Enhancement of Radiation Therapy through Blockade of the Immune Checkpoint, V-domain Ig Suppressor of T Cell Activation (VISTA), in Melanoma and Adenocarcinoma Murine Models.

Radiation therapy (RT) has recently demonstrated promise at stimulating an enhanced immune response. The recent success of immunotherapies, such as checkpoint inhibitors, CART cells, and other immune modulators, affords new opportunities for combination with radiation. The aim of this study is to evaluate whether and to what extent blockade of VISTA, an immune checkpoint, can potentiate the tumor control ability of radiation therapy. Our study is novel in that it is the first comparison of two VISTA-blocking methods (antibody inhibition and genetic knockout) in combination with RT. VISTA was blocked either through genetic knockout (KO) or an inhibitory antibody and combined with RT in two syngeneic murine flank tumor models (B16 and MC38). Selected mRNA, immune cell infiltration, and tumor growth delay were used to assess the biological effects. When combined with a single 15Gy radiation dose, VISTA blockade via genetic knockout in the B16 model and via anti-VISTA antibodies in the MC38 model significantly improved survival compared to RT alone by an average of 5.5 days and 6.3 days, respectively (p < 0.05). The gene expression data suggest that the mechanism behind the enhanced tumor control is primarily a result of increased apoptosis and immune-mediated cytotoxicity. VISTA blockade significantly enhances the anti-tumor effect of a single dose of 15Gy radiation through increased expression and stimulation of cell-mediated apoptosis pathways. These results suggest that VISTA is a biologically relevant immune promoter that has the potential to enhance the efficacy of a large single radiation dose in a synergic manner.

mNP hyperthermia and hypofractionated radiation activate similar immunogenetic and cytotoxic pathways.

OBJECTIVE: The goal of this study is to better understand the immunogenetic expression and related cytotoxic responses of moderate but clinically relevant doses of hypofractionated radiation (1x15 Gy and 3x8 Gy) and magnetic nanoparticle hyperthermia (mNPH, CEM43 30). METHODS: Genetic, protein, immunopathology and tumor growth delay assessments were used to determine the immune and cytotoxic responses following radiation and mNPH alone and in combination. Although the thermal dose used, 43 C°/30 min (CEM43 30), typically results in modest independent cytotoxicity, it has shown the ability to stimulate an immune response and enhance other cancer treatments. The radiation doses studied (15 Gy and 3x8 Gy) are commonly used in preclinical research and are effective in selected stereotactic and palliative treatment settings, however they are not commonly used as first-line primary tumor treatment regimens. RESULTS: Our RNA-based genetic results suggest that while many of the cytotoxic and immune gene and protein pathways for radiation and hyperthermia are similar, radiation, at the doses used, results in a more consistent and expansive anti-cancer immune/cytotoxic expression profile. These results were supported by immunohistochemistry based cytotoxic T-cell tumor infiltration and tumor growth delay studies. When used together radiation and hyperthermia led to greater immune and cytotoxic activity than either modality alone. CONCLUSION: This study clearly shows that modest, but commonly used hypofractionated radiation and hyperthermia doses share many important immune and cytotoxic pathways and that combining the treatments, as compared to either treatment alone, results in genetic and biological anti-cancer benefits.

Immunogenetic effects of low dose (CEM43 30) magnetic nanoparticle hyperthermia and radiation in melanoma cells.

Objective: In this in vitro study we have used an RNA quantification technique, nanoString, and a conventional protein analysis technique (Western Blot) to assess the genetic and protein expression of B16 murine melanoma cells following a modest magnetic nanoparticle hyperthermia (mNPH) dose equivalent to 30 minutes @ 43°C (CEM43 30) and/or a clinically relevant 8 Gy radiation dose.Methods: Melanoma cells with mNPs(2.5 µg Fe/106 cells) were pelleted and exposed to an alternating magnetic field (AMF) to generate the targeted thermal dose. Thermal dose was accurately monitored by a fiber optic probe and automatically maintained at CEM43 30. All cells were harvested 24 hours after treatment.Results: The mNPH dose demonstrated notable elevations in the thermotolerance/immunogenic HSP70 gene and a number of chemoattractant and toll-like receptor gene pathways. The 8 Gy dose also upregulated a number of important immune and cytotoxic genetic and protein pathways. However, the mNPH/radiation combination was the most effective stimulator of a wide variety of immune and cytotoxic genes including HSP70, cancer regulating chemokines CXCL10, CXCL11, the T-cell trafficking chemokine CXCR3, innate immune activators TLR3, TLR4, the MDM2 and mTOR negative regulator of p53, the pro-apoptotic protein PUMA, and the cell death receptor Fas. Importantly a number of the genetic changes were accurately validated by protein expression changes, i.e., HSP70, p-mTOR, p-MDM2.Conclusion: These results not only show that low dose mNPH and radiation independently increase the expression of important immune and cytotoxic genes but that the effect is greatly enhanced when they are used in combination.

Porcine-human glioma xenograft model. Immunosuppression and model reproducibility.

BACKGROUND: Glioblastoma is the most common primary malignant and treatment-resistant human brain tumor. Rodent models have played an important role in understanding brain cancer biology and treatment. However, due to their small cranium and tumor volume mismatch, relative to human disease, they have been less useful for translational studies. Therefore, development of a consistent and simple large animal glioma xenograft model would have significant translational benefits. METHODS: Immunosuppression was induced in twelve standard Yucatan minipigs. 3 pigs received cyclosporine only, while 9 pigs received a combined regimen including cyclosporine (55 mg/kg q12 h), prednisone (25 mg, q24 h) and mycophenolate (500 mg q24 h). U87 cells (2 × 106) were stereotactically implanted into the left frontal cortex. The implanted brains were imaged by MRI for monitoring. In a separate study, tumors were grown in 5 additional pigs using the combined regimen, and pigs underwent tumor resection with intra-operative image updating to determine if the xenograft model could accurately capture the spatial tumor resection challenges seen in humans. RESULTS: Tumors were successfully implanted and grown in 11 pigs. One animal in cyclosporine only group failed to show clinical tumor growth. Clinical tumor growth, assessed by MRI, progressed slowly over the first 10 days, then rapidly over the next 10 days. The average tumor growth latency period was 20 days. Animals were monitored twice daily and detailed records were kept throughout the experimental period. Pigs were sacrificed humanely when the tumor reached 1 - 2 cm. Some pigs experienced decreased appetite and activity, however none required premature euthanasia. In the image updating study, all five pigs demonstrated brain shift after craniotomy, consistent with what is observed in humans. Intraoperative image updating was able to accurately capture and correct for this shift in all five pigs. CONCLUSION: This report demonstrates the development and use of a human intracranial glioma model in an immunosuppressed, but nongenetically modified pig. While the immunosuppression of the model may limit its utility in certain studies, the model does overcome several limitations of small animal or genetically modified models. For instance, we demonstrate use of this model for guiding surgical resection with intraoperative image-updating technologies. We further report use of a surrogate extracranial tumor that indicates growth of the intracranial tumor, allowing for relative growth assessment without radiological imaging.

Comparison of Tumor Control and Skin Damage in a Mouse Model after Ultra-High Dose Rate Irradiation and Conventional Irradiation.

Recent studies suggest ultra-high dose rate radiation treatment (UHDR-RT) reduces normal tissue damage compared to conventional radiation treatment (CONV-RT) at the same dose. In this study, we compared first, the kinetics and degree of skin damage in wild-type C57BL/6 mice, and second, tumor treatment efficacy in GL261 and B16F10 dermal tumor models, at the same UHDR-RT and CONV-RT doses. Flank skin of wild-type mice received UHDR-RT or CONV-RT at 25 Gy and 30 Gy. Normal skin damage was tracked by clinical observation to determine the time to moist desquamation, an endpoint which was verified by histopathology. Tumors were inoculated on the right flank of the mice, then received UHDR-RT or CONV-RT at 1 × 11 Gy, 1 × 15, 1 × 25, 3 × 6 and 3 × 8 Gy, and time to tumor tripling volume was determined. Tumors also received 1 × 11, 1 × 15, 3 × 6 and 3 × 8 Gy doses for assessment of CD8+/CD4+ tumor infiltrate and genetic expression 96 h postirradiation. All irradiations of the mouse tumor or flank skin were performed with megavoltage electron beams (10 MeV, 270 Gy/s for UHDR-RT and 9 MeV, 0.12 Gy/s for CONV-RT) delivered via a clinical linear accelerator. Tumor control was statistically equal for similar doses of UHDR-RT and CONV-RT in B16F10 and GL261 murine tumors. There were variable qualitative differences in genetic expression of immune and cell damage-associated pathways between UHDR and CONV irradiated B16F10 tumors. Compared to CONV-RT, UHDR-RT resulted in an increased latent period to skin desquamation after a single 25 Gy dose (7 days longer). Time to moist skin desquamation did not significantly differ between UHDR-RT and CONV-RT after a 30 Gy dose. The histomorphological characteristics of skin damage were similar for UHDR-RT and CONV-RT. These studies demonstrated similar tumor control responses for equivalent single and fractionated radiation doses, with variable difference in expression of tumor progression and immune related gene pathways. There was a modest UHDR-RT skin sparing effect after a 1 × 25 Gy dose but not after a 1 × 30 Gy dose.

In Vitro and In Vivo Delivery of Magnetic Nanoparticle Hyperthermia using a Custom-Built Delivery System.

Hyperthermia has long been used in the treatment of cancer. Techniques have varied from the intra-tumoral insertion of hot iron rods, to systemically delivered tumor antibody-targeted magnetic nanoparticles, at temperatures from 39 ˚C (fever-level) to 1,000 ˚C (electrocautery) and treatment times from seconds to hours. The temperature-time relationship (thermal dose) dictates the effect with high thermal doses resulting in the tissue ablation and lower thermal doses resulting in sublethal effects such as increased blood flow, accumulation of drugs and immune stimulation. One of the most promising current medical therapies is magnetic nanoparticle hyperthermia  (mNPH). This technique involves activating magnetic nanoparticles, that can be delivered systemically or intratumorally, with a non-invasive, non-toxic alternating magnetic field. The size, construct and association of the magnetic nanoparticles and the frequency and field strength of the magnetic field are major heating determinants. We have developed sophisticated instrumentation and techniques for delivering reproducible magnetic nanoparticle hyperthermia in large and small animal models and cultured cells. This approach, using continuous, real time temperature monitoring in multiple locations, allows for the delivery of well-defined thermal doses to the target tissue (tumor) or cells while limiting non-target tissue heating. Precise control and monitoring of temperature, in multiple sites, and use of the industry standard algorithm (cumulative equivalent minutes at 43 ˚C /CEM43), allows for an accurate determination and quantification of thermal dose. Our system, which allows for a wide variety of temperatures, thermal doses, and biological effects, was developed through a combination of commercial acquisitions and inhouse engineering and biology developments. This system has been optimized in a manner that allows for the rapid conversion between ex vivo, in vitro, and in vivo techniques. The goal of this protocol is to demonstrate how to design, develop and implement an effective technique and system for delivering reproducible and accurate magnetic nanoparticle therapy (mNP) hyperthermia.

Cowpea Mosaic Virus Nanoparticle Enhancement of Hypofractionated Radiation in a B16 Murine Melanoma Model.

INTRODUCTION: Virus and virus-like nanoparticles (VNPs) have been used for a variety of preclinical treatments, including in situ anti-cancer vaccination. The Cowpea mosaic virus (CPMV) is a VNP that has shown the ability to stimulate an anti-cancer immune response. The hypothesis of this study is two-fold: that intratumoral CPMV enhances the immunogenetic and cytotoxic response of hypofractionated radiation (15 Gy or 3 x 8 Gy), and that the effect differs between fraction regimens in the murine B16 flank melanoma model. METHODS: CPMV nanoparticles were delivered intratumorally, 100 µg/tumor to B16 murine melanoma flank tumors alone, and in combination with either 15 Gy or 3 x 8 Gy (3 consecutive days). Tumors were assessed for immune and cytotoxic gene and protein expression, and cytotoxic T cell infiltration 4 days post treatment. Treatment based tumor control was assessed by a 3-fold tumor growth assay. RESULTS: Both CPMV and radiation alone demonstrated the activation of a number of important immune and cytotoxic genes including natural killer cell and T cell mediated cytotoxicity pathways. However, the combination treatment activated greater expression than either treatment alone. CPMV combined with a single dose of 15 Gy demonstrated greater immune and cytotoxic gene expression, protein expression, CD8+ T cell infiltration activity, and greater tumor growth delay compared to 3 x 8 Gy with CPMV. CONCLUSION: CPMV presents a unique and promising hypofractionated radiation adjuvant that leads to increased anti-tumor cytotoxic and immune signaling, especially with respect to the immune mediated cytotoxicity, immune signaling, and toll-like receptor signaling pathways. This improvement was greater with a single dose than with a fractionated dose.