Elimination of radiation-induced senescent cancer cells and stromal cells in vitro by near-infrared photoimmunotherapy.

INTRODUCTION: Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as a novel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. METHODS: Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. RESULTS: Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. CONCLUSION: NIR-PIT eliminates both senescent cancer and stromal cells in vitro suggesting it may be a novel strategy for tumor treatment.

TXNIP-mediated crosstalk between oxidative stress and glucose metabolism.

Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. TXNIP consists of two arrestin (ARR, N-ARR and C-ARR) domains at its amino-terminus and two PPxY (PY) motifs and a di-leucine (LL) motif for endocytosis at its carboxyl-terminus. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. While TXNIP functions as a gatekeeper of TRX by default, it robustly interacted with class I GLUTs through its C-ARR domain upon increase of intracellular reactive oxygen species. This interaction prompted the surface expression downregulation and lysosomal degradation of GLUTs by its carboxyl-terminal LL endocytic signaling motif to attenuate glucose uptake. Consequently, TXNIP expression significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and the pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP and provide a promising target for the drug development against GLUT-related metabolic disorders.

In vivo active-targeting fluorescence molecular imaging with adaptive background fluorescence subtraction.

Using active tumor-targeting nanoparticles, fluorescence imaging can provide highly sensitive and specific tumor detection, and precisely guide radiation in translational radiotherapy study. However, the inevitable presence of non-specific nanoparticle uptake throughout the body can result in high levels of heterogeneous background fluorescence, which limits the detection sensitivity of fluorescence imaging and further complicates the early detection of small cancers. In this study, background fluorescence emanating from the baseline fluorophores was estimated from the distribution of excitation light transmitting through tissues, by using linear mean square error estimation. An adaptive masked-based background subtraction strategy was then implemented to selectively refine the background fluorescence subtraction. First, an in vivo experiment was performed on a mouse intratumorally injected with passively targeted fluorescent nanoparticles, to validate the reliability and robustness of the proposed method in a stringent situation wherein the target fluorescence was overlapped with the strong background. Then, we conducted in vivo studies on 10 mice which were inoculated with orthotopic breast tumors and intravenously injected with actively targeted fluorescent nanoparticles. Results demonstrated that active targeting combined with the proposed background subtraction method synergistically increased the accuracy of fluorescence molecular imaging, affording sensitive tumor detection.

Boosted Radiation Bystander Effect of PSMA-Targeted Gold Nanoparticles in Prostate Cancer Radiosensitization.

Metal nanoparticles are effective radiosensitizers that locally enhance radiation doses in targeted cancer cells. Compared with other metal nanoparticles, gold nanoparticles (GNPs) exhibit high biocompatibility, low toxicity, and they increase secondary electron scatter. Herein, we investigated the effects of active-targeting GNPs on the radiation-induced bystander effect (RIBE) in prostate cancer cells. The impact of GNPs on the RIBE presents implications for secondary cancers or spatially fractionated radiotherapy treatments. Anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated with PEGylated GNPs through EDC-NHS chemistry. The media transfer technique was performed to induce the RIBE on the non-irradiated bystander cells. This study focused on the LNCaP cell line, because it can model a wide range of stages relating to prostate cancer progression, including the transition from androgen dependence to castration resistance and bone metastasis. First, LNCaP cells were pretreated with phosphate buffered saline (PBS) or PSMA-targeted GNPs (PGNPs) for 24 h and irradiated with 160 kVp X-rays (0-8 Gy). Following that, the collected culture media were filtered (sterile 0.45 µm polyethersulfone) in order to acquire PBS- and PGNP- conditioned media (CM). Then, PBS- and PGNP-CM were transferred to the bystander cells that were loaded with/without PGNPs. MTT, γ-H2AX, clonogenic assays and reactive oxygen species assessments were performed to compare RIBE responses under different treatments. Compared with 2 Gy-PBS-CM, 8 Gy-PBS-CM demonstrated a much higher RIBE response, thus validating the dose dependence of RIBE in LNCaP cells. Compared with PBS-CM, PGNP-CM exhibited lower cell viability, higher DNA damage, and a smaller survival fraction. In the presence of PBS-CM, bystander cells loaded with PGNPs showed increased cell death compared with cells that did not have PGNPs. These results demonstrate the PGNP-boosted expression and sensitivity of RIBE in prostate cancer cells.

Quantifying Radiosensitization of PSMA-Targeted Gold Nanoparticles on Prostate Cancer Cells at Megavoltage Radiation Energies by Monte Carlo Simulation and Local Effect Model.

Active targeting gold nanoparticles (AuNPs) are a very promising avenue for cancer treatment with many publications on AuNP mediated radiosensitization at kilovoltage (kV) photon energies. However, uncertainty on the effectiveness of AuNPs under clinically relevant megavoltage (MV) radiation energies hinders the clinical translation of AuNP-assisted radiation therapy (RT) paradigm. The aim of this study was to investigate radiosensitization mediated by PSMA-targeted AuNPs irradiated by a 6 MV radiation beam at different depths to explore feasibility of AuNP-assisted prostate cancer RT under clinically relevant conditions. PSMA-targeted AuNPs (PSMA-AuNPs) were synthesized by conjugating PSMA antibodies onto PEGylated AuNPs through EDC/NHS chemistry. Confocal fluorescence microscopy was used to verify the active targeting of the developed PSMA-AuNPs. Transmission electron microscopy (TEM) was used to demonstrate the intracellular biodistribution of PSMA-AuNPs. LNCaP prostate cancer cells treated with PSMA-AuNPs were irradiated on a Varian 6 MV LINAC under varying depths (2.5 cm, 10 cm, 20 cm, 30 cm) of solid water. Clonogenic assays were carried out to determine the in vitro cell survival fractions. A Monte Carlo (MC) model developed on TOPAS platform was then employed to determine the nano-scale radial dose distribution around AuNPs, which was subsequently used to predict the radiation dose response of LNCaP cells treated with AuNPs. Two different cell models, with AuNPs located within the whole cell or only in the cytoplasm, were used to assess how the intracellular PSMA-AuNP biodistribution impacts the prostate cancer radiosensitization. Then, MC-based microdosimetry was combined with the local effect model (LEM) to calculate cell survival fraction, which was benchmarked against the in vitro clonogenic assays at different depths. In vitro clonogenic assay of LNCaP cells demonstrated the depth dependence of AuNP radiosensitization under clinical megavoltage beams, with sensitization enhancement ratio (SER) of 1.14 ± 0.03 and 1.55 ± 0.05 at 2.5 cm depth and 30 cm depth, respectively. The MC microdosimetry model showed the elevated percent of low-energy photons in the MV beams at greater depth, consequently resulting in increased dose enhancement ratio (DER) of AuNPs with depth. The AuNP-induced DER reached ~5.7 and ~8.1 at depths of 2.5 cm and 30 cm, respectively. Microdosimetry based LEM accurately predicted the cell survival under 6 MV beams at different depths, for the cell model with AuNPs placed only in the cell cytoplasm. TEM results demonstrated the distribution of PSMA-AuNPs in the cytoplasm, confirming the accuracy of MC microdosimetry based LEM with modelled AuNPs distributed within the cytoplasm. We conclude that AuNP radiosensitization can be achieved under megavoltage clinical radiotherapy energies with a dependence on tumor depth. Furthermore, the combination of Monte Carlo microdosimetry and LEM will be a valuable tool to assist with developing AuNP-aided radiotherapy paradigm and drive clinical translation.

Data Processing of Product Ion Spectra: Quality Improvement by Averaging Multiple Similar Spectra of Small Molecules.

In metabolomics studies using high-resolution mass spectrometry (MS), a set of product ion spectra is comprehensively acquired from observed ions using the data-dependent acquisition (DDA) mode of various tandem MS. However, especially for low-intensity signals, it is sometimes difficult to distinguish artifact signals from true fragment ions derived from a precursor ion. Inadequate precision in the measured m/z value is also one of the bottlenecks to narrowing down the candidate compositional formula. In this study, we report that averaging multiple product ion spectra can improve m/z precision as well as the reliability of fragment ions that are observed in such spectra. A graph-based method was applied to cluster a set of similar spectra from multiple DDA data files resulting in creating an averaged product-ion spectrum. The error levels for the m/z values declined following the central limit theorem, which allowed us to reduce the number of candidate compositional formulas. The improved reliability and precision of the averaged spectra will contribute to a more efficient annotation of product ion spectral data.

Interactions of Urea-Based Inhibitors with Prostate-Specific Membrane Antigen for Boron Neutron Capture Therapy.

In this study, molecular interactions of prostate-specific membrane antigen (PSMA) with five chemically distinct urea-based boron-containing inhibitors have been investigated at the atomic level using molecular docking and molecular dynamics simulations. The PSMA-inhibitor complexations have been analyzed by comparing their binding modes, secondary structures, root-mean-square deviations, noncovalent interactions, principal components, and binding free energies. PSMA is a cell surface glycoprotein upregulated in cancerous cells and can be targeted by boron-labeled inhibitors for boron neutron capture therapy (BNCT). The effective BNCT requires the selective boron delivery to the tumor area and highly specific PSMA-mediated cellular uptake by tumor. Thus, a potent inhibitor must exhibit both high binding affinity and high boron density. The computational results suggest that the chemical nature of inhibitors affects the binding mode and their association with PSMA is primarily dominated by hydrogen bonding, salt bridge, electrostatic, and π-π interactions. The binding free energies (-28.0, -15.2, -43.9, -23.2, and -38.2 kcal/mol) calculated using λ-dynamics for all inhibitors (In1-5) predict preferential binding that is in accordance with experimental data. Among all inhibitors, In5 was found to be the best candidate for BNCT. The binding of this inhibitor to PSMA preserved its overall secondary structure. These results provide computational insights into the coordination flexibility of PSMA and its interaction with various inhibitors. They can be used for the design and synthesis of efficient BNCT agents with improved drug selectivity and high boron percentage.

Silinanyl Rhodamines and Silinanyl Fluoresceins for Super-Resolution Microscopy.

Single-molecule localization microscopy (SMLM) enables the visualization of biomolecules at unprecedented resolution and requires control of the fluorescent blinking (ON/OFF) states of fluorophores to detect single-molecule fluorescence without overlapping of the signals. Although SMLM probes based on the intramolecular spirocyclization of Si-xanthene fluorophores have been developed, fluorophores with lower ON/OFF ratios are required for SMLM visualization of high-density structures. Here, we describe a silinane structure that lowers the ON/OFF ratio of Si-xanthene fluorophores. On the basis of Mulliken population analysis, we replaced the dimethylsilane moiety in Si-rhodamine with a silinane moiety to increase the partial charge at the 9-position of the carbon atom in the Si-xanthene ring and to promote the ring-closure reaction. Evaluation of fluorescence properties in a solution and in single-molecule imaging indicated that introducing the silinane sufficiently stabilized the nonfluorescent spirocyclic forms, thus decreasing the fluorescence ON/OFF ratio. This novel substitution was applied to Si-rhodamines with various amine structures and to an Si-fluorescein to expand the color palette. We demonstrated SMLM observation of microtubules in fixed HeLa cells using the developed fluorophores in two color channels. The results demonstrated the feasibility of extending the design strategies of SMLM probes based on Si-xanthenes through modification of the substituents on the Si atom.

Prostate Cancer Targeted X-Ray Fluorescence Imaging via Gold Nanoparticles Functionalized With Prostate-Specific Membrane Antigen (PSMA).

PURPOSE: The gold nanoparticle (GNP) as a promising theranostic probe has been increasingly studied. The tumor-targeting efficiency of GNPs is crucial to increase the therapeutic ratio. In this study, we developed PSMA-targeted GNPs to enhance GNP uptake in prostate cancer and developed an x-ray fluorescence imaging system to noninvasively monitor and assess GNP delivery. METHODS AND MATERIALS: For targeted therapy of prostate cancer, anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PEGylated GNPs through 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC/NHS) chemistry. In vivo imaging was implemented using an in-house-developed dual-modality computed tomography (CT) and x-ray fluorescence CT (XFCT) system on mice bearing subcutaneous LNCaP prostate tumors. After intravenous administration of GNPs (15 mg/mL, 200 µL), the x-ray fluorescence signals from the tumor were collected at various time points (5 minutes to approximately 30 hours) for GNP pharmacokinetics analysis. At 24 hours after administration, x-ray fluorescence projection (XRFproj) and XFCT imaging were conducted to evaluate the prostate tumor uptake of active- and passive-targeting GNPs. Inductively coupled plasma mass spectrometry analysis was adopted as a benchmark to verify the quantification accuracy of XRFproj/XFCT imaging. RESULTS: Fluorescence microscopic imaging confirmed the enhanced (approximately 4 times) targeting efficiency of PSMA-targeted GNPs in vitro. The pharmacokinetics analysis showed enhanced tumor uptake/retention of PSMA-targeted GNPs and revealed that the peak tumor accumulation appeared at approximately 24 hours after intravenous administration. Both XRFproj and XFCT imaging presented their accuracy in quantifying GNPs within tumors noninvasively. Moreover, XFCT imaging verified its unique capabilities to simultaneously determine the heterogeneous spatial distribution and the concentration of GNPs within tumors in vivo. CONCLUSIONS: In conjunction with PSMA-targeted GNPs, XRFproj/XFCT would be a highly sensitive tool for targeted imaging of prostate cancer, benefiting the elucidation of mechanisms of GNP-assisted prostate-cancer therapy.

Development of Thermostable Lyophilized Sabin Inactivated Poliovirus Vaccine.

As oral poliovirus vaccine (OPV) causes vaccine-associated paralytic poliomyelitis, the polio endgame strategy introduced by the Global Polio Eradication Initiative calls for a phased withdrawal of OPV and an introduction of inactivated poliovirus vaccine (IPV). The introduction of IPV creates challenges in maintaining the cold chain for vaccine storage and distribution. Recent advances in lyophilization have helped in finding a temperature-stable formulation for multiple vaccines; however, poliovirus vaccines have yet to capture a stable, safe formula for lyophilization. In addition, efficient in vitro methods for antigen measurement are needed for screening stable vaccine formulations. Here, we report size exclusion high-performance liquid chromatography (SE-HPLC) as a reliable means to identify the leading lyophilized formulation to generate thermostable Sabin inactivated poliovirus vaccine (sIPV). High-throughput screening and SE-HPLC determined the leading formulation, resulting in 95% D-antigen recovery and low residual moisture content of sIPV following lyophilization. Furthermore, the lyophilized sIPV remained stable after 4 weeks of incubation at ambient temperature and induced strong neutralizing antibodies and full protection of poliovirus receptor transgenic mice against the in vivo challenge of wild-type poliovirus. Overall, this report describes a novel means for the high-throughput evaluation of sIPV antigenicity and a thermostable lyophilized sIPV with in vivo vaccine potency.IMPORTANCE Poliomyelitis is a highly contagious disease caused by the poliovirus. While the live attenuated OPV has been the vaccine of choice, a major concern is its ability to revert to a form that can cause paralysis, so-called vaccine-associated paralytic poliomyelitis. Therefore, the new endgame strategy of the Global Polio Eradication Initiative includes the introduction of an IPV. However, the feasibility of the use of current IPV formulations in developing countries is limited, because IPV is insufficiently stable to be purified, transported, and stored under unrefrigerated conditions. We successfully designed the sIPV for use in the dry state that maintains the full vaccine potency in animal models after incubation at ambient temperature. This report provides, for the first time, candidate formulations of sIPV that are stable at elevated temperatures.

Tracking the Oxygen Status in the Cell Nucleus with a Hoechst-Tagged Phosphorescent Ruthenium Complex.

Molecular oxygen in living cells is distributed and consumed inhomogeneously, depending on the activity of each organelle. Therefore, tractable methods that can be used to monitor the oxygen status in each organelle are needed to understand cellular function. Here we report the design of a new oxygen-sensing probe for use in the cell nucleus. We prepared "Ru-Hoechsts", each consisting of a phosphorescent ruthenium complex linked to a Hoechst 33258 moiety, and characterized their properties as oxygen sensors. The Hoechst unit shows strong DNA-binding properties in the nucleus, and the ruthenium complex shows oxygen-dependent phosphorescence. Thus, Ru-Hoechsts accumulated in the cell nucleus and showed oxygen-dependent signals that could be monitored. Of the Ru-Hoechsts prepared in this study, Ru-Hoechst b, in which the ruthenium complex and the Hoechst unit were linked through a hexyl chain, showed the most suitable properties for monitoring the oxygen status. Ru-Hoechsts are probes with high potential for visualizing oxygen fluctuations in the nucleus.

Water-soluble phosphorescent ruthenium complex with a fluorescent coumarin unit for ratiometric sensing of oxygen levels in living cells.

Dual emission was applied to a molecular probe for the ratiometric sensing of oxygen concentration in a living system. We prepared ruthenium complexes possessing a coumarin unit (Ru-Cou), in which the (3)MLCT phosphorescence of the ruthenium complex was efficiently quenched by molecular oxygen, whereas the coumarin unit emitted constant fluorescence independent of the oxygen concentration. The oxygen status could be determined precisely from the ratio of phosphorescence to fluorescence. We achieved the molecular imaging of cellular oxygen levels using Ru-Cou possessing an alkyl chain, which provided appropriate lipophilicity to increase cellular uptake.

Phosphorescent ruthenium complexes with a nitroimidazole unit that image oxygen fluctuation in tumor tissue.

Understanding oxygen fluctuation in a cancerous tumor is important for effective treatment, especially during radiotherapy. In this paper, ruthenium complexes bearing a nitroimidazole group are shown to report the oxygen status in tumor tissue directly. The nitroimidazole group was known to be accumulated in hypoxic tumor tissues. On the other hand, the ruthenium complex showed strong phosphorescence around 600 nm. The emission of ruthenium is quenched instantaneously by molecular oxygen due to energy transfer between triplet states of oxygen and ruthenium complex, but the emission is then recovered by the removal of oxygen. Thus, we could observe oxygen fluctuation in tumor tissue in a real-time manner by monitoring the phosphorescence of the ruthenium complex. The versatility of the probe is demonstrated by monitoring oxygen fluctuation in living cells and tumor tissue planted in mice. The ruthenium complex promptly penetrated plasma membrane and accumulated in cells to emit its oxygen-dependent phosphorescence. In vivo experiments revealed that the oxygen level in tumor tissue seems to fluctuate at the sub-minute timescale.