To identify key dosimetric parameters that have close associations with tumor treatment response and body weight change in SFRT treatments with a large range of spatial-fractionation scale at dose rates of several Gy/min. Methods Six study arms using uniform tumor radiation, half-tumor radiation, 2mm beam array radiation, 0.3mm minibeam radiation, and an untreated arm were used. All treatments were delivered on a 320kV x-ray irradiator. Forty-two female Fischer 344 rats with fibrosarcoma tumor allografts were used. Dosimetric parameters studied are peak dose and width, valley dose and width, peak-to-valley-dose-ratio (PVDR), volumetric average dose, percentage volume directly irradiated, and tumor-and normal-tissue EUD. Animal survival, tumor volume change, and body weight change (indicative of treatment toxicity) are tested for association with the dosimetric parameters using linear regression and Cox Proportional Hazards models. Results The dosimetric parameters most closely associated with tumor response are tumor EUD (R 2 = 0.7923, F-stat = 15.26*; z-test =-4.07***), valley (minimum) dose (R 2 = 0.7636, Fstat = 12.92*; z-test =-4.338***), and percentage tumor directly irradiated (R 2 = 0.7153, Fstat = 10.05*; z-test =-3.837***) per the linear regression and Cox Proportional Hazards models, respectively. Tumor response is linearly proportional to valley (minimum) doses

Measuring changes in tumor volume using anatomical imaging weeks to months post radiation therapy (RT) is currently the clinical standard for indicating treatment response to RT. For patients whose tumors do not respond successfully to treatment, this approach is suboptimal as timely modification of the treatment approach may lead to better clinical outcomes. We propose to use tumor microvasculature as a biomarker for early assessment of tumor response to RT. Acoustic angiography is a novel contrast ultrasound imaging technique that enables high-resolution microvascular imaging and has been shown to detect changes in microvascular structure due to cancer growth. Data suggest that acoustic angiography can detect longitudinal changes in the tumor microvascular environment that correlate with RT response.Methods: Three cohorts of Fisher 344 rats were implanted with rat fibrosarcoma tumors and were treated with a single fraction of RT at three dose levels (15 Gy, 20 Gy, and 25 Gy) at a dose rate of 300 MU/min. A simple treatment condition was chosen for testing the feasibility of our imaging technique. All tumors were longitudinally imaged immediately prior to and after treatment and then every 3 days after treatment for a total of 30 days. Both acoustic angiography (using in-house produced microbubble contrast agents) and standard b-mode imaging was performed at each imaging time point using a pre-clinical Vevo770 scanner and a custom modified dual-frequency transducer.Results: Results show that all treated tumors in each dose group initially responded to treatment between days 3-15 as indicated by decreased tumor growth accompanied with decreased vascular density. Untreated tumors continued to increase in both volume and vascular density until they reached the maximum allowable size of 2 cm in diameter. Tumors that displayed complete control (no tumor recurrence) continued to decrease in size and vascular density, while tumors that progressed after the initial response presented an increase in tumor volume and volumetric vascular density. The increase in tumor volumetric vascular density in recurring tumors can be detected 10.25 ± 1.5 days, 6 ± 0 days, and 4 ± 1.4 days earlier than the measurable increase in tumor volume in the 15, 20, and 25 Gy dose groups, respectively. A dose-dependent growth rate for tumor recurrence was also observed.Conclusions: In this feasibility study we have demonstrated the ability of acoustic angiography to detect longitudinal changes in vascular density, which was shown to be a potential biomarker for tumor response to RT.

Cancer affects 39.6% of Americans at some point during their lifetime. Solid tumor microenvironments are characterized by a disorganized, leaky vasculature that promotes regions of low oxygenation (hypoxia). Tumor hypoxia is a key predictor of poor treatment outcome for all radiotherapy (RT), chemotherapy and surgery procedures, and is a hallmark of metastatic potential. In particular, the radiation therapy dose needed to achieve the same tumor control probability in hypoxic tissue as in normoxic tissue can be up to 3 times higher. Even very small tumors (<2–3 mm3) comprise 10–30% of hypoxic regions in the form of chronic and/or transient hypoxia fluctuating over the course of seconds to days. We investigate the potential of recently developed lipid-stabilized oxygen microbubbles (OMBs) to improve the therapeutic ratio of RT. OMBs, but not nitrogen microbubbles (NMBs), are shown to significantly increase dissolved oxygen content when added to water in vitro and increase tumor oxygen levels in vivo in a rat fibrosarcoma model. Tumor control is significantly improved with OMB but not NMB intra-tumoral injections immediately prior to RT treatment and effect size is shown to depend on initial tumor volume on RT treatment day, as expected.

GRID directs alternating regions of high- and low-dose radiation at tumors. A large animal model mimicking the geometries of human treatments is needed to complement existing rodent systems (eg, microbeam) and clarify the physical and biological attributes of GRID. A pilot study was undertaken in pet dogs with spontaneous soft tissue sarcomas to characterize responses to GRID. Subjects were treated with either 20 Gy (3 dogs) or 25 Gy (3 dogs), delivered using 6 MV X-rays and a commercial GRID collimator. Acute toxicity and tumor responses were assessed 2, 4, and 6 weeks later. Acute Radiation Therapy Oncology Group grade I skin toxicity was observed in 3 of the 6 dogs; none experienced a measurable response, per Response Evaluation Criteria in Solid Tumors. Serum vascular endothelial growth factor, tumor necrosis factor α, and secretory sphingomyelinase were assayed at baseline, 1, 4, 24, and 48 hours after treatment. There was a trend toward platelet-corrected serum vascular endothelial growth factor concentration being lower 1 and 48 hours after GRID than at baseline. There was a significant decrease in secretory sphingomyelinase activity 48 hours after 25 Gy GRID (P = .03). Serum tumor necrosis factor α was quantified measurable at baseline in 4 of the 6 dogs and decreased in each of those subjects at all post-GRID time points. The new information generated by this study includes the observation that high-dose, single fraction application of GRID does not induce measurable reduction in volume of canine soft tissue sarcomas. In contrast to previously published data, these data suggest that GRID may be associated with at least short-term reduction in serum concentration of vascular endothelial growth factor and serum activity of secretory sphingomyelinase. Because GRID can be applied safely, and these tumors can be subsequently surgically resected as part of routine veterinary care, pet dogs with sarcomas are an appealing model for studying the radiobiologic responses to spatially fractionated radiotherapy.

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Background: Minibeam radiation therapy is an experimental radiation therapy utilizing an array of parallel submillimeter planar X-ray beams. In preclinical studies, minibeam radiation therapy has been shown to eradicate tumors and cause significantly less damage to normal tissue compared to equivalent radiation doses delivered by conventional broadbeam radiation therapy, where radiation dose is uniformly distributed. Methods: Expanding on prior studies that suggested minibeam radiation therapy increased perfusion in tumors, we compared a single fraction of minibeam radiation therapy (peak dose:valley dose of 28 Gy:2.1 Gy and 100 Gy:7.5 Gy) and broadbeam radiation therapy (7 Gy) in their ability to enhance tumor delivery of PEGylated liposomal doxorubicin and alter the tumor microenvironment in a murine tumor model. Plasma and tumor pharmacokinetic studies of PEGylated liposomal doxorubicin and tumor microenvironment profiling were performed in a genetically engineered mouse model of claudin-low triple-negative breast cancer (T11). Results: Minibeam radiation therapy (28 Gy) and broadbeam radiation therapy (7 Gy) increased PEGylated liposomal doxorubicin tumor delivery by 7.1-fold and 2.7-fold, respectively, compared to PEGylated liposomal doxorubicin alone, without altering the plasma disposition. The enhanced tumor delivery of PEGylated liposomal doxorubicin by minibeam radiation therapy is consistent after repeated dosing, is associated with changes in tumor macrophages but not collagen or angiogenesis, and nontoxic to local tissues. Our study indicated that the minibeam radiation therapy’s ability to enhance the drug delivery decreases from 28 to 100 Gy peak dose. Discussion: Our studies suggest that low-dose minibeam radiation therapy is a safe and effective method to significantly enhance the tumor delivery of nanoparticle agents.

2728 Abstract 29 Purpose: To identify key dosimetric parameters that have close associations with tumor treatment 30 response and body weight change in SFRT treatments with a large range of spatial-fractionation 31 scale at dose rates of several Gy/min. 32 Methods: Six study arms using uniform tumor radiation, half-tumor radiation, 2mm beam array 33 radiation, 0.3mm minibeam radiation, and an untreated arm were used. All treatments were 34 delivered on a 320kV x-ray irradiator. Forty-two female Fischer 344 rats with fibrosarcoma tumor 35 allografts were used. Dosimetric parameters studied are peak dose and width, valley dose and 36 width, peak-to-valley-dose-ratio, volumetric average dose, percentage volume directly irradiated, 37 and tumor-and normal-tissue EUD. Animal survival, tumor volume change, and body weight 38 change (indicative of treatment toxicity) are tested for association with the dosimetric parameters 39 using linear regression and Cox Proportional Hazards models.

Background: Recent meta-analyses have reported that the tumor delivery of nanoparticles (NPs) is lower than expected and inefficient. Thus, there is a strong need to develop new methods to enhance the tumor delivery of NPs without increasing toxicity. Microbeam radiation therapy (MRT) is an experimental therapy utilizing an array of parallel microplaner X-ray beams to deliver periodically oscillating high and low dose regions in the treatment volume. MRT has been shown to eradicate tumors and causes significantly less toxicity compared to equivalent radiation doses delivered by conventional broadbeam radiation therapy (BRT). Our prior studies reported that MRT significantly altered tumor microvasculature and enhanced the tumor delivery of PEGylated liposomal doxorubicin (PLD) compared to PLD alone in mice (Chang et al. AACR 2015). In this study we compared MRT and the clinically widely used BRT in their ability to enhance the tumor delivery of PLD in a preclinical murine tumor model. Methods: Plasma and tumor PK studies of PLD were performed in a genetically engineered mouse model of claudin-low triple-negative breast cancer (T11) in 4 treatment regimens: 1) PLD alone, 2) BRT 28 Gy + PLD, 3) MRT 28 Gy + PLD, 4) MRT 100 Gy + PLD. Mice received a single radiation treatment 24 h prior to administration of PLD at 6 mg/kg IVP x 1 via a tail vein. Following administration of PLD, mice were harvested at 5 min and 24 h. Encapsulated and released doxorubicin concentrations (conc) in plasma and sum total (encapsulated + released) doxorubicin conc in tumor were measured by HPLC with fluorescence. Results: The mean ± SD conc of sum total doxorubicin in tumor at 24 h after administration of PLD alone, BRT 28 Gy + PLD, MRT 28 Gy + PLD, and MRT 100 Gy + PLD were 2,575 ± 459, 8,601 ± 1,552*, 7,579 ± 4,428, and 12,911 ± 3,445* ng/mL, respectively (*P&lt;0.05 vs PLD alone). In addition, the ratio of tumor sum total conc to plasma encapsulated conc at 24 h for PLD alone, BRT 28 Gy + PLD, MRT 28 Gy + PLD, and MRT 100 Gy + PLD were 0.21 ± 0.04, 0.51 ± 0.17*, 0.39 ± 0.22, and 1.45 ± 0.70*, respectively (*P&lt;0.05 vs PLD alone). The exposures of sum total doxorubicin in tumor at 5 min were similar in all groups. The plasma PK of PLD was also similar in all groups. Conclusions: MRT and BRT administered 24 h prior to PLD increased the tumor exposure of sum total doxorubicin compared to PLD alone with 100Gy MRT having the greatest increase in tumor delivery of PLD. In addition, MRT exhibits significantly lower toxicity to normal tissues in comparison to BRT allowing for enhanced PLD tumor delivery with low toxicity. Studies are ongoing to evaluate the mechanism(s) for the enhanced tumor uptake of PLD induced by MRT. Future studies include investigating dose dependence of MRT-induced tumor delivery enhancement and effects on other NP agents and tumor models. Citation Format: Sha X. Chang, Judith N. Rivera, Leah B. Herity, Lauren S. Price, Andrew J. Madden, Jose R. Roques, Charlene Santos, David Darr, William C. Zamboni. Comparison of microbeam versus conventional broadbeam radiation therapy on tumor delivery enhancement of PEGylated liposomal doxorubicin in a triple negative breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5051. doi:10.1158/1538-7445.AM2017-5051