B Wang

Local Tumor Control and Normal Tissue Toxicity of Pulsed Low-Dose Rate Radiotherapy for Recurrent Lung Cancer An In Vivo Animal Study

Objectives: This study investigates (1) local tumor control and (2) normal tissue toxicity of pulsed low-dose rate radiotherapy (PLDR) for recurrent lung cancer. Methods: For study 1, nude mice were implanted with A549 tumors and divided into the following 3 groups: (1) control (n = 10), (2) conventional radiotherapy (RT; n = 10), and (3) PLDR (n = 10). Tumor-bearing mice received 2 Gy daily dose for 2 consecutive days. Weekly magnetic resonance imaging was used for tumor growth monitoring. For study 2, 20 mice received 8 Gy total body irradiation either continuously (n = 10) or 40 × 0.2 Gy pulses with 3-minute intervals (n = 10). Results: For study 1, both conventional RT and PLDR significantly inhibited the growth of A549 xenografts compared with the control group (>35% difference in the mean tumor volume; P < .05). The PLDR results were slightly better than conventional RT (8% difference in the mean tumor volume; P > .05). For study 2, the average weight was 20.94 ± 1.68 g and 25.69 ± 1.27 g and the survival time was 8 days and 12 days for mice treated with conventional RT and PLDR (P < .05), respectively. Conclusion: This study showed that PLDR could control A549 tumors as effectively as conventional RT, and PLDR induced much less normal tissue toxicity than conventional RT. Thus, PLDR would be a good modality for recurrent lung cancers. Advances in Knowledge: This article reports our results of an in vivo animal investigation of PLDR for the treatment of recurrent cancers, which may not be eligible for treatment because of the dose limitations on nearby healthy organs that have been irradiated in previous treatments. This was the first in vivo study to quantify the tumor control and normal tissue toxicities of PLDR using mice with implanted tumors, and our findings provided evidence to support the clinical trials that employ PLDR treatment techniques.

Tissue TGF-β expression following conventional radiotherapy and pulsed low-dose-rate radiation

ABSTRACT The release of inflammatory cytokines has been implicated in the toxicity of conventional radiotherapy (CRT). Transforming growth factor β (TGF-β) has been suggested to be a risk marker for pulmonary toxicity following radiotherapy. Pulsed low-dose rate radiotherapy (PLDR) is a technique that involves spreading out a conventional radiotherapy dose into short pulses of dose with breaks in between to reduce toxicities. We hypothesized that the more tolerable toxicity profile of PLDR compared with CRT may be related to differential expression of inflammatory cytokines such as TGF-β in normal tissues. To address this, we analyzed tissues from mice that had been subjected to lethal doses of CRT and PLDR by histology and immunohistochemistry (IHC). Equivalent physical doses of CRT triggered more cellular atrophy in the bone marrow, intestine, and pancreas when compared with PLDR as indicated by hematoxylin and eosin staining. IHC data indicates that TGF-β expression is increased in the bone marrow, intestine, and lungs of mice subjected to CRT as compared with tissues from mice subjected to PLDR. Our in vivo data suggest that differential expression of inflammatory cytokines such as TGF-β may play a role in the more favorable normal tissue late response following treatment with PLDR.

MO-FG-CAMPUS-JeP3-05: Evaluation of 4D CT-On-Rails Target Localization Methods for Free Breathing Liver Stereotactic Body Radiotherapy (SBRT)

PURPOSE Liver SBRT patients unable to tolerate breath-hold for radiotherapy are treated free-breathing with image guidance. Target localization using 3D CBCT requires extra margins to accommodate the respiratory motion. The purpose of this study is to evaluate the accuracy and reproducibility of 4D CT-on-rails in target localization for free-breathing liver SBRT. METHODS A Siemens SOMATOM CT-on-Rails 4D with Anzai Pressure Belt system was used both as the simulation and the localization CT. Fiducial marker was placed close to the center of the target prior to the simulation. Amplitude based sorting was used in the scan. Eight or sixteen phases of reconstructed CT sets (depends on breathing pattern) can be sent to Velocity to create the maximum intensity projection (MIP) image set. Target ITV and fiducial ITV were drawn based on the MIP image. In patient localization, a 4D scan was taken with the same settings as the sim scan. Images were registered to match fiducial ITVs. RESULTS Ten liver cancer patients treated for 50Gy over 5 fractions, with amplitudes of breathing motion ranging from 4.3-14.5 mm, were analyzed in this study. Results show that the Intra & inter fraction variability in liver motion amplitude significantly less than the baseline inter-fraction shifts in liver position. 90% of amplitude change is less than 3 mm. The differences in the D99 and D95 GTV dose coverage between the 4D CT-on-Rails and the CBCT plan were small (within 5%) for all the selected cases. However, the average PTV volume by using the 4D CT-on-Rails is 37% less than the CBCT PTV volume. CONCLUSION Simulation and Registration using 4D CT-on-Rails provides accurate target localization and is unaffected by larger breathing amplitudes as seen with 3D CBCT image registration. Localization with 4D CT-on-Rails can significantly reduce the PTV volume with sufficient tumor.

WE-AB-BRA-01: Biodistribution of Paclitaxel-Loaded Nanodroplets for Prostate Cancer Management Using Ultrasound-Mediated Drug Delivery Under MRGuidance

PURPOSE To study the biodistribution of paclitaxel-loaded nanodroplets in vivo in order to evaluate the efficacy of focused ultrasound (FUS)-mediated drug delivery under MR-guidance to prostate tumor. METHODS Poly {ethylene oxide}-co-poly {D, L-lactide} (PDLA) nanodroplets loaded with fluorescently labeled-paclitaxel (F-PTX) were synthesized using solid dispersion technique. Human prostate cancer, LNCaP cells were implanted orthotopically in prostates of male nude mice. Tumor-bearing mice (n=3) were injected with 0.1% F-PTX, 2% PDLA nanodroplets using tail vein. At chosen time points (30min; 2h; 4h; 6h; 12h; 24h) animals were anesthetized, blood was collected by eye bleed, animals were sacrificed, tumor and vital organs (liver, spleen, lung and heart) were excised, cut and weighted. Then the organ was homogenized, incubated in lysis buffer and centrifuged. The lysates were read using fluorescence spectrophotometer (Excitation- 496nm, Emission- 524nm). Fluorescence readings were compared with values from the standard calibration curve. Tumor-bearing mice (n=3) were also injected with 0.1% F-PTX solution (fluorescent drug in free form). RESULTS Quantitative analysis showed 20% of the injected drug-loaded nanodroplets in the tumor, 30 min after systemic injection; which increased to 30% after 2h; 35% after 4h; then decreased to 27% after 6h; 11% after 12h and 5% after 24h. When drug in free form was injected, drug accumulation in the tumor was 7% within 30mnt; 8% within 2h; 10% within 4h; 9% within 6h, 3% within 12h and 2% within 24h. Liver showed major drug accumulation for drug-loaded nanodroplets while heart and lung showed less. For drug in free form, liver and spleen showed maximum drug concentrations while heart showed concentrations similar to that in tumor. CONCLUSION Present work indicates that the optimum time for applying focused ultrasound is after 4h of systemically injecting drug-loaded nanodroplets. This would increase treatment efficacy and provide preclinical data for FUS-mediated drug delivery under MR-guidance.

WE-AB-BRA-02: Study of the Therapeutic Effects of Pulsed Focused Ultrasound Combined with Encapsulated Chemotherapeutic Agents for Prostate Cancer in Vivo

PURPOSE To investigate the improvement of prostate cancer inhibition by combining pulsed focused ultrasound (pFUS) exposures and chemotherapeutic agents encapsulated nanodroplets under MR guidance for prostate cancer therapy. METHODS Prostate cancer (LNCaP) cells were implanted orthotopically. First we developed nanodroplets encapsulated with chemotherapeutic agents for both paclitaxel (PTX) and docetaxel (DTX). Secondly, tumor-bearing mice were randomly divided into 5 groups (n=5). Group 1 animals were treated with an i.v injection of DTX-encapsulated nanodroplets (DTX-ND) + pFUS. Group 2 were treated with pFUS alone. Group 3 were injected (i.v) with DTX-ND alone, Group 4 received free DTX and Group 5 was used as control. Ultrasound treatment parameters were 1MHz, 25W acoustic power, 10% duty cycle and 60 seconds for each sonication. After treatment, animals were allowed to survive for 4 weeks. Tumor volumes were measured on MRI. Third, we repeated the experiment with PTX-ND. Finally we performed study on biodistribution of PTX-ND for prostate cancer and the treatment effects on tumor growth delay are being evaluated. RESULTS With DTX-ND, significant tumor growth delay was observed in Group 1 with p=0.039. There was no significant tumor growth delay observed for Group 2 (p=0.477), Group 3 (p=0.209) and Group 4 (p=0.476). The results are consistent with earlier PTX-ND studies in which significant tumor growth delay was observed in Group 1 with p=0.004. There was no significant tumor growth delay observed for Group 2 (p=0.285), Group 3 (p=0.452) and Group 4 (p=0.158). The peak of drug update in tumor appeared at 4h after injection in the biodistribution study. CONCLUSION Our results showed a great potential of targeted nanodroplets for prostate cancer therapy, which could be activated by pFUS. Our study also suggested that the optimal timing for applying pFUS is 4h after i.v injection of PTX-ND and the treatment effect is being evaluated.

SU-G-IeP4-11: Monitoring Tumor Growth in Subcutaneous Murine Tumor Model in Vivo: A Comparison Between MRI and Small Animal CT

PURPOSE The purpose of the study is to compare the volume measurement of subcutaneous tumors in mice with different imaging platforms, namely a GE MRI and a Sofie-Biosciences small animal CT scanner. METHODS A549 human lung carcinoma cells and FaDu human head and neck squamous cell carcinoma cells were implanted subcutaneously into flanks of nude mice. Three FaDu tumors and three A549 tumors were included in this study. The MRI scans were done with a GE Signa 1.5 Tesla MR scanner using a fast T2-weighted sequence (70mm FOV and 1.2mm slice thickness), while the CT scans were done with the CT scanner on a Sofie-Biosciences G8 PET/CT platform dedicated for small animal studies (48mm FOV and 0.2mm slice thickness). Imaging contrast agent was not used in this study. Based on the DICOM images from MRI and CT scans, the tumors were contoured with Philips DICOM Viewer and the tumor volumes were obtained by summing up the contoured area and multiplied by the slice thickness. RESULTS The volume measurements based on the CT scans agree reasonably with that obtained with MR images for the subcutaneous tumors. The mean difference in the absolute tumor volumes between MRI- and CT-based measurements was found to be -6.2% ± 1.0%, with the difference defined as (VMR - VCT)*100%/VMR. Furthermore, we evaluated the normalized tumor volumes, which were defined for each tumor as V/V0 where V0 stands for the volume from the first MR or CT scan. The mean difference in the normalized tumor volumes was found to be 0.10% ± 0.96%. CONCLUSION Despite the fact that the difference between normal and abnormal tissues is often less clear on small animal CT images than on MR images, one can still obtain reasonable tumor volume information with the small animal CT scans for subcutaneous murine xenograft models.

SU-F-T-674: In Vitro Study of 5-Aminolevulinic Acid-Mediated Photo Dynamic Therapy in Human Cancer Cell Lines

PURPOSE Photodynamic therapy (PTD) is a promising cancer treatment modality. 5-sminolevulinic acid (ALA) is a clinically approved photosensitizer. Here we studied the effect of 5-ALA administration with irradiation on several cell lines in vitro. METHODS Human head and neck (FaDu), lung (A549) and prostate (LNCaP) cancer cells (104/well) were seeded overnight in 96-well plates (Figure 1). 5-ALA at a range from 0.1 to 30.0mg/ml was added to confluent cells 3h before irradiation in 100ul of culture medium. 15MV photon beams from a Siemens Artiste linear accelerator were used to deliver 2 Gy dose in one fraction to the cells. Cell viability was evaluated by WST1 assay. The development of orange color was measured 3h after the addition of WST-1 reagent at 450nm on an Envision Multilabel Reader (Figure 2) and directly correlated to cell number. Control, untreated cells were incubated without 5-ALA. The experiment was performed twice for each cell line. RESULTS The cell viability rates for the head and neck cancer line are shown in Figure 3. FaDu cell viability was reduced significantly to 36.5% (5-ALA) and 18.1% (5-ALA + RT) only at the highest concentration of 5-ALA, 30mg/ml. This effect was observed in neither A549, nor LNCaP cell line. No toxicity was detected at lower 5-ALA concentrations. CONCLUSION Application of 5-ALA and subsequent PDT was found to be cytotoxic at the highest dose of the photosensitizer used in the FaDu head and neck cell line, and their effect was synergistic. Further efforts are necessary to study the potential therapeutic effects of 5-ALA PTD in vitro and in vivo. Our results suggest 5-ALA may improve the efficacy of radiotherapy by acting as a radiomediator in head and neck cancer.

SU‐F‐J‐225: Histology Study of MR Guided Pulsed Focused Ultrasound On Treatment of Prostate Cancer in Vivo

PURPOSE Our previous study demonstrated significant tumor growth delay in the mice treated with pulsed high intensity focused ultrasound (pHIFU). The purpose of this study is to understand the cell killing mechanisms of pHIFU. METHODS Prostate cancer cells (LNCaP), were grown orthotopically in 17 nude mice. Tumor-bearing mice were treated using pHIFU with an acoustic power of 25W, pulse width 100msec and 300 pulses in one sonication under MR guidance. Mutiple sonications were used to cover the whole tumor volume. The temperature (less than 40 degree centigrade in the focal spot) was monitored using MR thermometry. Animals were euthanized at pre-determined time points (n=2) after treatment: 0 hours; 6 hrs; 24 hrs; 48 hrs; 4 days and 7 days. Two tumorbearing mice were used as control. Three tumor-bearing mice were treated with radiation (RT, 2 Gy) using 6 MV photon beams. RT treated mice were euthanized at 0 hr, 6 hrs and 24 hrs. The tumors were processed for immunohistochemical (IHC) staining for PARP (a surrogate of apoptosis). A multispectral imaging analysis system was used to quantify the expression of PARP staining. Cell apoptosis was calculated based on the PARP expression level using the DAB analysis software. RESULTS Our data showed that PARP related apoptosis peaked at 48 hrs and 7 days in pHIFU treated mice, which is comparable to that for the RT group at 24 hrs. The preliminary results from this study were consistent with our previous study on tumor growth delay using pHIFU. CONCLUSION Our results demonstrated that non-thermal pHIFU increased apoptotic tumor cell death through the PARP related pathway. MR guided pHIFU may have a great potential as a safe, noninvasive treatment modality for cancer therapy. This treatment modality may synergize with PARP inhibitors to achieve better therapeutic result.

SU‐F‐J‐215: Non‐Thermal Pulsed High Intensity Focused Ultrasound Therapy Combined with 5‐Aminolevulinic Acid: An in Vivo Pilot Study

PURPOSE It has recently been shown that non-thermal pulsed high intensity focused ultrasound (pHIFU) has a cell-killing effect. The purpose of the study is to investigate the sonosensitizing effect of 5-Aminolevulinic Acid (5-ALA) in non-thermal pHIFU cancer therapy. METHODS FaDu human head and neck squamous cell carcinoma cells were injected subcutaneously in the flanks of nude mice. After one to two weeks, the tumors reached the volume of 112 ± 8 mm3 and were assigned randomly into a non-thermal pHIFU group (n=9) and a non-thermal sonodynamic therapy (pHIFU after 5-ALA administration) group (n=7). The pHIFU treatments (parameters: 1 MHz frequency; 25 W acoustic power; 0.1 duty cycle; 60 seconds duration) were delivered using an InSightec ExAblate 2000 system with a GE Signa 1.5T MR scanner. The mice in the non-thermal sonodynamic group received 5-ALA tail-vein injection 4 hours prior to the pHIFU treatment. The tumor growth was monitored using the CT scanner on a Sofie-Biosciences G8 PET/CT system. RESULTS The tumors in this study grew very aggressively and about 60% of the tumors in this study developed ulcerations at various stages. Tumor growth delay after treatments was observed by comparing the treated (n=9 in pHIFU group; n=7 in sonodynamic group) and untreated tumors (n=17). However, no statistically significant differences were found between the non-thermal pHIFU and non-thermal sonodynamic group. The mean normalized tumor volume of the untreated tumors on Day 7 after their first CT scans was 7.05 ± 0.54, while the normalized volume of the treated tumors on Day 7 after treatment was 5.89 ± 0.79 and 6.27 ± 0.47 for the sonodynamic group and pHIFU group, respectively. CONCLUSION In this study, no significant sonosensitizing effects of 5-ALA were obtained on aggressive FaDu tumors despite apparent tumor growth delay in some mice treated with non-thermal sonodynamic therapy.

SU‐F‐J‐216: MR‐Guided Non‐Thermal Pulsed Focused Ultrasound for Cancer Therapy

PURPOSE High-intensity focused ultrasound has been investigated for ablative therapy and drug enhancement for gene therapy and chemotherapy. The aim of this work is to explore the feasibility of pulsed focused ultrasound (pFUS) for non-thermal cancer therapy using an in vivo animal model. METHODS An InSightec ExAblate 2000 with a 1.5T GE MR scanner was used in this study. Suitable ultrasound parameters were investigated to perform non-thermal sonications. LNCaP human prostate tumor cells (106) were injected orthotopically in the prostates of nude mice (n = 16). When tumors reached the volume of 88 ± 18 mm3 as measured on MRI, the tumor-bearing mice (n = 10) were treated with pFUS (1 MHz frequency; 25 W acoustic power; 0.1 duty cycle; 60 sec duration) 4 times in 2 weeks. A total of 6-12 sonications were used to cover the entire tumor volume under MR image guidance. The animals were allowed to survive for 4 weeks after the initial treatment. The tumor volume was measured on MRI weekly post treatment and was compared with that of the control group (n = 6). RESULTS Significant tumor growth delay was observed in the tumor-bearing mice treated with pFUS. The mean tumor volume for the pFUS treated mice grew from 1 to 1.18 and 1.81 at 1 week and 2 weeks after the initial treatment, respectively, while the mean tumor volume of the control mice grew from 1 to 1.67 and 2.78, respectively, over the same time periods. These results are statistically significant and are consistent with our previous findings. CONCLUSION Our results demonstrated that non-thermal pFUS has a great potential for cancer therapy. Further experiments are needed to understand the cell killing mechanisms of pFUS and to derive optimal ultrasound parameters and fractionation schemes to maximize the therapeutic effect of pFUS.