Anesthetic Oxygen Use and Sex Are Critical Factors in the FLASH Sparing Effect.

Purpose: Ultra High Dose-Rate (UHDR) radiation has been reported to spare normal tissue, compared with Conventional Dose-Rate (CDR) radiation. However, important work remains to be done to improve the reproducibility of the FLASH effect. A better understanding of the biologic factors that modulate the FLASH effect may shed light on the mechanism of FLASH sparing. Here, we evaluated whether sex and/or the use of 100% oxygen as a carrier gas during irradiation contribute to the variability of the FLASH effect. Methods and Materials: C57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane mixed with either room air or 100% oxygen. Subsequently, the mice received 27 Gy of either 9 MeV electron UHDR or CDR to a 1.6 cm2 diameter area of the right leg skin using the Mobetron linear accelerator. The primary postradiation endpoint was time to full thickness skin ulceration. In a separate cohort of mice (4 male, 4 female), skin oxygenation was measured using PdG4 Oxyphor under identical anesthesia conditions. Results: Neither supplemental oxygen nor sex affected time to ulceration in CDR irradiated mice. In the UHDR group, skin damage occured earlier in male and female mice that received 100% oxygen compared room air and female mice ulcerated sooner than male mice. However, there was no significant difference in time to ulceration between male and female UHDR mice that received room air. Oxygen measurements showed that tissue oxygenation was significantly higher when using 100% oxygen as the anesthesia carrier gas than when using room air, and female mice showed higher levels of tissue oxygenation than male mice under 100% oxygen. Conclusions: The skin FLASH sparing effect is significantly reduced when using oxygen during anesthesia rather than room air. FLASH sparing was also reduced in female mice compared to male mice. Both tissue oxygenation and sex are likely sources of variability in UHDR studies. These results suggest an oxygen-based mechanism for FLASH, as well as a key role for sex in the FLASH skin sparing effect.

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.

Anesthetic oxygen use and sex are critical factors in the FLASH sparing effect.

Introduction: Ultra-high dose-rate (UHDR) radiation has been reported to spare normal tissue compared to conventional dose-rate (CDR) radiation. However, reproducibility of the FLASH effect remains challenging due to varying dose ranges, radiation beam structure, and in-vivo endpoints. A better understanding of these inconsistencies may shed light on the mechanism of FLASH sparing. Here, we evaluate whether sex and/or use of 100% oxygen as carrier gas during irradiation contribute to the variability of the FLASH effect. Methods: C57BL/6 mice (24 male, 24 female) were anesthetized using isoflurane mixed with either room air or 100% oxygen. Subsequently, the mice received 27 Gy of either 9 MeV electron UHDR or CDR to a 1.6 cm2 diameter area of the right leg skin using the Mobetron linear accelerator. The primary post-radiation endpoint was time to full thickness skin ulceration. In a separate cohort of mice (4 male, 4 female) skin oxygenation was measured using PdG4 Oxyphor under identical anesthesia conditions. Results: In the UHDR group, time to ulceration was significantly shorter in mice that received 100% oxygen compared to room air, and amongst them female mice ulcerated sooner compared to males. However, no significant difference was observed between male and female UHDR mice that received room air. Oxygen measurements showed significantly higher tissue oxygenation using 100% oxygen as the anesthesia carrier gas compared to room air, and female mice showed higher levels of tissue oxygenation compared to males under 100% oxygen. Conclusion: The FLASH sparing effect is significantly reduced using oxygen during anesthesia compared to room air. The FLASH sparing was significantly lower in female mice compared to males. Both tissue oxygenation and sex are likely sources of variability in UHDR studies. These results suggest an oxygen-based mechanism for FLASH, as well as a key role for sex in the FLASH skin sparing effect.

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.