Radiation-induced dormancy of intracerebral melanoma: endotoxin inflammation leads to both shortened tumor dormancy and long-term survival with localized senescence.

Radiation therapy (RT) treats approximately half of all cancers and most brain cancers. RT is variably effective at inducing a dormant tumor state i.e. the time between RT and clinical recurrence of tumor growth. Interventions that significantly lengthen tumor dormancy would improve long-term outcomes. Inflammation can promote the escape of experimental tumors from metastatic dormancy in the lung. Previously we showed intracerebral B16F10 melanoma dormancy varied with RT dose; 20.5 Gy induced dormancy lasted ~ 2 to 4 weeks-sufficient time to study escape from dormancy. Tumors were followed over time using bioluminescence. Surprisingly, some tumors in endotoxin-treated mice exited from dormancy slower; a large fraction of the mice survived more than 1-year. A cohort of mice also experienced an accelerated exit from dormancy and increased mortality indicating there might be variation within the tumor or inflammatory microenvironment that leads to both an early deleterious effect and a longer-term protective effect of inflammation. Some of the melanin containing cells at the site of the original tumor were positive for senescent markers p16, p21 and ßGal. Changes in some cytokine/chemokine levels in blood were also detected. Follow-up studies are needed to identify cytokines/chemokines or other mechanisms that promote long-term dormancy after RT.

Gold nanoparticle hyperthermia reduces radiotherapy dose.

Gold nanoparticles can absorb near infrared light, resulting in heating and ablation of tumors. Gold nanoparticles have also been used for enhancing the X-ray dose to tumors. The combination of hyperthermia and radiotherapy is synergistic, importantly allowing a reduction in X-ray dose with improved therapeutic results. Here we intratumorally infused small 15 nm gold nanoparticles engineered to be transformed from infrared-transparent to infrared-absorptive by the tumor, then heated by infrared followed by X-ray treatment. Synergy was studied using a very radioresistant subcutaneous squamous cell carcinoma (SCCVII) in mice. It was found that the dose required to control 50% of the tumors, normally 55 Gy, could be reduced to <15 Gy (a factor of >3.7). Gold nanoparticles therefore provide a method to combine hyperthermia and radiotherapy to drastically reduce the X-ray radiation needed, thus sparing normal tissue, reducing side effects, and making radiotherapy more effective. FROM THE CLINICAL EDITOR: Gold nanoparticles are known to enhance the efficacy of X-ray in tumor irradiation resulting in tumor heating and ablation. They also absorb near infrared light. This dual property was studied using a very radioresistant subcutaneous squamous cell carcinoma in mice, demonstrating that the dose required to control 50% of the tumors could be reduced by a factor of > 3.7, paving the way to potential future clinical applications.

Iodine Nanoparticles (Niodx™) for Radiotherapy Enhancement of Glioblastoma and Other Cancers: An NCI Nanotechnology Characterization Laboratory Study.

Effective and durable treatment of glioblastoma is an urgent unmet medical need. In this article, we summarize a novel approach of a physical method that enhances the effectiveness of radiotherapy. High atomic number nanoparticles that target brain tumors are intravenously administered. Upon irradiation, the nanoparticles absorb X-rays creating free radicals, increasing the tumor dose several fold. Radiotherapy of mice with orthotopic human gliomas and human triple negative breast cancers growing in the brain showed significant life extensions when the nanoparticles were included. An extensive study of the properties of the iodine-containing nanoparticle (Niodx) by the Nanotechnology Characterization Laboratory, including sterility, physicochemical characterization, in vitro cytotoxicity, in vivo immunological characterization, and in vivo toxicology, is presented. In summary, the iodine nanoparticle Niodx appears safe and effective for translational studies toward human use.

Accelerated brachytherapy with the Xoft electronic source used in association with iodine, gold, bismuth, gadolinium, and hafnium nano-radioenhancers.

PURPOSE: The current study was designed to calculate the dose enhancement factor (DEF) of iodine (I), gold (Au), bismuth (Bi), gadolinium (Gd), and hafnium (Hf) nanoparticles (NP)s by Monte Carlo (MC) modeling of an electronic brachytherapy source in resection cavities of breast tumors. METHODS AND MATERIALS: The GEANT4 MC code was used for simulation of a phantom containing a water-filled balloon and a Xoft source (50 kVp) to irradiate the margins of a resected breast tumor. NPs with a diameter of 20 nm and concentrations from 1 to 5% w/w were simulated in a tumor margin with 5 mm thickness as well as a hypothetical breast model consisting of spherical island-like residual tumor-remnants. The DEFs for all NPs were calculated in both models. RESULTS: In the margin-loaded model, for the concentration of 1% w/w heavy atom, DEFs of 2.5, 2.3, 2.1, 2, and 1.7 were calculated for Bi, Au, I, Hf, and Gd NPs (descending order), which increased, almost linearly with concentration for all NPs. Moreover, normal tissue dose behind the NP-loaded margin declined significantly depending on NP type and concentration. When modeling residual tumor islands, DEF values were very close to the margin-loaded values except for Bi and I, where DEFs of 2.55 and 1.7 were seen, respectively. CONCLUSIONS: Considerable dose enhancements were obtained for the heavy atom NPs studied in the partial breast brachytherapy with a Xoft electronic source. In addition, normal tissue doses were lowered in the points beyond the NP-loaded margin. The findings revealed promising outcomes and the probability of improved tumor control for NP-aided brachytherapy with the Xoft electronic source.

Novel Iodine nanoparticles target vascular mimicry in intracerebral triple negative human MDA-MB-231 breast tumors.

Triple negative breast cancer (TNBC), ~ 10-20% of diagnosed breast cancers, metastasizes to brain, lungs, liver. Iodine nanoparticle (INP) radioenhancers specifically localize to human TNBC MDA-MB-231 tumors growing in mouse brains after iv injection, significantly extending survival of mice after radiation therapy (RT). A prominent rim of INP contrast (MicroCT) previously seen in subcutaneous tumors but not intracerebral gliomas, provide calculated X-ray dose-enhancements up to > eightfold. Here, MDA-MB-231-cells, INPs, CD31 were examined by fluorescence confocal microscopy. Most INP staining co-localized with CD31 in the tumor center and periphery. Greatest INP/CD31 staining was in the tumor periphery, the region of increased MicroCT contrast. Tumor cells are seen to line irregularly-shaped spaces (ISS) with INP, CD31 staining very close to or on the tumor cell surface and PAS stain on their boundary and may represent a unique form of CD31-expressing vascular mimicry in intracerebral 231-tumors. INP/CD31 co-staining is also seen around ISS formed around tumor cells migrating on CD31+ blood-vessels. The significant radiation dose enhancement to the prolific collagen I containing, INP-binding ISS found throughout the tumor but concentrated in the tumor rim, may contribute significantly to the life extensions observed after INP-RT; VM could represent a new drug/NP, particularly INP, tumor-homing target.

Iodine nanoparticle radiotherapy of human breast cancer growing in the brains of athymic mice.

About 30% of breast cancers metastasize to the brain; those widely disseminated are fatal typically in 3-4 months, even with the best available treatments, including surgery, drugs, and radiotherapy. To address this dire situation, we have developed iodine nanoparticles (INPs) that target brain tumors after intravenous (IV) injection. The iodine then absorbs X-rays during radiotherapy (RT), creating free radicals and local tumor damage, effectively boosting the local RT dose at the tumor. Efficacy was tested using the very aggressive human triple negative breast cancer (TNBC, MDA-MB-231 cells) growing in the brains of athymic nude mice. With a well-tolerated non-toxic IV dose of the INPs (7 g iodine/kg body weight), tumors showed a heavily iodinated rim surrounding the tumor having an average uptake of 2.9% iodine by weight, with uptake peaks at 4.5%. This is calculated to provide a dose enhancement factor of approximately 5.5 (peaks at 8.0), the highest ever reported for any radiation-enhancing agents. With RT alone (15 Gy, single dose), all animals died by 72 days; INP pretreatment resulted in longer-term remissions with 40% of mice surviving 150 days and 30% surviving > 280 days.

Distributions of intravenous injected iodine nanoparticles in orthotopic u87 human glioma xenografts over time and tumor therapy.

Aim: To analyze the localization, distribution and effect of iodine nanoparticles (INPs) on radiation therapy (RT) in advanced intracerebral gliomas over time after intravenous injection. Materials & methods: Luciferase/td-tomato expressing U87 human glioma cells were implanted into mice which were injected intravenously with INPs. Mice with gliomas were followed for tumor progression and survival. Immune-stained mouse brain sections were examined and quantified by confocal fluorescence microscopy. Results: INPs injected intravenously 3 days prior to RT, compared with 1 day, showed greater association with CD31-staining structures, accumulated inside tumor cells more, covered more of the tumor cell surface and trended toward increased median survival. Conclusion: INP persistence and redistribution in tumors over time may enable greater RT enhancement and clinically relevant hypo-fractionated-RT and may enhance INP efficacy.

Roadmap for metal nanoparticles in radiation therapy: current status, translational challenges, and future directions.

This roadmap outlines the potential roles of metallic nanoparticles (MNPs) in the field of radiation therapy. MNPs made up of a wide range of materials (from Titanium, Z = 22, to Bismuth, Z = 83) and a similarly wide spectrum of potential clinical applications, including diagnostic, therapeutic (radiation dose enhancers, hyperthermia inducers, drug delivery vehicles, vaccine adjuvants, photosensitizers, enhancers of immunotherapy) and theranostic (combining both diagnostic and therapeutic), are being fabricated and evaluated. This roadmap covers contributions from experts in these topics summarizing their view of the current status and challenges, as well as expected advancements in technology to address these challenges.

Iodine nanoparticles enhance radiotherapy of intracerebral human glioma in mice and increase efficacy of chemotherapy.

Gliomas and other brain tumors have evaded durable therapies, ultimately causing about 20% of all cancer deaths. Tumors are widespread in the brain at time of diagnosis, limiting surgery and radiotherapy effectiveness. Drugs are also poorly effective. Radiotherapy (RT) is limited by dose to normal tissue. However, high-atomic-number elements absorb X-rays and deposit the absorbed dose locally, even doubling (or more) the local dose. Previously we showed that gold nanoparticles (AuNPs) with RT could eradicate some brain tumors in mice and many other preclinical studies confirmed AuNPs as outstanding radioenhancers. However, impediments to clinical translation of AuNPs have been poor clearance, skin discoloration, and cost. We therefore developed iodine nanoparticles (INPs) that are almost colorless, non-toxic, lower cost, and have reasonable clearance, thus overcoming major drawbacks of AuNPs. Here we report the use of iodine nanoparticle radiotherapy (INRT) in treating advanced human gliomas (U87) grown orthotopically in nude mice resulting in a more than a doubling of median life extension compared to RT alone. Significantly, INRT also enhanced the efficacy of chemotherapy when it was combined with the chemotherapeutic agent Doxil, resulting in some longer-term survivors. While ongoing optimization studies should further improve INRT, clinical translation appears promising.

Three-dimensional structure of human chromatin accessibility complex hCHRAC by electron microscopy.

ATP-dependent chromatin remodeling complexes modulate the dynamic assembly and remodeling of chromatin involved in DNA transcription, replication, and repair. There is little structural detail known about these important multiple-subunit enzymes that catalyze chromatin remodeling processes. Here we report a three-dimensional structure of the human chromatin accessibility complex, hCHRAC, using single particle reconstruction by negative stain electron microscopy. This structure shows an asymmetric 15x10x12nm disk shape with several lobes protruding out of its surfaces. Based on the factors of larger contact area, smaller steric hindrance, and direct involvement of hCHRAC in interactions with the nucleosome, we propose that four lobes on one side form a multiple-site contact surface 10nm in diameter for nucleosome binding. This work provides the first determination of the three-dimensional structure of the ISWI-family of chromatin remodeling complexes.

Gold nanoparticle size controlled by polymeric Au(I) thiolate precursor size.

We developed a method in preparing size-controllable gold nanoparticles (Au NPs, 2-6 nm) capped with glutathione by varying the pH (between 5.5 and 8.0) of the solution before reduction. This method is based on the formation of polymeric nanoparticle precursors, Au(I)-glutathione polymers, which change size and density depending on the pH. Dynamic light scattering, size exclusion chromatography, and UV-vis spectroscopy results suggest that lower pH values favor larger and denser polymeric precursors and higher pH values favor smaller and less dense precursors. Consequently, the larger precursors led to the formation of larger Au NPs, whereas smaller precursors led to the formation of smaller Au NPs. Using this strategy, Au NPs functionalized with nickel(II) nitriloacetate (Ni-NTA) group were prepared by a mixed-ligand approach. These Ni-NTA functionalized Au NPs exhibited specific binding to 6x-histidine-tagged Adenovirus serotype 12 knob proteins, demonstrating their utility in biomolecular labeling applications.

Radiotherapy enhancement with gold nanoparticles.

Gold is an excellent absorber of X-rays. If tumours could be loaded with gold, this would lead to a higher dose to the cancerous tissue compared with the dose received by normal tissue during a radiotherapy treatment. Calculations indicate that this dose enhancement can be significant, even 200% or greater. In this paper, the physical and biological parameters affecting this enhancement are discussed. Gold nanoparticles have shown therapeutic efficacy in animal trials and these results are reviewed. Some 86% long-term (>1 year) cures of EMT-6 mouse mammary subcutaneous tumours was achieved with an intravenous injection of gold nanoparticles before irradiation with 250-kVp photons, whereas only 20% were cured with radiation alone. The clinical potential of this approach is also discussed.

Small, Long Blood Half-Life Iodine Nanoparticle for Vascular and Tumor Imaging.

Standard clinical X-ray contrast agents are small iodine-containing molecules that are rapidly cleared by the kidneys and provide robust imaging for only a few seconds, thereby limiting more extensive vascular and tissue biodistribution imaging as well as optimal tumor uptake. They are also not generally useful for preclinical microCT imaging where longer scan times are required for high resolution image acquisition. We here describe a new iodine nanoparticle contrast agent that has a unique combination of properties: 20 nm hydrodynamic diameter, covalent PEG coating, 40 hour blood half-life, 50% liver clearance after six months, accumulation in tumors, and well-tolerated to at least 4 g iodine/kg body weight after intravenous administration in mice. These characteristics are unique among the other iodine nanoparticles that have been previously reported and provide extended-time high contrast vascular imaging and tumor loading. As such, it is useful for preclinical MicroCT animal studies. Potential human applications might include X-ray radiation dose enhancement for cancer therapy and vascular imaging for life-threatening situations where high levels of contrast are needed for extended periods of time.

Intravenously-injected gold nanoparticles (AuNPs) access intracerebral F98 rat gliomas better than AuNPs infused directly into the tumor site by convection enhanced delivery.

BACKGROUND: Intravenously (IV)-injected gold nanoparticles (AuNPs) powerfully enhance the efficacy of X-ray therapy of tumors including advanced gliomas. However, pharmacokinetic issues, such as slow tissue clearance and skin discoloration, may impede clinical translation. The direct infusion of AuNPs into the tumor might be an alternative mode of delivery. MATERIALS AND METHODS: Using the advanced, invasive, and difficult-to-treat F98 rat glioma model, we have studied the biodistribution of the AuNPs in the tumor and surrounding brain after either IV injection or direct intratumoral infusion by convection-enhanced delivery using light microscopy immunofluorescence and direct gold visualization. RESULTS: IV-injected AuNPs localize more specifically to intracerebral tumor cells, both in the main tumor mass and in the migrated tumor cells as well as the tumor edema, than do the directly infused AuNPs. Although some of the directly infused AuNPs do access the main tumor region, such access is largely restricted. CONCLUSION: These data suggest that IV-injected AuNPs are likely to have a greater therapeutic benefit when combined with radiation therapy than after the direct infusion of AuNPs.

Metallographic in situ hybridization.

Metallographic methods, in which a target is visualized using a probe or antibody that deposits metal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH) detection as well as for other labeling and detection methods. Autometallographically enhanced gold labeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens. Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directly from solution, has been used to develop bright-field ISH methods for HER2 gene determination in breast cancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both for visualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies to allow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics. An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staining instruments. Metallographic staining also provides excellent results for immunohistochemistry and may be combined with other staining procedures for the simultaneous detection of more than one gene or combinations of genes and proteins.

Biodistribution of gold nanoparticles in BBN-induced muscle-invasive bladder cancer in mice.

Bladder-sparing options are being developed for muscle-invasive bladder cancer in place of radical cystectomy, including the combination of chemotherapy and radiation therapy. We reasoned that improving the radiotherapy component of chemoradiation could improve the control of locally advanced disease. Previously, we showed that gold nanoparticles (AuNPs) are potent enhancers of radiation therapy. We hypothesized that if AuNPs were to preferentially localize to bladder tumors, they may be used to enhance the radiation component of muscle-invasive bladder tumor therapy. Mice were treated with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 17, 20, and 22 weeks - long enough to induce muscle-invasive tumors. Mice were then anesthetized and injected intravenously with 1.9 nm AuNPs of which most were rapidly cleared from the blood and excreted after a 30-50 minute residence time in the bladder. We found AuNPs distributed throughout the bladder wall, but most of the AuNPs were associated with the stroma surrounding the tumor cells or extracellular keratin produced by the tumor cells. There were relatively few AuNPs in the tumor cells themselves. The AuNPs therefore localized to tumor-associated stroma and this tumor specificity might be useful for specific X-ray dose enhancement therapy of muscle-invasive bladder carcinomas.

High-resolution immunophenotyping of subcellular compartments in tissue microarrays by enzyme metallography.

Ultrasensitive bright field in situ hybridization assays using enzyme metallography (EnzMet) have been developed and validated, but little is known regarding the applicability of EnzMet for immunophenotypic detection of protein via IHC. Superior resolution via discrete metallographic deposits offers the potential for enhancing high-resolution immunophenotyping. Using high-complexity tissue microarrays (TMAs), 88 common solid tumors were evaluated by automated EnzMet (Nanoprobes and Ventana). Targets were chosen to assess the ability of EnzMet to specifically localize encoded antigens in the nucleus (estrogen receptor), cytoplasm (cytokeratins), and cytoplasmic membrane (HER2) in TMAs. Results were compared with conventional IHC diaminobenzidine (DAB) immunostaining. There was full concordance between the EnzMet and conventional IHC results. Furthermore, the EnzMet reaction products did not appreciably diffuse, were dense and sharply defined, and provided excellent high-resolution differentiation of cellular compartments in paraffin sections for the nuclear, cytoplasmic, and cell membrane-localized antigens evaluated. The higher density of elemental silver deposited during enzyme metallography permitted evaluation of core immunophenotypes at a relatively low magnification, allowing more tissue to be screened in an efficient manner. This preliminary study shows the utility of using enzyme metallography for high-resolution immunophenotyping in TMAs.

FluoroNanogold: an important probe for correlative microscopy.

Correlative microscopy is a powerful imaging approach that refers to observing the same exact structures within a specimen by two or more imaging modalities. In biological samples, this typically means examining the same sub-cellular feature with different imaging methods. Correlative microscopy is not restricted to the domains of fluorescence microscopy and electron microscopy; however, currently, most correlative microscopy studies combine these two methods, and in this review, we will focus on the use of fluorescence and electron microscopy. Successful correlative fluorescence and electron microscopy requires probes, or reporter systems, from which useful information can be obtained with each of the imaging modalities employed. The bi-functional immunolabeling reagent, FluoroNanogold, is one such probe that provides robust signals in both fluorescence and electron microscopy. It consists of a gold cluster compound that is visualized by electron microscopy and a covalently attached fluorophore that is visualized by fluorescence microscopy. FluoroNanogold has been an extremely useful labeling reagent in correlative microscopy studies. In this report, we present an overview of research using this unique probe.

Fibrinogen assembly and crosslinking on a fibrin fragment E template.

There is an ongoing controversy concerning whether crosslinked gamma chains in fibrin are oriented "transversely" between fibril strands or "end-to-end" along fibril strands. From the latter viewpoint, Veklich et al. [Proc Natl Acad Sci (USA) 95: 1438, 1998] observed that fibrinogen fibrils that had been assembled on a fibrin fragment E template, cross-linked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end. This led to the conclusion that crosslinked gamma chains in fibrin under physiological conditions were also aligned end-to-end. To assess its validity we studied the assembly and organization of fibrinogen molecules on a des AB-fibrin fragment E (E-des AB) or a des A-fibrin fragment E (E-des A) template. We evaluated the roles of E polymerization sites E(A) and E(B), and D association sites gammaXL, Da, Db, betaC and alphaC in this process. E(A):Da interactions caused fibrinogen: E "DED" complexes to form, and markedly enhanced the gamma chain crosslinking rates of fibrinogen or des alphaC-fibrinogen. Fibrinogen crosslinking without added fibrin E was slower, and that of des alphaC-fibrinogen was still slower. These events showed that although alphaC domains promote fibrinogen fibril assembly and crosslinking, they contribute little to increasing the E(A):Da-dependent crosslinking rate. Electron microscopic (STEM) images of E-des AB and fibrinogen plus factor XIIIa showed single-, double-, and multistranded fibrils with interstrand DED complexes aligned side-to-side. This alignment was due to betaC:betaC contacts resulting from D subdomain rearrangements initiated by the E(B):Db interactions, and also occurred in mixtures of des alphaC-fibrinogen with E-des AB. In contrast, a mixture of fibrinogen and E-des A plus XIIIa revealed double-stranded fibrils with interstrand DED complexes in a half-staggered arrangement, an alignment that we attribute to crosslinking of gammaXL sites bridging between fibrils strands. These and other features of E-des A-based fibrinogen fibrils, including interstrand gamma chain bridges and early and extensive lateral fibril strand associations concomitant with accelerated gamma chain crosslinking, indicate that crosslinking of fibrin fibril strands takes place preferentially on transversely positioned gamma chains.

Murine EMT-6 carcinoma: high therapeutic efficacy of microbeam radiation therapy.

Microbeam radiation therapy is an experimental modality using parallel arrays of thin (<100 micro m) slices of synchrotron-generated X rays (microplanar beams, microbeams). We used EMT-6 murine mammary carcinoma subcutaneously inoculated in the hind legs of mice to compare the therapeutic efficacies of single-fraction, unidirectional (1) "co-planar" microbeams (an array of vertically oriented microplanar beams), (2) "cross-planar" microbeams (two arrays of parallel microbeams propagated in the same direction, one with vertically and the other with horizontally oriented microplanar beams), and (3) seamless (broad) beams from the same synchrotron source. The microbeams were 90 micro m wide and were spaced 300 micro m on center; the median energy in all beams was 100 or 118 keV. Tumor ablation rates were 4/8, 4/8 and 6/7 for a 410-, 520- and 650-Gy in-slice cross-planar microbeam dose, respectively, and 1/8, 3/8, 3/7 and 6/8 for a 23-, 30-, 38- and 45-Gy broad-beam dose, respectively. When the data were pooled from the three highest doses (same average tumor ablations of 50-60%), the incidences of normal-tissue acute toxicity (moist desquamation and epilation) and delayed toxicity (failure of hair regrowth) were significantly lower for cross-planar microbeams than broad beams (P < 0.025). Furthermore, for the highest doses in these two groups, which also had the same tumor ablation rate (>75%), not only were the above toxicities lower for the cross-planar microbeams than for the broad beams (P < 0.02), but severe leg dysfunction was also lower (P < 0.003). These findings suggest that single-fraction microbeams can ablate tumors at high rates with relatively little normal-tissue toxicity.