to maintain tumor control as in conventional proton therapy. The inherent advantages of protons in comparison to photons like a limited range that prevents a dosage of distal tissues are maintained by proton minibeams and can even be exploited for interlacing from different beam directions. A first animal study was conducted to systematically investigate and quantify the tissue-sparing effects of proton pencil minibeams as a function of beam size and dose distributions, using beam widths between σ = 95, 199, 306, 411, 561 and 883 µm (standard deviation) at a defined center-to-center beam distance (ctc) of 1.8 mm. The average dose of 60 Gy was distributed in 4x4 minibeams using 20 MeV protons (LET ~ 2.7 keV/µm). The induced radiation toxicities were measured by visible skin reactions and ear swelling for 90 days after irradiation. The largest applied beam size to ctc ratio (σ/ctc = 0.49) is similar to a homogeneous irradiation and leads to a significant 3-fold ear thickness increase compared to the control group. Erythema and desquamation was also increased significantly 3-4 weeks after irradiation. With decreasing beam sizes and thus decreasing σ/ctc, the maximum skin reactions are strongly reduced until no ear swelling or other visible skin reactions should occur for σ/ctc < 0.032 (extrapolated from data). These results demonstrate that proton pencil minibeam radiotherapy has better tissue-sparing for smaller σ/ctc, corresponding to larger peak-to-valley dose ratios PVDR, with the best effect for σ/ctc < 0.032. However, even quite large σ/ctc (e.g. σ/ctc = 0.23 or 0.31, i.e. PVDR = 10 or 2.7) show less acute side effects than a homogeneous dose distribution. This suggests that proton minibeam therapy spares healthy tissue not only in the skin but even for dose distributions appearing in deeper layers close to the tumor enhancing its benefits for clinical proton therapy.

Beam size limit for pencil minibeam radiotherapy determined from side effects in an in-vivo mouse ear model.

Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams ≥ 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Submillimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus.

Nucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1-100 carbon ions per point (about 0.3-30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.

Nanoscopic analysis of 53BP1, BRCA1 and Rad51 reveals new insights in temporal progression of DNA-repair and pathway choice.

The accumulation and spatial distribution of 53BP1, BRCA1 and Rad51, key proteins in DNA double-strand break (DSB) repair, was investigated with high temporal resolution over a time span of 24 h, using STED nanoscopy. DNA lesions were induced by irradiation with high-LET (linear energy transfer) α-particles. We show that 53BP1 IRIF formation occurs quickly in almost all cells and after about 6 h the fraction of 53BP1 IRIF positive cells slowly declines. Against the expectations BRCA1 and Rad51 IRIF formation is only shortly delayed but with the maximum of cells showing foci after 6 and 8 h after irradiation. At this stage, almost all IRIF in a given Rad51-positive cell show Rad51 accumulation, suggesting that repair via homologous recombination is attempted at almost all residual DSB sites. The frequency of BRCA1 IRIF positive cells increases much earlier and remains high after Rad51 positive cells start to decline, supporting models claiming that functional roles of BRCA1 change over time. Correlation analysis showed a high degree of correlation of Rad51 with BRCA1, while the exclusion of 53BP1 from the actual resection-zone is demonstrated by anti-correlation of Rad51 and 53BP1. Interestingly, these correlation and anti-correlation patterns exhibit complementary temporal variation.

Acute Skin Damage and Late Radiation-Induced Fibrosis and Inflammation in Murine Ears after High-Dose Irradiation.

The use of different scoring systems for radiation-induced toxicity limits comparability between studies. We examined dose-dependent tissue alterations following hypofractionated X-ray irradiation and evaluated their use as scoring criteria. Four dose fractions (0, 5, 10, 20, 30 Gy/fraction) were applied daily to ear pinnae. Acute effects (ear thickness, erythema, desquamation) were monitored for 92 days after fraction 1. Late effects (chronic inflammation, fibrosis) and the presence of transforming growth factor beta 1 (TGFß1)-expressing cells were quantified on day 92. The maximum ear thickness displayed a significant positive correlation with fractional dose. Increased ear thickness and erythema occurred simultaneously, followed by desquamation from day 10 onwards. A significant dose-dependency was observed for the severity of erythema, but not for desquamation. After 4 × 20 and 4 × 30 Gy, inflammation was significantly increased on day 92, whereas fibrosis and the abundance of TGFß1-expressing cells were only marginally increased after 4 × 30 Gy. Ear thickness significantly correlated with the severity of inflammation and fibrosis on day 92, but not with the number of TGFß1-expressing cells. Fibrosis correlated significantly with inflammation and fractional dose. In conclusion, the parameter of ear thickness can be used as an objective, numerical and dose-dependent quantification criterion to characterize the severity of acute toxicity and allow for the prediction of late effects.

Membrane Hsp70-supported cell-to-cell connections via tunneling nanotubes revealed by live-cell STED nanoscopy.

Heat shock protein Hsp70 (Hsp70) is found on the cell surface of a large variety of human and mouse tumor cell types including U87, GL261 glioblastoma, and 4T1 mammary carcinoma cells. We studied the role of membrane-bound Hsp70 (mHsp70) in the formation of cell-to-cell connections via tunneling nanotubes (TNTs) using live-cell STED nanoscopy. This technique allows the visualization of microstructures in the 100-nm range in the living cells. We could show that the presence of tumor-derived mHsp70 in TNTs with a diameter ranging from 120 to 140 nm predominantly originates from cholesterol-rich-microdomains containing the lipid compound globoyltriaosylceramide (Gb3). Under non-stress conditions, Hsp70 and Gb3 are structurally clustered in the membrane of TNTs of tumor cells that showed tumor type specific variations in the amount of cell-to-cell connection networks. Furthermore depletion of cholesterol and ionizing radiation as a stress factor results in a complete loss of Hsp70-containing TNTs.

DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage.

DNA double strand breaks (DSB) play a pivotal role for cellular damage, which is a hazard encountered in toxicology and radiation protection, but also exploited e.g. in eradicating tumors in radiation therapy. It is still debated whether and in how far clustering of such DNA lesions leads to an enhanced severity of induced damage. Here we investigate - using focused spots of ionizing radiation as damaging agent - the spatial extension of DNA lesion patterns causing cell inactivation. We find that clustering of DNA damage on both the nm and µm scale leads to enhanced inactivation compared to more homogeneous lesion distributions. A biophysical model interprets these observations in terms of enhanced DSB production and DSB interaction, respectively. We decompose the overall effects quantitatively into contributions from these lesion formation processes, concluding that both processes coexist and need to be considered for determining the resulting damage on the cellular level.

Live cell imaging of mitochondria following targeted irradiation in situ reveals rapid and highly localized loss of membrane potential.

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1 µm induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria's response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.