Evaluation of the Yield of DNA Double-Strand Breaks for Carbon Ions Using Monte Carlo Simulation and DNA Fragment Distribution.

PURPOSE: The aim of this work was to provide a method to evaluate the yield of DNA double-strand breaks (DSBs) for carbon ions, overcoming the bias in existing methods due to the nonrandom distribution of DSBs. METHODS AND MATERIALS: A previously established biophysical program based on the radiation track structure and a multilevel chromosome model was used to simulate DNA damage induced by x-rays and carbon ions. The fraction of activity retained (FAR) as a function of absorbed dose or particle fluence was obtained by counting the fraction of DNA fragments larger than 6 Mbp. Simulated FAR curves for the 250 kV x-rays and carbon ions at various energies were compared with measurements using constant-field gel electrophoresis. The doses or fluences at the FAR of 0.7 based on linear interpolation were used to estimate the simulation error for the production of DSBs. RESULTS: The relative difference of doses at the FAR of 0.7 between simulation and experiment was -8.5% for the 250 kV x-rays. The relative differences of fluences at the FAR of 0.7 between simulations and experiments were -17.5%, -42.2%, -18.2%, -3.1%, 10.8%, and -14.5% for the 34, 65, 130, 217, 2232, and 3132 MeV carbon ions, respectively. In comparison, the measurement uncertainty was about 20%. Carbon ions produced remarkably more DSBs and DSB clusters per unit dose than x-rays. The yield of DSBs for carbon ions, ranging from 10 to 16 Gbp-1Gy-1, increased with linear energy transfer (LET) but plateaued in the high-LET end. The yield of DSB clusters first increased and then decreased with LET. This pattern was similar to the relative biological effectiveness for cell survival for heavy ions. CONCLUSIONS: The estimated yields of DSBs for carbon ions increased from 10 Gbp-1Gy-1 in the low-LET end to 16 Gbp-1Gy-1 in the high-LET end with 20% uncertainty.

Label-Free Separation of Circulating Tumor Cells and Clusters by Alternating Frequency Acoustic Field in a Microfluidic Chip.

Circulating tumor cells (CTCs) play an important role in the prognosis and efficacy evaluation of metastatic tumors. Since CTCs are present in very low concentrations in the blood and the phenotype is dynamically changing, it is a great challenge to achieve efficient separation while maintaining their viability. In this work, we designed an acoustofluidic microdevice for CTCs separation based on the differences in cell physical properties of size and compressibility. Efficient separation can be achieved with only one piece of piezoceramic working on alternating frequency mode. The separation principle was simulated by numerical calculation. Cancer cells from different tumor types were separated from peripheral blood mononuclear cells (PBMCs), with capture efficiency higher than 94% and a contamination rate of about 1% was obtained. Furthermore, this method was validated to have no negative effect on the viability of the separated cells. Finally, blood samples from patients with different cancer types and stages were tested, with measured concentrations of 36-166 CTCs per milliliter. Effective separation was achieved even when the size of CTCs is similar to that of PBMCs, which has the prospect of clinical application in cancer diagnosis and efficacy evaluation.

Measurement of the Compressibility of Cell and Nucleus Based on Acoustofluidic Microdevice.

Cell mechanics play an important role in tumor metastasis, malignant transformation of cells, and radiosensitivity. During these processes, studying the mechanical properties of the cells is often challenging. Conventional measurement methods based on contact such as compression or stretching are prone to cause cell damage, affecting measurement accuracy and subsequent cell culture. Measurements in adherent state can also affect accuracy, especially after irradiation since ionizing radiation will flatten cells and enhance adhesion. Here, a cell mechanics measurement system based on acoustofluidic method has been developed. The cell compressibility can be obtained by recording the cell motion trajectory under the action of the acoustic force, which can realize fast and non-destructive measurement in suspended state. This paper reports in detail the protocols for chip design, sample preparation, trajectory recording, parameter extraction and analysis. The compressibility of different types of tumor cells was measured based on this method. Measurement of the compressibility of nucleus was also achieved by adjusting the resonance frequency of the piezoelectric ceramic and the width of the microchannel. Combined with the molecular level verification of immunofluorescence experiments, the cell compressibility before and after drug-induced epithelial to mesenchymal transition (EMT) were compared. Further, the change of cell compressibility after X-ray irradiation with different doses was revealed. The cell mechanics measurement method proposed in this paper is universal and flexible and has broad application prospects in scientific research and clinical practice.

Ionizing radiation-induced DNA damage responses affect cell compressibility.

Radiotherapy is an important treatment modality for cancer patients. Ionizing radiation kills cancer cells by inducing DNA damage. However, it remains unclear how this process affects the biomechanical properties of cells and how cellular mechanics affect DNA damage and repair. In this study, the effects of ionizing radiation on cell mechanics were investigated by precisely measuring the compressibility of cells in suspension using an in-house developed microfluidic device. We found that cell compressibility significantly increased after irradiation, depending on cell type and radiation dose. Radiation-induced DNA damage response (DDR) is an important process that alters cell mechanics, and this association was confirmed experimentally by inhibiting the DDR process. Furthermore, the mechanisms involved in changes in cell compressibility were investigated from the perspective of the nucleus and cytoplasm. Experiments showed that radiation reduced H3K9me3 staining intensity but had no effect on F-actin. This suggested that chromatin decondensation caused by radiation-induced DDR is the primary cause of changes in cell compressibility after radiation. The effects of cell mechanics on radiation-induced DNA damage were also investigated. The addition of blebbistatin reduced cytoskeletal forces on the nucleus, which resulted in a reduction in radiation-induced DNA damage. Collectively, this study elucidated reciprocal mechanisms of radiation-induced DNA damage and cell mechanics.

The potential of electrotrophic denitrification coupled with sulfur recycle in MFC and its responses to COD/SO42- ratios.

Electrotrophic denitrification is a promising novel nitrogen removal technique. In this study, the performance and the mechanism of electrotrophic denitrification coupled with sulfate-sulfide cycle were investigated under different anodic influent COD/SO42- ratios. The results showed that electrotrophic denitrification contributed to more than 22% total nitrogen removal in cathode chamber. Higher COD/SO42- ratios would deteriorate the sulfate reduction but enhance methane production. Further mass balance indicated that the electron flow utilized by methanogenic archaea (MA) increased while that utilized by sulfate-reducing bacteria (SRB) decreased as the COD/SO42- ratio increased from 0.44 to 1.11. However, higher COD/SO42- ratios would produce more electrons to strengthen electrotrophic denitrification. Microbial community analysis showed that the biocathode was predominantly covered by Thiobacillus that encoded with narG gene. These findings collectively suggest that electrotrophic denitrification could be a sustainable approach to simultaneously remove COD and nitrogen under suitable COD/SO42- ratio based on sulfur cycle in wastewater.

Measurement of cell compressibility changes during epithelial-mesenchymal transition based on acoustofluidic microdevice.

Epithelial-mesenchymal transition (EMT) confers migratory and invasiveness abilities on cancer cells, as well as leading to changes in biomechanical properties and cytoskeletal structure. Cell mechanical properties are considered to be promising label-free markers for diagnosis of cancer metastasis. In this work, cell compressibility, a novel and important parameter of cell mechanical properties, was measured directly and quickly using a specially designed acoustofluidic microdevice. The compressibilities of cells with different metastatic potentials were investigated. Based on a comparison of the measurement results, non-metastatic cells exhibited lower compressibility than metastatic cells. The correlation between cell compressibility and EMT status was further studied; the results showed that the acquisition of mesenchymal status was accompanied by an increase in cell compressibility. These findings imply strong correlations among cell compressibility, EMT status, and invasiveness. Therefore, cell compressibility represents a novel biomechanical marker for evaluating malignant transformation and metastasis of cancer.

Potential applications of endogenous sulfide for enhanced denitrification of low C/N domestic wastewater in anodic mixotrophic denitrification microbial fuel cell: The mechanism of electrons transfer and microbial community.

Anodic mixotrophic denitrification microbial fuel cell (MFC) was developed for pollutants removal and electricity generation in treatment of low C/N domestic wastewater. The experimental results show that the MFC achieved up to 100% of acetate, 100% of sulfide, and more than 91% of nitrate removal efficiency in all the MFCs. Particularly, thiosulfate was generated as the main intermediate of sulfide oxidation, and the sulfate generation ratio ranged from 66.93% to 73.76%. Those electrons produced during the acetate and sulfide oxidation were mainly used for denitrification and electricity generation. The microbial community analysis revealed that heterotrophic denitrifying bacteria (HDB) and sulfide-based autotrophic denitrifying bacteria (SADB) were the dominant bacteria for pollutants removal, and those facultative autotrophic bacterium (FAB) were key functional genera for high sulfate generation under both low and high sulfide concentrations. Meanwhile, the microbial functional prediction revealed that sulfide oxidation gene of Sqr and Sox were highly expressed. Moreover, a preliminary sulfide-based autotrophic denitrification (SAD) potential estimation indicated that the sulfide generated in the WWTPs had great potential for denitrification.

Characterizing the DNA damage response in fibrosarcoma stem cells by in-situ cell tracking.

PURPOSE: Radiotherapy works by generating large amounts of DNA double-strand breaks (DSBs). Cancer stem-like cells (CSCs) are the principal cause of tumor radioresistance. Therefore, investigating the dynamics and mechanisms of DNA damage response (DDR) in CSCs is of great importance. MATERIALS AND METHODS: Human fibrosarcoma cell line HT1080 stably transfected with 53BP1-GFP was used to investigate the real-time cellular response to DSBs induced by γ-rays. HT1080 CSCs were sorted based on aldehyde dehydrogenase (ALDH1) levels by flow cytometry and verified by mammosphere formation assay. We set the number, area and intensity of ionizing radiation-induced foci (IRIF) as endpoints. Using live-cell imaging to track single IRIF in-situ, we compared the IRIF induction and dispersal in HT1080 cells and CSCs. RESULTS: ALDH1+ cells showed much stronger mammosphere-forming capability, indicating the property of CSCs and could be considered as HT1080 CSCs. After γ-irradiation, CSCs had fewer IRIF number and smaller IRIF size than HT1080 cells. Different repair kinetics (with plateau and without plateau) were observed both in CSCs and HT1080 cells. Similar to HT1080 cells, IRIF with a plateau in CSCs showed higher intensity, larger area and slower decay rate of intensity than IRIF without plateau. Additionally, the level of IRIF merging in HT1080 cells was significantly higher than that in CSCs. CONCLUSIONS: CSCs have fewer and smaller IRIF, indicating the reduced complexity of DNA damage. This may contribute to tumor radioresistance. Heterogeneous repair kinetics (with and without plateau) were observed although the dynamics of IRIF with or without plateau in CSCs resemble the dynamics in HT1080 cells based on single IRIF analysis.

The effect of hyperthermia on the DNA damage response induced by γ-rays, as determined through in situ cell tracking.

Hyperthermia (HT) acts as a cancer treatment by direct cell killing, radiosensitization, and promotion of tumor reoxygenation. The sensor proteins of the DNA damage response (DDR) are the direct targets of HT. However, the spatiotemporal properties of sensor proteins under HT are still unclear. Therefore, investigating the impact of HT on sensor proteins is of great importance. In the present study, the human fibrosarcoma cell line HT1080 stably transfected with 53BP1-GFP [the DDR protein 53BP1 fused to green fluorescent protein (GFP)] was used to investigate the real-time cellular response to DNA double-strand breaks (DSBs) induced by γ-rays. Using live-cell imaging combined with HT treatment, the spatiotemporal properties of the 53BP1 protein were directly monitored and quantitatively studied. We found that HT could delay and decrease the formation of 53BP1 ionizing radiation-induced foci (IRIF). Moreover, through the in situ tracking of individual IRIF, it was found that HT resulted in more unrepaired IRIF over the period of observation compared with IR alone. Additionally, the unrepaired IRIF had a larger area, higher intensity, and slower repair rate. Indeed, almost every cell treated with HT had unrepaired IRIF, and the majority of these IRIF increased in area individually, while the rest increased in area by the merging of adjacent IRIF. In summary, our study demonstrated that HT could perturb the primary event in the DDR induced by IR, and this may have important implications for cancer treatment and heat radiosensitization.

Association of elevated reactive oxygen species and hyperthermia induced radiosensitivity in cancer stem-like cells.

Cancer stem-like cells (CSCs) are the principal causes of tumor radio-resistance, dormancy and recurrence after radiotherapy. Clinical trials show hyperthermia (HT) might be a potent radiation sensitizer. In this study, CSCs were found to be more susceptible to radiation when combined with HT treatment. Treated cells showed significantly reduced self-renewal, cell survival and proliferation in vitro, as well as significant reduced tumor formation in vivo. Further study demonstrated that the radiosensitization effect was associated with increased intracellular reactive oxygen species (ROS) level in CSCs, confirmed by modifying redox status in CSCs bidirectionally. Pharmacologic depletion of glutathione by buthionine sulphoximine mimicked HT induced radiosensitivity in CSCs. Antioxidant N-acetylcysteine could efficiently rescue HT induced radiosensitivity in CSCs. To our knowledge, this may be the first report suggesting the association between elevated intracellular ROS level and HT induced radiosensitization in human breast CSCs and pancreatic CSCs, which might provide new strategy for improving CSCs radiosensitivity.

Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions.

Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or 60Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

Rescue of Targeted Nonstem-Like Cells from Bystander Stem-Like Cells in Human Fibrosarcoma HT1080.

Cancer stem-like cells (CSCs) have been suggested to be the principal cause of tumor radioresistance, dormancy and recurrence after radiotherapy. However, little is known about CSC behavior in response to clinical radiotherapy, particularly with regard to CSC communication with bulk cancer cells. In this study, CSCs and nonstem-like cancer cells (NSCCs) were co-cultured, and defined cell types were chosen and irradiated, respectively, with proton microbeam. The bidirectional rescue effect in the combinations of the two cell types was then investigated. The results showed that out of all four combinations, only the targeted, proton irradiated NSCCs were protected by bystander CSCs and showed less accumulation of 53BP1, which is a widely used indicator for DNA double-strand breaks. In addition, supplementation with c-PTIO, a specific nitric oxide scavenger, can show a similar effect on targeted NSCCs. These results, showed that the rescue effect of CSCs on targeted NSCCs involves nitric oxide in the process, suggesting that the cellular communication between CSCs and NSCCs may be important in determining the survival of tumor cells after radiation therapy. To our knowledge, this is the first report demonstrating a rescue effect of CSCs to irradiated NSCCs that may help us better understand CSC behavior in response to cancer radiotherapy.

Atomic layer deposition modified track-etched conical nanochannels for protein sensing.

Nanopore-based devices have recently become popular tools to detect biomolecules at the single-molecule level. Unlike the long-chain nucleic acids, protein molecules are still quite challenging to detect, since the protein molecules are much smaller in size and usually travel too fast through the nanopore with poor signal-to-noise ratio of the induced transport signals. In this work, we demonstrate a new type of nanopore device based on atomic layer deposition (ALD) Al2O3 modified track-etched conical nanochannels for protein sensing. These devices show very promising properties of high protein (bovine serum albumin) capture rate with well time-resolved transport signals and excellent signal-to-noise ratio for the transport events. Also, a special mechanism involving transient process of ion redistribution inside the nanochannel is proposed to explain the unusual biphasic waveshapes of the current change induced by the protein transport.

Target irradiation induced bystander effects between stem-like and non stem-like cancer cells.

Tumors are heterogeneous in nature and consist of multiple cell types. Among them, cancer stem-like cells (CSCs) are suggested to be the principal cause of tumor metastasis, resistance and recurrence. Therefore, understanding the behavior of CSCs in direct and indirect irradiations is crucial for clinical radiotherapy. Here, the CSCs and their counterpart non stem-like cancer cells (NSCCs) in human HT1080 fibrosarcoma cell line were sorted and labeled, then the two cell subtypes were mixed together and chosen separately to be irradiated via a proton microbeam. The radiation-induced bystander effect (RIBE) between the CSCs and NSCCs was measured by imaging 53BP1 foci, a widely used indicator for DNA double strand break (DSB). CSCs were found to be less active than NSCCs in both the generation and the response of bystander signals. Moreover, the nitric oxide (NO) scavenger c-PTIO can effectively alleviate the bystander effect in bystander NSCCs but not in bystander CSCs, indicating a difference of the two cell subtypes in NO signal response. To our knowledge, this is the first report shedding light on the RIBE between CSCs and NSCCs, which might contribute to a further understanding of the out-of-field effect in cancer radiotherapy.

Dynamics between cancer cell subpopulations reveals a model coordinating with both hierarchical and stochastic concepts.

Tumors are often heterogeneous in which tumor cells of different phenotypes have distinct properties. For scientific and clinical interests, it is of fundamental importance to understand their properties and the dynamic variations among different phenotypes, specifically under radio- and/or chemo-therapy. Currently there are two controversial models describing tumor heterogeneity, the cancer stem cell (CSC) model and the stochastic model. To clarify the controversy, we measured probabilities of different division types and transitions of cells via in situ immunofluorescence. Based on the experiment data, we constructed a model that combines the CSC with the stochastic concepts, showing the existence of both distinctive CSC subpopulations and the stochastic transitions from NSCCs to CSCs. The results showed that the dynamic variations between CSCs and non-stem cancer cells (NSCCs) can be simulated with the model. Further studies also showed that the model can be used to describe the dynamics of the two subpopulations after radiation treatment. More importantly, analysis demonstrated that the experimental detectable equilibrium CSC proportion can be achieved only when the stochastic transitions from NSCCs to CSCs occur, indicating that tumor heterogeneity may exist in a model coordinating with both the CSC and the stochastic concepts. The mathematic model based on experimental parameters may contribute to a better understanding of the tumor heterogeneity, and provide references on the dynamics of CSC subpopulation during radiotherapy.

Impact of cell dissociation on identification of breast cancer stem cells.

BACKGROUND: Fluorescence-activated cell sorting was commonly used for identification of cancer stem cells (CSCs), which relied on specific cell surface markers. And this approach makes it possible for us to study characteristics of CSCs in vitro. However, the pattern of membrane protein including surface makers might be vitally influenced during the dissociation of the adherent cells, thus it might heavily impact the quantity and quality of CSCs identified by flow cytometry. METHODS: To address this question, in present study, three commonly used digestive reagents and two different temperatures were performed in MCF-7 cells to assay CD44(+)CD24(-) CSCs subpopulation. The potential of sorted CD44(+)CD24(-) cells from different digestion to form mammosphere in culture was also compared. RESULTS: The results showed that trypsin, a commonly used reagent in CSCs studies, most aggressively reduced antigenicity for surface markers and make part of CD44(+)CD24(-) CSCs subpopulation cleaved into CD44(+)CD24(-) non-stem cancer cells (NSCCs). And it also increased the mammosphere formation efficiency of CD44(-)CD24(-) subpopulation. This cleavage effect is especially serious when cells are digested at 37°C. While accutase, a purified collagenase/neutral protease cocktail, provides the best balance of dissociation efficiency and antigen retention. CONCLUSION: Taken together; these results indicate that enzymatic digestion process plays an important role in identification of CSCs with surface marker via flow cytometer, suggesting that researchers need to reconsider this process seriously.