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.

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.

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.

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.

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.

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.

Correction: Dependence of gold nanoparticle radiosensitization on cell geometry.

Correction for 'Dependence of gold nanoparticle radiosensitization on cell geometry' by Wonmo Sung, et al., Nanoscale, 2017, 9, 5843-5853.

Dependence of gold nanoparticle radiosensitization on cell geometry.

The radiosensitization effect of gold nanoparticles (GNPs) has been demonstrated both in vitro and in vivo in radiation therapy. The purpose of this study was to systematically assess the biological effectiveness of GNPs distributed in the extracellular media for realistic cell geometries. TOPAS-nBio simulations were used to determine the nanometre-scale radial dose distributions around the GNPs, which were subsequently used to predict the radiation dose response of cells surrounded by GNPs. MDA-MB-231 human breast cancer cells and F-98 rat glioma cells were used as models to assess different cell geometries by changing (1) the cell shape, (2) the nucleus location within the cell, (3) the size of GNPs, and (4) the photon energy. The results show that the sensitivity enhancement ratio (SER) was increased up to a factor of 1.2 when the location of the nucleus is close to the cell membrane for elliptical-shaped cells. Heat-maps of damage-likelihoods show that most of the lethal events occur in the regions of the nuclei closest to the membrane, potentially causing highly clustered damage patterns. The effect of the GNP size on radiosensitization was limited when the GNPs were located outside the cell. The improved modelling of the cell geometry was shown to be crucial because the dose enhancement caused by GNPs falls off rapidly with distance from the GNPs. We conclude that radiosensitization can be achieved for kV photons even without cellular uptake of GNPs when the nucleus is shifted towards the cell membrane. Furthermore, damage was found to concentrate in a small region of the nucleus in close proximity to the extracellular, GNP-laden region.

Increasing radiation dose improves immunotherapy outcome and prolongation of tumor dormancy in a subgroup of mice treated for advanced intracerebral melanoma.

Previously, we developed a clinically relevant therapy model for advanced intracerebral B16 melanomas in syngeneic mice combining radiation and immunotherapies. Here, 7 days after B16-F10-luc2 melanoma cells were implanted intracerebrally (D7), syngeneic mice with bioluminescent tumors that had formed (1E10(5) to 7E10(6) photons per minute (>1E10(6), large; <1E10(6), small) were segregated into large-/small-balanced subgroups. Then, mice received either radiation therapy alone (RT) or radiation therapy plus immunotherapy (RT plus IT) (single injection of mAbPC61 to deplete regulatory T cells followed by multiple injections of irradiated granulocyte macrophage colony stimulating factor transfected B16-F10 cells) (RT plus IT). Radiation dose was varied (15, 18.75 or 22.5 Gy, given on D8), while immunotherapy was provided similarly to all mice. The data support the hypothesis that increasing radiation dose improves the outcome of immunotherapy in a subgroup of mice. The tumors that were greatly delayed in beginning their progressive growth were bioluminescent in vivo-some for many months, indicating prolonged tumor "dormancy," in some cases presaging long-term cures. Mice bearing such tumors had far more likely received radiation plus immunotherapy, rather than RT alone. Radiotherapy is a very important adjunct to immunotherapy; the greater the tumor debulking by RT, the greater should be the benefit to tumor immunotherapy.

Cytomegalovirus-Based Vaccine Expressing a Modified Tumor Antigen Induces Potent Tumor-Specific CD8(+) T-cell Response and Protects Mice from Melanoma.

The presence of tumor-infiltrating CD8(+) T cells is associated with tumor regression and better prognosis. Cytomegalovirus (CMV) infection elicits a robust and long-lasting CD8(+) T-cell response, which makes CMV a potentially promising vaccine vector against cancer. In the current study, we used recombinant murine CMV (MCMV) strains as prophylactic and therapeutic vaccines in an aggressive B16 lung metastatic melanoma model. Immunization with MCMV-expressing ovalbumin (OVA) induced a potent OVA-specific CD8(+) T-cell response and was effective in protecting mice from OVA-expressing B16 melanoma in an antigen-dependent manner. We engineered MCMV to express a modified B16 melanoma antigen gp100 (MCMV-gp100KGP). Immunization with MCMV-gp100KGP was highly effective in overcoming immune tolerance to self-antigen and induced a strong, long-lasting gp100-specific CD8(+) T-cell response even in the presence of preexisting anti-CMV immunity. Furthermore, both prophylactic and therapeutic vaccinations of mice with MCMV-gp100KGP effectively protected mice from highly aggressive lung B16-F10 melanoma, and the protection was mediated by gp100-specific CD8(+) T cells. We showed that MCMV is a superior vaccine vector compared with a commonly used vesicular stomatitis virus vector. Collectively, our studies demonstrate that CMV is a promising vaccine vector to prevent and treat tumors.

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.

Infrared-transparent gold nanoparticles converted by tumors to infrared absorbers cure tumors in mice by photothermal therapy.

Gold nanoparticles (AuNPs) absorb light and can be used to heat and ablate tumors. The "tissue window" at ∼ 800 nm (near infrared, NIR) is optimal for best tissue penetration of light. Previously, large, 50-150 nm, gold nanoshells and nanorods that absorb well in the NIR have been used. Small AuNPs that may penetrate tumors better unfortunately barely absorb at 800 nm. We show that small AuNPs conjugated to anti-tumor antibodies are taken up by tumor cells that catalytically aggregate them (by enzyme degradation of antibodies and pH effects), shifting their absorption into the NIR region, thus amplifying their photonic absorption. The AuNPs are NIR transparent until they accumulate in tumor cells, thus reducing background heating in blood and non-targeted cells, increasing specificity, in contrast to constructs that are always NIR-absorptive. Treatment of human squamous cell carcinoma A431 which overexpresses epidermal growth factor receptor (EGFr) in subcutaneous murine xenografts with anti-EGFr antibodies conjugated to 15 nm AuNPs and NIR resulted in complete tumor ablation in most cases with virtually no normal tissue damage. The use of targeted small AuNPs therefore provides a potent new method of selective NIR tumor therapy.

Therapy model for advanced intracerebral B16 mouse melanoma using radiation therapy combined with immunotherapy.

A reproducible therapy model for advanced intracerebral B16 melanoma is reported. Implanted tumors (D0), suppressed by a single 15 Gy radiosurgical dose of 100 kVp X-rays (D8), were further suppressed by a single ip injection of a Treg-depleting mAb given 2 days prior to the initiation (D9) of four weekly then eight bi-monthly sc injections of GMCSF-transfected, mitotically disabled B16 cells. The trends of seven independent experiments were similar to the combined result: The median (days) [SD/total N] of survival went from 15[1.09/62] (no treatment control) to 35.8[8.8/58] (radiation therapy only) to 52.5[13.5/57] (radiation therapy plus immunotherapy). Within 2 weeks after immunization, tumors in mice receiving radiation therapy plus immunotherapy were significantly smaller than tumors in mice treated only with radiosurgery. Splenocytes and lymph node cells from immunized mice showed increased interferon γ production when cultured with syngeneic tumor cells. We suggest that our model will be useful for the development and testing of novel combination therapies for brain tumors.

Microlocalization of lipophilic porphyrins: non-toxic enhancers of boron neutron-capture therapy.

PURPOSE: To compare the macroscopic and microscopic distributions of the novel non-toxic lipophilic porphyrins copper (II) 5,10,15,20-tetrakis-(3-[1,2 dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin (CuTCPH), potentially useful for boron neutron-capture therapy (BNCT), with those of its zinc fluorescent congener zinc (II) 5,10,15,20-tetrakis-(3-[1,2 dicarba-closo-dodecaboranyl]methoxyphenyl)-porphyrin (ZnTCPH) in tissues of tumor-bearing mice. MATERIALS AND METHODS: ZnTCPH and CuTCPH were synthesized, then injected intraperitoneally (ip) into tumor-bearing mice. Macroscopic biodistribution was assessed by determining average boron concentrations in tumor, blood, brain, skin, and liver using atomic-emission spectrometry. The euthanized mice and their vital organs were photographed first under an ultraviolet lamp and then under a bright fluorescent lamp. Thin sections of liver and tumor were analyzed by confocal fluorescence microscopy (CFM). RESULTS: ZnTCPH-injected, but not CuTCPH-injected mice bearing subcutaneous tumors showed fluorescence from liver, spleen and tumors. Macrodistributions of boron in various tissues were similar in mice whether injected with ZnTCPH or CuTCPH. CFM of unfixed liver sections showed cytoplasmic fluorescence from Kupffer cells in a periportal lobular distribution evenly throughout the liver. In the tumors studied, such fluorescence was also cytoplasmic but unlike liver fluorescence, was macroscopically heterogeneous. CONCLUSION: ZnTCPH serves as a useful fluorescent experimental surrogate for CuTCPH to delineate its macroscopic and microscopic distributions in organs and tumors.

Gold nanoparticle imaging and radiotherapy of brain tumors in mice.

AIM: To test intravenously injected gold nanoparticles for x-ray imaging and radiotherapy enhancement of large, imminently lethal, intracerebral malignant gliomas. MATERIALS & METHODS: Gold nanoparticles approximately 11 nm in size were injected intravenously and brains imaged using microcomputed tomography. A total of 15 h after an intravenous dose of 4 g Au/kg was administered, brains were irradiated with 30 Gy 100 kVp x-rays. RESULTS: Gold uptake gave a 19:1 tumor to normal brain ratio with 1.5% w/w gold in tumor, calculated to increase local radiation dose by approximately 300%. Mice receiving gold and radiation (30 Gy) demonstrated 50% long term (>1 year) tumor-free survival, whereas all mice receiving radiation only died. CONCLUSION: Intravenously injected gold nanoparticles cross the blood-tumor barrier, but are largely blocked by the normal blood-brain barrier, enabling high-resolution computed tomography tumor imaging. Gold radiation enhancement significantly improved long-term survival compared with radiotherapy alone. This approach holds promise to improve therapy of human brain tumors and other cancers.