624 research outputs found

    How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

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    Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

    Developmental delays and subcellular stress as downstream effects of sonoporation

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    Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

    A study on the change in plasma membrane potential during sonoporation

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    Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

    Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

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    Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

    MRI-guided focused ultrasound surgery in musculoskeletal diseases: the hot topics

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    MRI-guided focused ultrasound surgery (MRgFUS) is a minimally invasive treatment guided by the most sophisticated imaging tool available in today's clinical practice. Both the imaging and therapeutic sides of the equipment are based on non-ionizing energy. This technique is a very promising option as potential treatment for several pathologies, including musculoskeletal (MSK) disorders. Apart from clinical applications, MRgFUS technology is the result of long, heavy and cumulative efforts exploring the effects of ultrasound on biological tissues and function, the generation of focused ultrasound and treatment monitoring by MRI. The aim of this article is to give an updated overview on a "new" interventional technique and on its applications for MSK and allied sciences

    Focused Ultrasound-Induced Cavitation Renders Cancer Cells Susceptible to Radiation Therapy, Hyperthermia and Testosterone Treatment: No

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    Focused ultrasound (FUS) is a less-invasive medical technique with the potential to improve the treatment outcome of many diseases by utilizing acoustic transducers to generate and concentrate the multiple intersecting ultrasonic waves on a targeted site in the body. The bio-effects induced by FUS are mostly classified into thermal and mechanical effects (mainly focus on cavitation effect). Cavitation is capable of disrupting tumor vasculature and cell membranes. Numerous studies reported that cavitation-induced sonoporation could provoke multiple anti-proliferative effects on cancer cells, including cell-cycle arrest, cell apoptosis, and clonogenicity suppression. Therefore, the combination of FUS-induced cavitation and other treatment modalities like radiation therapy is of great interest, but research in this field is inadequate. A special high-throughput FUS system was used for cancer cell treatment with a customized 1.467 MHz single focused transducer. Characterization of acoustic behavior of gas-filled cavities was performed via a fiber-optic hydrophone (FOH) system and chemical terephthalic acid method helped to define the acoustic parameters, which could lead to occurrence of cavitation at the bottom of 96-well cell culture plates where cancer cells were located. Cavitation occurs at and above the acoustic intensity of 344 W/ cm2 for the 1.467 MHz transducer. The short- and long-term effects of FUS-induced cavitation and adjuvant effects to radiation therapy, standard hyperthermia and testosterone treatment (only for prostate cancer) were investigated comprehensively at the cellular and molecular levels in human prostate cancer (PC-3 and LNCap), glioblastoma (T98G) and head and neck (FaDu) cells in vitro. The long-term additive effects of short FUS shots (with or without cavitation) to radiation therapy (RT) or hyperthermia (HT) were displayed by significantly reduced clonogenic survival in PC-3, T98G and FaDu cells compared to single treatments. The combination treatment of short FUS with cavitation (FUS-Cav) and RT led to a comparable radio-sensitization effect to HT at 45 °C for 30 min and showed a significant reduction in treatment duration, especially for PC-3 cells. The short-term additive effects of short FUS shots to RT or HT are manifested in reducing the potential of cells to invade and decreased metabolic activity. The induction of sonoporation by FUS-Cav was supposed to be the mechanism of cancer cell sensitization to other therapies at the cellular level. The dramatic decline of 5α-reductase type III (SRD5A3) level induced by combination treatment with FUS-Cav and HT is presumed as the underlying mechanism of additive effects of FUS-Cav to HT at the molecular level. Besides, testosterone solutions with normal physiological levels were discovered to inhibit the long-term metabolic activity of androgen-dependent prostate cancer cells LNCap in vitro, while short FUS shots displayed a long-term additive effect to the testosterone treatment. The presented multilevel study demonstrates that short FUS shots using FUS-induced cavitation carry the potential to sensitize cancer cells to other cancer treatment modalities precisely and less-invasively, providing a promising adjuvant therapy to cancer treatments in the future.:1 Abbreviations 2 Summary 3 Introduction 4 Medical and technical background 4.1 The biological basis of prostate cancer treatment 4.1.1 Androgen receptor: an essential signaling pathway for progression of prostate cancer 4.1.2 5α-reductase: a promising therapeutic target for prostate cancer therapy 4.1.3 Testosterone: duality effects in prostate cancer development 4.2 Advantages and disadvantages of current clinical treatments of prostate cancer 4.3 Basics of focused ultrasound (FUS) 4.3.1 Medical application of FUS-induced thermal effects 4.3.1.1 High-intensity focused ultrasound (HIFU) induced thermal ablation 4.3.1.2 Hyperthermia: an alternative heating strategy to sensitize cancer cells for radiation therapy and chemotherapy 4.3.1.3 FUS-induced hyperthermia triggered drug delivery with thermo-sensitive drug carriers 4.3.2 Medical application of FUS-induced mechanical/cavitation effects 4.3.2.1 Cell sonoporation for drug delivery 4.3.2.2 Sonoporation induced anti-proliferative effects for cancer cells 4.3.2.3 Histotripsy 4.3.2.4 Anti-vascular and anti-metastatic effects 4.3.3 The state of art of cavitation detection in medical application 4.3.3.1 Sonoluminescence and sonochemistry 4.3.3.2 Passive cavitation detection 4.3.3.3 Active cavitation detection 4.3.3.4 High-speed sequential photography of cavitation dynamics 4.3.3.5 Laser scattering technique 4.3.3.6 Synchrotron X-ray imaging technique 4.3.3.7 MRI techniques 5 Aims of the thesis 6 Materials and methods 6.1 Materials 6.1.1 Devices 6.1.2 Chemicals and reagents 6.1.3 Consumables 6.1.4 Human cancer cell lines 6.2 Methods 6.2.1 Composition of the FUS system for in vitro treatment of cells 6.2.2 Physical characterization of the in vitro FUS system 6.2.2.1 Setup of fiber-optic hydrophone system to characterize the FUS apparatus 6.2.2.2 Data analysis in MATLAB 6.2.3 Cavitation measurement with FOH system 6.2.4 Chemical cavitation measurement with terephthalic acid (TA) 6.2.5 Culture of human cancer cell lines 6.2.6 FUS treatment of cancer cells 6.2.7 Water bath hyperthermia treatment 6.2.8 Radiation therapy in vitro 6.2.9 The protocol of FUS\FUS-Cav combined with RT or HT 6.2.10 Evaluation of cell ability to reproduce with clonogenic assay 6.2.11 Measurement of cellular metabolic activity with WST-1 assay 6.2.12 Evaluation of cell invasion ability with Transwell® assay 6.2.13 Detection of sonoporation by cell staining with propidium iodide (PI) 6.2.14 Detection of SRD5A as a therapeutic target for prostate cancer with immunofluorescence microscopy 6.2.15 Quantification for the reduction of SRD5A proteins with flow cytometry 6.2.16 Testosterone treatment 6.2.17 FUS/FUS-Cav combined with testosterone treatment 7 Results 7.1 Physical characterization of the in vitro FUS system 7.2 Cavitation occurs at a certain level of acoustic intensity 7.2.1 Characteristics of ultrasonic spectrograms 7.2.2 Cavitation dose depends on the acoustic intensity 7.3 FUS/FUS-Cav supports RT to reduce the long-term clonogenic survival of cancer cells 7.4 FUS/FUS-Cav increases the effects of HT by reducing the long-term clonogenic survival of cancer cells 7.5 FUS/FUS-Cav enhances the suppressive effects of RT in short-term cell potential to invade and metabolic activity of prostate cancer cells 7.6 FUS/FUS-Cav supports HT to diminish the short-term cell potential to invade and metabolic activity of prostate cancer cells 7.7 FUS-Cav treatment immediately induces sonoporation effects in PC-3 and FaDu cells 7.8 FUS/FUS-Cav enhances the effects of HT by inhibiting the SRD5A protein level in prostate cancer cell lines 7.9 FUS-Cav enhances the effects of the testosterone treatment by reducing the long-term cell metabolic activity of androgen-dependent prostate cancer cell line 8 Discussion 8.1 Cavitation measurement in a defined 96-well plate by PCD technique and sonochemistry method 8.2 Short-term and long-term additive effects of FUS-Cav to RT or HT 8.3 Inhibitory effects of FUS-cav in the potential of prostate cancer cells to invade 8.4 The reduction of the long-term metabolic activity of androgen-dependent prostate cancer cells by the combination treatment of FUS-Cav and testosterone 9 Conclusion 10 References 11 Appendix 11.1 Erklärung über die eigenständige Abfassung der Arbeit 11.2 List of figures 11.3 List of tables 11.4 Curriculum vitae 11.5 Acknowledgment

    Thermal dosimetry for bladder hyperthermia treatment. An overview.

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    The urinary bladder is a fluid-filled organ. This makes, on the one hand, the internal surface of the bladder wall relatively easy to heat and ensures in most cases a relatively homogeneous temperature distribution; on the other hand the variable volume, organ motion, and moving fluid cause artefacts for most non-invasive thermometry methods, and require additional efforts in planning accurate thermal treatment of bladder cancer. We give an overview of the thermometry methods currently used and investigated for hyperthermia treatments of bladder cancer, and discuss their advantages and disadvantages within the context of the specific disease (muscle-invasive or non-muscle-invasive bladder cancer) and the heating technique used. The role of treatment simulation to determine the thermal dose delivered is also discussed. Generally speaking, invasive measurement methods are more accurate than non-invasive methods, but provide more limited spatial information; therefore, a combination of both is desirable, preferably supplemented by simulations. Current efforts at research and clinical centres continue to improve non-invasive thermometry methods and the reliability of treatment planning and control software. Due to the challenges in measuring temperature across the non-stationary bladder wall and surrounding tissues, more research is needed to increase our knowledge about the penetration depth and typical heating pattern of the various hyperthermia devices, in order to further improve treatments. The ability to better determine the delivered thermal dose will enable clinicians to investigate the optimal treatment parameters, and consequentially, to give better controlled, thus even more reliable and effective, thermal treatments

    Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

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    PURPOSE: High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. METHODS: An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). RESULTS: The FUS intensities of 213 (1.147 MHz) and 225 W/cm(2) (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. CONCLUSION: Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s00066-021-01774-5) contains supplementary material, which is available to authorized users

    VISUALIZATION OF ULTRASOUND INDUCED CAVITATION BUBBLES USING SYNCHROTRON ANALYZER BASED IMAGING

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    Ultrasound is recognized as the fastest growing medical modality for imaging and therapy. Being noninvasive, painless, portable, X-ray radiation-free and far less expensive than magnetic resonance imaging, ultrasound is widely used in medicine today. Despite these benefits, undesirable bioeffects of high-frequency sound waves have raised concerns; particularly, because ultrasound imaging has become an integral part of prenatal care today and is increasingly used for therapeutic applications. As such, ultrasound bioeffects must be carefully considered to ensure optimal benefits-to-risk ratio. In this context, few studies have been done to explore the physics (i.e. ‘cavitation’) behind the risk factors. One reason may be associated with the challenges in visualization of ultrasound-induced cavitation bubbles in situ. To address this issue, this research aims to develop a synchrotron-based assessment technique to enable visualization and characterization of ultrasound-induced microbubbles in a physiologically relevant medium under standard ultrasound operating conditions. The first objective is to identify a suitable synchrotron X-ray imaging technique for visualization of ultrasound-induced microbubbles in water. Two synchrotron X-ray phase-sensitive imaging techniques, in-line phase contrast imaging (PCI) and analyzer-based imaging (ABI), were evaluated. Results revealed the superiority of the ABI method compared to PCI for visualization of ultrasound-induced microbubbles. The second main objective is to employ the ABI method to assess the effects of ultrasound acoustic frequency and power on visualization and mapping of ultrasound-induced microbubble patterns in water. The time-averaged probability of ultrasound-induced microbubble occurrence along the ultrasound beam propagation in water was determined using the ABI method. Results showed the utility of synchrotron ABI for visualizing cavitation bubbles formed in water by clinical ultrasound systems working at high frequency and output powers as low as used for therapeutic systems. It was demonstrated that the X-ray ABI method has great potential for mapping ultrasound-induced microbubble patterns in a fluidic environment under different ultrasound operating conditions of clinical therapeutic devices. Taken together, this research represents an advance in detection techniques for visualization and mapping of ultrasound-induced microbubble patterns using the synchrotron X-ray ABI method without usage of contrast agents. Findings from this research will pave the road toward the development of a synchrotron-based detection technique for characterization of ultrasound-induced cavitation microbubbles in soft tissues in the future
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